DNA Sequence Of Gene Research Articles

Prevalence, Transmission and Genetic Diversity of Pyrazinamide Resistance Among Multidrug-Resistant Mycobacterium tuberculosis Isolates in Hunan, China.

China is a country with a burden of high rates of both TB and multidrug-resistant TB (MDR-TB). However, published data on pyrazinamide (PZA) resistance are still limited in Hunan province, China. This study investigated the prevalence, transmission, and genetic diversity of PZA resistance among multidrug-resistant Mycobacterium tuberculosis isolates in Hunan province. Drug susceptibility testing (DST) with the Bactec MGIT 960 PZA kit and pyrazinamidase (PZase) testing were conducted on all 298 MDR clinical isolates. Moreover, 24-locus MIRU-VNTR and DNA sequencing of pncA, rpsA, and panD genes were conducted on 180 PZA-resistant (PZA-R) isolates. The prevalence of PZA resistance among MDR-TB strains reached 60.4%. Newly diagnosed PZA-R TB patients and clustered isolates with identical pncA, rpsA, and panD mutations showed that transmission of PZA-R isolates played a significant role in the formation of PZA-R TB. Ninety-eight mutation patterns were observed in the pncA among 180 PZA-R isolates, and seventy-one (72.4%) were point mutations. Twenty-four of these mutations are new, including 2 base substitutions (V93G and T153S) and 22 nucleotide deletions or insertions. The W119C was found in PZA-S isolates, on the other hand, F94L and V155A mutations were found in both PZA resistant and susceptible isolates with positive PZase activity, indicating that they were not associated with PZA resistance. This is not entirely in line with the WHO catalogue. Ten novel rpsA mutations were found in 10 PZA-R isolates, which all combined with mutations in pncA. Thus, it is unpredictable whether these mutations in rpsA can impact PZA resistance. No panD mutation was found in all PZA-R isolates. DNA sequencing of pncA and PZase activity testing have great potential in predicting PZA resistance.

One novel single nucleotide polymorphism c.424A>G on A1.02 allele in ABO glycosyltransferases leads to Aweak phenotype

BackgroundThe dysfunction of the ABO glycosyltransferase (GT) enzyme, which is caused by mutations in the ABO gene, can lead to weak ABO phenotypes. In this study, we have discovered a novel weak ABO subgroup allele and investigated the underlying mechanism to causing its Aweak phenotype. Materials and methodsThe ABO phenotyping and genotyping were performed by serological studies and direct DNA sequencing of ABO gene. The role of the novel single nucleotide polymorphism (SNP) was evaluated by 3D model, predicting protein structure changes, and in vitro expression assay. The total glycosyltransferase transfer capacity in supernatant of transfected cells was examined. ResultsThe results of serological showed the subject was Aweak phenotype. A novel SNP c.424A > G (p. M142V) based on ABO∗A1.02 was identified, and the genotype of the subject was AW-var/O.01 according to the gene analysis. In silico analysis showed that the SNP c.424A > G on the A allele may change the local conformation by damaging the hydrogen bonds and reduce the stability of GT. In vitro expression study showed that SNP p.M142V impaired H to A antigen conversion, although it did not affect the generation of A glycosyltransferase (GTA). ConclusionOne novel AW allele was identified and the SNP c.424A > G (p.M142V) can cause the Aweak phenotype through damaging the hydrogen bonds and reducing stability of the GTA.

Serum metabolome and gut microbiome alterations are associated with low handgrip strength in older adults.

Handgrip strength (HGS), which represents global muscle strength, is a powerful indicator of disability and mortality in older adults; it is also used for the diagnosis of possible- or probable- sarcopenia and physical frailty. This study aimed to explore the metabolic mechanisms and potential biomarkers associated with declining HGS among older adults. We recruited 15 age- and environment-matched inpatients (age, 77-90 years) with low or normal HGS. Liquid chromatography-mass spectrometry (LC-MS) and 16S ribosomal DNA (rDNA) gene sequencing were performed to analyze the metabolome of serum and stool samples and the gut microbiome composition of stool samples. Spearman's correlation analysis was used to identify the potential serum and fecal metabolites associated with HGS. We assessed the levels of serum and fecal metabolites belonging to the class of cinnamic acids and derivatives and reported that the levels of carboxylic acids and their derivatives decreased in the low-HGS group. Serum levels of microbial metabolites, including cinnamoylglycine, 4-methoxycinnamic acid, and (e)-3,4,5-trimethoxycinnamic acid, were positively correlated with HGS. We found that gut microbial α-diversity was significantly higher in the low-HGS group, whereas higher β-diversity was observed in the normal group. The relative abundances of the genera Parabacteroides and Intestinibacter increased significantly in the low-HGS group and were negatively correlated with the serum levels of cinnamoylglycine. The identified metabolites whose levels were markedly altered, and intestinal flora associated with these metabolites suggest the potential metabolic underpinnings for HGS and provide a basis for the further identification of biomarkers of muscle strength decline in older adults.

First Report of Pseudomonas oryzihabitans Causing Walnut Leaf Spot Disease in China.

China ranks first in the production and harvest area of walnut (Juglans regia L.) worldwide. Currently, the poor health and low yield of walnut caused by pathogen infection is of concern. In 2022, severe walnut leaf spot disease was observed on the seedlings of four walnut nurseries (0.08 to 0.23 ha) in Liaocheng, Shandong, China, with an average incidence of 48.6% (from 34.6% to 65.3% on the cultivar Xiangling). From August to October, leaf spots mainly appeared on the edges of the leaflets, and occasionally between veins. The lesions were initially soft and rotten, and then light brown, round to semi-circular. Subsequently, the adjacent lesions fused, and the edges of the leaflets and entire leaflets showed symptoms of browning and wilting. For pathogen isolation, five leaflets with representative symptoms from one of the nurseries were collected and wiped three times with sterile absorbent cotton dipped in 75% alcohol and washed with distilled water. Leaflet pieces at the junction of the lesion and healthy tissues were removed, crushed in a sterile mortar, and soaked in a small amount of distilled water for 10 min. The diseased tissue suspension was streaked on a nutrient agar medium (NA) with a sterile inoculation ring and incubated at 28°C for 24 to 72 h. The bacterial colonies obtained were further cultured on NA. The purified colonies were uniform in shape, round, and yellow, with a raised, shiny surface and smooth margin. The isolates were Gram-negative, and the electron microscope analysis showed that the pathogens were short rods (0.35 to 0.52 × 0.90 to 1.24 μm, average = 0.44 ± 0.05 × 1.08 ± 0.11 μm, n = 25). For bacterial species identification, a single-colony culture was subjected to genomic DNA extraction and gene amplification and sequencing of 16S rRNA, rpoD, and gyrB. The universal primers 27F/1492R (Lane 1991) were used to amplify the 16S rRNA gene and the specific primers 70F/70R and UP-1E/APrU (Yamamoto et al. 2000) were used to amplify the rpoD and gyrB genes, respectively. In the BLAST analysis, the 16S rRNA sequence (GenBank OR195734) of the isolate shared 99% similarity (1409/1410 bp) with Pseudomonas oryzihabitans strain IAM 1568T (AM262973.1), and the rpoD (OR709708) and gyrB (OR709707) sequences showed >98% identity to rpoD (707/717 bp; FN554494.1) and gyrB (787/801 bp; FN554210.1) of P. oryzihabitans strain LMG 7040T. Based on the above results, the isolated bacterium was identified as P. oryzihabitans. For the pathogenicity test, healthy leaflets from 10 two-year-old potted walnut seedlings (cv. Xiangling) were used as inoculation materials. The leaflets were punctured with a sterile inoculation needle of 0.4 mm, and three small holes on each leaflet at an interval of about 5 mm were covered with a piece of sterile cotton. A bacterial suspension (1 ml) at 107 CFU/ml was spread onto the cotton, and wrapped with plastic film for 24 h. Water was used as a negative control. The inoculations were performed five times. Plants were grown outdoors at a daily average temperature of 22°C with relative humidity over 45%. Two days after inoculation, the disease began to develop in the leaflets with similar symptoms to those observed in the field. In contrast, control plants remained healthy and symptomless. Bacteria were reisolated from the inoculated walnut plants, and the morphology and 16S rRNA gene sequences of the isolates were the same as those of the original strains. Since it was discovered as an opportunistic human pathogenic bacterium in the 1970s (Keikha et al. 2019), P. oryzihabitans has also been shown to cause certain plant diseases, such as panicle blight and grain discoloration on rice (Hou et al. 2020), fruit black rot on prickly ash (Liu et al. 2021), and stem and leaf rot on muskmelon (Li et al. 2021). As far as we know, this is the first report of P. oryzihabitans causing walnut leaf spot disease in China. Leaf spot caused by P. oryzihabitans may be a threat to walnut cultivation, and this report of its occurrence is the first step in determining potential spread and effective control measures.

Next Generation Sequencing in Detecting Oral Cancer Due to Tobacco Consumption

DNA sequence DNA Sequencing is the first step in establishing phylogenetic trees, protein structure prediction, diagnosis of cancer, discovery of drugs and hence its importance cannot be underestimated. DNA sequencing finds its use in the diagnosis of oral squamous cell carcinoma (OSCC). Oral Cancer is the most common occurring malignancies in the world, especially in India where the prevalence for smoking, Areca nut chewing coupled with a lifestyle that encourages these two activities as fashion are left many people diagnosed with OSCC. Patients with this OSCC are more likely unaware of its side effects and over time might suffer from facial deformity. The importance to understanding the symptoms, prevention and treatment of oral cancer is very much essential today. In this paper, we looked at over 2000 odd papers published and look at the correlation between the next Generation DNA sequencing algorithms (NGS) play an important role in diagnosis of OSCC. This is a further study on some of the papers which have highlighted NGS role in OSCC Diagnosis. We did like to see a comprehensive review on the papers published so far. In the discussion, we will see frequently mutated genes in the OSCC, recent discoveries and OSCC treatment based on the findings.

Molecular characterization and phylogenetic position of the giant deep-sea oyster Neopycnodonte zibrowii Gofas, Salas & Taviani, 2009

The giant deep-sea oyster Neopycnodonte zibrowii Gofas, C. Salas & Taviani, 2009 is a keystone deep-sea habitat builder species. Discovered about fifteen years ago in the Azores, it has been described and assigned to the genus Neopycnodonte Fischer von Waldheim, 1835 based on morphological features. In this study, we generated DNA sequence data for both mitochondrial (COI and 16S) and nuclear (ITS2 and 28S) markers based on the holotype specimen of N. zibrowii to establish a molecular phylogenetic framework for the systematic assessment of this species and to provide a reliable (i.e., holotype-based) reference sequence set for multilocus DNA barcoding approaches. Molecular data provide compelling evidence that the giant deep-sea oyster is a distinct species, rather than a deep-water ecophenotype of Neopycnodonte cochlear (Poli, 1795), with extremely high genetic divergence from any other gryphaeid. Multilocus phylogenetic analyses place the giant deep-sea oyster within the clade “Neopycnodonte/Pycnodonte” with closer affinity to N. cochlear rather than to P. taniguchii Hayami & Kase, 1992, thus supporting its assignment to the genus Neopycnodonte. Relationships within this clade are not well supported because mitochondrial variation is inflated by saturation that eroded phylogenetic signal, implying an old split between taxa within this clade. Finally, the set of reference barcode sequences of N. zibrowii generated in this study will be useful for a wide plethora of barcoding applications in deep-sea biodiversity surveys. Molecular validation of recent records of deep-sea oysters from the Atlantic Ocean and the Mediterranean Sea will be crucial to clarify the distribution of N. zibrowii and assess the phenotypic variation and ecology of this enigmatic species.

Association between TP53 gain of function and loss of function mutational subgroups and survival in pancreatic adenocarcinoma.

694 Background: TP53 is a commonly mutated tumor suppressor in pancreatic adenocarcinoma (PDAC) but the clinical implications for different classes of TP53 variants remain unclear. In contrast to loss of function (LOF) mutations, TP53 gain of function (GOF) mutations alter DNA conformational binding (e.g. R175H, G245S, R249S, R282H) or modify DNA contact hotspots (e.g. R248Q, R248W, R273H) and are associated with aggressive phenotypes. Here, we analyze progression-free survival (PFS) on standard therapies and overall survival (OS) of PDAC patients (pts) within TP53 mutational subgroups: GOF, LOF, and wild type (WT). Methods: We analyzed longitudinal outcomes across 775 pts with next generation DNA sequencing (NGS) results from Perthera’s Real-World Evidence database who received at least 1 line of therapy in the advanced setting for PDAC. PFS was evaluated from initiation of 1st line for advanced disease until discontinuation. Hazard ratios and p-values were computed via Cox regression when comparing OS relative to date of advanced diagnosis and PFS on subsets who received 1st line FOLFIRINOX (FFX) or 1st line gemcitabine/nab-paclitaxel (GA). Differences in frequencies of genomic alterations between TP53 mutational subgroups were analyzed by Fisher’s exact test. Results: In the TP53 LOF subgroup, median PFS on 1st line GA was significantly worse than 1st line FFX (Table) but this difference was not observed in TP53 GOF or WT subgroups. Irrespective of 1st line therapy choice, median OS in the WT subgroup (2.1y [1.8-2.4]) was significantly longer (p<0.05) than pts with TP53 GOF (1.5y [1.3-2.1]) or TP53 LOF (1.4y [1.3-1.5]). KRAS mutations were enriched (unadjusted p<0.05) for co-occurrence with TP53 in GOF (93.7%) and LOF (93.3%) subgroups relative to WT (74.5%). Mutations in BRCA1/2 were less common in the TP53 GOF subgroup (2.5%) relative to LOF (5.1%) and WT (11.6%). ATM mutations as well as an expanded set of genomic alterations within the DDR pathway were also enriched with similar trends (GOF<LOF<WT). Other potentially confounding factors will be discussed in the context of multivariate OS/PFS analyses. Conclusions: TP53 mutations correlated with worse prognosis in advanced PDAC. Potential predictive associations favoring FFX over GA in the TP53 LOF subgroup (but not in GOF or WT subgroups) warrant further exploration. [Table: see text]

Tandem peptide lipid CRISPR-Cas9 complex combating APP and APOE4 gene abnormality in Alzheimer's disease

Alzheimer's attacks 24 million global population and dominates 60-80% of existing cases of dementia. It causes the accumulation of beta-amyloid (Aβ) plaques in the hippocampus and entorhinal cortex, resulting in decreased mass from the brain. Recent studies have shown that the manifestation of this disease is due to an overaccumulation of abnormal Aβ protein due to abnormalities in the APP and APOE4 genes. Point mutations in the APP gene will create the toxic form of Aβ protein, namely Aβ42, and the toxic APOE4 gene will accelerate the onset of Aβ42 deposition and pro-inflammatory activity that exacerbates the degenerative process of the brain. Gene editing as a potential definitive therapy was recently a concern by researchers. CRISPR-Cas9 repairs the APP gene and substitutes the APOE4 gene with APOE3 by modifying the gene's DNA sequence. Nano complex CRISPR-Cas9 tandem peptide lipid is a model for clinicians to target brain nerve cells. In vivo research on an Alzheimer's mouse model proved the potential of nano-complex-based peptides as carriers of CRISPR-Cas9 in brain nerve cells. This engineering technology offers satisfactory results with high precision, minimal side effects, and a relatively low price for long-term therapeutic effects and even a lifetime.

Bacterial diversity in a continuum from supraglacial habitats to a proglacial lake on the Tibetan Plateau.

Mountain glaciers are frequently assessed for their hydrological connectivity from glaciers to proglacial lakes. Ecological process on glacier surfaces and downstream ecosystems have often been investigated separately, but few studies have focused on the connectivity between the different glacial habitats. Therefore, it remains a limited understanding of bacterial community assembly across different habitats along the glacier hydrological continuum. In this study, we sampled along a glacial catchment from supraglacial snow, cryoconite holes, supraglacial runoff, ice-marginal moraine and proglacial lake on the Tibetan Plateau. The bacterial communities in these habitats were analyzed using high-throughput DNA sequencing of the 16S rRNA gene to determine the bacterial composition and assembly. Our results showed that each habitat hosted unique bacterial communities, with higher bacterial α-diversity in transitional habitats (e.g. runoff and ice-marginal moraine). Null model analysis indicated that deterministic processes predominantly shaped bacterial assembly in snow, cryoconite holes and lake, while stochastic process dominantly governed bacterial community in transitional habitats. Collectively, our findings suggest that local environment play a critical role in filtering bacterial community composition within glacier habitats. This study enhances our understanding of microbial assembly process in glacier environments and provides valuable insights into the factors governing bacterial community compositions across different habitats along the glacial hydrological continuum.

Electronic equipment and appliances in special wards of hospitals as a source of azole-resistant Aspergillus fumigatus: a multi-centre study from Iran

Azole-resistant Aspergillus fumigatus (ARAf), reported as a global public health concern, has been unexpectedly observed in different countries. To identify ARAf and detect azole resistance related to the CYP51A mutation in different hospital environmental samples. In this multi-centre study from Iran, surfaces of electronic equipment and appliances from different hospitals in Iran were sampled using cotton swabs. All samples were cultured using azole-containing agar plates (ACAPs). Recovered Aspergillus isolates were identified at the species level using partial DNA sequencing of the β-tubulin gene. The azole susceptibility testing of A.fumigatus isolates was performed using the Clinical and Laboratory Standards Institute M38-A3 guideline. The sequencing of the CYP51A gene was also performed to detect mutations related to resistance. Out of the 693 collected samples, 89 (12.8%) Aspergillus species were recovered from ACAPs. Aspergillus fumigatus (41.6%) was the most prevalent, followed by A.tubingensis (23.6%) and A.niger (15.6%). Among 37 isolates of A.fumigatus, 19 (51.3%) showed high minimum inhibitory concentration (MIC) values to at least one of the three azoles, voriconazole, itraconazole, and posaconazole. CYP51A polymorphisms were detected in all 19 isolates, of which 52.6% showed the TR34/L98H mutation. Other detected mutations were G432C, G448S, G54E/G138C, F46Y, and Y121F/M220I/D255E. T289F and G432C were the first reported mutations in ARAf. There was a considerable level of azole resistance in hospital environmental samples, a serious warning for patients vulnerable to aspergillosis. Our findings have also revealed a different mutation pattern in the CYP51A gene.

Machine learning empowered next generation DNA sequencing: perspective and prospectus.

The pursuit of ultra-rapid, cost-effective, and accurate DNA sequencing is a highly sought after aspect of personalized medicine development. With recent advancements, mainstream machine learning (ML) algorithms hold immense promise for high throughput DNA sequencing at the single nucleotide level. While ML has revolutionized multiple domains of nanoscience and nanotechnology, its implementation in DNA sequencing is still in its preliminary stages. ML-aided DNA sequencing is especially appealing, as ML has the potential to decipher complex patterns and extract knowledge from complex datasets. Herein, we present a holistic framework of ML-aided next-generation DNA sequencing with domain knowledge to set directions toward the development of artificially intelligent DNA sequencers. This perspective focuses on the current state-of-the-art ML-aided DNA sequencing, exploring the opportunities as well as the future challenges in this field. In addition, we provide our personal viewpoints on the critical issues that require attention in the context of ML-aided DNA sequencing.

Monitoring the prevalence of Pseudomonas fluorescens as a spoilage indicator in cow raw milk, teat surfaces, and milk tanks.

Milk and its products are very sensitive to spoilage if they are kept under unsuitable conditions which may provide favorable circumstances for the growth of specific spoilage organisms, Pseudomonas fluorescens accounted as the most dominant indicator for milk spoilage. This study highlights monitoring the prevalence of P. fluorescens as a spoilage indicator organism in cow raw milk and its contact surfaces represented by teat surfaces and milk tanks in Nineveh province. A total of 150 samples from cows' raw milk, teat surfaces, and milk tank swabs were collected from different locations in Nineveh province from October 2023 till February 2024. The Pseudomonas fluorescens were detected by using conventional cultivation methods supported by molecular detection of the target pathogen using the polymerase chain reaction technique. Out of 150 samples, 48 (32%) were positive for the prevalence of P. fluorescens by traditional methods, and 39 (26%) were positive using PCR assay according to the 16SPflu gene yielded a band at 850 bp. The P. fluorescens was recovered at 19 (38%) from raw milk. Teat surfaces revealed a higher isolation rate 11 (22%) compared to milk tanks 9 (18%). The mean counts of Pseudomonas in cows raw milk revealed 4.38, 6.29, and 7.37 log CFU/ml for the 0, 3, and 6 days of storage at chilling temperature. Results of DNA sequencing of the 16SrRNA gene revealed 12 strains recorded in the GenBank nucleotide sequence database. Our results shed light on the risk of P. fluorescens prevalence as a spoilage indicator in raw milk and surrounding surfaces which is inevitable to apply hygienic procedures during milk collecting, processing, and preservation to increase the shelf life of the products and ensure milk safety and consumer health.

Genetic identification and gut content of Gerong fish Osteochilus waandersii from Larai River

Gerong fish, or Osteochilus waandersii, is a popular freshwater fish among the Jakun tribe in Kampung Peta, Johor National Park of EndauRompin, particularly for its use in traditional medicine to alleviate dizziness by consuming its raw gut. Despite its cultural significance, the genetic and feeding behavior of Gerong fish remains unclear. This study aimed to amplify the mitochondrial DNA barcode region of Gerong fish for genetic identification and to identify its gut content. The DNA barcodes were produced using polymerase chain reaction and single-pass DNA sequencing of partial COI gene, and the gut content was observed using stereo microscope. Several five partial COI gene sequences were successfully obtained after DNA sequencing. In addition, the results of BLAST analysis with percent identities of 96.24% to 97.20% along with the phylogenetic inference of maximum likelihood analyses suggested the Gerong fish as Osteochilus waandersii or the Waanders's hard-lipped barb. The sequences were deposited in GenBank (accession numbers MN223396-MN223400). Microscopic observation of the fish guts revealed undigested worms and digested aquatic mosses, known as lumut batuk. These findings mark a significant step forward in understanding the genetics and feeding behavior of the Gerong fish.

Diversity and Species Composition of Midgut Symbiotic Bacteria in Culex quinquefasciatus Mosquitoes in Gampaha District, Sri Lanka.

Mosquitoes, notorious for their deadly impact as disease vectors, also hold economic value owing to their roles in disease transmission. The present study focuses on the importance of understanding mosquito gut microbiota for implementing innovative vector control strategies, thereby mitigating disease transmission. The study was conducted in the Gampaha Medical Office of Health (MOH) area of Sri Lanka with the focus of elucidating the microbial diversity within the midgut of Culex quinquefasciatus, a crucial step to support ongoing paratransgenesis efforts. Sampling was performed by utilizing standard mosquito sampling techniques and their midgut homogenates were plated on Plate Count Agar to isolate bacteria, which were then identified through biochemical tests. Subsequently, the most abundant bacterial families were subjected to DNA extraction, PCR amplification, and gene sequencing for species identification. The study revealed the presence of four bacterial families (Staphylococcaceae, Streptococcaceae, Neisseriaceae, and Moraxellaceae) in adult mosquitoes, while larvae harbored an additional family, Micrococcaceae. Interestingly, the relative distribution of midgut bacteria varied significantly among field-caught larval and adult strains from different study areas (chi-square = 1.673; P < 0.05), indicating similar bacterial flora across mosquito life stages and geographical locations. Of particular interest is the identification of Lysinibacillus sphaericus, a bacterium with potential for paratransgenesis applications. Given the high mosquito density in the study area, leveraging paratransgenesis for Cx. quinquefasciatus control is recommended. Furthermore, insights into gut microbes could inform the integration of gut microflora from modified strains into existing Sterile Insect Technique (SIT) and Incompatible Insect Technique (IIT) approaches in Sri Lanka.

A Microdissection Protocol for Proteogenomic Analysis of Histological Sections to Advance Drug Development.

Combining proteogenomics with laser capture microdissection (LCM) in cancer research offers a targeted way to explore the intricate interactions between tumor cells and the different microenvironment components. This is especially important for immuno-oncology (IO) research where improvements in the predictability of IO-based drugs are sorely needed, and depends on a better understanding of the spatial relationships involving the tumor, blood supply, and immune cell interactions, in the context of their associated microenvironments. LCM is used to isolate and obtain distinct histological cell types, which may be routinely performed on complex and heterogeneous solid tumor specimens. Once cells have been captured, nucleic acids and proteins may be extracted for in-depth multimodality molecular profiling assays. Optimizing the minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, RNA-seq may be performed for gene expression and DNA sequencing performed for the discovery and analysis of actionable mutations, copy number variation, methylation profiles, etc. However, there remains a need for highly sensitive proteomic methods targeting small-sized samples. A significant part of this protocol is an enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. Included is a detailed in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Described is a proteogenomic approach leveraged from microdissected fresh frozen tissue. The protocols may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.

Transcription Factor Binding Site Prediction Using CnNet Approach.

Controlling the gene expression is the most important development in a living organism, which makes it easier to find different kinds of diseases and their causes. It's very difficult to know what factors control the gene expression. Transcription Factor (TF) is a protein that plays an important role in gene expression. Discovering the transcription factor has immense biological significance, however, it is challenging to develop novel techniques and evaluation for regulatory developments in biological structures. In this research, we mainly focus on 'sequence specificities' that can be ascertained from experimental data with 'deep learning' techniques, which offer a scalable, flexible and unified computational approach for predicting transcription factor binding. Specifically, Multiple Expression motifs for Motif Elicitation (MEME) technique with Convolution Neural Network (CNN) named as CnNet, has been used for discovering the 'sequence specificities' of DNA gene sequences dataset. This process involves two steps: a) discovering the motifs that are capable of identifying useful TF binding site by using MEME technique, and b) computing a score indicating the likelihood of a given sequence being a useful binding site by using CNN technique. The proposed CnNet approach predicts the TF binding score with much better accuracy compared to existing approaches. The source code and datasets used in this work are available at https://github.com/masoodbai/CnNet-Approach-for-TFBS.git.

Clinicogenomic associations in patients with Langerhans cell histiocytosis: a cohort study

This article presents an analysis of genotype and phenotype correlations in a pediatric cohort with Langerhans cell histiocytosis (LCH). Approximately 60% of LCH patients carry BRAF V600E somatic point mutation. Numerous studies have demonstrated that the presence of this mutation is associated with a more severe course of the disease and a higher risk of relapse. Apart from BRAF V600E mutation, other mutations in genes involved in the RAS/RAF/MEK/ERK signaling pathway have been identified in LCH. Next generation sequencing of DNA with high coverage of target regions is considered an optimal approach to detect somatic mutations other than BRAF V600E. The identification of somatic driver mutations holds great potential for enhancing our understanding of the phenotypic heterogeneity of LCH and developing targeted therapeutic strategies. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology.

The occurrence of cyst nematodes in agricultural fields of Southern Manitoba

In southern Manitoba, Canada, a survey was carried out in 2012 and 2013 to determine the presence of Heteroderidae cyst-forming plant-parasitic nematodes, with a focus on the soybean cyst nematode (SCN) (Heterodera glycines). A total of 48 fields having grown soybean were sampled. A modified Fenwick elutriation-flotation technique was used to extract cysts with a 75% cyst recovery efficiency. Cyst population density averaged 0.9 cysts kg−1 soil, with a total of 65 cysts recovered. Preliminary screening of cysts, based on general body shape and vulval cone top structure, showed the presence of cysts belong to circumfenestrate, and ambifenestrate groups of cyst-forming nematodes. Limited morphological data was accessible due to poor quality or insufficient cysts for analysis; however, generated DNA sequences for nuclear rDNA ITS and D2-D3 expansion region of the 28S rRNA were obtained for four samples and matched sequences in GenBank for the cyst nematodes Cactodera milleri, C. torreyanae, C. weissi, C. estonica and unknown Cactodera species. Only one of the ambifenestrate cysts with a cone top structure of Heterodera species yielded DNA for analysis and its identification was ambiguous for soybean cyst nematode (SCN). None of the cysts were positive through SCN diagnostic PCR. Cactodera is not a pest of soybean or other crops in Manitoba. These cyst nematodes are likely to be naturally associated with weeds and grasses in the sampled fields or may be introduced from neighbouring states of the USA. Further annual surveys are needed and recommended in the near future to encompass more soybean fields and corroborate the absence of the pest.

Epigenetic variation mediated by lncRNAs accounts for adaptive genomic differentiation of the endemic blue mussel Mytiluschilensis

Epigenetic variation affects gene expression without altering the underlying DNA sequence of genes controlling ecologically relevant phenotypes through different mechanisms, one of which is long non-coding RNAs (lncRNAs). This study identified and evaluated the gene expression of lncRNAs in the gill and mantle tissues of Mytilus chilensis individuals from two ecologically different sites: Cochamó (41°S) and Yaldad (43°S), southern Chile, both impacted by climatic-related conditions and by mussel farming given their use as seedbeds. Sequences identified as lncRNAs exhibited tissue-specific differences, mapping to 3.54 % of the gill transcriptome and 1.96 % of the mantle transcriptome, representing an average of 2.76 % of the whole transcriptome. Using a high fold change value (≥|100|), we identified 43 and 47 differentially expressed lncRNAs (DE-lncRNAs) in the gill and mantle tissue of individuals sampled from Cochamó and 21 and 17 in the gill and mantle tissue of individuals sampled from Yaldad. Location-specific DE-lncRNAs were also detected in Cochamó (65) and Yaldad (94) samples. Via analysis of the differential expression of neighboring protein-coding genes, we identified enriched GO terms related to metabolic, genetic, and environmental information processing and immune system functions, reflecting how the impact of local ecological conditions may influence the M. chilensis (epi)genome expression. These DE-lncRNAs represent complementary biomarkers to DNA sequence variation for maintaining adaptive differences and phenotypic plasticity to cope with natural and human-driven perturbations.

Sequence Analysis of Prion Protein Gene in Bhagnari Cattle Breed from the Hottest Region of Sibi, Balochistan, Pakistan

Prion diseases are a type of neurodegenerative disorder caused by the transmission of specific pathogens containing prion proteins. Due to the unique structural characteristics of Prion proteins (PrP), which differ from other types of proteins, the extended incubation period observed in the transmission of specific Prions can be attributed to these differences, at least in part. Prions are found in several other mammals and animals. Prions are unique among false protein folding abnormalities because these are infections and contain various strains of contagious agents associated with a unique in vivo phenotype. They can be acquired either by inheritance or sporadically. There are two types: classical and typical BSP. The objective of this study was to observe the Bhagnari cattle breed of Balochistan, Pakistan, and compare the PrP gene sequence of the Bhagnari with other reported sequences from Pakistan and other parts of the world. This research collected 40 Bhagnari cattle blood samples from Tali, Bhag Nari, and Sibi district areas. DNA extraction of each sample was performed by inorganic method, and then DNA amplification and sequencing of PRNP Gene was performed. The results of this research work showed different polymorphic variations (SNPs) in 16 samples. In this study, while mutations causing prion diseases in cattle were detected in Italian and German breeds, none were identified in the PRNP gene of the cattle population investigated, despite its association with neurological disorders.