Extend the benchmarking indel set by manual review using the individual cell line sequencing data from the Sequencing Quality Control 2 (SEQC2) project.

Accurate indel calling plays an important role in precision medicine. A benchmarking indel set is essential for thoroughly evaluating the indel calling performance of bioinformatics pipelines. A reference sample with a set of known-positive variants was developed in the FDA-led Sequencing Quality Control Phase 2 (SEQC2) project, but the known indels in the known-positive set were limited. This project sought to provide an enriched set of known indels that would be more translationally relevant by focusing on additional cancer related regions. A thorough manual review process completed by 42 reviewers, two advisors, and a judging panel of three researchers significantly enriched the known indel set by an additional 516 indels. The extended benchmarking indel set has a large range of variant allele frequencies (VAFs), with 87% of them having a VAF below 20% in reference Sample A. The reference Sample A and the indel set can be used for comprehensive benchmarking of indel calling across a wider range of VAF values in the lower range. Indel length was also variable, but the majority were under 10 base pairs (bps). Most of the indels were within coding regions, with the remainder in the gene regulatory regions. Although high confidence can be derived from the robust study design and meticulous human review, this extensive indel set has not undergone orthogonal validation. The extended benchmarking indel set, along with the indels in the previously published known-positive set, was the truth set used to benchmark indel calling pipelines in a community challenge hosted on the precisionFDA platform. This benchmarking indel set and reference samples can be utilized for a comprehensive evaluation of indel calling pipelines. Additionally, the insights and solutions obtained during the manual review process can aid in improving the performance of these pipelines.

Phytochemicals and bioactive compounds effective against acute myeloid leukemia: A systematic review.

This systematic review identified various bioactive compounds which have the potential to serve as novel drugs or leads against acute myeloid leukemia. Acute myeloid leukemia (AML) is a heterogeneous hematopoietic malignancy that arises from the dysregulation of cell differentiation, proliferation, and cell death. The risk factors associated with the onset of AML include long-term exposure to radiation and chemicals such as benzene, smoking, genetic disorders, blood disorders, advancement in age, and others. Although novel strategies to manage AML, including a refinement of the conventional chemotherapy regimens, hypomethylating agents, and molecular targeted drugs, have been developed in recent years, resistance and relapse remain the main clinical problems. In this study, three databases, PubMed/MEDLINE, ScienceDirect, and Google Scholar, were systematically searched to identify various bioactive compounds with antileukemic properties. A total of 518 articles were identified, out of which 59 were viewed as eligible for the current report. From the data extracted, over 60 bioactive compounds were identified and divided into five major groups: flavonoids, alkaloids, organosulfur compounds, terpenes, and terpenoids, and other known and emerging bioactive compounds. The mechanism of actions of the analyzed individual bioactive molecules differs remarkably and includes disrupting chromatin structure, upregulating the synthesis of certain DNA repair proteins, inducing cell cycle arrest and apoptosis, and inhibiting/regulating Hsp90 activities, DNA methyltransferase 1, and histone deacetylase 1.

Clinical Profile of Adult Bronchial Asthma Patients Presenting at a Tertiary Care Teaching Institute in Northern India.

BACKGROUND: In the previous four decades there have been remarkable changes and development in the approach toward the diagnosis and management of asthma. There are wide variations in the clinical profile of asthma patients in different parts of a vast country like India due to significant variations in the geography, culture, ethnicity, and socioeconomic profile of the Indian population. In the present study, we have aimed to study the clinical profile of adult asthmatic patients in a tertiary care teaching institute in Northern India. METHODS: In this observational cross-sectional study, a total of 966 asthma patients were included from August 2020 to July 2021 after following strict inclusion and exclusion criteria. After a thorough history and clinical examination, patients were subjected to relevant investigations including spirometry. RESULTS: Our study showed slight female preponderance (51.7%) over males among asthma patients. A maximum number of patients were of comparatively younger age groups and urban. The most common symptom at the time of presentation was breathlessness (94.5%) followed by cough in about 59.8%. Family history was present in about 9.3% of patients. A maximum number of patients presented in the months of November and December and rhinitis was the most commonly associated atopic condition. The majority (65.28%) of previously diagnosed patients had uncontrolled asthma at the time of their first presentation in our department. CONCLUSION: Results of our study endorse the poor awareness in society towards education and management of asthma. Females and comparatively younger patients are more commonly affected. Significant differences in our study from previous studies in different parts of India confirm that the pattern and clinical profile of asthma patients in one region cannot be extrapolated to other regions and the need for future studies in other regions of our country is also required.

A Rare Case of Sternoclavicular Tuberculosis Diagnosed Using Microchip-Based Polymerase Chain Reaction in a Diabetic Female.

Osteoarticular tuberculosis (TB) accounts for almost 10% of all extrapulmonary TB cases. In the majority of cases, the spine, knee, hip, and large bones are involved; other sites like the sternoclavicular joint, elbow, wrist, and smaller joints are infrequently involved. Uncommon locations of extrapulmonary TB pose a challenge in diagnosis due to lack of clinical suspicion, non-availability of samples, and unavailability of suitable diagnostic modalities. Here we report a case of uncommon location of osteoarticular TB diagnosed through microchip-based real-time polymerase chain reaction (PCR).

Bioactive Compounds Effective Against Type 2 Diabetes Mellitus: A Systematic Review.

BACKGROUND: Type 2 diabetes (adult onset diabetes) is the most common type of diabetes, accounting for around 90% of all diabetes cases with insulin resistance and insulin secretion defect. The key goal of anti-diabetic therapy is to increase the development of insulin, immunity and/or decrease the amount of blood glucose. While many synthetic compounds have been produced as antidiabetic agents, due to their side effects and limited effectiveness, their usefulness has been hindered. METHODS: This systematic review investigated the bioactive compounds reported to possess activities against type 2 diabetes. Three (3) databases, PubMed, ScienceDirect and Google Scholar were searched for research articles published between January 2010 and October 2020. A total of 6464 articles were identified out of which 84 articles were identified to be elligible for the study. RESULT AND DISCUSSION: From the data extracted, it was found that quercetin, Kaempferol, Rosmarinic acid, Cyanidin, Rutin, Catechin, Luteolin and Ellagic acid were the most cited bioactive compounds which all falls within the class of polyphenolic compounds. The major sources of these bioactive compounds includes citrus fruits, grapes, onions, berries, cherries, broccoli, honey, apples, green tea, Ginkgo biloba, St. John's wort, green beans, cucumber, spinach, tea, Rosmarinus officinalis, Aloe vera, Moringa oleifera, tomatoes, potatoes, oregano, lemon balm, thyme, peppermint, Ocimum basilicum, red cabbage, pears, olive oil and walnut.

Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology.

Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments and proficiency testing on standardized, cell-line-derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas, below this limit, detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false negatives) were more common than erroneous candidates (false positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best practice guidelines and provides a resource for precision oncology.

Fluorescent carbon dots driven from ayurvedic medicinal plants for cancer cell imaging and phototherapy.

Ayurveda based nanomaterials are recently conceptualized phenomena for biomedical applications especially for imaging and treatment of in vitro cancer cell. Wide range florescent (blue to red emission) quantum dots are versatile materials for imaging and sensing applications. Various procedures and precursors of fluorescent carbon quantum dots (CQDs) are well established and documented in the literature. However, expensive precursors and production, and time consuming process limit their economical design that need to be addressed. Herein, we report a cost effective simple route for fluorescent CQDs by using affordable ayurvedic plant's precursors such as Azadirachta Indica, OcimumTenuiflorum and Tridax Procumbens. Obtained quantum dots from ayurvedic plant leaves namely CQDs-1 (AzadirachtaIndica), CQDs-2 (OcimumTenuiflorum) and CQDs-3 (TridaxProcumbens) showed homogeneous size distribution (∼6-12 nm) and green fluorescent nature, average photo-stability, biocompatibility (more than 85 %), cancer cell imaging and promising phototherapy for cancer and bacterial cell lines.

Hypomethylation coordinates antagonistically with hypermethylation in cancer development: a case study of leukemia.

BACKGROUND: Methylation changes are frequent in cancers, but understanding how hyper- and hypomethylated region changes coordinate, associate with genomic features, and affect gene expression is needed to better understand their biological significance. The functional significance of hypermethylation is well studied, but that of hypomethylation remains limited. Here, with paired expression and methylation samples gathered from a patient/control cohort, we attempt to better characterize the gene expression and methylation changes that take place in cancer from B cell chronic lymphocyte leukemia (B-CLL) samples. RESULTS: Across the dataset, we found that consistent differentially hypomethylated regions (C-DMRs) across samples were relatively few compared to the many poorly consistent hypo- and highly conserved hyper-DMRs. However, genes in the hypo-C-DMRs tended to be associated with functions antagonistic to those in the hyper-C-DMRs, like differentiation, cell-cycle regulation and proliferation, suggesting coordinated regulation of methylation changes. Hypo-C-DMRs in B-CLL were found enriched in key signaling pathways like B cell receptor and p53 pathways and genes/motifs essential for B lymphopoiesis. Hypo-C-DMRs tended to be proximal to genes with elevated expression in contrast to the transcription silencing-mechanism imposed by hypermethylation. Hypo-C-DMRs tended to be enriched in the regions of activating H4K4me1/2/3, H3K79me2, and H3K27ac histone modifications. In comparison, the polycomb repressive complex 2 (PRC2) signature, marked by EZH2, SUZ12, CTCF binding-sites, repressive H3K27me3 marks, and "repressed/poised promoter" states were associated with hyper-C-DMRs. Most hypo-C-DMRs were found in introns (36 %), 3' untranslated regions (29 %), and intergenic regions (24 %). Many of these genic regions also overlapped with enhancers. The methylation of CpGs from 3'UTR exons was found to have weak but positive correlation with gene expression. In contrast, methylation in the 5'UTR was negatively correlated with expression. To better characterize the overlap between methylation and expression changes, we identified correlation modules that associate with "apoptosis" and "leukocyte activation". CONCLUSIONS: Despite clinical heterogeneity in disease presentation, a number of methylation changes, both hypo and hyper, appear to be common in B-CLL. Hypomethylation appears to play an active, targeted, and complementary role in cancer progression, and it interplays with hypermethylation in a coordinated fashion in the cancer process.

A full Bayesian partition model for identifying hypo- and hyper-methylated loci from single nucleotide resolution sequencing data.

BACKGROUD: DNA methylation is an epigenetic modification that plays important roles on gene regulation. Study of whole-genome bisulfite sequencing and reduced representation bisulfite sequencing brings the availability of DNA methylation at single CpG resolution. The main interest of study on DNA methylation data is to test the methylation difference under two conditions of biological samples. However, the high cost and complexity of this sequencing experiment limits the number of biological replicates, which brings challenges to the development of statistical methods. RESULTS: Bayesian modeling is well known to be able to borrow strength across the genome, and hence is a powerful tool for high-dimensional-low-sample-size data. In order to provide accurate identification of methylation loci, especially for low coverage data, we propose a full Bayesian partition model to detect differentially methylated loci under two conditions of scientific study. Since hypo-methylation and hyper-methylation have distinct biological implication, it is desirable to differentiate these two types of differential methylation. The advantage of our Bayesian model is that it can produce one-step output of each locus being either equal-, hypo- or hyper-methylated locus without further post-hoc analysis. An R package named as MethyBayes implementing the proposed full Bayesian partition model will be submitted to the bioconductor website upon publication of the manuscript. CONCLUSIONS: The proposed full Bayesian partition model outperforms existing methods in terms of power while maintaining a low false discovery rate based on simulation studies and real data analysis including bioinformatics analysis.

PRIMEGENSw3: a web-based tool for high-throughput primer and probe design.

Highly specific and efficient primer and probe design has been a major hurdle in many high-throughput techniques. Successful implementation of any PCR or probe hybridization technique depends on the quality of primers and probes used in terms of their specificity and cross-hybridization. Here we describe PRIMEGENSw3, a set of web-based utilities for high-throughput primer and probe design. These utilities allow users to select genomic regions and to design primer/probe for selected regions in an interactive, user-friendly, and automatic fashion. The system runs the PRIMEGENS algorithm in the back-end on the high-performance server with the stored genomic database or user-provided custom database for cross-hybridization check. Cross-hybridization is checked not only using BLAST but also by checking mismatch positions and energy calculation of potential hybridization hits. The results can be visualized online and also can be downloaded. The average success rate of primer design using PRIMEGENSw3 is ~90 %. The web server also supports primer design for methylated sequences, which is used in epigenetic studies. Stand-alone version of the software is also available for download at the website.

Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing.

We applied a solution hybrid selection approach to the enrichment of CpG islands (CGIs) and promoter sequences from the human genome for targeted high-throughput bisulfite sequencing. A single lane of Illumina sequences allowed accurate and quantitative analysis of ~1 million CpGs in more than 21,408 CGIs and more than 15,946 transcriptional regulatory regions. Of the CpGs analyzed, 77-84% fell on or near capture probe sequences; 69-75% fell within CGIs. More than 85% of capture probes successfully yielded quantitative DNA methylation information of targeted regions. Differentially methylated regions (DMRs) were identified in the 5'-end regulatory regions, as well as the intra- and intergenic regions, particularly in the X-chromosome among the three breast cancer cell lines analyzed. We chose 46 candidate loci (762 CpGs) for confirmation with PCR-based bisulfite sequencing and demonstrated excellent correlation between two data sets. Targeted bisulfite sequencing of three DNA methyltransferase (DNMT) knockout cell lines and the wild-type HCT116 colon cancer cell line revealed a significant decrease in CpG methylation for the DNMT1 knockout and DNMT1, 3B double knockout cell lines, but not in DNMT3B knockout cell line. We demonstrated the targeted bisulfite sequencing approach to be a powerful method to uncover novel aberrant methylation in the cancer epigenome. Since all targets were captured and sequenced as a pool through a series of single-tube reactions, this method can be easily scaled up to deal with a large number of samples.

Homolog-specific PCR primer design for profiling splice variants.

To study functional diversity of proteins encoded from a single gene, it is important to distinguish the expression levels among the alternatively spliced variants. A variant-specific primer pair is required to amplify each alternatively spliced variant individually. For this purpose, we developed a new feature, homolog-specific primer design (HSPD), in our high-throughput primer and probe design software tool, PRIMEGENS-v2. The algorithm uses a de novo approach to design primers without any prior information of splice variants or close homologs for an input query sequence. It not only designs primer pairs but also finds potential isoforms and homologs of the input sequence. Efficiency of this algorithm was tested for several gene families in soybean. A total of 187 primer pairs were tested under five different abiotic stress conditions with three replications at three time points. Results indicate a high success rate of primer design. Some primer pairs designed were able to amplify all splice variants of a gene. Furthermore, by utilizing combinations within the same multiplex pool, we were able to uniquely amplify a specific variant or duplicate gene. Our method can also be used to design PCR primers to specifically amplify homologs in the same gene family. PRIMEGENS-v2 is available at: http://primegens.org.