Introduction: Lung TRACERx is a prominent study employing multi-region and longitudinal multi-omics sequencing to unravel the evolutionary trajectories of lung cancer. Aberrant DNA methylation patterns have been widely described in nearly all human cancers, yet their interplay with DNA mutations in lung cancer is not well understood. Incorporating the contribution of epigenetic modifications to cancer evolution trajectories within TRACERx could improve our understanding of the intricate relationship between genetic and epigenetic changes in non-small cell lung cancer (NSCLC) evolution. Methods: Multi-region sampling from 38 TRACERx patients including 112 tumor regions and 37 matched normal adjacent tissue samples was performed. Reduced representation bisulfite sequencing (RRBS) was performed to assess DNA methylation and the CAMDAC (Larose-Cadieux et al, 2020) was applied to estimate purified tumor methylation rates and correct for copy number changes. Whole exome sequencing and somatic copy number alterations (SCNAs) were inferred using the ASCAT tool (Van Loo et al, 2010) and Methsig (Pan et al, 2021) was performed to discover new methylation driver genes. Results: Using multi-region sequencing, we identified ubiquitous hypermethylation of 29 known driver genes in both lung adenocarcinoma (LUAD) and squamous cell lung cancer (LUSC), together with an additional 9 and 27 genes exclusive to LUSC and LUAD, respectively. We also identified 13 and 7 driver genes non-ubiquitously hypermethylated exclusively in LUSC and LUAD, respectively. Using a differential methylation based approach, we describe a method to determine the extent of intra-tumor methylation heterogeneity akin to established ITH scores based on genomics data. In addition, we report the identification of novel subtype-specific methylation driver genes enriched in HOX family members which are related to cancer progression. Through integration of DNA methylation and genomic sequencing data, we identify parallel mechanisms contributing towards ubiquitous tumor suppressor gene alterations. At the patient level, multiple driver genes such as NSD1, GATA3 and MGA were subject to repression by both copy number loss and DNA hypermethylation. Finally, we describe dosage-compensation of genes such as the Notch ligands JAG2 and DLK1 that are proximal to amplified oncogenes and hypermethylated during tumor evolution. Conclusion: We describe the contribution of DNA methylation and genomic alterations to altering the landscape of NSCLC. Leveraging DNA methylation, we can determine the extent of convergent repression mechanisms in different regions of the same tumor, assess DNA methylation heterogeneity, and discover DNA methylation-based driver genes in NSCLC. Citation Format: Francisco Gimeno-Valiente, Carla Castignani, Elizabeth Larose-Cadieux, Kezhong Chen, Nana Mensah, Olga Chervova, Thomas Watkins, Pawan Dhami, Heli Vaikkinen, Andrew Feber, TRACERx Consortium, Jonas Demeulemeester, Miljana Tanic, Stephan Beck, Peter Van Loo, Charles Swanton, Nnennaya Kanu. Identification of convergent gene repression mechanisms through integrative genomic and DNA methylation analysis in TRACERx [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5710.

Lung cancer is the leading cause of cancer-related death, accounting for nearly 1.4 million deaths worldwide every year. Lung TRACERx is a leading national study employing multi-region and longitudinal genome sequencing to determine the timing of somatic events in relation to distinct genome instability processes and to unravel the evolutionary trajectories of cancers. We have observed that chromosomal instability, rather than mutational heterogeneity, is associated with disease progression. Incorporating the contribution of epigenetic modifications to cancer evolution trajectories within TRACERx could improve our understanding of the intricate relationship between genetic and epigenetic changes in NSCLC evolution. Multi-region sampling from 38 TRACERx patients (24 LUAD and 14 LUSC) has been performed in the study. RNA has been extracted and RNAseq performed for gene expression profiling. DNA was processed for reduced representation bisulphite sequencing (RRBS) for assessment of DNA methylation. Whole exome sequencing was performed and somatic copy number alterations (SCNAs) were inferred. The overall percentage of hypermethylated differentially methylated positions (DMPs) was assessed at the tumour and regional level. We saw an enrichment in SCNAs at genomic loci harboring genes involved in DNA methylation maintenance such as DNMT1 and DNMT3. The presence of SCNAs at those loci were found to be significantly correlated with the extent of hypermethylated DMPs in tumours. This effect is also observed at the intratumoural level. In contrast, a significant correlation was also observed between the extent of copy number gains in the Tet methylcytosine dioxygenase 3 (TET3) and increased hypomethylation status. These copy number changes were functional as observed by changes in gene expression observed in the parallel RNA seq analysis. We have studied the role of SCNAs influencing expression of global methylation genes on tumour methylation heterogeneity. These data offer insight into the role of SCNAs and DNA methylation changes in lung cancer evolution.

Introduction: Treatment resistant depression (TRD) in youth is a debilitating disorder in which many of conventional therapies, such as antidepressants and cognitive behavioral therapy, show suboptimal efficacy, creating the need for novel treatments. Repetitive transcranial magnetic stimulation (rTMS) has been shown to be efficacious in the treatment of depression in adults and youth. However, it is imperative to understand the neurophysiology of the developing brain as it relates to youth depression and its treatment, which may provide insight into the pathophysiology of youth TRD. Here, we investigated TMS-EEG measures of connectivity, inhibition, and excitability in a group of youth with TRD to healthy controls before they underwent rTMS therapy.

Glycyrrhiza glabra is very well known medicinal plant used in Ayuveda. The present study was conducted to evaluate the in vitro antibacterial properties of roots of G. glabra against multidrug resistant clinical isolates of E. coli. The antibacterial activity was carried out with agar well diffusion method. The results of the study revealed that ethanol extract has broad spectrum and strong activity than aqueous extract and ether extract. Its effectiveness against drug resistant bacteria pathogens provides hope that it can serve as an alternative therapeutic agent to treat many diseases.

Indiscriminate uses of antibiotics have caused microbial resistance and also lead to many side effects. To overcome from such situation plants and animal materials are widely used for the treating various ailments due its antimicrobial properties. In Ayurveda, cow urine has been used to improve general health of an individual. Therefore, present study is undertaken to study in vitro antibacterial potential of cow urine against various pathogenic bacteria. The method employed to study antibacterial activity is agar well diffusion technique. A reference standard was also employed along with the test during the experimental study. The results showed good antibacterial activity of cow urine against most of the test bacterial strains by exhibiting zone of inhibition. These results thus proves that cow urine possess good inhibitory activities against various clinical bacterial strains and can be used to control infectious diseases.

The reference sequence for each human chromosome provides the framework for understanding genome function, variation and evolution. Here we report the finished sequence and biological annotation of human chromosome 1. Chromosome 1 is gene-dense, with 3,141 genes and 991 pseudogenes, and many coding sequences overlap. Rearrangements and mutations of chromosome 1 are prevalent in cancer and many other diseases. Patterns of sequence variation reveal signals of recent selection in specific genes that may contribute to human fitness, and also in regions where no function is evident. Fine-scale recombination occurs in hotspots of varying intensity along the sequence, and is enriched near genes. These and other studies of human biology and disease encoded within chromosome 1 are made possible with the highly accurate annotated sequence, as part of the completed set of chromosome sequences that comprise the reference human genome.

The development of high-throughput screening methods such as array-based comparative genome hybridization (array CGH) allows screening of the human genome for copy-number changes. Current array CGH strategies have limits of resolution that make detection of small (less than a few tens of kilobases) gains or losses of genomic DNA difficult to identify. We report here a significant improvement in the resolution of array CGH, with the development of an array platform that utilizes single-stranded DNA array elements to accurately measure copy-number changes of individual exons in the human genome. Using this technology, we screened 31 patient samples across an array containing a total of 162 exons for five disease genes and detected copy-number changes, ranging from whole-gene deletions and duplications to single-exon deletions and duplications, in 100% of the cases. Our data demonstrate that it is possible to screen the human genome for copy-number changes with array CGH at a resolution that is 2 orders of magnitude higher than that previously reported.

We present a detailed in vivo characterization of hepatocyte transcriptional regulation in HepG2 cells, using chromatin immunoprecipitation and detection on PCR fragment-based genomic tiling path arrays covering the encyclopedia of DNA element (ENCODE) regions. Our data suggest that HNF-4 alpha and HNF-3 beta, which were commonly bound to distal regulatory elements, may cooperate in the regulation of a large fraction of the liver transcriptome and that both HNF-4 alpha and USF1 may promote H3 acetylation to many of their targets. Importantly, bioinformatic analysis of the sequences bound by each transcription factor (TF) shows an over-representation of motifs highly similar to the in vitro established consensus sequences. On the basis of these data, we have inferred tentative binding sites at base pair resolution. Some of these sites have been previously found by in vitro analysis and some were verified in vitro in this study. Our data suggests that a similar approach could be used for the in vivo characterization of all predicted/uncharacterized TF and that the analysis could be scaled to the whole genome.

The finished sequence of human chromosome 10 comprises a total of 131,666,441 base pairs. It represents 99.4% of the euchromatic DNA and includes one megabase of heterochromatic sequence within the pericentromeric region of the short and long arm of the chromosome. Sequence annotation revealed 1,357 genes, of which 816 are protein coding, and 430 are pseudogenes. We observed widespread occurrence of overlapping coding genes (either strand) and identified 67 antisense transcripts. Our analysis suggests that both inter- and intrachromosomal segmental duplications have impacted on the gene count on chromosome 10. Multispecies comparative analysis indicated that we can readily annotate the protein-coding genes with current resources. We estimate that over 95% of all coding exons were identified in this study. Assessment of single base changes between the human chromosome 10 and chimpanzee sequence revealed nonsense mutations in only 21 coding genes with respect to the human sequence.

Chromosome 6 is a metacentric chromosome that constitutes about 6% of the human genome. The finished sequence comprises 166,880,988 base pairs, representing the largest chromosome sequenced so far. The entire sequence has been subjected to high-quality manual annotation, resulting in the evidence-supported identification of 1,557 genes and 633 pseudogenes. Here we report that at least 96% of the protein-coding genes have been identified, as assessed by multi-species comparative sequence analysis, and provide evidence for the presence of further, otherwise unsupported exons/genes. Among these are genes directly implicated in cancer, schizophrenia, autoimmunity and many other diseases. Chromosome 6 harbours the largest transfer RNA gene cluster in the genome; we show that this cluster co-localizes with a region of high transcriptional activity. Within the essential immune loci of the major histocompatibility complex, we find HLA-B to be the most polymorphic gene on chromosome 6 and in the human genome.

Chromosome 13 is the largest acrocentric human chromosome. It carries genes involved in cancer including the breast cancer type 2 (BRCA2) and retinoblastoma (RB1) genes, is frequently rearranged in B-cell chronic lymphocytic leukaemia, and contains the DAOA locus associated with bipolar disorder and schizophrenia. We describe completion and analysis of 95.5 megabases (Mb) of sequence from chromosome 13, which contains 633 genes and 296 pseudogenes. We estimate that more than 95.4% of the protein-coding genes of this chromosome have been identified, on the basis of comparison with other vertebrate genome sequences. Additionally, 105 putative non-coding RNA genes were found. Chromosome 13 has one of the lowest gene densities (6.5 genes per Mb) among human chromosomes, and contains a central region of 38 Mb where the gene density drops to only 3.1 genes per Mb.

Chromosome 9 is highly structurally polymorphic. It contains the largest autosomal block of heterochromatin, which is heteromorphic in 6–8% of humans, whereas pericentric inversions occur in more than 1% of the population. The finished euchromatic sequence of chromosome 9 comprises 109,044,351 base pairs and represents >99.6% of the region. Analysis of the sequence reveals many intra- and interchromosomal duplications, including segmental duplications adjacent to both the centromere and the large heterochromatic block. We have annotated 1,149 genes, including genes implicated in male-to-female sex reversal, cancer and neurodegenerative disease, and 426 pseudogenes. The chromosome contains the largest interferon gene cluster in the human genome. There is also a region of exceptionally high gene and G + C content including genes paralogous to those in the major histocompatibility complex. We have also detected recently duplicated genes that exhibit different rates of sequence divergence, presumably reflecting natural selection.

Chromosome 6 is a metacentric chromosome that constitutes about 6% of the human genome. The finished sequence comprises 166,880,988 base pairs, representing the largest chromosome sequenced so far. The entire sequence has been subjected to high-quality manual annotation, resulting in the evidence-supported identification of 1,557 genes and 633 pseudogenes. Here we report that at least 96% of the protein-coding genes have been identified, as assessed by multi-species comparative sequence analysis, and provide evidence for the presence of further, otherwise unsupported exons/genes. Among these are genes directly implicated in cancer, schizophrenia, autoimmunity and many other diseases. Chromosome 6 harbours the largest transfer RNA gene cluster in the genome; we show that this cluster co-localizes with a region of high transcriptional activity. Within the essential immune loci of the major histocompatibility complex, we find HLA-B to be the most polymorphic gene on chromosome 6 and in the human genome.

We have recently mapped a locus for hereditary prostate cancer (termed HPCX) to the long arm of the X chromosome (Xq25–q27) through a genome-wide linkage study. Here we report the construction of an ∼9-Mb sequence-ready bacterial clone contig map of Xq26.3–q27.3. The contig was constructed by screening BAC/PAC libraries with markers spaced at ∼85-kb intervals. We identified overlapping clones by end-sequencing framework clones to generate 407 new sequence-tagged sites, followed by PCR verification of overlaps. Contig assembly was based on clone restriction fingerprinting and the landmark information. We identified a minimal overlap contig for genomic sequencing, which has yielded 7.7 Mb of finished sequence and 1.5 Mb of draft sequence. The transcriptional mapping effort localized 57 known and predicted genes by database searching, STS content mapping, and sequencing, followed by sequence annotation. These transcriptional units represent candidate genes for HPCX and multiple other hereditary diseases at Xq26.3–q27.3.