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Abstract The Human Cell Atlas (HCA) is a global partnership “to create comprehensive reference maps of all human cells—the fundamental units of life – as a basis for both understanding human health and diagnosing, monitoring, and treating disease.” ( https://www.humancellatlas.org/ ) The atlas shall characterize cells from diverse individuals across the globe to better understand human biology. HCA proactively considers the priorities of, and benefits accrued to, contributing communities. Here, we lay out principles and action items that have been adopted to affirm HCA’s commitment to equity so that the atlas is beneficial to all of humanity.

Abstract Human papillomavirus (HPV) drives cervical cancer (CaCx) pathogenesis and viral oncoproteins jeopardize global gene expression in such cancers. In this study, our aim was to identify differentially expressed coding (DEcGs) and long noncoding RNA genes (DElncGs) specifically sense intronic and Natural Antisense Transcripts as they are located in the genic regions and may have a direct influence on the expression pattern of their neighbouring coding genes. We compared HPV16-positive CaCx patients (N = 44) with HPV-negative normal individuals (N = 34) by employing strand-specific RNA-seq and determined the relationships between DEcGs and DElncGs and their clinical implications. By performing Gene set enrichment and protein–protein interaction (PPI) analyses of DEcGs, we identified enrichment of processes crucial for abortive virus life cycle and cancer progression. The DEcGs formed 16 gene clusters which we identified through Molecular Complex Detection (MCODE) plugin of Cytoscape. All the gene clusters portrayed cancer-related functions. We recorded significantly correlated expression levels of 79 DElncGs with DEcGs at proximal genomic loci based on Pearson’s Correlation coefficients. Of these gene pairs, 24 pairs portrayed significantly altered correlation coefficients among patients, compared to normal individuals. Of these, 6 DEcGs of 6 such gene pairs, belonged to 5 of the identified gene clusters, one of which was survival-associated. Out of the 24 correlated DEcG: DElncG pairs, we identified 3 pairs, where expression of both members was significantly associated with patient overall survival. The findings justify the cooperative roles of these gene pairs, in patient prognostication, thereby bearing immense potential for translation. Thus, elucidation of correlative strengths between paired DElncGs and DEcGs in patient and normal samples, could serve as a foundation for identification of therapeutic and prognostic targets of HPV16-positive CaCx. Graphical abstract

Abstract No biomarker has yet been identified that allows accurate diagnosis and prognosis of oral cancers. In this study, we investigated the presence of key metabolites in oral cancer using proton nuclear magnetic resonance (NMR) spectroscopy to identify metabolic biomarkers of gingivobuccal oral squamous cell carcinoma (GB-OSCC). NMR spectroscopy revealed that uracil was expressed in 83.09% of tumor tissues and pyrimidine metabolism was active in GB-OSCC; these results correlated well with immunohistochemistry (IHC) and RNA sequencing data. Based on further gene and protein analyses, we proposed a pathway for the production of uracil in GB-OSCC tissues. Uridinetriphosphate (UTP) is hydrolyzed to uridine diphosphate (UDP) by CD39 in the tumor microenvironment (TME). We hypothesized that UDP enters the cell with the help of the UDP-specific P2Y6 receptor for further processing by ENTPD4/5 to produce uracil. As the ATP reserves diminish, the weakened immune cells in the TME utilize pyrimidine metabolism as fuel for antitumor activity, and the same mechanism is hijacked by the tumor cells to promote their survival. Correspondingly, the differential expression of ENTPD4 and ENTPD5 in immune and tumor cells, respectively, indicatedtheir involvement in disease progression. Furthermore, higher uracil levels were detected in patients with lymph node metastasis, indicating that metastatic potential is increased in the presence of uracil. The presence of uracil and/or expression patterns of intermediate molecules in purine and pyrimidine pathways, such asCD39, CD73, and P2Y6 receptors together with ENTPD4 and ENTPD5, hold promise as biomarker(s) for oral cancer diagnosis and prognosis.

Abstract Background For various cancers, differences in response to treatment and subsequent survival period have been reported to be associated with variation in immune contextures. Aim We sought to identify whether such association exists in respect of gingivobuccal oral cancer. Materials and methods We performed deep immune profiling of tumor and margin tissues collected from 46 treatment naïve, Human Papillomavirus (HPV) negative, patients. Each patient was followed for 24 months and prognosis (recurrence/death) noted. Key findings were validated by comparing with TCGA‐HNSC cohort data. Results About 28% of patients showed poor post‐treatment prognosis. These patients exhibited a high probability of recurrence even within 1 year and death within 2 years. There was restricted immune cell infiltration in tumor, but not in margin, among these patients. Reduced expression of eight immune‐related genes (IRGs) (NT5E, THRA, RBP1, TLR4, ITGA6, BMPR1B, ITGAV, SSTR1) in tumor strongly predicted better quality of prognosis, both in our patient cohort and in TCGA‐HNSC cohort. Tumors of patients with better prognosis were associated with (a) lower CD73+ cells with concomitant lower expression level of NT5E/CD73, (b) higher proportions of CD4+ and CD8+ T cells, B cells, NK cells, M1 macrophages, (c) higher %Granzyme+ cells, (d) higher TCR and BCR repertoire diversities. CD73 expression in tumor was associated with low CD8+ and CD4+ T cells, low immune repertoire diversity, and advanced cancer stage. Discussion and conclusion High infiltration of anti‐tumor immune cells in both tumors and margins results in good prognosis, while in patients with minimal infiltration in tumors in spite of high infiltration in margins results in poor prognosis. Targeted CD73 immune‐checkpoint inhibition may improve clinical outcome.

NorthEast India, with its unique geographic location in the midst of the Himalayas and Bay of Bengal, has served as a passage for the movement of modern humans across the Indian subcontinent and East/Southeast Asia. In this study we look into the population genetics of a unique population called the Khasi, speaking a language (also known as the Khasi language) belonging to the Austroasiatic language family and residing amidst the Tibeto-Burman speakers as an isolated population. The Khasi language belongs to one of the three major broad classifications or phyla of the Austroasiatic language and the speakers of the three sub-groups are separated from each other by large geographical distances. The Khasi speakers are separated from their nearest Austroasiatic language-speaking sub-groups: the “Mundari” sub-family from East and peninsular India and the “Mon-Khmers” in Mainland Southeast Asia. We found the Khasi population to be genetically distinct from other Austroasiatic speakers, i.e. Mundaris and Mon-Khmers, but relatively similar to the geographically proximal Tibeto Burmans. The possible reasons for this genetic-linguistic discordance lie in the admixture history of different migration events that originated from East Asia and proceeded possibly towards Southeast Asia. We found at least two distinct migration events from East Asia. While the ancestors of today’s Tibeto-Burman speakers were affected by both, the ancestors of Khasis were insulated from the second migration event. Correlating the linguistic similarity of Tibeto-Burman and Sino-Tibetan languages of today’s East Asians, we infer that the second wave of migration resulted in a linguistic transition while the Khasis could preserve their linguistic identity.

Abstract Oral squamous cell carcinoma of the gingivo-buccal region (OSCC-GB) accounts for the highest cancer morbidity and mortality among men in India. It has been observed that about one-third of individuals with oral leukoplakia, a dysplastic precancerous lesion in the oral cavity, progress to oral cancer. We aimed to identify systematic transcriptomic changes as a normal tissue in the oral cavity progresses to frank OSCC-GB. Seventy-two OSCC-GB patients, from multiple hospitals, were recruited, and transcriptome analysis of tumor and adjacent normal tissue (of all patients) and adjacent leukoplakia tissue (of a subset of 25 unselected patients with concomitant leukoplakia) was performed. We have identified many differences in the transcriptomic profiles between OSCC-GB and squamous cell carcinoma of the head and neck regions. Compared to the normal/precancerous tissue, significant enrichment of ECM−receptor interaction, PI3K-Akt signaling, cytokine−cytokine receptor interaction, focal adhesion, and cell cycle pathways were observed in OSCC-GB. Using gene set enrichment analysis, we identified a profound role of interferon receptor signaling in tumor growth by activating immune evasion mechanisms. The role of tumor-infiltrating immune cells further supported the growth and immunosuppressive mechanism of tumor tissues. Some immune evasion genes—CD274, CD80, and IDO1—were found to be activated even in the precancerous tissue. Taken together, our findings provide a clear insight into the sequential genetic dysregulation associated with progression to oral cancer. This insight provides a window to the development of predictive biomarkers and therapeutic targets for gingivo-buccal oral cancer.

Abstract Background Studies of epigenomic alterations associated with diseases primarily focus on methylation profiles of promoter regions of genes, but not of other genomic regions. In our past work (Das et al. 2019) on patients suffering from gingivo-buccal oral cancer – the most prevalent form of cancer among males in India – we have also focused on promoter methylation changes and resultant impact on transcription profiles. Here, we have investigated alterations in non-promoter (gene-body) methylation profiles and have carried out an integrative analysis of gene-body methylation and transcriptomic data of oral cancer patients. Methods Tumor and adjacent normal tissue samples were collected from 40 patients. Data on methylation in the non-promoter (gene-body) regions of genes and transcriptome profiles were generated and analyzed. Because of high dimensionality and highly correlated nature of these data, we have used Random Forest (RF) and other data-analytical methods. Results Integrative analysis of non-promoter methylation and transcriptome data revealed significant methylation-driven alterations in some genes that also significantly impact on their transcription levels. These changes result in enrichment of the Central Carbon Metabolism (CCM) pathway, primarily by dysregulation of (a) NTRK3, which plays a dual role as an oncogene and a tumor suppressor; (b) SLC7A5 (LAT1) which is a transporter dedicated to essential amino acids, and is overexpressed in cancer cells to meet the increased demand for nutrients that include glucose and essential amino acids; and, (c) EGFR which has been earlier implicated in progression, recurrence, and stemness of oral cancer, but we provide evidence of epigenetic impact on overexpression of this gene for the first time. Conclusions In rapidly dividing cancer cells, metabolic reprogramming from normal cells takes place to enable enhanced proliferation. Here, we have identified that among oral cancer patients, genes in the CCM pathway – that plays a fundamental role in metabolic reprogramming – are significantly dysregulated because of perturbation of methylation in non-promoter regions of the genome. This result compliments our previous result that perturbation of promoter methylation results in significant changes in key genes that regulate the feedback process of DNA methylation for the maintenance of normal cell division.

Introduction and objectives: Non-alcoholic fatty liver disease (NAFLD) is multistage with heterogeneous outcomes. We studied the influence of insulin resistance (IR) on the hepatic transcriptome of early NAFLD stages, to understand disease development. Materials and methods: In this cross-sectional study, possible clinicopathological risk factors were compared between mild-NAFL (N = 72) and non-alcoholic steatohepatitis (NASH; N = 51) patients. Liver tissue-transcriptome difference was studied between a subset of 25 mild-NAFL and 20 NASH biopsies and validated on another subset of 12 mild-NAFL and 13 NASH biopsies, using RT-PCR. The relationship between IR driven gene expression changes with fibrosis in NASH was investigated. Results: Significantly higher weight (p = 0.005) and elevated levels of HbA1c (p = 0.009), FBG (p = 0.03) and HOMA-IR (p = 0.009) were found in NASH patients. Five differentially expressed genes (DEGs, fold change > 1.5) were identified in NASH-FABP4, FABP5L2, CD24, PRAP1, and SPP1. The DEGs were positively associated with disease severity and HOMA-IR, and were found to be efficient classifiers of mild-NAFL and NASH. Additional 1218 genes identified related to IR (IrCGs), which can classify NASH-with-fibrosis patients separately from mild-NAFL and NASH patients. IrCGs can promote intra-hepatic fat accumulation, dysregulation of the lipid metabolism, lipotoxicity, and activation of cell survival pathways including activation of cell proliferation and differentiation pathways. Conclusions: Hepatic expression of genes associated with insulin resistance may drive NAFLD development and progression.

BackgroundThe incidence of preterm birth (PTB) in India is around 13%. Specific bacterial communities or individual taxon living in the vaginal milieu of pregnant women is a potential risk factor for PTB and may play an important role in its pathophysiology. Besides, bacterial taxa associated with PTB vary across populations.ObjectiveConduct a comparative analysis of vaginal microbiome composition and microbial genomic repertoires of women who enrolled in the Interdisciplinary Group for Advanced Research on Birth Outcomes – A DBT India Initiative (GARBH-Ini) pregnancy cohort to identify bacterial taxa associated with term birth (TB) and PTB in Indian women.MethodsVaginal swabs were collected during all three trimesters from 38 pregnant Indian women who delivered spontaneous term (n=20) and preterm (n=18) neonates. Paired-end sequencing of V3-V4 region of 16S rRNA gene was performed using the metagenomic DNA isolated from vaginal swabs (n=115). Whole genome sequencing of bacterial species associated with birth outcomes was carried out by shotgun method. Lactobacillus species were grown anaerobically in the De Man, Rogosa and Sharpe (MRS) agar culture medium for isolation of genomic DNA and whole genome sequencing.ResultsVaginal microbiome of both term and preterm samples reveals similar alpha diversity indices. However, significantly higher abundance of Lactobacillus iners (p-value All_Trimesters

Background: Atopic Dermatitis (AD) has been associated with the loss of function (LoF) mutations in Filaggrin (FLG) gene and increase in relative abundance of specific microbes in the lesional skin, predominantly in Caucasians. Our study aims to determine, in Indian AD patients, (a) the prevalence of FLG LoF and missense mutations, and (b) the nature and extent of dysbiosis and altered microbial pathways with and without mutations in FLG. AD patients (n = 34) and healthy controls (n = 54) were recruited from India in this study and shotgun sequencing was carried out in a subset of samples with adequate microbiome DNA concentration. Host DNA from the same subset of samples was subjected to FLG coding region sequencing and host-microbiome association was estimated.Results: The prevalence of FLG LoFs that are associated with AD globally were significantly lesser in our cases and controls (8.6%, 0%) than those reported in Europeans (27%, 2.6%). Staphylococcus aureus was present only on AD skin [abundance in Pediatric AD: 32.86%; Adult AD: 22.17%], but not on healthy skin on which Staphylococcus hominis (Adult controls: 16.43%, Adult AD: 0.20%; p = 0.002), Cutibacterium acnes (Adult controls:10.84%, Adult AD: 0.90%; p = 0.02), and Malassezia globosa (Adult controls: 8.89%, Adult AD: 0.005%; p = 0.001) were significantly more abundant. Microbial pathways mostly associated with skin barrier permeability, ammonia production and inflammation (Arginine and Proline metabolism, Histidine Metabolism and Staphylococcus aureus infection) were significantly enriched on AD skin metagenome. These pathways are also reported to impair antimicrobial peptide activity. Among AD patients with missense single nucleotide polymorphisms harboring “potentially damaging” alleles in FLG gene, damaging allele dosage was significantly (p < 0.02) positively correlated with relative abundance of phylum_Proteobacteria up to order_Pseudomonadales and negatively correlated with phylum_Firmicutes up to species_Staphylococcus aureus.Conclusion: Our study has provided evidence that host DNA profile is significantly associated with microbiome composition in the development of AD. Species and strain level analysis showed that the microbial pathways enriched in AD cases were mostly found in MRSA strains. These evidences can be harnessed to control AD by modulating the microbiome using a personalized strategy. Our findings on the association of FLG genotypes with the microbiome dysbiosis may pave the way for a personalized strategy to provide a more effective control of AD.

India and Southeast Asia are home to diverse linguistic groups; the Austroasiatic language group being one of them. The Austroasiatic speakers live in scattered settlements in these regions. What led to such dispersed distribution over this vast geographical space is yet to be resolved. Our work is aimed at reconstructing the migration route of early Austroasiatic settlers and examines their relationship with other linguistic groups. We genotyped 511 unrelated individuals from India and Malaysia out of which 189 were Austroasiatic. The rest belonged to Indo-European, Dravidian, Tibeto-Burman and Austronesian language families. Jarawa and Onge populations from Andaman and Nicobar Islands were also included. Our genotype data was combined with that of 940 individuals from HGDP dataset. We analyzed nearly 0.3 million autosomal SNPs and found that allele frequency correlation between Malaysian Austroasiatics and Indian Tibeto-Burmans was slightly higher (R2= 0.77) than with Indian Austroasiatics (R2= 0.72). Principal Components Analysis revealed that Malaysian Austroasiatic clustered closer to Tibeto-Burman than to Indian Austroasiatic. Similar clustering pattern was obtained by fineSTRUCTURE cluster dendrogram. The ADMIXTURE analysis inferred genetic component that is modal to the Malaysian Austroasiatic, is also significantly higher amongst Tibeto-Burman than Indian Austroasiatic (P < 2.117e-10), indicating that genetic distance correlates better with geography than language. Studying segments which were Identity by descent between individuals belonging to two different linguistic groups; i.e. Austroasiatic and Tibeto-Burman, we found Tibeto-Burman sharing larger number of segments with Malaysian Austroasiatic, but overall smaller in size. On the other hand the segments shared between the two Austroasiatic populations (India and Malaysia) are comparatively larger in size (P= 0.034) but smaller in number. Our analyses indicate that Malaysian Austroasiatic and Tibeto-Burman initially split from a common ancestor. Then a small group of individuals separated from Malaysian Austroasiatic giving rise to the present day Indian Austroasiatic. Treemix and D-statistics analysis provided evidence for gene flow between Malaysian Austroasiatic and Tibeto-Burman post split. Meanwhile, the southward migration of East Asians resulted in an extensive genetic exchange between East Asians and Tibeto-Burman as was evident in our ADMIXTURE analysis. This subsequent genetic exchange might have shaped the present day language structure.

It is hypothesized that same driver gene mutations should be present in both oral leukoplakia and cancer tissues. So, we attempted to find out mutations at one of the driver genes, CASP8, in cancer and adjacent leukoplakia tissues. Patients (n = 27), affected by both of cancer and adjacent leukoplakia, were recruited for the study. Blood and tissue DNA samples were used to identify somatic mutations at CASP8 by next generation sequencing method. In total, 56% (15 out of 27) cancer and 30% (8 out of 27) leukoplakia tissues had CASP8 somatic mutations. In 8 patients, both cancer and adjacent leukoplakia tissues, located within 2-5 cm of tumor sites, had identical somatic mutations. But, in 7 patients, cancer samples had somatic mutations but none of the leukoplakia tissues, located beyond 5cm of tumor sites, had somatic mutations. Mutated allele frequencies at CASP8 were found to be more in cancer compared to adjacent leukoplakia tissues. This study provides mutational evidence that oral cancer might have progressed from previously grown leukoplakia lesion. Leukoplakia tissues, located beyond 5cm of cancer sites, were free from mutation. The study implies that CASP8 mutation could be one of the signatures for some of the leukoplakia to progress to oral cancer.

Memory T and B lymphocyte numbers are thought to be regulated by recent and cumulative microbial exposures. We report here that memory-phenotype lymphocyte frequencies in B, CD4 and CD8 T-cells in 3-monthly serial bleeds from healthy young adult humans were relatively stable over a 1-year period, while Plasmablast frequencies were not, suggesting that recent environmental exposures affected steady state levels of recently activated but not of memory lymphocyte subsets. Frequencies of memory B and CD4 T cells were not correlated, suggesting that variation in them was unlikely to be determined by cumulative antigenic exposures. Immunophenotyping of adult siblings showed high concordance in memory, but not of recently activated lymphocyte subsets. To explore the possibility of cell-intrinsic regulation of T cell memory, we screened effector memory-phenotype T cell (TEM) frequencies in common independent inbred mice strains. Using two pairs from these strains that differed predominantly in either CD4 TEM and/or CD8 TEM frequencies, we constructed bi-parental bone marrow chimeras in F1 recipient mice, and found that memory T cell frequencies in recipient mice were determined by donor genotypes. Together, these data suggest cell-autonomous determination of memory T niche size, and suggest mechanisms maintaining immune variability.