L-Cysteine functionalized magnetite nanoparticle adorned Ti3C2-MXene nanohybrid based screen printed immunosensor for oral cancer biomarker detection.

Nanohybrid based non-invasive biosensing platforms are emerging as promising alternatives to detect biomarkers in complex and diverse bio-fluids toward ultrasensitive point-of-care diagnostics. Herein, we report the development of a highly sensitive, facile, non-invasive, label free, affordable, and innovative electrochemical screen printed immunosensor for identifying CYFRA 21-1, an established and crucial biomarker for oral cancer. Until now, no work has been reported utilizing a titanium carbide Ti3C2 MXene nanosheet and L-cysteine (L-Cyst) functionalized magnetite nanoparticle (MNPs) nanohybrid based immunosensor for electrochemical detection of CYFRA 21-1. The L-Cyst@MNPs/Ti3C2-MXene nanohybrid was synthesized via the co-precipitation method and later deposited on a gold screen printed electrode (GSPE) offering enhanced surface area and electrochemical properties. The nanohybrid modified GSPE was then surface immobilized with monoclonal antibodies (anti-CYFRA-21-1) to fabricate an anti-CYFRA-21-1/L-Cyst@MNPs/Ti3C2-MXene/GSPE immunoelectrode and the non-specific locations of the immunoelectrode were covered with bovine serum albumin (BSA). The spectroscopic, morphological, and structural analyses of the synthesized nanohybrid and the fabricated electrodes were performed using different analytical techniques. The electrochemical studies of modified electrodes were evaluated using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). The fabricated BSA/anti-CYFRA-21-1/L-Cyst@MNPs/Ti3C2-MXene/GSPE immunosensor has shown an excellent limit of detection of 0.023 ng mL-1, a linear detection range of (0.5-30) ng mL-1, a sensitivity of 277.28 µA (ng mL-1)-1 cm-2 and a lower limit of quantification of 0.618 ng mL-1 for electrochemical CYFRA 21-1 determination. Hence, this L-Cyst@MNPs/Ti3C2-MXene nanohybrid could also be explored as a potential candidate for determining other cancer biomarkers.

Salivary microbial dysbiosis may predict lung adenocarcinoma: A pilot study.

BACKGROUND: Adenocarcinoma is a more common type of Non-small cell lung cancer (NSCLC). Lung cancer showed a statistically significant increment in the Kamrup Urban district of Assam, Tripura, Sikkim, and Manipur of India. The goal of our pilot study is to identify non-invasive microbial biomarkers to detect lung adenocarcinoma (LAC). MATERIAL AND METHODS: DNA extraction from saliva samples of five LAC patients and five healthy controls was performed by Qiagen DNeasy blood and tissue kit using Lysozyme (3mg/ml) treatment. 16S rRNA genes of distinct regions (V3-V4) were amplified from saliva DNA by PCR. Paired-end sequencing targeting the V3-V4 region of the 16S rRNA gene has been performed on the Illumina MiSeq platform. Raw sequences were analyzed using the QIIME(Quantitative Insights Into Microbial Ecology) software package. RESULTS: Our preliminary results showed that Rothia mucilaginosa, Veillonella dispar, Prevotella melaninogenica, Prevotella pallens, Prevotella copri, Haemophilus parainfluenzae, Neisseria bacilliformis and Aggregatibacter segnis were significantly elevated in saliva of LAC which may serve as potential non-invasive biomarkers for LAC detection. Functional prediction analysis showed that bacterial genes involved in glycosyltransferase, peptidases, amino sugar, and nucleotide sugar metabolism, starch and sucrose metabolism were significantly enriched in LAC. CONCLUSION: These salivary bacteria may contribute to the development of LAC by increasing expression of glycosyltransferase and peptidases. However to understand their role in pathobiology, studies are required to perform in large cohort.

RUNX1T1, a potential prognostic marker in breast cancer, is co-ordinately expressed with ERα, and regulated by estrogen receptor signalling in breast cancer cells.

BACKGROUND: RUNX1T1 is extensively studied in the context of AML1-RUNX1T1 fusion protein in acute myeloid leukemia. Little is known about the function of RUNX1T1 itself, although data on its function and regulation have begun to emerge from clinical, and in vitro studies. It is a putative tumor suppressor, whose expression is altered in a variety of solid tumors. Recently, reduced expression of RUNX1T1 in triple-negative breast tumors, and its influence on prognosis was reported. METHODS AND RESULTS: The Kaplan-Meier Plotter online tool was used to study the relationship between RUNX1T1 expression and survival of breast cancer patients. High RUNX1T1 expression was associated with longer overall survival (OS), relapse-free survival (RFS) and distant metastasis free survival (DMFS). RUNX1T1 expression positively and negatively influenced OS of patients with ERα-positive and ERα-negative breast tumors, respectively. It was also associated with prolonged RFS, and DMFS in tamoxifen-treated patients. Expression of RUNX1T1 and ERα mRNA was analyzed in 40 breast tumor samples, and breast cancer cell lines using RT-PCR. TCGA-BRCA data was mined to study the relationship between RUNX1T1 and ERα mRNA expression. ERα-positive breast tumors showed significantly higher RUNX1T1 mRNA expression compared to ERα-negative tumors. RUNX1T1 mRNA expression was analyzed by qRT-PCR in MCF-7 or T47D cells, which were treated with 17ß-estradiol, or the ERα agonist PPT, alone or in combination with 4-hydroxytamoxifen. Effect of ERα knockdown was also investigated. Results indicate that estrogen downmodulated RUNX1T1 mRNA expression via ERα. CONCLUSION: Higher expression of RUNX1T1 in breast tumors is associated with favourable prognosis. RUNX1T1 and ERα show co-ordinated expression in breast tumors, and breast cancer cell lines. Estrogen-ERα signalling downmodulates the expression of RUNX1T1 mRNA in ERα-positive breast cancer cells. In-depth investigations on the interaction between RUNX1T1 and ERα are warranted to unravel the role and relevance of RUNX1T1 in breast cancer.

Dysbiosis of salivary microbiome and cytokines influence oral squamous cell carcinoma through inflammation.

Advanced combinatorial treatments of surgery, chemotherapy, and radiotherapy do not have any effect on the enhancement of a 5-year survival rate of oral squamous cell carcinoma (OSCC). The discovery of early diagnostic non-invasive biomarkers is required to improve the survival rate of OSCC patients. Recently, it has been reported that oral microbiome has a significant contribution to the development of OSCC. Oral microbiome induces inflammatory response through the production of cytokines and chemokines that enhances tumor cell proliferation and survival. The study aims to develop saliva-based oral microbiome and cytokine biomarker panel that screen OSCC patients based on the level of the microbiome and cytokine differences. We compared the oral microbiome signatures and cytokine level in the saliva of OSCC patients and healthy individuals by 16S rRNA gene sequencing targeting the V3/V4 region using the MiSeq platform and cytokine assay, respectively. The higher abundance of Prevotella melaninogenica, Fusobacterium sp., Veillonella parvula, Porphyromonas endodontalis, Prevotella pallens, Dialister, Streptococcus anginosus, Prevotella nigrescens, Campylobacter ureolyticus, Prevotella nanceiensis, Peptostreptococcus anaerobius and significant elevation of IL-8, IL-6, TNF-α, GM-CSF, and IFN-γ in the saliva of patients having OSCC. Oncobacteria such as S. anginosus, V. parvula, P. endodontalis, and P. anaerobius may contribute to the development of OSCC by increasing inflammation via increased expression of inflammatory cytokines such as IL-6, IL-8, TNF-α, IFN-γ, and GM-CSF. These oncobacteria and cytokines panels could potentially be used as a non-invasive biomarker in clinical practice for more efficient screening and early detection of OSCC patients.

Accumulating Impact of Smoking and Co-morbidities on Severity and Mortality of COVID-19 Infection: A Systematic Review and Meta-analysis.

Background: High prevalence, severity, and formidable morbidity have marked the recent emergence of the novel coronavirus disease (COVID-19) pandemic. The significant association with the pre-existing co-morbid conditions has increased the disease burden of this global health emergency, pushing the patients, healthcare workers and facilities to the verge of complete disruption. Methods: Meta-analysis of pooled data was undertaken to assess the cumulative risk assessment of multiple co-morbid conditions associated with severe COVID-19. PubMed, Scopus, and Google Scholar were searched from January 1st to June 27th 2020 to generate a well-ordered, analytical, and critical review. The exercise began with keying in requisite keywords, followed by inclusion and exclusion criteria, data extraction, and quality evaluation. The final statistical meta-analysis of the risk factors of critical/severe and non-critical COVID-19 infection was carried out on Microsoft Excel (Ver. 2013), MedCalc (Ver.19.3), and RevMan software (Ver.5.3). Results: We investigated 19 eligible studies, comprising 12037 COVID-19 disease patients, representing the People's Republic of China (PRC), USA, and Europe. 18.2% (n = 2200) of total patients had critical/severe COVID-19 disease. The pooled analysis showed a significant association of COVID-19 disease severity risk with cardiovascular disease (RR: 3.11, p < 0.001), followed by diabetes (RR: 2.06, p < 0.001), hypertension (RR: 1.54, p < 0.001), and smoking (RR: 1.52, p < 006). Conclusion: The review involved a sample size of 12037 COVID-19 patients across a wide geographical distribution. The reviewed reports have focussed on the association of individual risk assessment of co-morbid conditions with the heightened risk of COVID-19 disease. The present meta-analysis of cumulative risk assessment of co-morbidity from cardiovascular disease, diabetes, hypertension, and smoking signals a novel interpretation of inherent risk factors exacerbating COVID-19 disease severity. Consequently, there exists a definite window of opportunity for increasing survival of COVID-19 patients (with high risk and co-morbid conditions) by timely identification and implementation of appropriately suitable treatment modalities.

Alterations of salivary microbial community associated with oropharyngeal and hypopharyngeal squamous cell carcinoma patients.

The highest number (35.1% of global incident cases) of new oropharyngeal (OP) and hypopharyngeal (HP) cancer cases was reported in South-Central Asia. The highest incidence of HP cancer in India was reported in East Khasi Hills District of Meghalaya, Aizawl District of Mizoram, and Kamrup Urban District of Assam. HP and OP cancer showed the highest mortality rate, worst prognoses and the highest rate of nodal metastases and distant metastases. Thus, research is required to detect specific biomarkers for early prevention and diagnosis for these cancers. Oral microbiome signatures in saliva are considered as a potential diagnostic biomarker for OP and HP cancer. Bacterial profile alterations in OP and HP cancer have not been reported in India population, to establish the association of oral bacteria in the progression of OP and HP cancer; we studied bacterial communities in saliva of eight OP and seven HP cancer patients as compared to healthy controls using 16S rRNA V3-V4 region sequencing. The higher abundance of Haemophilus parainfluenzae, Haemophilus influenzae and Prevotella copri and lower abundance of Rothia mucilaginosa, Aggregatibacter segnis, Veillonella dispar, Prevotella nanceiensis, Rothia aeria, Capnocytophaga ochracea, Neisseria bacilliformis, Prevotella nigrescens and Selenomonas noxia in saliva of OP and HP cancer patients may be considered as a non-invasive diagnostic biomarker for OP and HP cancer patients. Streptococcus anginosus may be considered as a non-invasive diagnostic biomarker for OP cancer patients only. Therefore, evaluation of salivary microbial biomarkers may be informative to understand the pathobiology and carcinogenesis of OP and HP cancer.

Epigenetic deregulations of Wnt/ß-catenin and transforming growth factor beta-Smad pathways in esophageal cancer: Outcome of DNA methylation.

BACKGROUND: Promoter methylation of tumor suppressor genes (TSGs) is a well-reported portent in carcinogenesis; hence, it is worthy to investigate this in high-risk Northeast population of India. The study was designed to investigate methylation status of 94 TSGs in esophageal squamous cell carcinoma (ESCC). Further, the effect of OPCML promoter methylation on gene expression was analyzed by immunohistochemistry. Moreover, in silico protein-protein interactions were examined among 8 TSGs identified in the present study and 23 epigenetically regulated genes reported previously by our group in ESCC. MATERIALS AND METHODS: Methylation profiling was carried out by polymerase chain reaction array and OPCML protein expression was examined by tissue microarray-based immunohistochemistry. RESULTS: OPCML, NEUROG1, TERT, and WT1 genes were found hypermethylated and SCGB3A1, CDH1, THBS1, and VEGFA were hypomethylated in Grade 2 tumor. No significant change in OPCML expression was observed among control, Grade 1, and Grade 2 tumor. Conclusively, hypermethylation of the studied OPCML promoter in Grade 2 tumor produced no effect on expression. Unexpectedly, OPCML expression was downregulated in Grade 3 tumor in comparison to other groups signifying that downregulation of OPCML expression may lead to higher grade of tumor formation at the time of diagnosis of ESCC in patients. Significant interactions at protein level were found as VEGFA:PTK2, CTNNB1:CDH1, CTNNB1:VEGFA, CTNNB1:NEUROG1, CTNND2:CDH1, and CTNNB1:TERT. These interactions are pertinent to Wnt/ß-catenin and TGF-ß-Smad pathways. CONCLUSIONS: Deranged OPCML expression may lead to high-grade ESCC as well as epigenetically regulated genes, that is, CDH1, CTNNB1, CTNND2, THBS1, PTK2, WT1, OPCML, TGFB1, and SMAD4 may alter the Wnt/ß-catenin and TGF-ß-Smad pathways in ESCC. Further study of these genes could be useful to understand the molecular pathology of ESCC with respect to epithelial-mesenchymal transition (EMT) mediated by Wnt/ß-catenin and TGF-ß signaling pathways.

Esophageal Cancer Epigenomics and Integrome Analysis of Genome-Wide Methylation and Expression in High Risk Northeast Indian Population.

Esophageal cancer is a major global health burden with a strong host-environment interaction component and epigenomics underpinnings that remain to be elucidated further. Certain populations such as the Northeast Indians suffer at a disproportionately higher rate from this devastating disease. Promoter methylation is correlated with transcriptional silencing of various genes in esophageal cancer. Very few studies on genome-wide methylation for esophageal cancer exist and yet, no one has carried out an integromics analysis of methylation and gene expression. In the present study, genome-wide methylation was measured in samples collected from the Northeast Indian population by Infinium 450k array, and integration of the methylation data was performed. To prepare a network of genes displaying enriched pathways, together with the list of genes exhibiting promoter hypermethylation or hypomethylation with inversely correlated expression, we performed an integrome analysis. We identified 23 Integrome network enriched genes with relevance to tumor progression and associated with the processes involved in metastasis such as cell adhesion, integrin signaling, cytoskeleton, and extracellular matrix organizations. These included four genes (PTK2, RND1, RND3, and UBL3) with promoter hypermethylation and downregulation, and 19 genes (SEMG2, CD97, CTNND2, CADM3, OMD, NEFM, FBN2, CTNNB1, DLX6, UGT2B4, CCDC80, PZP, SERPINA4, TNFSF13B, NPC1, COL1A1, TAC3, BMP8A, and IL22RA2) with promoter hypomethylation and upregulation. A Methylation Efficiency Index was further calculated for these genes; the top five gene with the highest index were COL1A1, TAC3, SERPINA4, TNFSF13B, and IL22RA2. In conclusion, we recommend that the circulatory proteins IL22RA2, TNFSF13B, SERPINA4, and TAC3 in serum of patients and disease-free healthy controls can be examined in the future as putative noninvasive biomarkers.

Alcohol and Tobacco Increases Risk of High Risk HPV Infection in Head and Neck Cancer Patients: Study from North-East Region of India.

BACKGROUND: Human papilloma virus (HPV) associated Head and Neck Cancers (HNCs) have generated significant amount of research interest in recent times. Due to high incidence of HNCs and lack of sufficient data on high-risk HPV (hr-HPV) infection from North -East region of India, this study was conceived to investigate hr-HPV infection, its types and its association with life style habits such as tobacco, alcohol consumption etc. METHODS: A total of one hundred and six primary HNC tumor biopsy specimens were collected. These samples were analyzed for hr-HPV DNA (13 HPV types) using hybrid capture 2 (HC2) assay and genotyping was done by E6 nested multiplex PCR (NMPCR). RESULTS: The presence of hr-HPV was confirmed in 31.13% (n = 33) and 24.52% (n = 26) of the HNC patients by nested multiplex PCR (NMPCR) and HC2 assay respectively. Among hr-HPV positive cases, out of thirteen hr- HPV types analyzed, only two prevalent genotypes, HPV-16 (81.81%) followed by HPV-18 (18.18%) were found. Significant association was observed between hr-HPV infection with alcohol consumption (p <0.001) and tobacco chewing (p = 0.02) in HNC cases. Compared to HPV-18 infection the HPV-16 was found to be significantly associated with tobacco chewing (p = 0.02) habit. CONCLUSIONS: Our study demonstrated that tobacco chewing and alcohol consumption may act as risk factors for hr-HPV infection in HNCs from the North-East region of India. This was the first study from North-East India which also assessed the clinical applicability of HC2 assay in HNC patient specimens. We suggest that alcohol, tobacco and hr- HPV infection act synergistically or complement each other in the process of HNC development and progression in the present study population.

Hybrid capture 2 assay based evaluation of high-risk HPV status in healthy women of north-east India.

BACKGROUND: High risk HPV (HR-HPV) testing has been recommended as an effective tool along with cytology screening in identification of cervical intraepithelial lesions (CINs) and prevention of their progress towards invasive cervical cancer. The aim of this study was to assess the HR-HPV DNA status by Hybrid Capture 2 (HC2) assay in healthy asymptomatic women of North-East India. MATERIALS AND METHODS: This study examined cervical cell samples of forty three (n=43) healthy women by HC2 assay. A High Risk HPV DNA kit (Qiagen) was used which can detect 13 high risk HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. RESULTS: The mean relative light units (RLU) for samples was in the range of 141-5, 94, 619. HR-HPV DNA was confirmed in 16% (7/43) of participant women samples. Among demographic and clinical parameters, menstrual irregularity (p=0.039) and infection history (p=0.028) has shown statistically significant differences between the HR-HPV-positive and negative groups. In the HR-HPV positive group, two women were confirmed for CINs after colposcopy and histopathologic examination. CONCLUSIONS: We suggest that there may be an association between irregular menstruation and infection history of the urogenital tract with HR-HPV DNA prevalence in North-East Indian asymptomatic women. HC2 assay can be a valuable tool for HR-HPV screening.

Nested multiplex PCR based detection of human papillomavirus in cervical carcinoma patients of North- East India.

BACKGROUND: Persistent infection of one or more of about 15 high-risk human papillomaviruses (HR-HPVs), most commonly HPV types 16/18, has a significant role in cervical cancer initiation and progression. There are limited data available from north-east India about HPV prevalence though this region has high incidence rates of cervical cancer. The aim of this study was to investigate the HPV genotypes prevalent in cervical cancer patients of north-east India. MATERIALS AND METHODS: We analyzed 107 cervical cancer patient samples. Nested multiplex PCR assays were employed for detection of 13 high risk and 5 low risk HPV types. RESULTS: HPV was confirmed in 105 samples. The presence of 6 'carcinogenic' HPV types, HPV-16 (88%), -18 (15%), -31(4%) ,-45 (3%), -59 (4%), -58(1%), and one non carcinogenic, HPV-6/11 (6%), was recorded. Among various demographic and clinical factors only tumour stage showed a statistically significant association with HPV type infection (P=0.019). CONCLUSIONS: We suggest that the most prevalent genotype is HPV-16 followed by HPV-18 in cervical carcinoma patients of the north-eastern region of India. Advanced tumour stage may be associated with increased possibility of harbouring multiple HPV genotypes.