Infectious Disease Biology

COVID – 19

Deep proteomic analysis of COVID-19 Nasopharyngeal swab for identification of host prognosis markers. We performed a comprehensive proteomics-based investigation of nasopharyngeal swab samples from COVID-19 patients to identify host and viral proteins by employing simple extraction strategies. Using high-resolution mass spectrometry, we identify viral proteins and also developed targeted Selected Reaction Monitoring (SRM) assays (Pat #202021034687). Swab proteomics identified neutrophil degranulation, IL-12 and mRNA translation of proteins pathways in COVID-19 patients, and host proteins like AST, LDH, IL-6, FTH and CRP as biomarkers of infection (iScience, 2021). Further, LDH, CRP, AST and ALT were also validated in clinical settings. Thus, the mass-spectrometry identified host proteins from swab samples can be routinely monitored in clinics for the disease severity progression.COVID-19 infected host response study for severity prediction We employed a mass spectrometry-based label-free quantitative (LFQ) proteomics approach to study alteration in plasma proteome in a cohort of 73 patients to understand the disease progression. Of the 1200 proteins detected in the patient plasma, 38 proteins were differentially expressed between non-severe and severe groups Using proteomics, metabolomics & ML approaches, he discovered classifiers of COVID-19 severity using patient plasma, such as AGT, FGG, APOB and SERPINA3 and developed SRM assays for clinical translation (Pat # 202023054753). The altered plasma proteome of COVID-19 severe patients revealed dysregulation of peptidase activity, regulated exocytosis and myeloid leukocyte activation pathways (Front. Physiol. 2021). Interestingly, previous studies have indicated that the increasing level of FGG correlated with the high level of D-dimers observed in the COVID-19 patients with the worst outcomes. Thus, we certainly foresee the potential for these panel of host prognosis makers for clinical translation.

Malaria

Clinical samples are collected from various endemic regions of the country. The whole blood samples are used for parasite isolation to study the parasite protein based diagnostics and host based prognostic candidates to create a multiplex kit for clinics.
Parasite pellets from P. vivax infected whole blood samples are used to obtain parasite proteome profile which is further explored and validated in the plasma samples for diagnostics purposes (Venkatesh et. al. 2017; 2020). The target parasite proteins are currently being cloned, purified and tested for immunoassays for translational purposes.
Vivax and falciparum infected plasma samples are also used to perform high throughput screening of immunogenic parasite proteins that might not be protective in nature but capable of engendering the immune response in the infected host. The target proteins can be taken forward for vaccine development and therapeutic purposes.
Infected host plasma samples are utilized for prognostic candidate search using multiomic approach such as proteomics and metabolomics using high through put techniques like Mass spectrometry, Raman spectroscopy and NMR. Raman spectroscopy (RS) and Mass spectrometry (MS)-based approaches were taken to identify altered metabolites of plasma samples of dengue, malaria and healthy individuals and developed a model to differentiate the diseases with an overall precision of 0.9–1(Patel et. al. 2019). A labeled proteomics approach was used to analyze plasma samples of severe, non-severe vivax malaria infected patient and healthy individuals to understand the altered biomolecules and pathways related to the different levels of severity. Further, elastic net regularization logistic regression model was used to explore panel of proteins and selected proteins to differentiate malaria from dengue, vivax from falciparum and also differentiate between different severe form of falciparum conditions. (Kumar et. al. 2020).

Cancer Biology

Breast Cancer

Breast cancer continues to be the most diagnosed cancer worldwide and the second leading cause of death in women. Triple negative breast cancer is the most aggressive subtype of breast cancer among all, which lacks all three molecular markers (ER, PR, and Her2 Chemotherapy is usually used to treat it. However, local and distant recurrence after successful chemotherapy is still a major hurdle for a small cohort. The main cause of the recurrence of tumour is the development of chemotherapy resistance in triple negative breast cancer patients. A Key challenge is cellular pathways and multiple crosstalk mechanisms in cells that pave the way for the development of resistance. Our group is working on multi-omics characterization of TNBC in collaboration with Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Mumbai. This study aims to integrate the multi-omics data in conjunction with a robust statistical interpretation which will help to identify altered biological pathways and provide a more complete understanding of the chemo-resistance mechanism. Translating these omics data into clinical practice would improve triple negative breast cancer patient treatment towards a new era of personalized medicine.

Cervical Cancer

Cervical cancer remains to be a worldwide epidemic where more than 5,00,000 cases are reported every year, globally. In India, it is the second most common gynaecological cancer. In less developed countries, there is a higher incidence of locally advanced cases including FIGO stage IB2 and IIA2. The total number of cervical cancer incidents tends to slow down with the progression of modern-aged targeted therapy. However, local and distant recurrences after chemo-radiation therapy are still encountered in a small cohort. This happens majorly due to rising chemoresistance in cervical cancer patients. The appearance of therapy resistance (both chemo and radio) in locally advanced cervical cancer patients is still under investigation and the pathobiological reasons are not so clear to date. Also, cervical cancer is one of the very less explored domains of cancer research where both basic and application-based studies have not been done much. One of the reasons could be that it is very hard to obtain such invasive cancer sample types where multiple ethics-related barriers are there to overcome to proceed with further research. The unavailability of data and information in this domain is one of the biggest challenges. When it comes to omics-based studies in both cervical cancer and cervical cancer chemoradioresistance, very little knowledge is available, where some of the sub-domains (eg, Proteogenomics studies in cervical cancer chemoresistance) are left unveiled.

Our study aims to investigate the causes of chemoradioresistance using a multi omics-based followed by several bioinformatics-based approaches to find out the pathobiology behind cervical cancer therapy resistance. We will investigate the same on a very small cohort of patient samples, which will majorly be extrapolated by multiple cell-line-based models to investigate, verify and validate potential biomarkers, driver gene mutations, and target pathways leading to chemoresistance in cervical cancer.

Colorectal Cancer

Colorectal cancer (CAC) has been reported as the second leading cause of cancer death worldwide. The 5-year annual survival is around 50%, mainly due to late diagnosis, striking necessity for early detection. This study aims to identify autoantibody in patients’ sera for early screening of cancer. The study used a high-density human proteome array with approximately 17,000 recombinant proteins. Screening of sera from healthy individuals, CAC from Indian origin, and CAC from middle-east Asia origin were performed. Bio-statistical analysis was performed to identify significant autoantibodies altered. Pathway analysis is performed to explore the underlying mechanism of the disease. The comprehensive proteomic analysis revealed dysregulation of 15 panels of proteins including CORO7, KCNAB1, WRAP53, NDUFS6, KRT30, and COLGALT2. Further biological pathway analysis for the top dysregulated autoantigenic proteins revealed perturbation in important biological pathways such as ECM degradation and cytoskeletal remodeling etc. The generation of an autoimmune response against cancer-linked pathways could be linked to the screening of the disease. The process of immune surveillance can be detected at an early stage of cancer. Moreover, AAbs can be easily extracted from blood serum through the least invasive test for disease screening.

Glioma and Glioblastoma

Malignant gliomas are highly lethal tumors originating in the central nervous system. High grade gliomas including glioblastoma are the most lethal form of the primary brain tumors. Omics technologies have greatly contributed to our current level of understanding of the disease. Genome, Epigenome, Transcriptome, Proteome and Metabolome, each level, have their own importance and significantly contribute to the overall manifestation of the disease. We can see how different “omics” technologies have individually contributed to the diagnosis and prognosis of gliomas. In one way, we can say that starting from the identification of biomarkers to the development of immunotherapy-based treatments have all been possible with the help of omics-based technologies. With all the achievements and advancements of technologies, the new field of -omics is rapidly evolving, and it holds all the potential to decipher the unknown and novel signatures. A recent study on the Proteogenomic and metabolomic characterization of 99 glioblastoma tissues (without treatment) could reveal immune-based subtypes, the role of DNA repair pathways, the role of phosphorylation in signaling in receptor tyrosine kinase (RTK) altered tumors, and how acetylation of histone (H2B) is associated with the glioblastoma. This shows how the Proteogenomic and metabolomic approaches can become a valuable resource to decipher the pathophysiology of gliomas/glioblastoma and help in effective screening and treatment.

Medulloblastoma

Radiological image of a Pediatric patient with Medulloblastoma

Brain cancers are the top reason for cancer related deaths in children. Medulloblastoma is the most common posterior brain tumor in children. It is molecularly classified into four subgroups viz. WNT, SHH, Group 3 and Group 4 based on the differences in their genomic and transcriptomic landscapes. The pathobiology for group 3 and group 4 remains a bit obscure till now. WHO has updated a broad classification of medulloblastoma, based on recent molecular evidence but clinical management strategies still need to account for these changes. Even in this molecular era with several advancements in the field, targeted therapy remains to be a challenge in medulloblastoma.

The proteomics community has just started exploring into this field, but shows promise. Literature shows that post transcriptional regulations are involved in the pathobiology of Medulloblastoma. And yet proteomic studies on the Indian cohort are largely limited due to the small sample size. To this end, we are working on techniques to reliably extract proteins from FFPE samples for the proteomic studies.

Mitigation of the treatment strategies by considering molecular characteristics of each subgroup has been possible for SHH and WNT. Much is still unknown for Group 3 and Group 4 tumors, that form the majority of MB population. The inherent heterogeneity in these subgroups makes targeting treatment strategies more complicated. The in vitro studies on cell lines, aim to understand and elucidate the pathobiology of Group 3 Medulloblastomas from a proteomic perspective and identifying targets that can have clinical implications in therapy.

Meningioma

Meningiomas are benign tumours that arise from non-neuroepithelial progenitor cells called arachnoid cap cells. Cerebrospinal fluid resorption is mediated by arachnoidal cap cells, a morphologically distinct and metabolically active subgroup of arachnoidal cells. They are frequently located within a dural sinus and are present at the apex of Pacchionian bodies, exposing to venous blood flow. Meningiomas make up 13 to 26% of all cerebral tumours, according to estimates. The vast majority of meningiomas are benign, with a WHO histopathologic grade of I. Meningiomas become more common as people become older, culminating in their sixth and seventh decades. In adults, there is a significant gender bias, with a female:male ratio ranging from 3:2 to 2:1, and the ratio for spinal lesions is 9:1. Meningiomas are thought to occur 2–7/100.000 times in women each year and 1–5 times in men per year. Meningiomas are becoming more common, especially in the older population. A deletion of the neurofibromatosis type 2 gene, which causes an autosomal dominant condition that is commonly accompanied with a cytogenetically detectable deletion of chromosome 22’s long arm at q12, is one risk factor. The occurrence of multiple schwannomas, meningiomas, and gliomas in affected patients distinguishes neurofibromatosis type 2. The NF2 gene produces the protein merlin, which acts as a tumour suppressor (schwannoma). Merlin resembles cytoskeleton proteins such as protein 4.1, talin, moesin, ezrin, radixin, and tyrosine phosphatases in many ways. The protein’s function is thought to be to connect the cell membrane to the actin cytoskeleton(Marosi et al., 2008).

Oral Cancer

Oral cancer is the most common across the world and accounts 3% of the death of all diseases. India, Pakistan, Brazil, and France shows the most elevated occurrence of oral cancer across the world. Tobacco smoking, chewing of betel quid, and alcohol consumption are the major risk factors for the oral cancer. However, in some population human papilloma virus (HPV) infection is also acts as a risk factor. Despite the fact that the oral cavity is a possibly open site for assessment or examination of oral cancer. But, more than 50% of oral cancer cases are not identified until the tumor reached to the advanced stages. Screening and identification of tumor at early-stage increases the chances of survivability of the patients. So, there is an urgent need to develop or identify the marker which are involved in the early-stage of the oral cancer.

The main aim of this project is to identification and validation of the potential diagnosis and prognosis biomarkers which are involved in the early-stage of oral cancer and from the identified biomarker to develop diagnostic kit which help in the identification of early-stage of oral cancer.

Pituitary Cancer

The pituitary is commonly known as the master gland and is located in the bony pouch (sella turcica), having a significant role in controlling nearly all endocrine glands and itself is regulated by the hypothalamus. Most pituitary tumours originate from the two-thirds of the frontal area of the pituitary gland known as adenohypophysis or anterior lobe, which includes about 80% of the pituitary gland and is known for producing crucial hormones such as (growth hormone, prolactin, adrenocorticotropin, thyrotropin, luteinizing hormone and follicular stimulating hormone). Tumours rarely develop from the rear one-third area of the pituitary, known as neurophysis or posterior pituitary, which secretes oxytocin and vasopressin into the blood. These Pituitary Adenomas such as Acromegaly, Cushing’s and NFPA is considered as one of the most frequent intracranial tumours having salient impacts on human health such as mass effects, hypopituitarism and visual defects etc. In the past few decades, there has been enormous advancement in mass spectrometry-based proteomics. However, very little is known about the molecular pathogenesis of pituitary adenomas in the context of Omics. In our lab we study on various tissue (FF /FFPE) and serum samples of the Pituitary Adenomas (Acromegaly, Cushing’s and NFPA) with various omics technologies like Mass Spectrometry, Spectroscopy and NGS, validate the key identified dysregulated proteins with BLI, ELISA and Targeted Mass Spectroscopy assay to decipher the disease pathobiology and identify surrogate protein markers. Results outcomes have to be tabulated and contended in form of Pituitary Adenomas database and made available to scientific community.

Other Technical & Proteome profiling Projects

Chronic Obstructive Pulmonary Disease (COPD)

As per the Global Initiative for Chronic Obstructive Lung Disease (GOLD), a person having forced expiratory volume 1% (FEV1%) < 0.7 is regarded as COPD.  Chronic Obstructive Pulmonary Disease (COPD) is an airway disease wherein a person feels difficulty while exhaling the air out of the lungs. COPD is currently the 3rd leading cause of death globally and cigarette smoke is considered as the significant cause of COPD. We have synthesized a drug loaded nanoemulsions to target the cigarette smoke rat COPD model. With the help of the OMICS approach, we are trying to decipher the pathways, genes, and the metabolites involved in COPD and the treated groups so that different therapeutic strategies can be developed.

Human Brain Project (BrainProt)

The human brain has always been a black box full of mysteries. The correlation of the proteome level expression in different anatomical regions with their physiological function is an area to explore deep.   Here, we attempt to investigate the brain proteome focusing on inter-hemispheric and inter-region based differences in collaboration with Navarra Institute for Health Research, Spain. An extensive mass spectrometry-based analysis of 19 brain regions from both left and right hemispheres measured more than 3000 proteins and 38000 peptides.  This high-resolution data provides a comprehensive coverage of experimentally measured (non-hypothetical) proteins across various regions to characterize inter-hemispheric differences. The study reveals different region specific markers of Olfactory Bulb, Basal Ganglia and Cerebellar vermis. We also tried to understand the role of brain proteins in terms of synapse biology and their expression in different region and sub-region of the brain using the in-silico approach. The study has attempted to investigate the expression of neuroanatomical allied region and brain disorder protein markers in 19 region and sub-region of the brain. Furthermore, we have developed a Brain Proteome Database, based on our and publicly available curated data representing more than 9000 proteins (with isoforms) and around 90000 peptides at www.brainprot.org, which can aid in understanding the human brain’s complexity. 

Fish Project (Fishprot)

Labeo rohita (rohu), one of the Indian major carps (IMCs) and the most cultivable fish in India, accounts for a major portion of aquaculture revenue generation. Omics information for these aquaculture species is very limited. This study aimed at developing proteomic and peptide information for Labeo rohita which could help in studying several aquaculture issues like disease pathogenesis in fish. Towards this end, a reference proteome map was developed for rohu through proteomic profiling of multiple histologically normal fish samples. The data acquired in the study would provide a basis for further omics research and can help in elucidating biological processes in healthy or diseased fish.

COVID-19 Project (CovProt)

The world’s first endeavor at the curation of mass spectrometry-based host proteomics of SARS-CoV-2 for the user-friendly search, data visualization, and easy data accessibility to the scientific and medical community. This portal has been developed by Proteomics Laboratory, IIT Bombay. Currently, it contains information on 2263 different proteins and 33397 different peptides from 5 mass spectrometry datasets covering a total of 172 clinical samples across the globe. We believe that CoVProt will become a community resource for proteomic applications and will widely support clinical studies to identify biomarkers and therapeutics with translational potential.