School of Biotechnology (Jan – Mar 2023)

314

JOURNAL PAPERS:

  1. Butti, R., Kapse, P., Bhadauriya, G., Ahmad, S., Chaubal, R., Parab, P., Kadam, R., Mahapatra, S. S., Shet, T., Dutt, A., Gupta, S., & Kundu, G. C. (2023). Development and characterization of a patient‑derived orthotopic xenograft of therapy‑resistant breast cancer. Oncology reports49(5), 99. https://doi.org/10.3892/or.2023.8536

Abstract

Numerous years of cell line‑based studies have enhanced the current understanding of cancer and its treatment. However, limited success has been achieved in treating hormone receptor‑positive, HER2‑negative metastatic breast cancers that are refractory to treatment. The majority of cancer cell lines are unsuitable for use as pre‑clinical models that mimic this critical and often fatal clinical type, since they are derived from treatment‑naive or non‑metastatic breast cancer cases. The aim of the present study was to develop and characterize patient‑derived orthotopic xenografts (PDOXs) from patients with endocrine hormone receptor‑positive, HER2‑negative metastatic breast cancer who had relapsed on therapy. A patient who progressed on endocrine hormone therapy provided her tumor via a biobank. This tumor was implanted in mice. It was then serially passaged by implanting PDOX tumor fragments into another set of mice to develop further generations of PDOXs. These tissues were characterized using various histological and biochemical techniques. Histological, immunofluorescence and western blot analyses indicated that the PDOX tumors retained a similar morphology, histology and subtype‑specific molecular features to that of the patient’s tumor. The present study successfully established PDOXs of hormone‑resistant breast cancer and characterized them in comparison with those derived from the original breast cancer tissue of the patient. The data highlight the reliability and usefulness of PDOX models for studies of biomarker discovery and preclinical drug screening. The present study was registered with the clinical trial registry of India (CTRI; registration no. CTRI/2017/11/010553; registered on 17/11/2017).


  1. Lavudi, K., Harika, G. V. S., Thirunavukarasou, A., Govindarajan, G., Patnaik, S., Golla, N., Kotakadi, V. S., Penchalaneni, J. (2023). Green Synthesis of Tecoma stans Flower and Leaf Extracts: Characterization and Anti-Proliferative Activity in Colorectal Cancer Cell Lines. (2022). Letters in Applied NanoBioScience, 12(3), 61. https://doi.org/10.33263/lianbs123.061

Abstract

Since times immemorial, many plant species have been utilized to cure severe diseases. A wide range of diversification has been observed in various medicinal plants, which are indeed able to cure several deadly diseases. The presence of secondary metabolites is a high priority for their medicinal characteristics. This study focused on T. stans (Yellow bells), a shrub that grows profoundly in tropical and sub-tropical regions. Although many studies have been done on the medicinal value of this plant, not much has been done on cancer treatment and nanomedicine. Green synthesized silver nanoparticles are an eco-friendly approach to delivering the drug to the target size. Nano appearance is an add-on advantage of these compounds. Hence, it is emerging in medicine. Colorectal cancer is the fourth deadliest one globally. Hence the synthesized silver nanoparticles of T. stans flower and leaf extracts showed cytotoxically and wound healing properties on colorectal cancer cell lines (HCT 116 and SW 480). Synthesis of silver nanoparticles confirmation is done by Ultra Violet Visible spectrophotometry and Particle size analyzer. All the results showcase the beneficial effects of silver nanoparticles synthesized plant extracts and may be used as a novel medicine in the field of chemotherapy.


  1. Sinha, A., Vaggu, R. G., Swain, R., & Patnaik, S. (2023). Repurposing of RAS-Pathway Mediated Drugs for Intestinal Inflammation Related Diseases for Treating SARS-CoV-2 Infection. Current Microbiology, 80(6). https://doi.org/10.1007/s00284-023-03304-1

Abstract
Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is an emerging zoonotic virus, which causes Coronavirus Disease 2019 (COVID-19). Entry of coronaviruses into the cell depends on binding of the viral spike (S) proteins to cellular receptors Angiotensin-converting enzyme 2 (ACE2). The virus-mediated reduction of ACE2/Ang1-7 causes flooding of inflammatory cytokines. A similar scenario of hyper immunologic reaction has been witnessed in the context of Intestinal Inflammatory Diseases (IIDs) with the deregulation of ACE2. This review summarizes several IIDs that lead to such susceptible conditions. It discusses suitable mechanisms of how ACE2, being a crucial regulator of the Renin-Angiotensin System (RAS) signaling pathway, can affect the physiology of intestine as well as lungs, the primary site of SARS-CoV-2 infection. ACE2, as a SARS-CoV-2 receptor, establishes a critical link between COVID-19 and IIDs. Intercessional studies targeting the RAS signaling pathway in patients may provide a novel strategy for addressing the COVID-19 crisis. Hence, the modulation of these key RAS pathway members can be beneficial in both instances. However, it’s difficult to say how beneficial are the ACE inhibitors (ACEI)/ Angiotensin II type-1 receptor blockers (ARBs) during COVID-19. As a result, much more research is needed to better understand the relationship between the RAS and SARS-CoV-2 infection.


  1.  Esmaeilniakooshkghazi, A., Pham, E., George, S. P., Ahrorov, A., Villagomez, F. R., Byington, M., Mukhopadhyay, S., Patnaik, S., Conrad, J. C., Naik, M., Ravi, S., Tebbutt, N., Mooi, J., Reehorst, C. M., Mariadason, J. M., & Khurana, S. (2023). In colon cancer cells fascin1 regulates adherens junction remodeling. The FASEB Journal, 37(3). https://doi.org/10.1096/fj.202201454r

Abstract

Adherens junctions (AJs) are a defining feature of all epithelial cells. They regulate epithelial tissue architecture and integrity, and their dysregulation is a key step in tumor metastasis. AJ remodeling is crucial for cancer progression, and it plays a key role in tumor cell survival, growth, and dissemination. Few studies have examined AJ remodeling in cancer cells consequently, it remains poorly understood and unleveraged in the treatment of metastatic carcinomas. Fascin1 is an actin-bundling protein that is absent from the normal epithelium but its expression in colon cancer is linked to metastasis and increased mortality. Here, we provide the molecular mechanism of AJ remodeling in colon cancer cells and identify for the first time, fascin1’s function in AJ remodeling. We show that in colon cancer cells fascin1 remodels junctional actin and actomyosin contractility which makes AJs less stable but more dynamic. By remodeling AJs fascin1 drives mechanoactivation of WNT/β-catenin signaling and generates “collective plasticity” which influences the behavior of cells during cell migration. The impact of mechanical inputs on WNT/β-catenin activation in cancer cells remains poorly understood. Our findings highlight the role of AJ remodeling and mechanosensitive WNT/β-catenin signaling in the growth and dissemination of colorectal carcinomas. © 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.


  1. Mahapatra, S. R., Dey, J., Raj, T. K., Misra, N., & Suar, M. (2023). Designing a Next-Generation Multiepitope-Based Vaccine against Staphylococcus aureus Using Reverse Vaccinology Approaches. Pathogens (Basel, Switzerland)12(3), 376. https://doi.org/10.3390/pathogens12030376

Abstract

Staphylococcus aureus is a human bacterial pathogen that can cause a wide range of symptoms. As virulent and multi-drug-resistant strains of S. aureus have evolved, invasive S. aureus infections in hospitals and the community have become one of the leading causes of mortality and morbidity. The development of novel techniques is therefore necessary to overcome this bacterial infection. Vaccines are an appropriate alternative in this context to control infections. In this study, the collagen-binding protein (CnBP) from S. aureus was chosen as the target antigen, and a series of computational methods were used to find epitopes that may be used in vaccine development in a systematic way. The epitopes were passed through a filtering pipeline that included antigenicity, toxicity, allergenicity, and cytokine inducibility testing, with the objective of identifying epitopes capable of eliciting both T and B cell-mediated immune responses. To improve vaccine immunogenicity, the final epitopes and phenol-soluble modulin α4 adjuvant were fused together using appropriate linkers; as a consequence, a multiepitope vaccine was developed. The chosen T cell epitope ensemble is expected to cover 99.14% of the global human population. Furthermore, docking and dynamics simulations were used to examine the vaccine’s interaction with the Toll-like receptor 2 (TLR2), revealing great affinity, consistency, and stability between the two. Overall, the data indicate that the vaccine candidate may be extremely successful, and it will need to be evaluated in experimental systems to confirm its efficiency.


  1. Bose, S., Singh, D. V., Adhya, T. K., & Acharya, N. (2023). Escherichia coli, but Not Staphylococcus aureus, Functions as a Chelating Agent That Exhibits Antifungal Activity against the Pathogenic Yeast Candida albicans. Journal of fungi (Basel, Switzerland), 9(3), 286. https://doi.org/10.3390/jof9030286

Abstract

Humans are colonized by diverse populations of microbes. Infections by Candida albicans, an opportunistic fungal pathogen, are a result of imbalances in the gut microbial ecosystem and are due to the suppressed immunity of the host. Here, we explored the potential effects of the polymicrobial interactions of C. albicans with Staphylococcus aureus, a Gram-positive bacterium, and Escherichia coli, a Gram-negative bacterium, in dual and triple in vitro culture systems on their respective growth, morphology, and biofilms. We found that S. aureus promoted the fungal growth and hyphal transition of C. albicans through cell-to-cell contacts; contrarily, both the cell and cell-free culture filtrate of E. coli inhibited fungal growth. A yet to be identified secretory metabolite of E. coli functionally mimicked EDTA and EGTA to exhibit antifungal activity. These findings suggested that E. coli, but not S. aureus, functions as a chelating agent and that E. coli plays a dominant role in regulating excessive growth and, potentially, the commensalism of C. albicans. Using animal models of systemic candidiasis, we found that the E. coli cell-free filtrate suppressed the virulence of C. albicans. In general, this study unraveled a significant antimicrobial activity and a potential role in the nutritional immunity of E. coli, and further determining the underlying processes behind the E. coli-C. albicans interaction could provide critical information in understanding the pathogenicity of C. albicans.


  1. Das, M., Sethy, C., Kundu, C. N., & Tripathy, J. (2023). Synergetic reinforcing effect of graphene oxide and nanosilver on carboxymethyl cellulose/sodium alginate nanocomposite films: Assessment of physicochemical and antibacterial properties. International journal of biological macromolecules, 239, 124185. https://doi.org/10.1016/j.ijbiomac.2023.124185

Abstract

Incorporating single or combined nanofillers in polymeric matrices is a promising approach for developing antimicrobial materials for applications in wound healing and packaging etc. This study reports a facile fabrication of antimicrobial nanocomposite films using biocompatible polymers sodium carboxymethyl cellulose (CMC) and sodium alginate (SA) reinforced with nanosilver (Ag) and graphene oxide (GO) using the solvent casting approach. Eco-friendly synthesis of Ag nanoparticles within a size range of 20-30 nm was carried out within the polymeric solution. GO was introduced into the CMC/SA/Ag solution in different weight percentages. The films were characterized by UV-Vis, FT-IR, Raman, XRD, FE-SEM, EDAX, and TEM. The results indicated the enhanced thermal and mechanical performance of CMC/SA/Ag-GO nanocomposites with increased GO weight %. The antibacterial efficacy of the fabricated films was evaluated on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The CMC/SA/Ag-GO2% nanocomposite exhibited the highest zone of inhibition of 21.30 ± 0.70 mm against E. coli and 18.00 ± 1.00 mm against S. aureus. The CMC/SA/Ag-GO nanocomposites exhibited excellent antibacterial activity as compared to CMC/SA and CMC/SA-Ag due to the synergetic bacterial growth inhibition activities of the GO and Ag. The cytotoxic activity of the prepared nanocomposite films was also assessed to investigate their biocompatibility.


  1. Chakraborthy, M., & Rao, A. (2023). A Feedback Loop between TGF-β1 and ATG5 Mediated by miR-122-5p Regulates Fibrosis and EMT in Human Trabecular Meshwork Cells. Current issues in molecular biology45(3), 2381–2392. https://doi.org/10.3390/cimb45030154

Abstract

Autophagy is a cell’s evolutionary conserved process for degrading and recycling cellular proteins and removing damaged organelles. There has been an increasing interest in identifying the basic cellular mechanism of autophagy and its implications in health and illness during the last decade. Many proteinopathies such as Alzheimer’s and Huntington’s disease are reported to be associated with impaired autophagy. The functional significance of autophagy in exfoliation syndrome/exfoliation glaucoma (XFS/XFG), remains unknown though it is presumed to be impaired autophagy to be responsible for the aggregopathy characteristic of this disease. In the current study we have shown that autophagy or ATG5 is enhanced in response to TGF-β1 in human trabecular meshwork (HTM) cells and TGF-β1 induced autophagy is necessary for increased expression of profibrotic proteins and epithelial to mesenchymal (EMT) through Smad3 that lead to aggregopathy. Inhibition of ATG5 by siRNA mediated knockdown reduced profibrotic and EMT markers and increased protein aggregates in the presence of TGF-β1 stimulation. The miR-122-5p, which was increased upon TGF exposure, was also reduced upon ATG5 inhibition. We thus conclude that TGF-β1 induces autophagy in primary HTM cells and a positive feedback loop exists between TGF-β1 and ATG5 that regulated TGF downstream effects mainly mediated by Smad3 signaling with miR-122-5p also playing a role.


  1. Behera, A., Biswas, M., Ergün, S., Karnati, S., & Sonawane, A. (2023). Detection of Peroxisomal Proteins During Mycobacterial Infection. Methods in molecular biology (Clifton, N.J.), 2643, 123–134. https://doi.org/10.1007/978-1-0716-3048-8_9

Abstract

Peroxisomes are ubiquitous organelles with essential roles in lipid and reactive oxygen species (ROS) metabolism. They are involved in modulating the immune responses during microbial infection, thus having major impact on several bacterial and viral infectious diseases including tuberculosis. Intracellular pathogens such as Mycobacterium tuberculosis (M. tb) employ various strategies to suppress the host oxidative stress mechanisms to avoid killing by the host. Peroxisome-mediated ROS balance is crucial for innate immune responses to M. tb. Therefore, peroxisomes represent promising targets for host-directed therapeutics to tuberculosis. Here, we present protocols used in our laboratory for the culture of M. tb and detection of peroxisomal proteins in M. tb infected macrophages.


  1. Charles, S., Edgar, M. P., & Mahapatra, R. K. (2023). Artificial intelligence based virtual screening study for competitive and allosteric inhibitors of the SARS-CoV-2 main protease. Journal of biomolecular structure & dynamics, 1–19. https://doi.org/10.1080/07391102.2023.2188419

Abstract

SARS-CoV-2 is a highly contagious and dangerous coronavirus that first appeared in late 2019 causing COVID-19, a pandemic of acute respiratory illnesses that is still a threat to health and the general public safety. We performed deep docking studies of 800 M unique compounds in both the active and allosteric sites of the SARS-COV-2 Main Protease (Mpro) and the 15 M and 13 M virtual hits obtained were further taken for conventional docking and molecular dynamic (MD) studies. The best XP Glide docking scores obtained were -14.242 and -12.059 kcal/mol by CHEMBL591669 and the highest binding affinities were -10.5 kcal/mol (from 444215) and -11.2 kcal/mol (from NPC95421) for active and allosteric sites, respectively. Some hits can bind both sites making them a great area of concern. Re-docking of 8 random allosteric complexes in the active site shows a significant reduction in docking scores with a t-test P value of 2.532 × 10-11 at 95% confidence. Some specific interactions have higher elevations in docking scores. MD studies on 15 complexes show that single-ligand systems are stable as compared to double-ligand systems, and the allosteric binders identified are shown to modulate the active site binding as evidenced by the changes in the interaction patterns and stability of ligands and active site residues. When an allosteric complex was docked to the second monomer to check for homodimer formation, the validated homodimer could not be re-established, further supporting the potential of the identified allosteric binders. These findings could be important in developing novel therapeutics against SARS-CoV-2.Communicated by Ramaswamy H. Sarma.


  1. Dash, S. R., Das, B., Das, C., Sinha, S., Paul, S., Pradhan, R., & Kundu, C. N. (2023). Near-infrared enhances antiangiogenic potentiality of quinacrine-gold hybrid nanoparticles in breast cancer stem cells via deregulation of HSP-70/TGF-β. Nanomedicine (London, England), 10.2217/nnm-2022-0243. https://doi.org/10.2217/nnm-2022-0243

Abstract

Aim: This study aimed to explore the antiangiogenic mechanism of quinacrine-gold hybrid nanoparticle (QAuNP) and near-infrared (NIR) radiation in patient-derived primary breast cancer stem cells. Materials & methods: Various cell-based in ovo angiogenesis and in vivo patient-derived xenograft mouse systems were used as models for the study. Results: The experimental results showed that QAuNP + NIR treatment deregulated the HSP-70/TGF-β physical interaction in primary breast cancer stem cells. Reduced TGF-β secretion in the tumor microenvironment inhibited angiogenesis activation in endothelial cells by deregulating the TGF-β-mediated PI3K/AKT/mTOR cascade. Conclusion: This study revealed that QAuNP + NIR irradiation downregulated HSP-70 expression, inhibited the HSP-70/TGF-β interaction, reduced the secretion of TGF-β in the tumor microenvironment and ultimately inhibited TGF-β-mediated angiogenesis.


  1. Kumari, K., Behera, H. T., Nayak, P. P., Sinha, A., Nandi, A., Ghosh, A., Saha, U., Suar, M., Panda, P. K., Verma, S. K., & Raina, V. (2023). Amelioration of lipopeptide biosurfactants for enhanced antibacterial and biocompatibility through molecular antioxidant property by methoxy and carboxyl moieties. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 161, 114493. https://doi.org/10.1016/j.biopha.2023.114493

Abstract

Biosurfactants having surface-active biomolecules have been the cynosure in environment research due to their vast application. However, the lack of information about their low-cost production and detailed mechanistic biocompatibility limits the applicability. The study explores techniques for the production and design of low-cost, biodegradable, and non-toxic biosurfactants from Brevibacterium casei strain LS14 and excavates the mechanistic details of their biomedical properties like antibacterial effects and biocompatibility. Taguchi’s design of experiment was used to optimize for enhancing biosurfactant production by optimal factor combinations like Waste glycerol (1%v/v), peptone (1%w/v), NaCl 0.4% (w/v), and pH 6. Under optimal conditions, the purified biosurfactant reduced the surface tension to 35 mN/m from 72.8 mN/m (MSM) and a critical micelle concentration of 25 mg/ml was achieved. Spectroscopic analyses of the purified biosurfactant using Nuclear Magnetic Resonance suggested it as a lipopeptide biosurfactant. The evaluation of mechanistic antibacterial, antiradical, antiproliferative, and cellular effects indicated the efficient antibacterial activity (against Pseudomonas aeruginosa) of biosurfactants due to free radical scavenging activity and oxidative stress. Moreover, the cellular cytotoxicity was estimated by MTT and other cellular assays revealing the phenomenon as the dose-dependent induction of apoptosis due to free radical scavenging with an LC50 of 55.6 ± 2.3 mg/ml.


  1. Subhadra, S., Sreenivasulu, D., Pattnaik, R., Panda, B. K., & Kumar, S. (2023). Bluetongue virus: Past, present, and future scope. Journal of infection in developing countries, 17(2), 147–156. https://doi.org/10.3855/jidc.16947

Abstract

Bluetongue (BT), once considered a disease of sheep confined to the southern African region, has spread all over the world. BT is a viral disease caused by the bluetongue virus (BTV). BT is regarded as an economically important disease in ruminants of compulsory notification to OIE. BTV is transmitted by the bite of Culicoides species. Research over the years has led to a better understanding of the disease, the nature of the virus life cycle between ruminants and Culicoides species, and its distribution in different geographical regions. Advances have also been made in understanding the molecular structure and function of the virus, the biology of the Culicoides species, its ability to transmit the disease, and the persistence of the virus inside the Culicoides and the mammalian hosts. Global climate change has enabled the colonization of new habitats and the spread of the virus into additional species of the Culicoides vector. This review highlights some of the current findings on the status of BT in the world based on the latest research on disease aspects, virus-host-vector interactions, and the different diagnostic approaches and control strategies available for BTV.


  1. Sarangi, P. K., Mishra, S., Mohanty, P., Singh, P. K., Srivastava, R. K., Pattnaik, R., Adhya, T. K., Das, T., Lenka, B., Gupta, V. K., Sharma, M., & Sahoo, U. K. (2023). Food and fruit waste valorisation for pectin recovery: Recent process technologies and future prospects. International journal of biological macromolecules, 235, 123929. https://doi.org/10.1016/j.ijbiomac.2023.123929

Abstract

Pectin possesses a dual property of resistance and flexibility and thus has diverse commercial value which has generated research interest on this versatile biopolymer. Formulated products using pectin could be useful in food, pharma, foam, plasticiser and paper substitute industries. Pectin is structurally tailor-made for greater bioactivity and diverse applications. Sustainable biorefinery leaves greener footprints while producing high-value bioproducts like pectin. The essential oils and polyphenols obtained as byproducts from a pectin-based biorefinery are useful in cosmetics, toiletries and fragrance industries. Pectin can be extracted from organic sources following eco-friendly strategies, and the extraction techniques, structural alterations and the applications are continually being upgraded and standardized. Pectin has great applications in diverse areas, and its green synthesis is a welcome development. In future, growing industrial application of pectin is anticipated as research orients on biopolymers, biotechnologies and renewable source-based processes. As the world is gradually adopting greener strategies in sync with the global sustainable development goal, active involvement of policy makers and public participation are prime. Governance and policy framing are essential in the transition of the world economy towards circularity since green circular bioeconomy is ill-understood among the public in general and within the administrative circles in particular. Concerted efforts by researchers, investors, innovators, and policy and decision makers to integrate biorefinery technologies as loops within loop of biological structures and bioprocesses is suggested. The review focusses on generation of the different nature of food wastes including fruits and vegetables with cauterization of their components. It discusses the innovative extraction and biotransformation approaches for these waste conversions into value-added products at cost-effective and eco-friendly way. This article compiles numerous effective and efficient and green way pectin extraction techniques with their advantages with varying success in an integrated manner.


  1. Chakraborty, A., Roy, S., Hande, M. P., & Banerjee, B. (2023). Telomere attrition and genomic instability in unexplained recurrent pregnancy loss in humans: A preliminary study. Mutation research. Genetic toxicology and environmental mutagenesis, 886, 503580. https://doi.org/10.1016/j.mrgentox.2022.503580

Abstract

Genome instability is defined as an elevated rate of DNA damage and mutations as a result of exposure to potential direct and indirect mutagens. This current investigation was designed to elucidate the genomic instability among couples experiencing unexplained recurrent pregnancy loss (uRPL). A cohort of 1272 individuals with history of unexplained RPL with normal karyotype was retrospectively screened for levels of intracellular ROS production, baseline genomic instability and telomere functionality. The experimental outcome was compared with 728 fertile control individuals. In this study, it was perceived that individuals with uRPL exhibited higher intracellular oxidative stress, along with higher basal levels of genomic instability as compared with the fertile controls. This observation elucidates the role of genomic instability as well as involvement of telomeres in cases of uRPL. It was also observed that higher oxidative stress might be associated with DNA damage and telomere dysfunction resulting in genomic instability among subjects with unexplained RPL. This study highlighted the assessment of genomic instability status in individuals experiencing uRPL.


  1. Sarkar, K., Dutta, K., Chatterjee, A., Sarkar, J., Das, D., Prasad, A., Chattopadhyay, D., Acharya, K., Das, M., Verma, S. K., & De, S. (2023). Nanotherapeutic potential of antibacterial folic acid-functionalized nanoceria for wound-healing applications. Nanomedicine (London, England), 10.2217/nnm-2022-0233. https://doi.org/10.2217/nnm-2022-0233

Abstract:

Aim: The functionalization and characterization of antibacterial nanoceria with folic acid (FA) and elucidation of their in vivo wound healing application. Materials & methods: Functionalization of nanoceria were done with FA using a chemical method and their antibacterial activity, cellular biocompatibility and in vivo wound healing application were evaluated. Results: The functionalization of nanoceria with FA was done with 10 to 20 nm size and -20.1 mV zeta potential. The nanoformulation showed a bacteriostatic effect along with biocompatibility to different cell lines; 0.1% w/v spray of FA-nanoceria demonstrated excellent wound healing capacity within 14 days in a Wister rat model. Conclusion: The antioxidant and reactive oxygen species scavenging activity of the FA-nanoceria make it a promising therapeutic agent as a unique spray formulation in wound healing applications.


  1. Kumari, K., Nandi, A., Sinha, A., Ghosh, A., Sengupta, S., Saha, U., Singh, P. K., Panda, P. K., Raina, V., & Verma, S. K. (2023). The paradigm of prophylactic viral outbreaks measures by microbial biosurfactants. Journal of infection and public health, 16(4), 575–587. https://doi.org/10.1016/j.jiph.2023.02.016

Abstract

The recent emergence and outbreak of the COVID-19 pandemic confirmed the incompetence of countries across the world to deal with a global public health emergency. Although the recent advent of vaccines is an important prophylactic measure, effective clinical therapy for SARS-Cov-2 is yet to be discovered. With the increasing mortality rate, research has been focused on understanding the pathogenic mechanism and clinical parameters to comprehend COVID-19 infection and propose new avenues for naturally occurring molecules with novel therapeutic properties to alleviate the current situation. In accordance with recent clinical studies and SARS-CoV-2 infection markers, cytokine storm and oxidative stress are entwined pathogenic processes in COVID-19 progression. Lately, Biosurfactants (BSs) have been studied as one of the most advanced biomolecules of microbial origin with anti-inflammatory, antioxidant, antiviral properties, antiadhesive, and antimicrobial properties. Therefore, this review inspects available literature and proposes biosurfactants with these properties to be encouraged for their extensive study in dealing with the current pandemic as new pharmaceutics in the prevention and control of viral spread, treating the symptoms developed after the incubation period through different therapeutic approaches and playing a potential drug delivery model.


  1. Mandal, D., Lohan, S., Sajid, M. I., Alhazza, A., Tiwari, R. K., Parang, K., & Montazeri Aliabadi, H. (2023). Modified Linear Peptides Effectively Silence STAT-3 in Breast Cancer and Ovarian Cancer Cell Lines. Pharmaceutics, 15(2), 666. https://doi.org/10.3390/pharmaceutics15020666

Abstract

RNA interference (RNAi) has drawn enormous attention as a powerful tool because of its capability to interfere with mRNA and protein production. However, designing a safe and efficient delivery system in RNAi therapeutics remains challenging. Herein, we have designed and synthesized several linear peptides containing tryptophan (W) and arginine (R) residues separated by the β-alanine (βA) spacer and attached to a lipophilic fatty acyl chain, cholesterol, or PEG. The peptide backbone sequences were: Ac-C-βA-βA-W4-βA-βA-R4-CO-NH2 and Ac-K-βA-βA-W4-βA-βA-R4-CO-NH2, with only a difference in N-terminal amino acid. The cysteine side chain in the first sequence was used for the conjugation with PEG2000 and PEG550. Alternatively, the side chain of lysine in the second sequence was used for conjugation with cholesterol or oleic acid. We hypothesized that amphiphilic peptides and optimum fatty acyl chain or PEG could function as an effective siRNA carrier by complementing each structural component’s self-assembly and membrane internalization properties. None of the designed peptides showed cytotoxicity up to 10 µM. Serum stability studies suggested that the newly designed peptides efficiently protected siRNA against early degradation by nucleases. Flow cytometry analysis indicated 50-90% cellular uptake of siRNA using the newly developed modified linear peptides (MLPs). Western blot results revealed more than 90% protein downregulation after targeting STAT3 in MDA-MB-231 and SKOV-3 cell lines. In summary, a new peptide class was developed to safely and efficiently deliver siRNA.


  1. Basu, J., Madhulika, S., Murmu, K. C., Mohanty, S., Samal, P., Das, A., Mahapatra, S., Saha, S., Sinha, I., & Prasad, P. (2023). Molecular and epigenetic alterations in normal and malignant myelopoiesis in human leukemia 60 (HL60) promyelocytic cell line model. Frontiers in cell and developmental biology, 11, 1060537. https://doi.org/10.3389/fcell.2023.1060537

Abstract

In vitro cell line model systems are essential in supporting the research community due to their low cost, uniform culturing conditions, homogeneous biological resources, and easy experimental design to study the cause and effect of a gene or a molecule. Human leukemia 60 (HL60) is an in-vitro hematopoietic model system that has been used for decades to study normal myeloid differentiation and leukemia biology. Here, we show that IMDM supplemented with 20% FBS is an optimal culturing condition and induces effective myeloid differentiation compared with RPMI supplemented with 10% FBS when HL60 is induced with 1α,25-dihydroxyvitamin D3 (Vit D3) and all-trans retinoic acid (ATRA). The chromatin organization is compacted, and the repressive epigenetic mark H3K27me3 is enhanced upon HL60-mediated terminal differentiation. Differential gene expression analysis obtained from RNA sequencing in HL60 cells during myeloid differentiation showed the induction of pathways involved in epigenetic regulation, myeloid differentiation, and immune regulation. Using high-throughput transcriptomic data (GSE74246), we show the similarities (genes that did not satisfy |log2FC|>1 and FDR<0.05) and differences (FDR <0.05 and |log2FC|>1) between granulocyte-monocyte progenitor vs HL60 cells, Vit D3 induced monocytes (vMono) in HL60 cells vs primary monocytes (pMono), and HL60 cells vs leukemic blasts at the transcriptomic level. We found striking similarities in biological pathways between these comparisons, suggesting that the HL60 model system can be effectively used for studying myeloid differentiation and leukemic aberrations. The differences obtained could be attributed to the fact that the cellular programs of the leukemic cell line and primary cells are different. We validated several gene expression patterns for different comparisons with CD34+ cells derived from cord blood for myeloid differentiation and AML patients. In addition to the current knowledge, our study further reveals the significance of using HL60 cells as in vitro model system under optimal conditions to understand its potential as normal myeloid differentiation model as well as leukemic model at the molecular level.


  1. Singh, A., Muduli, C., Senanayak, S. P., & Goswami, L. (2023). Graphite nanopowder incorporated xanthan gum scaffold for effective bone tissue regeneration purposes with improved biomineralization. International journal of biological macromolecules, 234, 123724. https://doi.org/10.1016/j.ijbiomac.2023.123724

Abstract

In the current work, biomaterial composed of Xanthan gum and Diethylene glycol dimethacrylate with impregnation of graphite nanopowder filler in their matrices was fabricated successfully for their potential usage in the engineering of bone defects. Various physicochemical properties associated with the biomaterial were characterized using FTIR, XRD, TGA, SEM etc. The biomaterial rheological studies imparted the better notable properties associated with the inclusion of graphite nanopowder. The biomaterial synthesized exhibited a controlled drug release. Adhesion and proliferation of different secondary cell lines do not generate ROS on the current biomaterial and thus show its biocompatibility and non-toxic nature. The synthesized biomaterial’s osteogenic potential on SaOS-2 cells was supported by increased ALP activity, enhanced differentiation and biomineralization under osteoinductive circumstances. The current biomaterial demonstrates that in addition to the drug-delivery applications, it can also be a cost-effective substrate for cellular activities and has all the necessary properties to be considered as a promising alternative material suitable for repairing and restoring bone tissues. We propose that this biomaterial may have commercial importance in the biomedical field.


  1. Patel, P., Nandi, A., Verma, S. K., Kaushik, N., Suar, M., Choi, E. H., & Kaushik, N. K. (2023). Zebrafish-based platform for emerging bio-contaminants and virus inactivation research. The Science of the total environment, 872, 162197. https://doi.org/10.1016/j.scitotenv.2023.162197

Abstract

Emerging bio-contaminants such as viruses have affected health and environment settings of every country. Viruses are the minuscule entities resulting in severe contagious diseases like SARS, MERS, Ebola, and avian influenza. Recent epidemic like the SARS-CoV-2, the virus has undergone mutations strengthen them and allowing to escape from the remedies. Comprehensive knowledge of viruses is essential for the development of targeted therapeutic and vaccination treatments. Animal models mimicking human biology like non-human primates, rats, mice, and rabbits offer competitive advantage to assess risk of viral infections, chemical toxins, nanoparticles, and microbes. However, their economic maintenance has always been an issue. Furthermore, the redundancy of experimental results due to aforementioned aspects is also in examine. Hence, exploration for the alternative animal models is crucial for risk assessments. The current review examines zebrafish traits and explores the possibilities to monitor emerging bio-contaminants. Additionally, a comprehensive picture of the bio contaminant and virus particle invasion and abatement mechanisms in zebrafish and human cells is presented. Moreover, a zebrafish model to investigate the emerging viruses such as coronaviridae and poxviridae has been suggested.


  1. Samanta, I., Roy, P. C., Das, E., Mishra, S., & Chowdhary, G. (2023). Plant Peroxisomal Polyamine Oxidase: A Ubiquitous Enzyme Involved in Abiotic Stress Tolerance. Plants (Basel, Switzerland), 12(3), 652. https://doi.org/10.3390/plants12030652

Abstract

Polyamines (PAs) are positively charged amines that are present in all organisms. In addition to their functions specific to growth and development, they are involved in responding to various biotic and abiotic stress tolerance functions. The appropriate concentration of PA in the cell is maintained by a delicate balance between the catabolism and anabolism of PAs, which is primarily driven by two enzymes, namely diamine oxidase and polyamine oxidase (PAO). PAOs have been found to be localized in multiple subcellular locations, including peroxisomes. This paper presents a holistic account of peroxisomal PAOs. PAOs are flavin adenine dinucleotide-dependent enzymes with varying degrees of substrate specificity. They are expressed differentially upon various abiotic stress conditions, namely heat, cold, salinity, and dehydration. It has also been observed that in a particular species, the various PAO isoforms are expressed differentially with a spatial and temporal distinction. PAOs are targeted to peroxisome via a peroxisomal targeting signal (PTS) type 1. We conducted an extensive bioinformatics analysis of PTS1s present in various peroxisomal PAOs and present a consensus peroxisome targeting signal present in PAOs. Furthermore, we also propose an evolutionary perspective of peroxisomal PAOs. PAOs localized in plant peroxisomes are of potential importance in abiotic stress tolerance since peroxisomes are one of the nodal centers of reactive oxygen species (ROS) homeostasis and an increase in ROS is a major indicator of the plant being in stress conditions; hence, in the future, PAO enzymes could be used as a key candidate for generating abiotic stress tolerant crops.


  1. Kumar Panda, R., Darshana Patra, S., Kumar Mohakud, N., Ranjan Sahu, B., Ghosh, M., Misra, N., & Suar, M. (2023). Draft genome of clinical isolate Salmonella enterica Typhimurium ms204 from Odisha, India, reveals multi drug resistance and decreased virulent gene expression. Gene, 863, 147248. https://doi.org/10.1016/j.gene.2023.147248

Abstract

Salmonellosis, a food-borne illnesses caused by enteropathogenic bacterium Salmonella spp., is a continuous concern in both developed and developing countries. This study was carried out to perform an in-depth examination of an MDR Salmonella strain isolated from gastroenteritis patients in Odisha, India, in order to understand the genomic architecture, distribution of pathogenic island regions, and virulence factor diversity. Fecal samples were obtained from individuals with acute gastroenteritis and further subjected to panel of biochemical tests. The IlluminaHiSeq X sequencer system was used to generate whole-genome sequencing. The draft genome was submitted to gene prediction and annotation using RAST annotation system. Pathogenicity Island database and bioinformatics pipeline were used to find Salmonella pathogenicity islands (SPI) from the built scaffold. The gene expression in SPI1 and SPI2 encoded regions was investigated using qRT-PCR. The taxonomic position of Salmonella enterica subsp. enterica serovar Typhimurium was validated by serotype analysis and 16S rRNA based phylogenetic analysis. The de-novo genome assembly showed total length of 5,034,110 bp and produced 37 contigs. There are nine prophage areas, comprising of 12 regions and scaffold 8 contained a single plasmid, IncFIB. The isolate contains six known SPI genes content which was shown to be largely conserved from SPI1 to SPI2. We identified the sit ABCD cluster regulatory cascade and acquired antibiotic resistance genes in S. enterica Typhimurium ms204. Further research may aid in the correct diagnosis and monitoring of MDR Salmonella strains with a variety of physiological activities.


  1. Dash, S. R., & Kundu, C. N. (2023). Cancer-induced pain management by nanotechnology-based approach. Current pharmaceutical biotechnology, 10.2174/1389201024666230123150856. https://doi.org/10.2174/1389201024666230123150856

Abstract

Cancer patients frequently report experiencing pain as one of their symptoms. Cancerrelated pain is often caused by the tumor itself, especially when the tumor is pressing on nerves. In addition to the pain caused by the tumor itself, patients also experience discomfort from the treatment, such as surgery, chemotherapy, radiation therapy, and the diagnostic procedures. The majority of today’s pain therapies rely on opioid analgesics, which have not been shown to be effective. The adverse effects of opioids and their addictive properties call for the development of innovative treatment techniques. Nanotechnology offers answers to the issues raised above, which are related to the utilization of more conventional modes of therapy. These nanotechnology-based nanotherapeutics reduce the systemic toxicity, offering outstanding selectiveness and prolonged release of the analgesic drugs at the target site. Thus, these reduce cancer-induced pain in the patients. In this article, we will explain the mechanism behind the most common types of pain that are caused by cancer, including neuropathic, somatic, and visceral pain. In addition, a comprehensive discussion is held on the use of various nanotherapeutics as analgesic drug carriers, as well as on their impacts and the potential opportunities that lie ahead in the field of cancer pain treatment.


  1. Jaiswal, K. S., Malka, O., Shauloff, N., Bersudsky, M., Voronov, E., Gupta, B., & Jelinek, R. (2023). Genistein carbon dots exhibit antioxidant and anti-inflammatory effects in vitro. Colloids and surfaces. B, Biointerfaces, 223, 113173. https://doi.org/10.1016/j.colsurfb.2023.113173

Abstract

Genistein, an isoflavone from soybean, has attracted attention due to its health benefits, particularly antioxidant and anti-inflammatory activities. Clinical applications of genistein, however, have been limited due to the considerable hydrophobicity and lower bioavailability of the molecule. In this study, carbon dots (C-dots) synthesized from genistein as the carbonaceous precursor exhibit antioxidant properties in test-tube and cell experiments. Anti-inflammatory activity of the genistein-C-dots was also recorded in LPS stimulated macrophages, manifested in inhibition of pro-inflammatory cytokine levels and enhancement anti-inflammatory cytokine expression. The antioxidant and anti-inflammatory effects of the genistein-C-dots, particularly in comparison to the parent genistein molecules, likely account to the display of functional genistein residues on the C-dots’ surfaces, and low band gap energy facilitating electron scavenging. Importantly, the genistein-C-dots featured biocompatibility and low cytotoxicity, underlining their potential as a therapeutic vehicle against inflammatory conditions.


  1. Chatterjee, R., Mahapatra, S. R., Dey, J., Raj Takur, K., Raina, V., Misra, N., & Suar, M. (2023). An immunoinformatics and structural vaccinology study to design a multi-epitope vaccine against Staphylococcus aureus infection. Journal of molecular recognition: JMR, 36(4), e3007. https://doi.org/10.1002/jmr.3007

Abstract

Staphylococcus aureus has been widely reported to be majorly responsible for causing nosocomial infections worldwide. Due to an increase in antibiotic-resistant strains, the development of an effective vaccine against the bacteria is the most viable alternative. Therefore, in the current work, an effort has been undertaken to develop a novel peptide-based vaccine construct against S aureus that can potentially evoke the B and T cell immune responses. The fibronectin-binding proteins are an attractive target as they play a prominent role in bacterial adherence and host cell invasion and are also well conserved among rapidly mutating pathogens. Therefore, highly immunogenic linear B lymphocytes (LBL), cytotoxic T lymphocytes (CTL), and helper T lymphocytes (HTL) epitopes were identified from the antigenic fibronectin-binding proteins A and B (FnBPA and FnBPB) of S aureus using immunoinformatics approaches. The selected peptides were confirmed to be non-allergenic, non-toxic, and with a high binding affinity to the majority of human leukocyte antigens (HLA) alleles. Consequently, the multi-peptide vaccine construct was developed by fusing the screened epitopes (three LBL, five CTL, and two HTL) together with the suitable adjuvant and linkers. In addition, the tertiary conformation of the peptide construct was modeled and later docked to the Toll-like receptor 2. Subsequently, a molecular dynamics simulation of 100 ns was employed to corroborate the stability of the designed vaccine-receptor complex. Besides exhibiting high immunogenicity and conformational stability, the developed vaccine was observed to possess wide population coverage of 99.51% worldwide. Additional in vivo and in vitro validation studies would certainly corroborate the designed vaccine construct to have improved prophylactic efficacy against S aureus.


  1. Kode, J., Maharana, J., Dar, A. A., Mukherjee, S., Gadewal, N., Sigalapalli, D. K., Kumar, S., Panda, D., Ghosh, S., Keshry, S. S., Mamidi, P., Chattopadhyay, S., Pradhan, T., Kailaje, V., Inamdar, S., & Gujjarwar, V. (2023). 6-Shogaol Exhibits Anti-viral and Anti-inflammatory Activity in COVID-19-Associated Inflammation by Regulating NLRP3 Inflammasomes. ACS omega, 8(2), 2618–2628. https://doi.org/10.1021/acsomega.2c07138

Abstract

Recent global health concern motivated the exploration of natural medicinal plant resources as an alternative target for treating COVID-19 infection and associated inflammation. In the current study, a phytochemical, 6-shogaol [1-(4-hydroxy-3-methoxyphenyl)dec-4-en-3-one; 6-SHO] was investigated as a potential anti-inflammatory and anti-COVID-19 agent. In virus release assay, 6-SHO efficiently (94.5%) inhibited SARS-CoV2 replication. When tested in the inflammasome activation model, 6-SHO displayed mechanistic action by regulating the expression of the inflammasome pathway molecules. In comparison to the existing drugs, remdesivir and hydroxy-chloroquine, 6-SHO was not only found to be as effective as the standard anti-viral drugs but also much superior and safe in terms of predicted physicochemical properties and clinical toxicity. Comparative molecular dynamics simulation demonstrated a stable interaction of 6-SHO with NLRP3 (the key inflammasome regulator) in the explicit water environment. Overall, this study provides important cues for further development of 6-SHO as potential anti-inflammatory and anti-viral therapeutic agents.


  1. Sinha, A., Bhattacharjee, R., Bhattacharya, B., Nandi, A., Shekhar, R., Jana, A., Saha, K., Kumar, L., Patro, S., Panda, P. K., Kaushik, N. K., Suar, M., & Verma, S. K. (2023). The paradigm of miRNA and siRNA influence in Oral-biome. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 159, 114269. https://doi.org/10.1016/j.biopha.2023.114269

Abstract

Short nucleotide sequences like miRNA and siRNA have attracted a lot of interest in Oral-biome investigations. miRNA is a small class of non-coding RNA that regulates gene expression to provide effective regulation of post-transcription. On contrary, siRNA is 21-25 nucleotide dsRNA impairing gene function post-transcriptionally through inhibition of mRNA for homologous dependent gene silencing. This review highlights the application of miRNA in oral biome including oral cancer, dental implants, periodontal diseases, gingival fibroblasts, oral submucous fibrosis, radiation-induced oral mucositis, dental Pulp, and oral lichenoid disease. Moreover, we have also discussed the application of siRNA against the aforementioned disease along with the impact of miRNA and siRNA to the various pathways and molecular effectors pertaining to the dental diseases. The influence of upregulation and downregulation of molecular effector post-treatment with miRNA and siRNA and their impact on the clinical setting has been elucidated. Thus, the mentioned details on application of miRNA and siRNA will provide a novel gateway to the scholars to not only mitigate the long-lasting issue in dentistry but also develop new theragnostic approaches.


  1. Panda, S., Singh, P. K., Mishra, S., Mitra, S., Pattnaik, P., Adhikary, S. D., & Mohapatra, R. K. (2023). Indian Biosimilars and Vaccines at Crossroads-Replicating the Success of Pharmagenerics. Vaccines, 11(1), 110. https://doi.org/10.3390/vaccines11010110

Abstract

The global pharma sector is fast shifting from generics to biologics and biosimilars with the first approval in Europe in 2006 followed by US approval in 2015. In the form of Hepatitis B vaccine, India saw its first recombinant biologics approval in 2000. Around 20% of generic medications and 62% of vaccines are now supplied by the Indian pharmaceutical industry. It is this good position in biologics and biosimilars production that could potentially improve healthcare via decreased treatment cost. India has witnessed large investments in biosimilars over the years. Numerous India-bred new players, e.g., Enzene Biosciences Ltd., are keen on biosimilars and have joined the race alongside the emerging giants, e.g., Biocon and Dr. Reddy’s. A very positive sign was the remarkable disposition during the COVID-19 pandemic by Bharat Biotech and the Serum Institute of India. India’s biopharmaceutical industry has been instrumental in producing and supplying preventives and therapeutics to fight COVID-19. Despite a weak supply chain and workforce pressure, the production was augmented to provide reasonably priced high-quality medications to more than 133 nations. Biosimilars could cost-effectively treat chronic diseases involving expensive conventional therapies, including diabetes, respiratory ailments, cancer, and connective tissue diseases. Biologics and biosimilars have been and are being tested to treat and manage COVID-19 symptoms characterized by inflammation and respiratory distress.


  1. Li, C. P., Wu, D. H., Huang, S. H., Meng, M., Shih, H. T., Lai, M. H., Chen, L. J., Jena, K. K., Hechanova, S. L., Ke, T. J., Chiu, T. Y., Tsai, Z. Y., Chen, G. K., Tsai, K. C., & Leu, W. M. (2023). The Bph45 Gene Confers Resistance against Brown Planthopper in Rice by Reducing the Production of Limonene. International journal of molecular sciences, 24(2), 1798. https://doi.org/10.3390/ijms24021798

Abstract

Brown planthopper (BPH), a monophagous phloem feeder, consumes a large amount of photoassimilates in rice and causes wilting. A near-isogenic line ‘TNG71-Bph45’ was developed from the Oryza sativa japonica variety ‘Tainung 71 (TNG71) carrying a dominant BPH-resistance locus derived from Oryza nivara (IRGC 102165) near the centromere of chromosome 4. We compared the NIL (TNG71-Bph45) and the recurrent parent to explore how the Bph45 gene confers BPH resistance. We found that TNG71-Bph45 is less attractive to BPH at least partially because it produces less limonene. Chiral analysis revealed that the major form of limonene in both rice lines was the L-form. However, both L- and D-limonene attracted BPH when applied exogenously to TNG71-Bph45 rice. The transcript amounts of limonene synthase were significantly higher in TNG71 than in TNG71-Bph45 and were induced by BPH infestation only in the former. Introgression of the Bph45 gene into another japonica variety, Tainan 11, also resulted in a low limonene content. Moreover, several dominantly acting BPH resistance genes introduced into the BPH-sensitive IR24 line compromised its limonene-producing ability and concurrently decreased its attractiveness to BPH. These observations suggest that reducing limonene production may be a common resistance strategy against BPH in rice.


  1. Bhal, S., & Kundu, C. N. (2023). Targeting crosstalk of signaling pathways in cancer stem cells: a promising approach for development of novel anti-cancer therapeutics. Medical oncology (Northwood, London, England), 40(2), 82. https://doi.org/10.1007/s12032-022-01905-7

Abstract

Wnt, Hedgehog (Hh), and Notch signaling pathways are the evolutionarily conserved signaling pathways that regulate the embryonic development and also play crucial role in maintaining stemness properties of cancer stem cells (CSCs) and inducing epithelial-to-mesenchymal transition (EMT), metastasis, and angiogenesis. It has been highly challenging to inhibit the CSCs growth and proliferation as these are capable of evading chemotherapeutic drugs and cause cancer recurrence through multiple signaling pathways. Therefore, novel therapeutic strategies to target the key players involved in the crosstalk of these signaling pathways need to be developed. In this review, we have identified the interacting molecules of Wnt, Hh, and Notch pathways responsible for enhancing the malignant properties of CSCs. Analyzing the functions of these crosstalk molecules will help us to find an approach toward the development of new anti-cancer drugs for inhibition of CSCs growth and progression. Long non-coding RNAs (LncRNAs) play a significant role in various cellular processes, like chromatin remodeling, epigenetic modifications, transcriptional, and post-transcriptional regulations. Here, we have highlighted the research findings suggesting the involvement of LncRNAs in maintenance of the stemness properties of CSCs through modulation of the above-mentioned signaling pathways. We have also discussed about the different therapeutic approaches targeting those key players responsible for mediating the crosstalk between the pathways. Overall, this review article will surely help the cancer biologists to design novel anti-CSCs agents that will open up a new horizon in the field of anti-cancer therapeutics.


  1. Husain, S., Nandi, A., Simnani, F. Z., Saha, U., Ghosh, A., Sinha, A., Sahay, A., Samal, S. K., Panda, P. K., & Verma, S. K. (2023). Emerging Trends in Advanced Translational Applications of Silver Nanoparticles: A Progressing Dawn of Nanotechnology. Journal of functional biomaterials, 14(1), 47. https://doi.org/10.3390/jfb14010047

Abstract

Nanoscience has emerged as a fascinating field of science, with its implementation in multiple applications in the form of nanotechnology. Nanotechnology has recently been more impactful in diverse sectors such as the pharmaceutical industry, agriculture sector, and food market. The peculiar properties which make nanoparticles as an asset are their large surface area and their size, which ranges between 1 and 100 nanometers (nm). Various technologies, such as chemical and biological processes, are being used to synthesize nanoparticles. The green chemistry route has become extremely popular due to its use in the synthesis of nanoparticles. Nanomaterials are versatile and impactful in different day to day applications, resulting in their increased utilization and distribution in human cells, tissues, and organs. Owing to the deployment of nanoparticles at a high demand, the need to produce nanoparticles has raised concerns regarding environmentally friendly processes. These processes are meant to produce nanomaterials with improved physiochemical properties that can have significant uses in the fields of medicine, physics, and biochemistry. Among a plethora of nanomaterials, silver nanoparticles have emerged as the most investigated and used nanoparticle. Silver nanoparticles (AgNPs) have become vital entities of study due to their distinctive properties which the scientific society aims to investigate the uses of. The current review addresses the modern expansion of AgNP synthesis, characterization, and mechanism, as well as global applications of AgNPs and their limitations.


  1. Das, A., Sinha, T., Mishra, S. S., Das, D., & Panda, A. C. (2023). Identification of potential proteins translated from circular RNA splice variants. European journal of cell biology, 102(1), 151286. https://doi.org/10.1016/j.ejcb.2023.151286

Abstract

Circular RNAs (circRNAs) are covalently closed RNA molecules generated from precursor RNAs by the head-to-tail backsplicing of exons. Hundreds of studies demonstrated that circRNAs are ubiquitously expressed and regulate cellular events by modulating microRNA (miRNA) and RNA-binding protein (RBP) activities. A few circRNAs are also known to translate into functional polypeptides regulating cellular physiology. All these functions primarily depend on the full-length sequence of the circRNAs. CircRNA backsplice junction sequence is the key to identifying circRNAs and their full-length mature sequence. However, some multi-exonic circRNAs exist in different isoforms sharing identical backsplice junction sequences and are termed circRNA splice variants. Here, we analyzed the previously published HeLa cell RNA-seq datasets to identify circRNA splice variants using the de novo module of the CIRCexplorer2 circRNA annotation pipeline. A subset of circRNAs with splice variants was validated by the circRNA-rolling circle amplification (circRNA-RCA) method. Interestingly, several validated circRNAs were predicted to translate into proteins by the riboCIRC database. Furthermore, polyribosome fractionation followed by quantitative PCR confirmed the association of a subset of circRNAs with polyribosome supporting their protein-coding potential. Finally, bioinformatics analysis of proteins derived from splice variants of circCORO1C and circASPH suggested altered protein sequences and structures that could affect their physiological functions. Together, our study identified novel circRNA splice variants and their potential translation into protein isoforms which may regulate various physiological processes.


  1. Chanda Roy, P., & Chowdhary, G. (2023). Molecular cloning of glutathione reductase from Oryza sativa, demonstrating its peroxisomal localization and upregulation by abiotic stresses. Acta biochimica Polonica, 70(1), 175–181. https://doi.org/10.18388/abp.2020_6441

Abstract

Abiotic stress is a major constraint on crop productivity and in the agricultural field, multiple abiotic stresses act synchronously leading to substantial damage to plants. A common after-effect of abiotic stress-induced damage in plants is an increased concentration of reactive oxygen species (ROS) leading to oxidative damage. Glutathione reductase (GR) plays a significant role in curtailing ROS. Apart from the GR enzyme, the peroxisome as an organelle also plays a significant role in ROS homeostasis. Here, we report a peroxisome localized GR, whose expression was found to be upregulated by various abiotic stresses. The in silico analysis also revealed that the peroxisomal localization of GR could be a common phenomenon in angiosperms, suggesting that it could be a suitable candidate against abiotic stress combinations.


  1. Behera, C. K., Gyandeep, G., Mishra, R., Mohanty, R. P., Pal, A., Behera, J., Samal, S., & Das, B. (2023). Genetic analysis of a Fanconi anemia case revealed the presence of FANCF mutation (exon 1;469>C-T) with implications to develop acute myeloid leukemia. Molecular biology reports, 50(1), 931–936. https://doi.org/10.1007/s11033-022-08071-z

Abstract

Background: Fanconi anemia (FA) is a rare genetic disorder and one of the most common inherited forms of aplastic anemia. FA is an autosomal recessive or X-linked genetic disorder that is characterized by typical physical malformations and haematopoietic anomalies. In most cases of FA, patients harbor homozygous or double heterozygous mutations in the FANCA (60-65%), FANCC (10-15%), FANCG (~ 10%), FANCD2 (3-6%) or FANCF (2%) genes in different ethnic populations, which leads to inherited bone marrow failure (IBMF). Hence, it is important to screen such mutations in correlation with clinical manifestations of FA in various ethnic populations. Approach: An 11 year old female pediatric patient of an East India family was presented with febrile illness, having thrombocytopenia with positive dengue IgM (Immunoglobulin M) and treated as a case of dengue hemorrhagic fever at the initial stage of diagnosis. Chromosomal breakage study was performed based on the abnormal physical examination, which showed 100% breaks, triradials, and quadrilaterals in mitomycin (MMC)-induced peripheral blood lymphocyte culture. Importantly, conventional cytogenetic assay in most of the bone marrow cells revealed an additional gain in chromosome 3q+ [46,XX,add(3)(q25)] and terminal loss in chr8p- [46,XX,del(8)(p23)], which might have a prognostic relevance in the outcomes of the FA patient. The bone marrow aspiration and biopsy were repeated and the results showed acute leukemia with 39% blast cells. Whole-genome sequencing analysis of the patient confirmed the presence of (exon 1; 496 > C-T) non-sense mutation leading to a truncated FANCF protein attributed to a stop codon at the amino acid position 166. Conclusion: The study reported the presence of a homozygous C-T exon 1 mutation in FANCF gene in the female pediatric patient from Odisha, India associated with FA. Furthermore, both parents were found to be carriers of FANCF gene mutation, as this allele was found to be in heterozygous state upon genome sequencing. The pathogenicity of the agent was robustly supported by the clinical phenotype and biochemical observations, wherein the patient eventually developed acute myeloid leukemia. The findings of the study infer the importance of early detection of FA and the associated mutations, which might lead to the development of acute myeloid leukemia.


  1. Sahu, B., & Bal, N. C. (2023). Adipokines from white adipose tissue in regulation of whole body energy homeostasis. Biochimie, 204, 92–107. https://doi.org/10.1016/j.biochi.2022.09.003

Abstract

Diseases originating from altered energy homeostasis including obesity, and type 2 diabetes are rapidly increasing worldwide. Research in the last few decades on animal models and humans demonstrates that the white adipose tissue (WAT) is critical for energy balance and more than just an energy storage site. WAT orchestrates the whole-body metabolism through inter-organ crosstalk primarily mediated by cytokines named “Adipokines”. The adipokines influence metabolism and fuel selection of the skeletal muscle and liver thereby fine-tuning the load on WAT itself in physiological conditions like starvation, exercise and cold. In addition, adipokine secretion is influenced by various pathological conditions like obesity, inflammation and diabetes. In this review, we have surveyed the current state of knowledge on important adipokines and their significance in regulating energy balance and metabolic diseases. Furthermore, we have summarized the interplay of pro-inflammatory and anti-inflammatory adipokines in the modulation of pathological conditions.


  1. Sahu, B., Tikoo, O., Pati, B., Senapati, U., & Bal, N. C. (2023). Role of Distinct Fat Depots in Metabolic Regulation and Pathological Implications. Reviews of physiology, biochemistry and pharmacology, 186, 135–176. https://doi.org/10.1007/112_2022_73

Abstract

People suffering from obesity and associated metabolic disorders including diabetes are increasing exponentially around the world. Adipose tissue (AT) distribution and alteration in their biochemical properties play a major role in the pathogenesis of these diseases. Emerging evidence suggests that AT heterogeneity and depot-specific physiological changes are vital in the development of insulin resistance in peripheral tissues like muscle and liver. Classically, AT depots are classified into white adipose tissue (WAT) and brown adipose tissue (BAT); WAT is the site of fatty acid storage, while BAT is a dedicated organ of metabolic heat production. The discovery of beige adipocyte clusters in WAT depots indicates AT heterogeneity has a more central role than hither to ascribed. Therefore, we have discussed in detail the current state of understanding on cellular and molecular origin of different AT depots and their relevance toward physiological metabolic homeostasis. A major focus is to highlight the correlation between altered WAT distribution in the body and metabolic pathogenesis in animal models and humans. We have also underscored the disparity in the molecular (including signaling) changes in various WAT tissues during diabetic pathogenesis. Exercise-mediated beneficial alteration in WAT physiology/distribution that protects against metabolic disorders is evolving. Here we have discussed the depot-specific biochemical adjustments induced by different forms of exercise. A detailed understanding of the molecular details of inter-organ crosstalk via substrate utilization/storage and signaling through chemokines provide strategies to target selected WAT depots to pharmacologically mimic the benefits of exercise countering metabolic diseases including diabetes.


  1. Simnani, F. Z., Singh, D., Choudhury, A., & Akhtar, A. (2023). Impact of COVID-19 on Brain and Psychological Health, its Possible Mechanisms, and Coping Strategies. Recent patents on biotechnology, 17(1), 62–79. https://doi.org/10.2174/1872208316666220617110402

Abstract

COVID-19 pandemic has been depicted to possess a robust association with psychological disorders. SARS-CoV-2 is the most recent virus of the coronavirus family and has the potential to bind the angiotensin-converting enzyme (ACE) receptor. The receptor is mainly present peripherally and to some extent in the brain. Different psychological and neurodegenerative disorders can arise due to the peripheral origin of destruction. These triggers could be inflammatory pathways releasing pro-inflammatory cytokines reaching the brain and causing neuroinflammation. In continuation with traditional viruses, SARS-CoV-2 too might lead to brain diseases like meningitis, encephalitis, etc. Besides, several peripheral hormonal changes like cortisol can influence neurochemical alterations, thereby inflicting mood-related activities and psychological phenomena. In this regard, health care workers, frontline line warriors and relatives of COVID-19 patients can be the secondary victims; however, patients with COVID-19 remain the primary ones prone to neurological health problems. Several strategies like socialization, engagement, physical activity, etc., are well-opted measures to get relief from and check psychiatric disturbances. The worth of this review can be attributed to the understanding of brain-related mechanisms of COVID-19 in the context of its mechanism of action to create the pathology pertaining to brain disorder, precisely psychological devastation. Likewise, its epidemiological relevance has concisely been mentioned. Furthermore, different categories and classes of people prone to psychological deterioration are briefly elaborated on. Lastly, some coping strategies and approaches have been discussed to minimize or combat mental health problems.


  1. Panda, J., Sahoo, T., Swain, J., Panda, P. K., Tripathy, B. C., Samantaray, R., & Sahu, R. (2023). The Journey from Porous Materials to Metal-organic Frameworks and their Catalytic Applications: A Review. Current organic synthesis, 20(2), 220–237. https://doi.org/10.2174/1570179419666220223093955

Abstract

Metal-Organic Frameworks (MOFs), a class of inorganic-organic hybrid materials, have been at the center of material science for the past three decades. They are synthesized by metal ions and organic linker precursors and have become very potential materials for different applications ranging from sensing, separation, catalytic behaviour to biomedical applications and drug delivery, owing to their structural flexibility, porosity and functionality. They are also very promising in heterogeneous catalysis for various industrial applications. These catalysts can be easily synthesized with extremely high surface areas, tunable pore sizes, and incorporation of catalytic centers via post-synthetic modification (PSM) or exchange of their components as compared to traditional heterogeneous catalysts, which is the preliminary requirement of a better catalyst. Here, in this review, we have presented the history of MOFs, different synthesis procedures, and MOFcatalysed reactions; for instance, coupling reactions, condensation reactions, Friedel-Crafts reaction, oxidation, etc. Special attention has been given to MOFs containing different catalytic centers, including open metal sites, incorporation of catalytic centers through PSM, and bifunctional acidbase sites. The important role of catalytic centers present in MOFs and reaction mechanisms have also been outlined with examples.


  1. Panda, C., & Mahapatra, R. K. (2023). Bi-Directional Relationship Between Autophagy and Inflammasomes in Neurodegenerative Disorders. Cellular and molecular neurobiology, 43(1), 115–137. https://doi.org/10.1007/s10571-021-01184-2

Abstract

The innate immune system, as the first line of cellular defense, triggers a protective response called inflammation when encountered with invading pathogens. Inflammasome is a multi-protein cytosolic signaling complex that induces inflammation and is critical for inflammation-induced pyroptotic cell death. Inflammasome activation has been found associated with neurodegenerative disorders (NDs), inflammatory diseases, and cancer. Autophagy is a crucial intracellular quality control and homeostasis process which removes the dysfunctional organelles, damaged proteins, and pathogens by sequestering the cytosolic components in a double-membrane vesicle, which eventually fuses with lysosome resulting in cargo degradation. Autophagy disruption has been observed in many NDs presented with persistent neuroinflammation and excessive inflammasome activation. An interplay between inflammation activation and the autophagy process has been realized over the last decade. In the case of NDs, autophagy regulates neuroinflammation load and cellular damage either by engulfing the misfolded protein deposits, dysfunctional mitochondria, or the inflammasome complex itself. A healthy two-way regulation between both cellular processes has been realized for cell survival and cell defense during inflammatory conditions. Therefore, clinical interest in the modulation of inflammasome activation by autophagy inducers is rapidly growing. In this review, we discuss the structural basis of inflammasome activation and the mechanistic ideas of the autophagy process in NDs. Along with comments on multiple ways of neuroinflammation regulation by microglial autophagy, we also present a perspective on pharmacological opportunities in this molecular interplay pertaining to NDs.


  1. Samanta, I., Chaturvedi, S., Roy, P. C., & Chowdhary, G. (2023). Molecular cloning, subcellular localization, and abiotic stress induction analysis of a polyamine oxidase gene from Oryza sativa. International journal of Agronomy, 5686484, 1-9, https://doi.org/10.1155/2023/5686484.

Abstract

Abiotic stress is a major bottleneck for crop productivity. To counter abiotic stresses, plants have developed several strategies, and the accumulation of polyamine (PA) serving as an osmolyte is one of them. The cellular pool of PAs is primarily regulated by polyamine oxidases (PAOs) either by terminal catabolism or by back conversion (BC) of polyamines. The role of PAO in abiotic stress tolerance has also been reported. Polyamine oxidases are primarily localized in the cytosol, cell wall, and apoplastic regions; however, lately, their peroxisomal localization has also been demonstrated. In this study, we reported the detection of polyamine oxidase isoform 3 from Oryza sativa (OsPAO3) in peroxisome as early as 12 h post-transformation under in vitro conditions using fluorescent microscopy. The gene was also found to be significantly upregulated by salinity, dehydration, cold, and heat stress. We have also demonstrated that the expression of OsPAO3 gene was mediated by a second messenger, calcium. The upregulation of OsPAO3 by salinity, dehydration, cold, and heat stresses suggests that it could be a suitable candidate for providing tolerance to plants against abiotic stress combination or stress matrix, which is a common feature in agricultural field conditions. Furthermore, the data provided here would be valuable in understanding the abiotic stress-mediated signal transduction network of PAOs.


  1. Choudhari, J., Nimma, R., Nimal, S. K., Totakura Venkata, S. K., Kundu, G. C., & Gacche, R. N. (2023). Prosopis juliflora (Sw.) DC phytochemicals induce apoptosis and inhibit cell proliferation signaling pathways, EMT, migration, invasion, angiogenesis and stem cell markers in melanoma cell lines. Journal of ethnopharmacology312, 116472. https://doi.org/10.1016/j.jep.2023.116472

Abstract

Ethnopharmacological relevance: Prosopis juliflora (Sw.), DC is a xerophytic plant species that extensively grow in Asia, Africa, Australia, and Brazil. From ancient time P. juliflora is being utilized in various folk remedies for example in wound healing, fever, inflammation, measles, excrescences, diarrhea and dysentery. Traditionally, gum, paste, and smoke obtained from the leaves and pods are applied for anticancer, antidiabetic, anti-inflammatory, and antimicrobial purposes.

Aim of the study: Our previous studies have demonstrated the promising potential of Prosopis Juliflora leaves methanol extract (PJLME) against breast cancer, and suggested its possible integration as a complementary medicine for the effective management of breast cancer. However, evidence against how PJLME mechanistically target the cancer proliferative pathways and other targets is poorly understood. The basic aim of the present study was to understand the anti-melanoma potential of PJLME against B16f10 cells with possible mechanisms of action.

Materials and methods: MTT assay was used to determine cell viability. Wound and transwell migration assay was performed to check migration potential of cells after PJLME treatment, while clonogenic assay was carried out to understand its colony inhibition actvity. Flow cytometry was used to perform annexin V/PI assay (apoptosis assay), ROS assay, cell cycle analysis. In-vitro angiogenesis assay was performed to check formation of capillary like vascular structure after PJLME treatment. Apoptotic genes, signaling pathways markers, EMT markers and stem cell markers were determined by western blotting. In-vivo BALB/C mice xenograft model study was performed to check the effect of PJLME on in-vivo melanoma tumor growth.

Results: The experimental outcome of the present study has clearly demonstrated the inhibition of growth, migration, invasion, colony formation and apoptosis inducing potential of PJLME against mouse melanoma cancer cells. Treatment of B16F10 melanoma cells with PJLME resulted in arrest of cell cycle at G0/G1 phase. Annexin V-FITC/PI assay confirmed the apoptosis inducing potential of PJLME in B16F10 and A375 melanoma cells. Furthermore, Western blot experiments confirmed that the treatment of PJLME downregulates the expression of anti-apoptotic gene like Bcl2 and increase the expression profile of pro-apoptotic genes like Bax, Bad, and Bak in B16F10 melanoma cells. HUVEC (Human umbilical vein endothelial cells) tube formation assay clearly demonstrated the anti-angiogenic potential of PJLME. The study also revealed that PJLME has potential to inhibit the Akt and Erk signaling pathways which are participating in cancer cell proliferation, migration, invasion etc. The outcome of qRT-PCR and immunoblotting analysis clearly unveiled that PJLME treatment leads to downregulation of epithelial-mesenchymal transition (EMT) as well as stem cell markers. Finally, the in-vivo animal xenograft model study also revealed the anti-melanoma potential of PJLME by significantly inhibiting the B16F10 melanoma tumor growth in BALB/c mice model. The LC-ESI-MS/MS analysis of PJLME showed the presence of variety of bioactive molecules associated with anticancer effects.

Conclusion: The outcome of the present investigation clearly demonstrated the anti-melanoma potential of PJLME against B16f10 melanoma cells. PJLME can be explored as an adjuvant or complementary therapy against melanoma cancer, however further studies are required to understand the clinical efficacy of PJLME. Nevertheless, it can be further explored as a promising resource for identification of novel anticancer candidate drug.


  1. Betallu, M. A., Bhalara, S. R., Sapnar, K. B., Tadke, V. B., Meena, K., Srivastava, A., Kundu, G. C., & Gorain, M. (2023). Hybrid Inorganic Complexes as Cancer Therapeutic Agents: In-vitro Validation. Nanotheranostics7(3), 270–280. https://doi.org/10.7150/ntno.81557

Abstract

A series of novel mixed transition metal-Magnesium tartarate complexes of general formulation [MMg(C4H4O6)2 .xH2O] (where M = Mn, Fe, Co, Ni, Cu and Zn) is prepared with bidentate tartarate ligand. The synthesized complexes (C1 to C6) are characterized by various analytical techniques such as Elemental analysis, Thermo gravimetric analysis, FT-IR Spectroscopy, X-ray Diffraction, Magnetic susceptibility study etc. All complexes exhibit the composition MMgL2 where M = Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II) and L = bidentate tartarate ligand. Analytical data reveals all complexes possesses 1:1 (metal: ligand) ratio. FT-IR spectral study shows that bidentate tartarate ligand coordinate with metal ion in a bidentate manner through two oxygen atoms. Thermo gravimetric analysis of all complexes shows that degradation curves of complexes agrees with recommended formulae of the complexes. X-ray diffraction technique suggests that all complexes (C1 to C6) are polycrystalline in nature. All newly synthesized metal tartarate complexes and ligand were screened in vitro for their anticancer activity against human breast cancer (MDA-MB-231) cell line. The bioassays of all these complexes showed C3 (Co) and C5 (Cu) Mg-tartarate complexes contains maximum antiproliferative activity at 200 µg/ml concentration on MDA-MB-231 cells as compared to other complexes. MDA-MB-231 cells treated with C3 (Co) and C5 (Cu) Mg-tartarate complexes also showed inhibition in cell migration.


  1. Nasker, S. S., Ajayan, P. M., & Nayak, S. (2023). Emerging Trends and Future Direction of Graphene Family of Materials as Potential Antimicrobials: A Critical Review. ACS Materials Letters, 5(3), 673-693. DOI: 10.1021/acsmaterialslett.2c01116

Abstract: Graphene research has progressed at an unprecedented rate since 2004 when Novoselov and Geim isolated and described a single sheet of graphene. In fact, the relentless progress in graphene literature over the past decades makes it challenging to diversify research efforts in varied directions. The superior optical, electrical, thermal, and mechanical properties of graphene usher in a broad spectrum of applications that attracts the interest of various scientific domains, including material scientists, physicists, chemists, and biologists. These exceptional properties of the graphene family of materials (Gfam) have inspired researchers to explore a cornucopia of potential applications surrounding graphene and its derivatives in the realm of bacterial, fungal, and viral cells. Herein, we provide an exhaustive discussion of the antimicrobial mechanism of Gfam against different pathogen types: bacteria, fungi, and viruses. In addition, we present the physicochemical differences among members of Gfam and the correlation of their germicidal activities to material properties. A comparative analysis of Gfam’s activities pertaining to bare metals and the enhanced broad-spectrum antimicrobial action of graphene family-based nanocomposites as well as surface coatings are also described. The review analyzes and discusses the present constraints and anticipated future directions that would enable graphene-based nanomaterials to advance as high-performance antimicrobial structures. Thus, Gfam as a robust biocidal material of interest can effectively bridge the gap between academia and industry.

BOOKS/BOOK CHAPTERS:

  1. Mitra, T., Mahapatra, S. S., Mishra, B., Panda, V. K., Malhotra, D., Roychowdhury, A., Kundu G.C., Hypoxia in Cancer: Significance and Impact on Cancer Therapy. Springer Nature Singapore. https://doi.org/10.1007/978-981-99-0313-9_1.
  1. Adebo, O. A., Chinma, C. E., Obadina, A. O., Soares, A. G., Panda, S. K., & Gan, R. Y.  (Eds.) (2023). Indigenous Fermented Foods for the Tropics. Academic Press, USA.
  1. Sahu, S., Parija, T., Panda, S.K. (2023). Starter cultures: an insight into specific applications in flavoring and health promotion. In Adebo, O. A., Chinma, C. E., Obadina, A. O., Soares, A. G., Panda, S. K., & Gan, R. Y.  (Eds.). Indigenous Fermented Foods for the Tropics. Academic Press, USA, 409-418.

4. PATENT FILED/PUBLISHED/GRANTED:

  1. Dr. Bandana Panda, Dr. Gaurav Patri, Dr. Chanakya Nath Kundu, Mr. Biswajit Das. Patent title: A novel application of I -PRF for human dental pulp stem cells proliferation. (India Patent Application no. 202331016204)

Abstract: The present invention relates to a preparation of traditional Indian herbal powder for dental care and its evaluations. Standardization of tooth powder were done by testing pH, tapped density, ash values, moisture content, phytochemical screening, microbial contamination. The herbs used in the powder may contain secondary plant metabolites, so the developed combination may prove efficient for dental care and the various therapeutic uses. Preparation method was simple and cost- effective. The prepared combination is efficient in curing the yellow teeth, dental caries, and gingivitis. The dental care powder is recommended to use infants, i.e the powder is high in safety and free of side effects.

RESEARCH FUNDING

Title of the Sanctioned Project: Quinacrine inhibits metastasis and angiogenesis in oral cancer stem cells through the inhibition of tumour-associated macrophages derived inflammatory cytokines

Principal Investigator’s Name: Prof. Chanakya Nath Kundu

Co-Principal Investigator’s Name: Dr. Tithi Parija

Name of Funding Agency: Indian Council of Medical Research (ICMR), Govt. of India

Abstract of the Project:

Oral cancer is one of the leading causes of cancer related death in men and women. The progression of this cancer is associated with inflammation via the secretion of various cytokines. Tumor associated macrophages (TAMs) are the major cell types within the tumor microenvironment (TME), secrete multiple cytokines, which amplifies tumor aggressiveness through invasion, progression, and metastasis. Although multiple chemotherapeutic drugs have been developed to reduce the tumor growth by depleting the TAMs derived cytokines but these drugs are associated with many drawbacks. Drug resistance, toxicity and requirement of higher doses and are the major issues in clinic. Quinacrine (QC) is a well-known anti-cancer agent, which exhibits various pharmacological properties and anti-cancer potentiality against many cancers. Till now, no study shows the anti-metastatic and anti-angiogenic potential of QC in patient derived TME through cytokines dependent phenomena. Herein, we will systematically study the potentiality of cytokines for the formation of cancer stem cells (CSCs) in patient derived oral cancer cells and evaluated the mechanism of QC mediated-reduction of CSCs properties in cells. To address this issue, cytokines-enriched conditioned medium (CM) will be generated by treating fixed doses of PMA and LPS in co-cultured of SCC-9+THP-1 cells. A CSCs populated environment will be created after addition of cytokine enriched-CM to patient derived primary oral cancer cells. After incubation with CM, induction of inflammation, CSCs growth, metastasis and angiogenesis will be found. QC will reduce the metastasis and angiogenesis by inhibition of inflammatory process through reduction of cytokines in patient derived CSCs.

Ph.D. DEGREE AWARDED (PROVISIONAL)

Student’s Name:  Dr. Soumya Prakash Biswal

Dr. Soumya Prakash Biswal

Supervisor’s Name: Dr Raghavendra Samantaray, Assistant Professor, School of Biotechnology, KIIT Deemed to be University, Bhubaneswar.

Co-Supervisor’s Name: Dr. Rojalin Sahu, Associate Professor, School of Applied Sciences, KIIT Deemed to be University, Bhubaneswar. 

Thesis Title: Design and Synthesis of Metal Organic Frameworks (MOFs) and Metal Complexes for Optical, Catalytic, and Biomedical applications.

Abstract of the Thesis:

With a goal to realise the application potentials of hybrid Metal Organic frameworks (MOFs)in the field of catalytic, environmental and biological research, we have synthesised a seriesof MOFs and critically analysed their structure-function relationship. Briefly, MOF hybridsare a blend of organic and inorganic materials with a crystalline and porous structure. InMOFs, organic linker molecules surround the positively arranged metal ions or clusters.Owing to their higher surface area, porous nature, and post-synthesis modification (PSM)potential in the last decades, MOFs have been extensively used in separation, adsorption,sensing, bio-medical, and environmental applications. We considered synthesising robustUiO-66 MOFs series and perform PSM on them to evaluate their structural diversity and theunderlying optical behavior. The Uio-66 MOF and its PSM with various anhydrides ((Maleic,Acetic, Valeric, Phthalic, and 2,3-Diphenylmaleic (stilbene)) were synthesised viahydrothermal process and characterized by a range of imaging and spectroscopic techniquessuch as diffused reflectance UV-Vis spectroscopy, Powder X-ray Diffraction, ScanningElectron Microscopy imaging, and Transmission Electron Microscopy imaging. The PSM onUiO-66 series, indeed, revealed new optical absorptions in these modified frameworks, andthe new optical absorptions are critically analysed by both experiments and theory (DFTcalculations) from the view point of Electron Withdrawing (EWG) and Electron DonatingGroup (EDG) behavior of the added functionalities in the UiO-66. Apart from these noveloptical variations in UiO-66 series, we assumed the catalytic potentiality in theseframeworks. Upon undertaking the catalytic study, UiO-66 proved themselves as excellentcatalyst for the model hydrogenation reduction of 4-Nitro Phenol (4-NP) to 4-Amino Phenol(4-AP), where NaBH 4 was used as a reducer. The study results implicate excellent catalyticbehaviour of UiO-66 due to the Lewis acid nature at metal nodes and larger surface area. Themost interesting finding of the study was the excellent recyclability ability of UiO-66confirmed by repetitive consecutive use in our study. We have also developed nano-composite of metal oxides for exploring their diverse applications, such as catalyst action andbiological activities. Overall, the current study encompasses synthesis, characterisation, bio-informatics structural elucidation, and application studies giving a rich insight into the Metal-Organic Framework and Nano-composites developments.


Student’s Name:  Dr. Bipasa Kar

Supervisor’s Name: Dr. Debdutta Bhattacharya, Scientist-D, ICMR – Regional Medical Research Center, Bhubaneswar.

Co-Supervisor’s Name: Dr. C.N.Kundu, Professor Research Dean, School of Biotechnology, KIIT Deemed to be University, Bhubaneswar, Odisha. 

Thesis Title:Identification of phyto – compounds as antimicrobials against beta-lactamase-producing gram-negative bacteria: in silico, in vitro study.

Abstract of the Thesis:

Over the past few years antimicrobial resistance is a huge concern. In the prime threat pathogen list, beta-lactamase producing gram-negative pathogens takes the top position. To tackle this antimicrobial resistance, both epidemiological study and drug discovery are important. Here we have screened bacterial clinical isolates from NE India and performed a prevalence study for beta-lactamase genes. This study showed that, 38% were only ESBL producers and 24% were only MBL producers in those regions. Next we selected CTX-M-3, CTX-M-15, NDM-1 and VIM-1 as representatives for searching alternative drugs. We have performed molecular docking and pharmacokinetic property screening to select best phyto-compounds. Next we selected four compounds against CTX-M-3 and CTX-M-15 proteins and four against NDM-1, VIM-1 protein. Among the best phyto-compounds silibinin against CTX-M proteins and hesperetin against NDM-1, VIM-1 proteins showed highest binding affinity, stability throughout the MD run. Next, the compounds were purchased to screen theirin vitro anti-bacterial effects. The compounds alone and in combination with conventional antibiotics acted as expected. Anti-bacterial assay revealed the compounds are bactericidal in nature. Both the compounds showed significant beta-lactamase inhibitory activity. Thus, both in silico and in vitro study confirmed the beta-lactamase inhibitory activity of silibinin and hesperetin and they both can be further promoted for in vivo clinical trials. 


Student’s Name:  Dr. Akashi

Supervisor’s Name: Dr. Sam J Mathew, Associate Professor, Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad, Haryana 

Co-Supervisor’s Name: Dr. Mrutyunjay Suar, Professor and Director General R&D KIIT, School of Biotechnology, KIIT Deemed to be University, Bhubaneswar, Odisha.

Thesis Title: Characterization of the role of Myosin Heavy chains (MyHCs) in Mammalian Ske letal Muscle Development and Disease.

Dr. Akashi

Abstract of the Thesis:

Skeletal muscle contractility is essential for fundamental functions such as posture maintenance, locomotion and metabolic homeostasis. Interaction between the actin and myosin filaments of the functional unit of muscle, the sarcomere, is responsible for the contraction. Muscle myosins are hexameric ATPase dependent motors comprising one pair of myosin heavy chains (MyHCs) and two pairs of myosin light chains. Apart from the MyHC isoforms expressed in adult muscle fibers, three developmental MyHC isoforms, namely, MyHC-embryonic, MyHC-slow and MyHC-perinatal are expressed during embryonic development and muscle regeneration. Mutations in genes encoding these developmental MyHCs have been reported to lead to several myopathies, cardiomyopathies and contracture syndromes, suggesting their crucial role in embryonicdevelopment. However, the precise function of these developmental isoforms is not clear. Therefore, we have investigated the functional significance of MyHC-embryonic, -slow and perinatal using in-vitro and in-vivo models of myogenic differentiation. Using Myh3 loss of function mice, we found that the lack of MyHC-embryonic leads to mis-regulation of other MyHCs, alterations in fiber size, fiber number and fiber type at neonatal stages. We also find that loss of MyHC-embryonic leads to mis-regulation of genes involved in muscle differentiation. Interestingly, the loss of MyHC-embryonic has distinct effects on different muscles, possibly reflecting the unique fiber type composition of different muscles. Most significantly, our results indicate that MyHC-embryonic is required during embryonic and fetal myogenesis to regulate myogenic progenitors and myoblastdifferentiation through paracrine signaling. Using C2C12 mouse myoblasts cell line as an in-vitro model of myogenic differentiation, we find that loss of MyHC-perinatal leads to altered expression dynamics of transcription factors regulating myogenesis, precocious differentiation of myoblasts by activating caspase3-mediated differentiation program, cellular metabolism shifts towards glycolytic type and non-cell autonomous regulation of myoblasts and muscle precursors. Remarkably, siRNA mediated knockdown of MyHC-embryonic or MyHC-perinatal results in distinct effects on MyHC expression. Absence of MyHC-embryonic leads to compensatory upregulation of MyHC-slow isoform, a slow muscle specific isoform. However, MyHC-perinatal knockdown results in compensatory upregulation of MyHC-IIb isoform, a fast muscle specific isoform. This indicates both MyHC-embryonic and MyHC-perinatal have specific roles in the regulation of MyHC isoform expression. We have also investigated the crucial role of MyHC-perinatal in muscle regeneration post injury. Our work suggests that the knockdown of MyHC-perinatal in regenerating muscle leads to alterations in fiber morphology, reduction in adult muscle stem cell numbers and induces fibrosis at 5 days post injury. Preliminary experiments of the MyHC-slow specific knockdown during C2C12 myogenic differentiation have not shown significant effects on the expression dynamics of myogenic regulatory factors. Overall, our data reveals the crucial roles of developmental MyHC isoforms in proper muscle formation.


Student’s Name: Dr. Aniruddha Das

Supervisor’s Name: Dr. Amaresh C. Panda, Scientist-D, Institute of Life Sciences, Bhubaneswar, Odisha.

Co-Supervisor’s Name: Dr. Selvakumar Elangovan, Associate Professor, School of Biotechnology, KIIT Deemed to be University, Bhubaneswar, Odisha.  

Thesis Title:Identification and characterization of circular RNA splice variants.

Abstract of the Thesis:

Circular RNAs (circRNAs) are a class of RNA molecules with regulatory functions. Circular RNAs are covalently closed RNA molecules generated by a process known as backsplicing. CircRNAs with no free ends make them a very stable form of RNA resistant to exonuclease degradation. Circular RNAs have also been shown to play a role in several disease conditions such as cancer, cardiovascular diseases, diabetes mellitus, and neurological disorders. CircRNAs affect cellular pathways through miRNA sponging or RBP sponging activity or by translating into protein. These functions primarily depend on the circRNA sequence, which accounts for its association with miRNAs/RBPs and determines their translation into proteins.


The unique chimeric backsplice junction gives a unique feature to circRNAs, separating them from their linear counterpart and essential for their detection and quantification. Recent advances in high-throughput RNA sequence strategies involving circRNA enrichment followed by sequencing could detect and quantify these circular RNAs with higher accuracy. However, it is technically challenging and error-prone to derive the full-length sequence of circRNA splice variants from RNA sequencing due to their low abundance, similarity with linear mRNA counterpart, and alternative splicing. Since circRNA sequence determines the function of circRNA, it becomes increasingly important to devise a method for accurate and efficient identification of the full-length sequence of circRNA and the function of circRNA splice variants in a given cell.


In this study, we sought to develop a method where total RNA was enriched for circRNA using RNase R exonuclease treatment followed by rolling-circle cDNA synthesis using H-minus reverse transcriptase resulting in tandem repeats of circRNA cDNA. First, the full-length cDNA was PCR amplified using one primer over the unique backspliced junction region and the reverse primer immediately upstream of the forward primer. These full-length PCR products were then resolved on an agarose gel followed by Sanger sequencing to reveal the different splice variants of circRNAs. Furthermore, we identified that the differential association of miRNAs and RBPs with different circRNA sequences leads to altered regulation of genes that eventually affect the regulation of various cellular processes.
In addition, the computational analysis identified altered open reading frames (ORFs) in circRNA splice variants. Furthermore, several circRNAs in HeLa cells were predicted to be translated into peptides spanning backsplice junctions and were supported by mass spectrometry analysis. Further, polyribosome analysis followed by quantitative-PCR confirmed the association of a subset of circRNAs with polyribosome supporting their protein-coding potential. Finally, bioinformatics analysis of proteins derived from splice variants of circASPH, circCORO1C, circASXL1, and circZNF148 suggested altered domains and structures which could have different physiological functions. However, further studies are required to explore the physiological functions of these novel proteins identified here. Together, our study identified novel protein isoforms translated from circRNAs which may regulate different physiological processes.


Student’s Name: Dr. Gyan Prakash Mishra

Supervisor’s Name:Dr. Sunil K Raghav, Scientist-F, DBT – Institute of Life Sciences, Bhubaneswar, Odisha. 

Co-Supervisor’s Name:Dr.Bhawna Gupta, Associate Professor, School of Biotechnology, KIIT Deemed to be University, Bhubaneswar, Odisha. 

Thesis Title: A multi-omics approach to understand the host response upon pathogen infection.

Abstract of the Thesis:

Pathogen infection such as bacterial or viral in both human and mice elicit host response through activation of cross-antigen presenting cells such as dendritic cells (DCs). DCs generate various immune responses and also educate naive T cells to differentiate into effector T cells to clear the pathogen. Transcription of immune response genes in these DCs is tightly controlled to maintain the balance between multiple immune responses such as inflammatory, anti-inflammatory and antiviral. However, how these responses are regulated at the molecular level through epigenetic regulation of gene expression is largely unknown. To understand DCs response against bacteria or viruses, here in this study, we focused on the murine conventional type -I dendritic cells (cDC1) activated with various Toll-like receptors (TLR) that recognize pathogen associated molecular pattern (PAMPs) associated with bacteria or viruses. We utilized the multi-omics approach to capture the transcriptome and genome wide enhancer activity through RNA-seq and H3K27ac ChIP-seq respectively in TLR9, TLR3 stimulation conditions. To understand the epigenetic control of various immune responses in cDC1, we focused on the global co-repressor protein NCoR1. We integrated genome-wide binding of NCoR1 using ChIP-seq in TLR activation condition along with RNA-seq and H3K27ac ChIP-seq in NCoR1 depleted condition to understand the overall impact on enhancer activity and gene expression upon TLR activation. Comprehensive analysis of the multi-omics datasets revealed spatio-temporal activity of TLR9 and TLR3 specific enhancers. Moreover, NCoR1 depletion increased enhancer activity and expression of genes belonging to inflammatory, anti-inflammatory as well as antiviral response in TLR9 activation whereas TLR3 showed decreased enhancer activity and expression of antiviral genes. The differential effect of NCoR1 in TLR3 versus TLR9, we mapped several publicly available ChIP-seq datasets. We found IRF3, an important transcription factor under TLR3 activation, showed decreased phosphorylation as well as binding at key enhancer regions of important antiviral genes. Furthermore, the decreased transcription of antiviral genes after NCoR1 knock-down translated into expected increase in Sendai virus (SeV) load. Overall, our study showed that the role of NCoR1 as corepressor is biased or skewed towards TLR9 as compared to TLR3.


During the analysis of multi-omics datasets that we generated from NGS experiments, a large number of genomic regions, such as transcription factor binding sites, chromatin marks were identified and the overlap of these genomics regions were performed to answer a variety of biological questions. Although several command-line tools are available to perform such an analysis, we observed there is a notable lack of an integrated web server application to perform genomic region intersections, generate publication-ready plots and perform functional annotation. Hence to address this gap, in this study we also developed a comprehensive and user-friendly web server application called BedSect.


Another host response particularly against RNA viruses is through molecular editing by host deaminase enzymes such as APOBEC and ADAR proteins. APOBEC and ADAR lead to C-to-T/G-to-A and A-to-G/T-to-C transition respectively. To understand the role of RNA editing, we studied the transcriptome of SARS-Cov2 virus isolated from 246 COVID-19 positive patients in India till Jun’ 2020 and 1106 samples between Nov’ 20 to May 2021. We performed intra-host variability (iSNV) analysis to identify mutations in the viral genome and found a skewed distribution of APOBEC and ADAR specific mutations compared to other mutations. Interestingly, many samples carried iSNVs at positions which defined the B.1 lineage and the India specific B.6 lineage. We further confirmed RNA editing in SARS-CoV2 viral genome by an observed high and constitutive ADARB1 expression in SARS-CoV-2 infected Vero cells. Further, we observed significant accumulation of iSNVs in Vero cells infected with SARS-CoV-2 from 11 samples at the seventh-passage compared to the original viral isolate. Overall, our study indicates that the host response to virus infection through RNA editing may lead to accumulation of variants in spatio-temporal scales and that might increase the fitness of the strains by influencing its virulence, infectivity or transmissibility that may be both beneficial or detrimental to the virus.


In summary, we employed the state-of-the-art multi-omics approach to understand the host response upon pathogen infection in murine dendritic cells and humans infected with SARS-CoV2 virus. Our study deciphered several molecular mechanisms such as epigenetic and RNA editing regulation as a host response toward pathogen infection.


Student’s Name:  Dr. Madhurima Chatterjee

Supervisor’s Name: Dr. Gopal Chowdhary, Associate Professor, School of Biotechnology, KIIT Deemed to be University, Bhubaneswar, Odisha

Co-Supervisor’s Name: Dr. Uma Rao, Former Head, Division of Nematology, ICAR-Indian Agricultural Research Institute, PUSA, New Delhi

Thesis Title: Exogenous and host-mediated RNAi gene silencing of functionally different genes in spotted pod borer Maruca vitrata (Lepidoptera: Crambidae)

Dr. Madhurima Chatterjee

Abstract of the Thesis:

The productivity of grain legumes is critically constrained due to several biotic and abiotic stresses resulting tremendous yield and economic losses every year worldwide. The larval stages of Lepidopteran spotted pod borer, Maruca vitrata is one of the most serious herbivores damaging more than 50% of annual pigeonpea production by attacking various parts of the plant including flowers and pods. Moreover, synthetic insecticide resistance has become the major limitation in managing these insect pests. In this study, the possibility of gene silencing is exhibited in M. vitrata through exogenous administration of target gene specific dsRNA and also host-delivered RNAi in pigeonpea.
To begin with, an array of functionally important genes, associated with the midgut, chemosensory, cell signalling and development, named Serine Protease 33 (SP33), H+ transporting ATP synthase delta subunit (HTAS), Triosephosphate Isomerse (TIM), Acyl-CoA delta-9 desaturase (ACCOA), Glutamate Receptor Ionotropic, Kainate 2-like (GLUR), Cadherin-like protein (CDH), non-visual Arrestin (ARR2) were selected to standardize efficient dsRNA delivery strategy. The study could establish the effect of gene silencing in M. vitrata through haemolymph injection as well as ingestion. Efficient transcript perturbation of target genes, higher transcript accumulation of key RNAi pathway gene DICER and detection of siRNA in dsRNA injected larvae authenticated successful gene silencing in M. vitrata. Also, identification of inhibitor molecules like morpholine, piperidine, carboxamide and piperidine–carboxamide through in silico analysis for blocking the function of SP33 established the utility of functional genomics.
The second part of the research work deals with the successful demonstration of gene silencing in M. vitrata through host-delivered RNAi (HDRNAi) in the highly recalcitrant pigeonpea for three functionally different target genes, Alpha-amylase (α-amylase), Chymotrypsin-like serine protease (CTLP) and Tropomyosin (TPM). In continuation of the first part showing the effect of gene silencing by haemolymph injection, the onset of HDRNAi has been validated in pigeonpea by introducing RNAi-hairpin constructs of the three selected target genes using shoot apical meristem-targeted in planta transformation strategy. Evaluation of transgenic events at plant level bioassays revealed reduced insect feeding as well as larval mortality compared to the wild type control plants. Additionally, molecular evidences for down regulation of the target genes in the insects that fed on the respective transgenic plants further authenticated successful HDRNAi.


NATIONAL/INTERNATIONAL AWARD RECEIVED BY THE FACULTY MEMBER

Name of the Faculty: Prof. Gopal C. Kundu

Title of the Award: Fellow of The Royal Society of Chemistry (FRSC)

Award Received: Name of the Agency Royal Society of Chemistry, Place United Kingdom

About the Award:

Prof. Gopal C. Kundu, Director (R&D), KIIT Deemed to be University (KIIT DU), Professor, KIIT School of Biotechnology, and Professor, Medicine and Molecular Research, KIMS has recently been admitted to a prestigious Fellow of the Royal Society of Chemistry (FRSC), a professional society based in the United Kingdom for his outstanding contribution in the area of drug discovery and breast cancer research. This Society with over 55,000 members across the world is an internationally renowned not-for-profit publishing and knowledge business, and has a reputation as an influential champion for the Chemical Sciences.

Through the ‘Leaders in the Field’ scheme, the Society identifies outstanding individuals in the chemical science community. Fellows of the RSC have the opportunity to channel their expertise and commitment to promoting the value of chemistry and chemical biology, through the international framework of the Society. The Royal Society of Chemistry (RSC) was established in 1841. RSC partners with industry and academia, advises governments on policy, and promotes the talent, collaboration, innovation, information and ideas that lead to great advances in science


Name of the Faculty: Dr K Sony Reddy

Title of the Award: INSA Medal for Young Scientists 2022

Award Received: Honorarium of INR 1,00,000

Name of the Agency: Indian National Science Academy, DST, Place: New Delhi

About the Award:

The Indian National Science Academy (INSA), established in 1935 with the objective to promote science in India and harness scientific knowledge for human and national welfare. Indian National Science Academy (INSA), New Delhi is an autonomous institution of Dept. Science & Technology, Govt. of India. It is recognized by the Government of India, as a Premier National Scientific Society. A Government of India’s decision in 1968 has mandated INSA to represent India in all the international science fora. It also publishes journals, holds scientific conferences, and publishes proceedings and monographs. To encourage the pursuit of excellence in the field of ‘Science and Technology,’ the academy established four categories of awards: International Awards, General Medal & Lecture Awards, Subject-wise Medals/Lectures, and Awards for Young Scientists.
The INSA Young Scientist Award, instituted in 1974, is considered as the highest recognition of promise, creativity and excellence. The award is made annually to the individuals below the age of 40 years, and being distinguished for these attributes. The research carried out in India by the nominees is been taken into consideration for the award. In 2022, 4 scientists (including Dr. K Sony Reddy), working in the field of Health Sciences throughout India, have been awarded.


ANY NATIONAL/INTERNATIONAL AWARD OR PUBLICATION (R&D RELATED) RECEIVED BY STUDENTS

Name of the Student: Saptarshi Sinha

Title of the Award: Research Excellence Award 2022

Award Received: Institute of Scholars (InSc), India

Sanctioned Amount of Project or Award: Not applicable

About the Award:

Research Excellence Award 2022 for the research work titled “Olaparib enhances the Resveratrol-mediated Apoptosis in Breast Cancer Cells by inhibiting the Homologous Recombination Repair pathway”, published in Experimental Cell Research, Elsevier.

Although sensitization of BRCA-mutated, homologous recombination (HR)-deficient breast cancer cells through PARP inhibitor is widely studied, not much is known about the treatment of BRCA-wild-type, HR-proficient breast cancer. Our aim was to investigate whether a natural bioactive compound, Resveratrol (RES), can induce DNA double-strand breaks in HR-proficient breast cancer cells and Olaparib (OLA), a PARP inhibitor, can enhance the RES-mediated apoptosis by deregulating the HR repair pathway. The detailed mechanism of anti-cancer action of RES+OLA combination in breast cancer has been evaluated using in vitro (MCF-7 and T47D cell lines), ex vivo (patient-derived primary breast cancer cells), and in vivo (xenograft mice) preclinical model systems. OLA increased RES-mediated DNA damage, downregulated the HR proteins, caused a late S/G2 arrest, enhanced apoptosis and cell death in RES pre-treated breast cancer cells at much lower concentrations than their individual treatments. Direct measurement of HR pathway activity using a GFP plasmid-based assay demonstrated reduced HR efficiency in I-SceI endonuclease-transfected cells treated with OLA. Moreover, RES+OLA treatment also caused significant reduction in PARP1-mediated PARylation and efficiently trapped PARP1 at the DNA damage site. Upon RES treatment, PARylated PARP1 was found to interact with BRCA1, which then activated other HR proteins. But after addition of OLA in RES pre-treated cells, PARP1 could not interact with BRCA1 due to inhibition of PARylation. This resulted in deregulation of HR pathway. To further confirm the role of BRCA1 in PARP1-mediated HR pathway activation, BRCA1 was knocked down that caused complete inhibition of HR pathway activity, and further enhanced apoptosis after RES+OLA treatment in BRCA1-silenced cells. In agreement with in vitro data, similar experimental results were obtained in ex vivo patient-derived breast cancer cells and in vivo xenograft mice. Thus, RES+OLA combination treatment enhanced breast cancer cell death by causing excessive DNA damage and also by simultaneously inhibiting the HR pathway.

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