Aurora Kinase A Is Overexpressed in Human Retinoblastoma and Correlates with Histopathologic High-Risk Factors: Implications for Targeted Therapy.

Retinoblastoma (RB) is an intraocular malignancy initiated by loss of RB1 function and/or dysregulation of MYCN oncogene. RB is primarily treated with chemotherapy; however, systemic toxicity and long-term adverse effects remain a significant challenge necessitating the identification of specific molecular targets. Aurora kinase A (AURKA), a critical cell cycle regulator, contributes to cancer pathogenesis, especially in RB1-deficient and MYCN-dysregulated tumors. Our immunohistochemistry study in patient specimens (n = 67) discovered that AURKA is overexpressed in RB, and elevated expression correlates with one or more histopathologic high-risk factors, such as tumor involvement of the optic nerve, choroid, sclera, and/or anterior segment. More specifically, AURKA is ubiquitously expressed in most advanced-stage RB tumors that show a suboptimal response to chemotherapy. shRNA-mediated depletion/pharmacologic inhibition studies in cell lines, patient-derived cells, in vivo xenografts, and enucleated patient specimens confirm that RB cells are highly sensitive to a lack of functional AURKA. In addition, we deciphered that AURKA and MYCN associate with each other to regulate their levels in RB cells. Overall, our results demonstrate a previously unknown up-regulation of AURKA in RB, facilitated by its crosstalk with MYCN, and elevated levels of this kinase may indicate unfavorable prognosis in tumors refractory to chemotherapy. This study provides a rationale and confirms that therapeutic targeting of elevated AURKA in RB could be a potential treatment approach.

SOX4 induces cytoskeleton remodeling and promotes cell motility via N-wasp/ARP2/3 pathway in colorectal cancer cells.

Filopodia are thin, actin-rich projection from the plasma membrane that promote cancer cell invasion and migration. Sex-determining region Y-related high-mobility group-box 4 (SOX4) is a crucial transcription factor that plays a role in the development and metastasis of colorectal cancer (CRC). However, the involvement of SOX4 in cytoskeleton remodeling in CRC remains unknown. For the first time, we demonstrate that SOX4 is a potent regulator of filopodia formation in CRC cells. Overexpression of SOX4 protein enhances both migration and invasion ability of HCT116, and CACO2 cells, which is relevant to the metastasis. Furthermore, through phalloidin staining, cytoskeleton re-assembly was observed in SOX4-modified cell lines. Enhanced expression of SOX4 increased the number and length of filopodia on cell surface. In contrast, silencing SOX4 in SW620 cells with higher endogenous expression of SOX4, impeded the filopodia formation. Moreover, SOX4 was found to be positively regulating the expression of central regulators of actin cytoskeleton - N-Wiskott-Aldrich syndrome protein (N-WASP); WAVE2; Actin related proteins, ARP2 and ARP3. Inhibiting the N-WASP/ARP2/3 pathway diminishes the filopodia formation and the migration of CRC cells. These results indicate the crucial role of SOX4 in the regulation of filopodia formation mediated by N-WASP/ARP2/3 pathway in CRC cells.

Biogenic Zinc Oxide Nanoparticles synthesized from Tinospora Cordifolia induce oxidative stress, mitochondrial damage and apoptosis in Colorectal Cancer.

Cancer chemotherapy remains a serious challenge, and new approaches to therapy are urgently needed to build novel treatment regimens. The methanol extract of the stem of Tinospora Cordifolia was used to synthesize biogenic zinc oxide nanoparticles (ZnO-NPs) that display anticancer activities against colorectal cancer. Biogenic ZnO-NPs synthesized from methanol extract of Tinospora Cordifolia stem (ZnO-NPs TM) were tested against HCT-116 cell lines to assess anticancer activity. UV-Vis, FTIR, XRD, SEM, and TEM analysis characterized the biogenic ZnO-NPs. To see how well biogenic ZnO-NPs fight cancer, cytotoxicity, AO/EtBr staining, Annexin V/PI staining, mitochondrial membrane potential (MMP), generation of reactive oxygen species (ROS) analysis, and caspase cascade activity analysis were performed to assess the anticancer efficacy of biogenic ZnO-NPs. The IC50 values of biogenic ZnO-NPs treated cells (HCT-116 and Caco-2) were 31.419 ± 0.682µg/ml and 36.675 ± 0.916µg/ml, respectively. qRT-PCR analysis showed that cells treated with biogenic ZnO-NPs Bax and P53 mRNA levels increased significantly (p ≤ 0.001). It showed to have impaired MMP and increased ROS generation. In a corollary, our in vivo study showed that biogenic ZnO-NPs have an anti-tumour effect. Biogenic ZnO-NPs TM showed both in vitro and in vivo anticancer effects that could be employed as anticancer drugs.

Actionable mechanisms of drug tolerance and resistance in Mycobacterium tuberculosis.

The emergence of antimicrobial resistance (AMR) across bacterial pathogens presents a serious threat to global health. This threat is further exacerbated in tuberculosis (TB), mainly due to a protracted treatment regimen involving a combination of drugs. A diversity of factors contributes to the emergence of drug resistance in TB, which is caused by the pathogen Mycobacterium tuberculosis (Mtb). While the traditional genetic mutation-driven drug resistance mechanisms operate in Mtb, there are also several additional unique features of drug resistance in this pathogen. Research in the past decade has enriched our understanding of such unconventional factors as efflux pumps, bacterial heterogeneity, metabolic states, and host microenvironment. Given that the discovery of new antibiotics is outpaced by the emergence of drug resistance patterns displayed by the pathogen, newer strategies for combating drug resistance are desperately needed. In the context of TB, such approaches include targeting the efflux capability of the pathogen, modulating the host environment to prevent bacterial drug tolerance, and activating the host anti-mycobacterial pathways. In this review, we discuss the traditional mechanisms of drug resistance in Mtb, newer understandings and the shaping of a set of unconventional approaches to target both the emergence and treatment of drug resistance in TB.

Erratum: Antibacterial Efficacy of ZnO/Bentonite (Clay) Nanocomposites against Multidrug-Resistant Escherichia coli.

[This corrects the article DOI: 10.1021/acsomega.3c07950.].

SOX4/HDAC2 Axis Enhances Cell Survivability and Reduces Apoptosis by Activating AKT/MAPK Signaling in Colorectal Cancer.

BACKGROUND: Increased SOX4 (SRY-related HMG-box) activity aids cellular transformation and metastasis. However, its specific functions and downstream targets remain to be completely elusive in colorectal cancer (CRC). AIMS: To investigate the role of SOX4 in CRC progression and the underlying mechanism. METHODS: In the current study, online available datasets of CRC patients were explored to check the expression status of SOX4. To investigate the further functions, SOX4 was overexpressed and knocked down in CRC cells. Colony formation assay, flowcytometry analysis, and MTT assay were used to check for proliferation and apoptosis. Acridine orange staining was done to check the role of SOX4 in autophagy induction. Furthermore, western blot, qRT-PCR, and bioinformatic analysis was done to elucidate the downstream molecular mechanism of SOX4. RESULTS: GEPIA database showed enhanced expression of SOX4 mRNA in CRC tumor, and the human protein atlas (HPA) showed strong staining of SOX4 protein in tumor when compared to the normal tissue. Ectopic expression of SOX4 enhanced colony formation ability as well as rescued cells from apoptosis. SOX4 overexpressed cells showed the formation of acidic vesicular organelles (AVOs) which indicated autophagy. Further results revealed the activation of p-AKT/MAPK molecules upon overexpression of SOX4. SOX4 expression was found to be positively correlated with histone deacetylase 2 (HDAC2). Knockdown of SOX4 or HDAC2 inhibition induced apoptosis, revealed by decrease in BCL2 and increase in BAX expression, and inactivated the p-AKT/MAPK signaling. CONCLUSION: The study uncovers that SOX4/HDAC2 axis improves cell survivability and reduces apoptosis via activation of the p-AKT/MAPK pathway.

Antibacterial Efficacy of ZnO/Bentonite (Clay) Nanocomposites against Multidrug-Resistant Escherichia coli.

The emergence of multidrug-resistant (MDR) bacteria has spurred the exploration of therapeutic nanomaterials such as ZnO nanoparticles. However, the inherent nonspecific toxicity of ZnO has posed a significant obstacle to their clinical utilization. In this research, we propose a novel approach to improve the selectivity of the toxicity of ZnO nanoparticles by impregnating them onto a less toxic clay mineral, Bentonite, resulting in ZB nanocomposites (ZB NCs). We hypothesize that these ZB NCs not only reduce toxicity toward both normal and carcinogenic cell lines but also retain the antibacterial properties of pure ZnO nanoparticles. To test this hypothesis, we synthesized ZB NCs by using a precipitation technique and confirmed their structural characteristics through X-ray diffraction and Raman spectroscopy. Electron microscopy revealed composite particles in the size range of 20-50 nm. The BET surface area of ZB NCs, within a relative pressure (P/P0) range of 0.407-0.985, was estimated to be 31.182 m2/g. Notably, 50 mg/mL ZB NCs demonstrated biocompatibility with HCT 116 and HEK 293 cell lines, supported by flow cytometry and fluorescence microscopy analysis. In vitro experiments further confirmed a remarkable five-log reduction in the population of MDR Escherichia coli in the presence of 50 mg/mL of ZB NCs. Antibacterial activity of the nanocomposites was also validated in the HEK293 and HCT 116 cell lines. These findings substantiate our hypothesis and underscore the effectiveness of ZB NCs against MDR E. coli while minimizing nonspecific toxicity toward healthy cells.

Transgenic mouse models support a protective role of type I IFN response in SARS-CoV-2 infection-related lung immunopathology and neuroinvasion.

Type I interferon (IFN-I) response is the first line of host defense against invading viruses. In the absence of definite mouse models, the role of IFN-I in SARS-CoV-2 infection remains perplexing. Here, we develop two mouse models, one with constitutively high IFN-I response (hACE2; Irgm1-/-) and the other with dampened IFN-I response (hACE2; Ifnar1-/-), to comprehend the role of IFN-I response. We report that hACE2; Irgm1-/- mice are resistant to lethal SARS-CoV-2 infection. In contrast, a severe SARS-CoV-2 infection along with immune cell infiltration, cytokine storm, and enhanced pathology is observed in the lungs and brain of hACE2; Ifnar1-/- mice. The hACE2; Irgm1-/-Ifnar1-/- double-knockout mice display loss of the protective phenotype observed in hACE2; Irgm1-/- mice, suggesting that heightened IFN-I response accounts for the observed immunity. Taking the results together, we demonstrate that IFN-I protects from lethal SARS-CoV-2 infection, and Irgm1 (IRGM) could be an excellent therapeutic target against SARS-CoV-2.

Targeting the retinoic acid signaling pathway as a modern precision therapy against cancers.

Retinoic acid (RA) is a vital metabolite derived from vitamin A. RA plays a prominent role during development, which helps in embryological advancement and cellular differentiation. Mechanistically, RA binds to its definite nuclear receptors including the retinoic acid receptor and retinoid X receptor, thus triggering gene transcription and further consequences in gene regulation. This functional heterodimer activation later results in gene activation/inactivation. Several reports have been published related to the detailed embryonic and developmental role of retinoic acids and as an anti-cancer drug for specific cancers, including acute promyelocytic leukemia, breast cancer, and prostate cancer. Nonetheless, the other side of all-trans retinoic acid (ATRA) has not been explored widely yet. In this review, we focused on the role of the RA pathway and its downstream gene activation in relation to cancer progression. Furthermore, we explored the ways of targeting the retinoic acid pathway by focusing on the dual role of aldehyde dehydrogenase (ALDH) family enzymes. Combination strategies by combining RA targets with ALDH-specific targets make the tumor cells sensitive to the treatment and improve the progression-free survival of the patients. In addition to the genomic effects of ATRA, we also highlighted the role of ATRA in non-canonical mechanisms as an immune checkpoint inhibitor, thus targeting the immune oncological perspective of cancer treatments in the current era. The role of ATRA in activating independent mechanisms is also explained in this review. This review also highlights the current clinical trials of ATRA in combination with other chemotherapeutic drugs and explains the future directional insights related to ATRA usage.

Phytoconstituents from Urtica dioica (stinging nettle) of Sikkim Himalaya and their molecular docking interactions revealed their nutraceutical potential as α-amylase and α-glucosidase inhibitors.

In this study, antioxidative methanolic leaf extract (MeOH-SIS) of Urtica dioica was characterized for anti-diabetic activity. The extract was purified on a column to yield seven homogenous fractions (F1-F7) which were further determined for DPPH radical scavenging activity. MeOH-SIS and the fraction F1 (selected based on % yield and activity) were evaluated for their in vitro α-amylase and α-glucosidase inhibitory activity. The results showed inhibition of both enzymes in a dose dependent manner and F1 exhibited relatively higher inhibition than its mother extract MeOH-SIS. GC-MS analyses of both the extracts identified 24 major compounds among which 10 were previously described as bioactive compounds. Among all, 5 compounds demonstrated to have quality pharmacokinetics profiles and were examined for possible binding affinity against the active sites of α-amylase and α-glucosidase using molecular docking. The binding interaction of 2R-acetoxymethyl-1,3,3-trimethyl-4 T-(3-methyl-2-buten-1-yl)-1 T-cyclohexanol within the active sites of the target receptors was found to be significant among others, and can be developed as a potential inhibitor of α-amylase and α-glucosidase. The leaf extract can be utilized to develop food additive for the control and management of oxidative stress induced diabetes.

ALDH1A1 promotes PARP inhibitor resistance by enhancing retinoic acid receptor-mediated DNA polymerase θ expression.

Poly (ADP-ribose) Polymerase (PARP) inhibitors (PARPi) have been approved for both frontline and recurrent setting in ovarian cancer with homologous recombination (HR) repair deficiency. However, more than 40% of BRCA1/2-mutated ovarian cancer lack the initial response to PARPi treatment, and the majority of those that initially respond eventually develop resistance. Our previous study has demonstrated that increased expression of aldehyde dehydrogenase 1A1 (ALDH1A1) contributes to PARPi resistance in BRCA2-mutated ovarian cancer cells by enhancing microhomology-mediated end joining (MMEJ) but the mechanism remains unknown. Here, we find that ALDH1A1 enhances the expression of DNA polymerase θ (Polθ, encoded by the POLQ gene) in ovarian cancer cells. Furthermore, we demonstrate that the retinoic acid (RA) pathway is involved in the transcription activation of the POLQ gene. The RA receptor (RAR) can bind to the retinoic acid response element (RARE) located in the promoter of the POLQ gene, promoting transcription activation-related histone modification in the presence of RA. Given that ALDH1A1 catalyzes the biosynthesis of RA, we conclude that ALDH1A1 promotes POLQ expression via the activation of the RA signaling pathway. Finally, using a clinically-relevant patient-derived organoid (PDO) model, we find that ALDH1A1 inhibition by the pharmacological inhibitor NCT-505 in combination with the PARP inhibitor olaparib synergistically reduce the cell viability of PDOs carrying BRCA1/2 mutation and positive ALDH1A1 expression. In summary, our study elucidates a new mechanism contributing to PARPi resistance in HR-deficient ovarian cancer and shows the therapeutic potential of combining PARPi and ALDH1A1 inhibition in treating these patients.

Repurposing of RAS-Pathway Mediated Drugs for Intestinal Inflammation Related Diseases for Treating SARS-CoV-2 Infection.

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.

In colon cancer cells fascin1 regulates adherens junction remodeling.

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/ß-catenin signaling and generates "collective plasticity" which influences the behavior of cells during cell migration. The impact of mechanical inputs on WNT/ß-catenin activation in cancer cells remains poorly understood. Our findings highlight the role of AJ remodeling and mechanosensitive WNT/ß-catenin signaling in the growth and dissemination of colorectal carcinomas.

Clonal evolution and expansion associated with therapy resistance and relapse of colorectal cancer.

Colorectal cancer (CRC) arises by a continuous process of genetic diversification and clonal evolution. Multiple genes and pathways have a role in tumor initiation and progression. The gradual accumulation of genetic and epigenetic processes leads to the establishment of adenoma and cancer. The important 'driver' mutations in tumor suppressor genes (such as TP53, APC, and SMAD4) and oncogenes (such as KRAS, NRAS, MET, and PIK3CA) confer selective growth advantages and cause CRC advancement. Clonal evolution induced by therapeutic pressure, as well as intra-tumoral heterogeneity, has been a great challenge in the treatment of metastatic CRC. Tumors often develop resistance to treatments as a result of intra-tumor heterogeneity, clonal evolution, and selection. Hence, the development of a multidrug personalized approach should be prioritized to pave the way for therapeutics repurposing and combination therapy to arrest tumor progression. This review summarizes how selective drug pressure can impact tumor evolution, resulting in the formation of polyclonal resistance mechanisms, ultimately promoting cancer progression. Current strategies for targeting clonal evolution are described. By understanding sources and consequences of tumor heterogeneity, customized and effective treatment plans to combat drug resistance may be devised.

Selective autophagy of RIPosomes maintains innate immune homeostasis during bacterial infection.

The NOD1/2-RIPK2 is a key cytosolic signaling complex that activates NF-κB pro-inflammatory response against invading pathogens. However, uncontrolled NF-κB signaling can cause tissue damage leading to chronic diseases. The mechanisms by which the NODs-RIPK2-NF-κB innate immune axis is activated and resolved remain poorly understood. Here, we demonstrate that bacterial infection induces the formation of endogenous RIPK2 oligomers (RIPosomes) that are self-assembling entities that coat the bacteria to induce NF-κB response. Next, we show that autophagy proteins IRGM and p62/SQSTM1 physically interact with NOD1/2, RIPK2 and RIPosomes to promote their selective autophagy and limit NF-κB activation. IRGM suppresses RIPK2-dependent pro-inflammatory programs induced by Shigella and Salmonella. Consistently, the therapeutic inhibition of RIPK2 ameliorates Shigella infection- and DSS-induced gut inflammation in Irgm1 KO mice. This study identifies a unique mechanism where the innate immune proteins and autophagy machinery are recruited together to the bacteria for defense as well as for maintaining immune homeostasis.

2-Dimensional in vitro culture assessment of ovarian cancer cell line using cost effective silver nanoparticles from Macrotyloma uniflorum seed extracts.

Our research focused on generating AgNPs using Macrotyloma uniflorum (MU) seed extracts and studied their efficacy in combating tumor growth using the 2-Dimensional method for ovarian cancer cell line-PA-1. Characterization studies including a UV-visible spectrophotometer confirmed the surface plasmon resonance peak of 436 nm. Particle size determination data validated the nanoparticle diameter of 91.8 nm. Synthesized AgNPs possess a negative charge of -28.0 mV, which was confirmed through the zeta potential study. Structural characterization studies including XRD determined the crystal phase of AgNPs at four distant peaks at 2θ (38.17, 44.36, 64.52, and 77.46) and were assigned to 111, 200, 220, and 311 planes of the FCC. FTIR studies have confirmed the presence of O-H, N-H, C=O, ethers, C-Br, and C-I groups in AgNPs respectively. DPPH study has confirmed the presence of free radicles and we observed that at 500 µg/ml concentration, 76.08% of free radicles were formed which shows their efficiency. MTT assay shows the efficacy of MU-AgNPs in reducing the cell viability. At lower concentrations of MU-AgNP, 66% viability was observed and 9% of viability was observed at higher dose. ROS production (21%) was observed using MU-AgNPs with respect to 0.45% in controls, which affirms the capacity to induce DNA damage via apoptosis. Standard drug camptothecin generated 26% of ROS production which confirms higher potential of AgNPs in inducing DNA damage in tumor cells without causing lethality to the healthy cells. Further, the Fluorescence-activated cell sorting (FACS) study using a standard Caspase-3 marker confirms the generation of apoptotic bodies using two different concentrations of MU-AgNPs. At 40 µg, 64% of apoptotic cell death was observed, whereas, using 20 µg, 23% of apoptosis was recorded via fluorescent intensity. Propidium iodide-based Cell cycle study has shown a significant decrease in G0/G1 phase compared to control (88.8%), which further confirmed the apoptotic induction. Matrix metalloproteinases (MMP) studies using JC-1 dye, showed a significant increase in green fluorescence owing to lowered membrane potential, thus ensuring the breakdown of mitochondrial potential compared to untreated and standard drugs. With the obtained results, we are concluding that MU-AgNPs has a tremendous capacity to suppress the ovarian cancer cell proliferation in vitro by inducing DNA damage and apoptosis.

A Novel Estrogen Receptor ß Agonist Diminishes Ovarian Cancer Stem Cells via Suppressing the Epithelial-to-Mesenchymal Transition.

Epithelial ovarian cancer is the most lethal malignancy of the female reproductive tract. A healthy ovary expresses both Estrogen Receptor α (ERα) and ß (ERß). Given that ERα is generally considered to promote cell survival and proliferation, thereby, enhancing tumor growth, while ERß shows a protective effect against the development and progression of tumors, the activation of ERß by its agonists could be therapeutically beneficial for ovarian cancer. Here, we demonstrate that the activation of ERß using a newly developed ERß agonist, OSU-ERb-12, can impede ovarian cancer cell expansion and tumor growth in an ERα-independent manner. More interestingly, we found that OSU-ERb-12 also reduces the cancer stem cell (CSC) population in ovarian cancer by compromising non-CSC-to-CSC conversion. Mechanistically, we revealed that OSU-ERb-12 decreased the expression of Snail, a master regulator of the epithelial-to-mesenchymal transition (EMT), which is associated with de novo CSC generation. Given that ERα can mediate EMT and facilitate maintenance of the CSC subpopulation and that OSU-ERb-12 can block the transactivity of ERα, we conclude that OSU-ERb-12 reduces the CSC subpopulation by inhibiting EMT in an ERα-dependent manner. Taken together, our data indicate that the ERß agonist OSU-ERb-12 could be used to hinder tumor progression and limit the CSC subpopulation with the potential to prevent tumor relapse and metastasis in patients with ovarian cancer.

Antiproliferative and apoptotic potential of methotrexate lipid nanoparticles in a murine breast cancer model.

Aim: To evaluate the efficacy of novel methotrexate-loaded nanoparticles (MTX-NPs) in vitro and in vivo in the treatment of breast cancer. Materials & methods: MTX-NPs were tested for cellular uptake, cell viability, cell cycle, cellular wound migration and changes in tumor volume using characterized NPs. Results: The solid lipid NPs (SLNPs) showed strong cellular uptake, increased apoptosis, controlled cytotoxicity at lower IC50 of methotrexate and a sizable reduction in tumor burden. Conclusion: MTX-NP oral formulation can be a promising candidate in breast cancer treatment with improved cellular uptake and in vivo efficacy.

Antioxidant Potential of Selected Wild Edible Leafy Vegetables of Sikkim Himalayan Region: Effects of Cooking Methods and Gastrointestinal Digestion on Activity.

Green leafy vegetables or GLVs are one of the main attractions in the local vegetable market and are widely consumed as the main course and side dish in the Sikkim Himalayan region (SHR). This study evaluated the total phenolic (TPC) and flavonoid contents (TFC) and antioxidant potential in different extracts such as methanolic (MeOH), ethyl acetate (EtOAC), and hexane extracts of selected GLVs followed by changes in the antioxidant activity on cooking and stimulated gastrointestinal (GI) digestion. The MeOH extracts of Urtica dioica L. (Sisnu), Nasturtium officinale W. T. Aiton (Simrayo), Diplazium esculentum Retz. Sw. (Ningro), and Chenopodium album L. (Bethu) were estimated to have higher TPC [22.73-45.84 µg gallic acid equivalent (GAE)/mg of extract]. In contrast, the plant extracts prepared using EtOAC (except for N. officinale, where TFC was found to be higher in hexane extract) were found to contain higher TFC (3.42-14.86 µg quercetin equivalent (QE)/mg of extract). The MeOH extracts also exhibited higher 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity (9.55-18.67 µg ascorbic acid equivalent (AAE)/mg of extract), total antioxidant activity (TAA) (0.27-0.32 mg AAE/mg of extract), and reducing power potential (RPP) (1.6-9.9 µg AAE/mg of extract). Among the test MeOH extracts, U. dioica demonstrated relatively higher antioxidant activities and was selected for cooking experiments followed by simulated GI digestion. The findings revealed that the loss of antioxidant activity was minimal in steam-cooked leaves (3.5% in 40 min) as compared to the boiled ones (18% in 10 min). The simulated GI (simulated salivary, gastric, and intestinal) digestion performed on raw, steam cooked, and boiled U. dioica leaves showed substantial enhancement of antioxidant properties (by 64.63%) through steam cooking in comparison to the raw leaves. Overall the study concludes that higher antioxidant properties can be achieved on the consumption of steam-cooked U. dioica leaves.

A bioinformatic approach of targeting SARS-CoV-2 replication by silencing a conserved alternative reserve of the orf8 gene using host miRNAs.

The causative agent of the COVID-19 pandemic, the SARS-CoV-2 virus has yielded multiple relevant mutations, many of which have branched into major variants. The Omicron variant has a huge similarity with the original viral strain (first COVID-19 strain from Wuhan). Among different genes, the highly variable orf8 gene is responsible for crucial host interactions and has undergone multiple mutations and indels. The sequence of the orf8 gene of the Omicron variant is, however, identical with the gene sequence of the wild type. orf8 modulates the host immunity making it easier for the virus to conceal itself and remain undetected. Variants seem to be deleting this gene without affecting the viral replication. While analyzing, we came across the conserved orf7a gene in the viral genome which exhibits a partial sequence homology as well as functional similarity with the SARS-CoV-2 orf8. Hence, we have proposed here in our hypothesis that, orf7a might be an alternative reserve of orf8 present in the virus which was compensating for the lost gene. A computational approach was adopted where we screened various miRNAs targeted against the orf8 gene. These miRNAs were then docked onto the orf8 mRNA sequences. The same set of miRNAs was then used to check for their binding affinity with the orf7a reference mRNA. Results showed that miRNAs targeting the orf8 had favorable shape complementarity and successfully docked with the orf7a gene as well. These findings provide a basis for developing new therapeutic approaches where both orf8 and orf7a can be targeted simultaneously.