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Abstract In vitro models are necessary to study the pathophysiology of the disease and the development of effective, tailored treatment methods owing to the complexity and heterogeneity of breast cancer and the large population affected by it. The cellular connections and tumor microenvironments observed in vivo are often not recapitulated in conventional two-dimensional (2D) cell cultures. Therefore, developing 3D in vitro models that mimic the complex architecture and physiological circumstances of breast tumors is crucial for advancing our understanding of the illness. A 3D scaffold-free in vitro disease model mimics breast cancer pathophysiology by allowing cells to self-assemble/pattern into 3D structures, in contrast with other 3D models that rely on artificial scaffolds. It is possible that this model, whether applied to breast tumors using patient-derived primary cells (fibroblasts, endothelial cells, and cancer cells), can accurately replicate the observed heterogeneity. The complicated interactions between different cell types are modelled by integrating critical components of the tumor microenvironment, such as the extracellular matrix, vascular endothelial cells, and tumor growth factors. Tissue interactions, immune cell infiltration, and the effects of the milieu on drug resistance can be studied using this scaffold-free 3D model. The scaffold-free 3D in vitro disease model for mimicking tumor pathophysiology in breast cancer is a useful tool for studying the molecular basis of the disease, identifying new therapeutic targets, and evaluating treatment modalities. It provides a more physiologically appropriate high-throughput platform for screening large compound library in a 96–384 well format. We critically discussed the rapid development of personalized treatment strategies and accelerated drug screening platforms to close the gap between traditional 2D cell culture and in vivo investigations. Graphical Abstract

The pathophysiology of depression is heavily dependent on inflammation. Evidence suggests that the etiology of depression is linked with NLRP3 inflammasome-induced inflammation. Therefore, blocking the activated NLRP3 inflammasome may be beneficial for treating depression. Due to the limitations of currently available antidepressants, it is necessary to develop novel, safe, and affordable drugs for the treatment of depression. A natural coumarin derivative named 4-methylesculetin (4-MESC) possesses anti-inflammatory properties. However, the role of 4-MESC as an antidepressant has not been elucidated. Therefore, in this study, we explored the antidepressant-like effects of 4-MESC and its underlying molecular mechanism through the modulation of the NLRP3 inflammasome. The docking and molecular dynamic simulation studies revealed that 4-MESC has a higher affinity for the NLRP3 PYD. Blood–brain barrier permeability was confirmed using the SwissADME pharmacokinetic tool. High doses (50 mg/kg) of 4-MESC significantly reduced the immobility duration in the tail-suspension test (TST) and forced swim test (FST) without changing the overall locomotor activity in the female Swiss albino mice that were subjected to lipopolysaccharide (LPS). LPS-induced pro-inflammatory cytokines such as IL-6 and TNF-α were reduced in serum and brain tissues using 4-MESC. 4-MESC’s neuroprotective effects are mediated by increased brain-derived neurotrophic factor (BDNF) and decreased cortisol levels. 4-MESC markedly reduced LPS-induced elevated levels of ROS and lipid peroxidation (malondialdehyde levels) and enhanced the superoxide dismutase (SOD) activity and glutathione levels, which revealed its anti-oxidant potential against oxidative stress. 4-MESC diminished the expression levels of NF-κBp65, IL-6, NLRP3, caspase-1, gasdermin D, and IL-1β in the hippocampus. These findings demonstrated that 4-MESC exhibited antidepressant-like effects by inhibiting the NLRP3 inflammasome. However, other antidepressant mechanisms might also be involved which require further studies.

Metabolic compromise is crucial in aggravating age-associated chronic inflammation, oxidative stress, mitochondrial damage, increased LDL and triglycerides, and elevated blood pressure. Excessive adiposity, hyperglycemia, and insulin resistance due to aging are associated with elevated levels of damaging free radicals, inducing a proinflammatory state and hampering immune cell activity, leading to a malfunctioning cardiometabolic condition. The age-associated oxidative load and redox imbalance are contributing factors for cardiometabolic morbidities via vascular remodelling and endothelial damage. Recent evidence has claimed the importance of gut microbiota in maintaining regular metabolic activity, which declines with chronological aging and cardiometabolic comorbidities. Genetic mutations, polymorphic changes, and environmental factors strongly correlate with increased vulnerability to aberrant cardiometabolic changes by affecting key physiological pathways. Numerous studies have reported a robust link between biological aging and cardiometabolic dysfunction. This review outlines the scientific evidence exploring potential mechanisms behind the onset and development of cardiovascular and metabolic issues, particularly exacerbated with aging. [ABSTRACT FROM AUTHOR]

Introduction: Glioblastoma, which affects a large number of patients every year and has an average overall lifespan of around 14.6 months following diagnosis stands out as the most lethal primary invasive brain tumor. Currently, surgery, radiation, and chemotherapy with temozolomide (TMZ) are the three major clinical treatment approaches. However, the ability to treat patients effectively is usually limited by TMZ resistance. Naringin, a bioflavonoid with anti-cancer, antioxidant, metal-chelating, and lipid-lowering effects, has emerged as a promising therapeutic option. Methods: To explore the targets and pathways of naringin and TMZ in glioblastoma network pharmacology, cell line-based ELISA, flow cytometry, immunocytochemistry, western blotting, and LC-HRMS based metabolomics study were used. Results: The findings through the network pharmacology suggested that the key targets of naringin in the chemosensitization of glioblastoma would be Poly [ADP-ribose] polymerase 1 (PARP-1), O-6-Methylguanine-DNA Methyltransferase (MGMT), and caspases. The functional enrichment analysis revealed that these targets were significantly enriched in important pathways such as p53 signaling, apoptosis, and DNA sensing. Further, the results of the in-vitro study in U87-MG and T98-G glioblastoma cells demonstrated that TMZ and naringin together significantly reduced the percentage of viability and inhibited the DNA repair enzymes PARP-1 and MGMT, and PI3K/AKT which led to chemosensitization and, in turn, induced apoptosis, which was indicated by increased p53, caspase-3 expression and decreased Bcl2 expression. Additionally, a metabolomics study in T98-G glioblastoma cells using liquid chromatography high-resolution mass spectrometry (LC-HRMS) revealed downregulation of C8-Carnitine (-2.79), L-Hexanoylcarnitine (-4.46), DLCarnitine (-2.46), Acetyl-L-carnitine (-3.12), Adenine (-1.3), Choline (-2.07), Propionylcarnitine (-1.69), Creatine (-1.33), Adenosine (-0.84), Spermine (-1.42), and upregulation of Palmitic Acid (+1.03) and Sphingosine (+0.89) in the naringin and TMZ treatment groups. Discussion: In conclusion, it can be said that naringin in combination with TMZ chemosensitized TMZ antiglioma response and induced apoptosis in tumor cells. [ABSTRACT FROM AUTHOR]

Rationale: N‐Nitroso dimethylamine (NDMA) is a mutagenic impurity detected in several ranitidine products. The amino functional group of ranitidine is a risk factor for classical nitrosation‐induced NDMA formation in ranitidine drug products during storage conditions. The United States Food and Drug Administration (US FDA) recommended the use of antioxidants to control NDMA in drug products. Considering the need for sensitive analytics, a liquid chromatography/high‐resolution mass spectrometry (LC–HRMS) method was developed and validated to detect NDMA in this pilot study to demonstrate the antioxidants as inhibitors of nitrosation reactions. Methods: The method, utilizing an EC‐C18 column and tuned to atmospheric pressure chemical ionization/selected ion monitoring (APCI/SIM) mode, separated NDMA (m/z: 75.0553; tR: 3.71 min) and ranitidine (m/z: 315.1485; tR: 8.61 min). APCI mode exhibited four times higher sensitivity to NDMA than electrospray ionization (ESI) mode. Classical nitrosation of the dimethyl amino group of ranitidine was studied with sodium nitrite in solid pellets. Antioxidants (alpha‐tocopherol, ascorbic acid, and trolox) were evaluated as NDMA attenuators in ranitidine pellets under vulnerable storage conditions. The developed method quantified NDMA levels in samples, extracted with methanol through vortex shaking for 45 min. Results: The method achieved a limit of detection (LOD) and limit of quantitation (LOQ) of 0.01 and 0.05 ng/mL, respectively, with linearity within 1–5000 ng/mL (R1: 0.9995). It demonstrated good intra‐day and inter‐day precision (% RSD [relative standard deviation]: <2) and accuracy (96.83%–101.72%). Nitrosation of ranitidine induced by nitrite was significant (p < 0.001; R2 = 0.9579) at various sodium nitrite levels. All antioxidants efficiently attenuated NDMA formation during ranitidine nitrosation. Ascorbic acid exhibited the highest NDMA attenuation (96.98%), followed by trolox (90.58%). This study recommends 1% ascorbic acid and trolox as potent NDMA attenuators in ranitidine drug products. Conclusions: This study compared the effectiveness of antioxidants as NDMA attenuators in ranitidine under storage conditions susceptible to NDMA generation. The study concluded that ascorbic acid and trolox are potent inhibitors of NDMA formation and nitrosation attenuators in ranitidine drug products. [ABSTRACT FROM AUTHOR]

The discovery of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated Cas endonucleases in bacterial and archaeal species allowed scientists to modify, utilized, and revolutionize this tool for genetic alterations in any species. Especially the type II CRISPR-Cas9 system has been extensively studied and utilized for precise and efficient DNA manipulation in plant and mammalian systems over the past few decades. Further, the discovery of the type V CRISPR-Cas12 (Cpf1) system provides more flexibility and precision in DNA manipulation in prokaryotes, plants, and animals. However, much effort has been made to employ and utilize the above CRISPR tools for RNA manipulation but the ability of Cas9 and Cas12 to cut DNA involves the nuisance of off-target effects on genes and thus may not be employed in all RNA-targeting applications. Therefore, the search for new and diverse Cas effectors which can precisely detect and manipulate the targeted RNA begins and this led to the discovery of a novel RNA targeting class 2, type VI CRISPR-Cas13 system. The CRISPR-Cas13 system consists of single RNA-guided Cas13 effector nucleases that solely target single-stranded RNA (ssRNA) in a programmable way without altering the DNA. The Cas13 effectors family comprises four subtypes (a-d) and each subtype has distinctive primary sequence divergence except the two consensuses Higher eukaryotes and prokaryotes nucleotide-binding domain (HEPN) that includes RNase motifs i.e. R-X4-6-H. These two HEPN domains are solely responsible for executing targetable RNA cleavage activity with high efficiency. Further, recent studies have shown that Cas13d exhibits higher efficiency and specificity in cleaving targeted RNA in the mammalian system compared to other Cas13 endonucleases of the Cas13 enzyme family. In addition to that, Cas13d has shown additional advantages over other Cas13 variants, structurally as well as functionally which makes it a prominent and superlative tool for RNA engineering and editing. Therefore considering the advantages of Cas13d over previously characterized Cas13 subtypes, in this review, we encompass the structural and mechanistic properties of type VI CRISPR-Cas13d systems, an overview of the current reported various applications of Cas13d, and the prospects to improve Cas13d based tools for diagnostic and therapeutic purposes.

Natural products are being targeted as alternative anticancer agents due to their non-toxic and safe nature. The present study was conducted to explore the in vitro anticancer potential of Justicia adhatoda (J. adhatoda) leaf extract. The methanolic leaf extract was prepared, and the phytochemicals and antioxidant potential were determined by LCMS analysis and DPPH radical scavenging assay, respectively. A docking study performed with five major alkaloidal phytoconstituents showed that they had a good binding affinity towards the active site of NF-κB. Cell viability assay was carried out in five different cell lines, and the extract exhibited the highest cytotoxicity in MCF-7, a breast cancer cell line. Extract-treated cells showed a significant increase in nitric oxide and reactive oxygen species production. Cell cycle analysis showed an arrest in cell growth at the Sub-G0 phase. The extract successfully inhibited cell migration and colony formation and altered mitochondrial membrane potential. The activities of superoxide dismutase and glutathione were also found to decrease in a dose-dependent manner. The percentage of apoptotic cells was found to increase in a dose-dependent manner in MCF-7 cells. The expressions of caspase-3, Bax, and cleaved-PARP were increased in extract-treated cells. An increase in the expression of NF-κB was found in the cytoplasm in extract-treated cells. J. adhatoda leaf extract showed a potential anticancer effect in MCF-7 cells.

The anti-oxidant and anti-inflammatory effect of beta-glucogallin (BGG), a plant-derived natural product, was evaluated in both in vitro and in vivo studies. For the in vitro study, the ability of BGG pre-treatment to quench LPS-induced effects compared to LPS alone in macrophages was investigated. It was found that BGG pre-treatment showed a significant decrease in ROS, NO, superoxide, and pro-inflammatory cytokines (TNF-alpha, IL-4, IL-17, IL-1β, and IL-6) and increased reduced glutathione coupled with the restoration of mitochondrial membrane potential. Gene profiling and further validation by qPCR showed that BGG pre-treatment downregulated the LPS-induced expression of c-Fos, Fas, MMP-9, iNOS, COX-2, MyD88, TRIF, TRAF6, TRAM, c-JUN, and NF-κB. We observed that BGG pre-treatment reduced nuclear translocation of LPS-activated NF-κB and thus reduced the subsequent expressions of NLRP3 and IL-1β, indicating the ability of BGG to inhibit inflammasome formation. Molecular docking studies showed that BGG could bind at the active site of TLR4. Finally, in the LPS-driven sepsis mouse model, we showed that pre-treatment with BGG sustained toxic shock, as evident from their 100% survival. Our study clearly showed the therapeutic potential of BGG in toxic shock syndrome.

Chronic arsenic (As) poisoning is mostly due to subsoil water contaminated with As and its salts. Exposure to As has been found to cause an elevation in reactive oxygen species (ROS), leading to the damage of DNA and proteins, and it also causes immunotoxicity. Treatment regimens are primarily based on chelation therapy and amino acid and vitamin supplementations. Recent studies have established that natural products display effective and progressive relief from arsenicosis without any side effects. β-glucogallin (BGG), a gallo-tannin natural product, is reported to possess anti-oxidant and anti-inflammatory properties. In the present study, we aim to observe the protective role of BGG against As-induced cytotoxicity, apoptosis, mitochondrial dysfunction, and the underlying mechanisms in RAW 264.7 macrophage cells. We found that BGG alleviates As-induced ROS, apoptosis, and mitochondrial dysfunction in RAW 264.7 macrophage cells. Thus, BGG can be used therapeutically to prevent As-induced toxicity.

Background Coronavirus disease 2019 (COVID-19) is a potentially fatal disease caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Several studies have shown that hydroxychloroquine (HCQ) significantly inhibits SARS-CoV-2 infections in vitro. Objective Since the phytoconstituents of Cinchona officinalis (CO) are similar to those of HCQ, the objective of this study was to test the antiviral potential of different homeopathic formulations of CO. Methods An analysis of the molecular composition of CO was carried out using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, followed by a detailed docking study. The constituents of CO were docked against various targets of SARS-CoV-2, and the binding potential of the phytoconstituents was compared and quantified. The ligand with the lowest Glide docking score is considered to have the best binding affinity. The cytotoxicity of several homeopathic formulations, including CO mother tincture (CO-MT), was also checked on VeroE6 cells. A known antiviral, remdesivir, was used as a positive control for the in vitro assays to evaluate the effects of CO-MT against SARS-CoV-2-infected VeroE6 cells. Results Molecular docking studies showed that constituents of CO exhibited binding potential to various targets of SARS-CoV-2, including Mpro, PLpro, RdRp, nucleocapsid protein, ACE2 (in host) and spike protein. Quinoline, one of the constituents of CO, can potentially bind the spike protein of SARS-CoV-2. Quinic acid showed better binding capabilities with Mpro, PLpro RdRp, nucleocapsid protein and ACE2 (allosteric site) than other constituents. Quinidine exhibited better binding to ACE2. Compared to HCQ, other phytoconstituents of CO had the equivalent potential to bind the RNA-dependent RNA polymerase, nucleocapsid protein, Mpro, PLpro and spike protein of SARS-CoV-2. In vitro assays showed that homeopathic CO-MT was not cytotoxic and that CO-MT and remdesivir respectively caused 89% and 99% inhibition of SARS-CoV-2 infection in VeroE6 cells. Conclusion Based on this in silico and in vitro evidence, we propose CO-MT as a promising antiviral medicine candidate for treating COVID-19. In vivo investigation is required to clarify the therapeutic potential of CO-MT in COVID-19. [ABSTRACT FROM AUTHOR]

Garcinol is an active constituent of Garcinia indica and Garcinia cambogia. Recent studies have proven that garcinol has anti-inflammatory, anti-cancer, and anti-oxidant activities. The objective of this study was to evaluate the inhibitory effects of garcinol on the activities of the drug metabolizing cytochrome P450 (CYP) isozymes to predict potential herb-drug interactions with co-administered drugs. Garcinol was incubated with a mixture of rat liver microsomes and eight CYP probe substrate cocktail under optimized incubation conditions and the samples were analyzed using a validated method on LC-MS/MS. Garcinol showed strong inhibition with IC50 values of CYP1A2 (7.6 µM), CYP2C9 (8.0 µM), CYP2B6 (2.1 µM), CYP2D6 (9.5 µM), and CYP3A4 (5.1 µM), respectively, and moderate inhibition towards CYP2C19 (16.4 µM) and CYP2E1 (19.0 µM). Molecular docking studies were performed on garcinol against the active sites of CYP2B6 and CYP3A4 proteins. These results further confirmed that the inhibitory activity of garcinol occurred by occupying the active sites of these human CYPs and by making favorable interactions with its key residues. In-vivo CYP inhibition studies were carried out in Sprague-Dawley rats. These results suggest garcinol may cause herb-drug interactions, mediated by inhibition of CYPs involved in drug metabolism in-vivo by altering the pharmacokinetic parameters like AUC and Cmax in a clinically significant manner. Garcinol was found to upregulate the expression and activity of P-gp in western blotting study and P-gp inhibition study in-vivo. These findings give a clear understanding to predict potential herb-drug/drug-drug interactions of garcinol for safe clinical use in future.