Aspartame Causes Developmental Defects and Teratogenicity in Zebra Fish Embryo: Role of Impaired SIRT1/FOXO3a Axis in Neuron Cells.

Aspartame, a widely used artificial sweetener, is present in many food products and beverages worldwide. It has been linked to potential neurotoxicity and developmental defects. However, its teratogenic effect on embryonic development and the underlying potential mechanisms need to be elucidated. We investigated the concentration- and time-dependent effects of aspartame on zebrafish development and teratogenicity. We focused on the role of sirtuin 1 (SIRT1) and Forkhead-box transcription factor (FOXO), two proteins that play key roles in neurodevelopment. It was found that aspartame exposure reduced the formation of larvae and the development of cartilage in zebrafish. It also delayed post-fertilization development by altering the head length and locomotor behavior of zebrafish. RNA-sequencing-based DEG analysis showed that SIRT1 and FOXO3a are involved in neurodevelopment. In silico and in vitro analyses showed that aspartame could target and reduce the expression of SIRT1 and FOXO3a proteins in neuron cells. Additionally, aspartame triggered the reduction of autophagy flux by inhibiting the nuclear translocation of SIRT1 in neuronal cells. The findings suggest that aspartame can cause developmental defects and teratogenicity in zebrafish embryos and reduce autophagy by impairing the SIRT1/FOXO3a axis in neuron cells.

Paraquat (herbicide) as a cause of Parkinson's Disease.

The four features of Parkinson's disease (PD), which also manifests other non-motor symptoms, are bradykinesia, tremor, postural instability, and stiffness. The pathogenic causes of Parkinsonism include Lewy bodies, intracellular protein clumps of αsynuclein, and the degeneration of dopaminergic neurons in the substantia nigra's pars compacta region. The pathophysiology of PD is still poorly understood due to the complexity of the illness. The apoptotic cell death of neurons in PD, however, has been linked to a variety of intracellular mechanisms, according to a wide spectrum of study. The endoplasmic reticulum's stress, decreased levels of neurotrophic factors, oxidative stress, mitochondrial dysfunction, catabolic alterations in dopamine, and decreased activity of tyrosine hydroxylase are some of these causes. The herbicide paraquat has been used in laboratory studies to create a variety of PD pathological features in numerous in-vitro and in-vivo animals. Due to the unique neurotoxicity that paraquat causes, understanding of the pathophysiology of PD has changed. Parkinson's disease (PD) is more likely to develop among people exposed to paraquat over an extended period of time, according to epidemiological studies. Thanks to this paradigm, the hunt for new therapy targets for PD has expanded. In both in-vitro and in-vivo models, the purpose of this study is to summarise the relationship between paraquat exposure and the onset of Parkinson's disease (PD).

Abdominal obesity in India: analysis of the National Family Health Survey-5 (2019-2021) data.

Background: The ever-growing trend of abdominal obesity worldwide has garnered global attention over the past three decades. In India, BMI has conventionally been used to measure obesity. National Family Health Survey (NFHS) is the largest demographic and health survey (DHS) in India. For the first time, the NFHS conducted the fifth round in 2019-21 which assessed abdominal obesity through waist circumference. The objective of the current study was to determine the prevalence of abdominal obesity and explore the associated socioeconomic factors. Methods: The prevalence of abdominal obesity in India was determined using the NFHS-5 dataset, where waist circumference was used as a measure. Multivariable binary logistic regression was then employed to examine the association of different socioeconomic factors with abdominal obesity. Findings: The prevalence of abdominal obesity in the country was found to be 40% in women and 12% in men. The findings show that 5-6 out of 10 women between the ages of 30-49 are abdominally obese. The association of abdominal obesity in women is stronger with older age groups, urban residents, wealthier sections, and non-vegetarians. For those practising the Sikh religion, the prevalence is higher in both men and women. Abdominal obesity is also on the rise in rural areas and is penetrating lower and middle socioeconomic sections of society. Interpretation: The findings of the current study highlight the need for the government and other stakeholders to proactively design targeted interventions for abdominal obesity, especially for women in their thirties and forties in India. Further research is recommended to understand the driving factors of abdominal obesity, their inter-operability, and the disease risk associated with this type of obesity. Funding: None.

Fabrication, characterization, and interventions of protein, polysaccharide and lipid-based nanoemulsions in food and nutraceutical delivery applications: A review.

The fabrication and application of nanoemulsions for incorporating and delivering diverse bioactive compounds, particularly hydrophobic substances, is becoming an increasing focus of research with the potential to improve the nutritional and health status of individuals. Constant advancements in nanotechnological approaches aid in the creation of nanoemulsions using diverse biopolymers such as proteins, peptides, polysaccharides, and lipids to improve the stability, bioactivity, and bioavailability of active hydrophilic and lipophilic compounds. This article provides a comprehensive overview of various techniques used to create and characterize nanoemulsions as well as theories for understanding their stability. The article also highlights the advancement of nanoemulsions in boosting the bioaccessibility of nutraceuticals to help advance their potential use in various food and pharmaceutical formulations.

Upregulation of miR-22-3p contributes to plumbagin-mediated inhibition of Wnt signaling in human colorectal cancer cells.

The emergence of resistance and side effects for currently available drugs warrant the need for an alternative treatment regime for colorectal cancer (CRC). Several natural compounds, including plumbagin, have exhibited promising anti-cancer effects in different cancer models, although the molecular mechanisms underlying their effects remain at large. We previously reported plumbagin-mediated inhibition of Wnt signaling in CRC cells and to unravel the actual mechanism, we hypothesized the involvement of miRNAs as they have emerged as critical regulators of gene expression. We performed miRNA-microarray to check if plumbagin-mediated effects are through alteration of miRNA expression and found 32 DE-miRNAs (11 upregulated and 21 downregulated), and KEGG enrichment analysis indicated that their target mRNAs are associated with several pathways relevant to CRC and Wnt signaling. miRNA-mRNA network analysis by miRNET revealed a highly upregulated miRNA upon plumbagin treatment, miR-22-3p, having multiple Wnt-associated mRNAs interacting partners. 3'UTR luciferase assays revealed that miR-22-3p directly targeted the 3'UTR of BCL9L (a coactivator of CTNNB1). miR-22-3p inhibited Wnt signaling by downregulating the levels of BCL9L and that of CTNNB1 and several Wnt-associated proteins (TCF4, c-Myc, CCND1, Snail, Slug, and vimentin) in CRC cells. We also demonstrated that both miR-22-3p and plumbagin reduced colony formation and caused apoptotic cell death (detected by Annexin V/PI dual staining), possibly through increased ROS (detection by Dihydroethidium staining) and decreased MMP (detection by MitoTracker™ Orange staining) in CRC cells. These effects of plumbagin were partially rescued by antimiR-22-3p, suggesting the involvement of miR-22-3p in plumbagin-mediated effects. The present study revealed that alteration in miR-22-3p levels by plumbagin contributes to the induction of apoptosis and its inhibitory effects on Wnt signaling and colony formation, thus providing a mechanistic basis behind its anti-cancer potential.

HMGB3 inhibition by miR-142-3p/sh-RNA modulates autophagy and induces apoptosis via ROS accumulation and mitochondrial dysfunction and reduces the tumorigenic potential of human breast cancer cells.

AIMS: High mobility group box (HMGB) family proteins, HMGB1, HMGB2, HMGB3, and HMGB4 are oncogenic. The oncogenic nature of HMGB1 is characterized by its association with autophagy, ROS, and MMP. Since HMGB3 is its paralog, we hypothesized that it might also modulate autophagy, ROS, and MMP. Hence, we targeted HMGB3 using its shRNA or miR-142-3p and assessed the changes in autophagy, ROS, MMP, and tumorigenic properties of human breast cancer cells. MAIN METHODS: Cell viability was assessed by resazurin staining and annexin-V/PI dual staining was used for confirming apoptosis. Colony formation, transwell migration, invasion and luciferase reporter (for miRNA-target validation) assays were also performed. ROS and MMP were detected using DHE and MitoTracker dyes, respectively. A zebrafish xenograft model was used to assess the role of miR-142-3p on in vivo metastatic potential of breast cancer cells. KEY FINDINGS: Breast cancer tissues from Indian patients and TCGA samples exhibit overexpression of HMGB3. miR-142-3p binds to 3' UTR of HMGB3, leading to its downregulation that subsequently inhibits colony formation and induces apoptosis involving increased ROS accumulation and decreased MMP, phospho-mTOR and STAT3. Our findings show that HMGB3 is directly involved in the miR-142-3p-mediated disruption of autophagy and induction of apoptotic cell death via modulation of LC3, cleaved PARP and Bcl-xL. In addition, miR-142-3p inhibited migration, invasion and metastatic potential of breast cancer cells. SIGNIFICANCE: Our findings highlighted the role of HMGB3, for the first time, in the modulation of autophagy and apoptosis in human breast cancer cells, and these results have therapeutic implications.

Design, synthesis and evaluation of 4,7-disubstituted 8-methoxyquinazoline derivatives as potential cytotoxic agents targeting ß-catenin/TCF4 signaling pathway.

Overactivation of Wnt/ß-catenin signaling by accumulated ß-catenin in the nucleus has been shown to play a crucial role in the etiology of cancer. Interaction of ß-catenin with Transcription factor 4 (TCF4) is a key step for the activation of Wnt genes in response to upstream signals of the Wnt/ß-catenin pathway. Hence, down regulation of Wnt/ß-catenin signaling or targeting downstream events by selective ß-catenin/TCF4 protein-protein interaction inhibitors could be a potential therapeutic strategy against such cancers. In this study structure-based drug design approach was followed to design novel 4,7-disubstituted 8-methoxyquinazoline-based derivatives which could act as potential cytotoxic agents inhibiting the ß-catenin/TCF4 protein-protein interactions. Fifteen compounds possessing 4,7-disubstituted 8-methoxyquinazoline scaffold were synthesized. Cytotoxic potential of the synthesised derivatives were determined against constitutively activated ß-catenin/TCF4 signaling pathway cancer cells (HCT116 and HepG2) using the sulforhodamine B assay. The most potent compound (18B) was selected for detailed biological evaluation. Cell morphology, Hoechst 33342 and Annexin V/PI staining were used to detect apoptosis, while inhibition of cell migration was assessed by in vitro wound healing assay against HCT116 and HepG2 cells. Effect on ß-catenin/TCF mediated transcriptional activity was assessed by TOPFlash/FOPFlash assay, TCF4 and ß-catenin protein expression by immunocytofluorescence, and Wnt target genes (like c-MYC and Cyclin D1) mRNA levels by RT-PCR against HCT116 cells. Cytotoxic potency of the most potential compound (18B) against primary human gallbladder cancer cells was also evaluated. The derivatives showed interactions with active site residues of ß-catenin and were capable of hindering the TCF4 binding, thereby disrupting ß-catenin/TCF4 interactions. Cytotoxic potencies (IC50) of these derivatives ranged from 5.64 ± 0.68 to 23.18 ± 0.45 µM against HCT116 and HepG2 cells respectively. Compound (18B), the most potent compound among the series, induced apoptosis and inhibited cell migration against HCT116 and HepG2 cells. Mechanistic studies indicated that compound (18B) downregulated ß-catenin/TCF4 signaling pathway, ß-catenin and TCF4 protein expression, and mRNA levels of c-MYC andCyclin D1 in HCT116 cells and showed cytotoxicity against primary human gallbladder cancer cells with IC50 value of 8.50 ± 1.44 µM. Thus, novel 4,7-disubstituted 8-methoxyquinazoline derivatives were identified as potential cytotoxic agents with potencies comparable to that of imatinib mesylate. Compound (18B) represents a promising lead molecule as anticancer agent against colon, hepatocellular and gallbladder cancers targeting ß-catenin/TCF4 signaling pathway.

RETRA induces necroptosis in cervical cancer cells through RIPK1, RIPK3, MLKL and increased ROS production.

Cervical cancer is the fourth most prevalent cancer in women worldwide, predominantly infected with human papillomavirus (HPV). The current chemo and radiotherapies are mostly futile due to acquired resistance to apoptosis and warrant new therapeutic approaches targeting potent non-apoptotic cell death pathways to eliminate cervical cancer cells. Induction of necroptosis by pharmaceutical interventions is emerging as a promising tool in multiple apoptotic resistant cancer cells. RETRA (REactivation of Transcriptional Reporter Activity) is a small molecule known to induce expression of p53 regulated genes in mutant (mt) p53 cells but, detailed mechanisms of its anticancer effects are poorly known. The present study investigated the potentials of RETRA as an anticancer agent and found that it induces necroptosis selectively in cervical cancer cells irrespective of p53 status through the phosphorylation of receptor-interacting protein kinase 1,3 (RIPK1, RIPK3) and mixed lineage kinase domain-like protein (MLKL) with no cytotoxic effects in normal human peripheral blood mononuclear cells (PBMCs). RETRA-treated cells also displayed necroptotic morphology of disintegrated plasma membranes with intact nuclei and also showed cell cycle arrest at the S phase with the upregulation of p21 and downregulation of cyclin-D3. Intriguingly, the combinatorial approach of using RETRA with Necrostain-1, a known inhibitor of necroptosis, reversed the effect of RETRA and rescued cell death. Moreover, induction of necroptosis by RETRA is associated with mitochondrial hyperpolarization and elevated ROS production. Collectively, these findings suggest that RETRA induces cell death via necroptosis with increased production of ROS, accentuating the therapeutic implication of RETRA in cervical cancer cells.

miR-142-3p simultaneously targets HMGA1, HMGA2, HMGB1, and HMGB3 and inhibits tumorigenic properties and in-vivo metastatic potential of human cervical cancer cells.

AIMS: High-mobility group (HMG) proteins are oncogenic in different cancers, including cervical cancer; silencing their individual expression using sh-RNAs, siRNAs, and miRNAs has had anti-tumorigenic effects, but the consequences of their collective downregulation are not known. Since multiple gene targeting is generally very effective in cancer therapy, the present study highlighted the consequences of silencing the expression of HMGA1, A2, B1, and B3 using sh-RNAs or miR-142-3p (that can potentially target HMGA1, A2, B1, and B3) in cervical cancer cell lines. MAIN METHODS: 3' UTR luciferase reporter assays were performed to validate HMGA1, A2, B1, and B3 as targets of miR-142-3p in human cervical cancer cells. Annexin V/PI dual staining and flow cytometry analyses were used to detect apoptotic cells. miR-142-3p-mediated regulation of cell death, colony formation, migration, and invasion was investigated in human cervical cancer cells together with in vivo metastasis in zebrafish. KEY FINDINGS: Concurrent knockdown of HMGA1, A2, B1, and B3 through their corresponding sh-RNAs inhibited cell viability and colony formation but induced apoptosis, and these effects were relatively reduced upon their individual knockdown. miR-142-3p targeted HMGA1, A2, B1, and B3 by binding to their 3'UTRs and induced apoptosis but inhibited proliferation, migration, and invasion of human cervical cancer cells. In addition, miR-142-3p expression decreased phospho-p65 and EMT-related proteins in cervical cancer cells and their in vivo metastatic potential upon implantation in zebrafish. SIGNIFICANCE: These findings suggest that miR-142-3p acts as a tumor-suppressive miRNA by targeting HMGA1, A2, B1, and B3 and may serve as a potential therapeutic agent in human cervical cancer.

SLC7A11/ xCT is a target of miR-5096 and its restoration partially rescues miR-5096-mediated ferroptosis and anti-tumor effects in human breast cancer cells.

Breast cancer cells evade cell death by overexpressing SLC7A11, which functions by transporting cystine into cells in exchange for intracellular glutamate facilitating glutathione synthesis and reducing reactive oxygen species (ROS)-mediated stress. Using an in silico approach, we predicted an miRNA (miR-5096) that can target and downregulate SLC7A11. We demonstrated SLC7A11 as a target of miR-5096 by 3'UTR luciferase assay and further validated it by identifying reduced mRNA and protein levels of SLC7A11 upon miR-5096 overexpression. miR-5096-induced ferroptotic cell death in human breast cancer cells was confirmed by concurrently increased ROS, OH-, lipid ROS, and iron accumulation levels and decreased GSH and mitochondrial membrane potential (MitoTracker™ Orange) with mitochondrial shrinkage and partial cristae loss (observed by TEM). miR-5096 inhibited colony formation, transwell migration, and breast cancer cell invasion, whereas antimiR-5096 promoted these tumorigenic properties. Ectopic expression of SLC7A11 partly reversed miR-5096-mediated effects on cell survival, ROS, lipid peroxides, iron accumulation, GSH, hydroxyl radicals, mitochondrial membrane potential, and colony formation. miR-5096 modulated the expression of epithelial-mesenchymal transition markers in vitro and inhibited the metastatic potential of MDA-MB-231 cells in a tumor xenograft model of zebrafish larvae. Our results demonstrate that miR-5096 is a tumor-suppressive miRNA in breast cancer cells, and this paper discusses its therapeutic implications.

Entrainment of superoxide rhythm by menadione in HCT116 colon cancer cells.

Reactive oxygen species (ROS) are primary effectors of cytotoxicity induced by many anti-cancer drugs. Rhythms in the pseudo-steady-state (PSS) levels of particular intracellular ROS in cancer cells and their relevance to drug effectiveness are unknown thus far. We report that the PSS levels of intracellular superoxide (SOX), an important ROS, exhibit an inherent rhythm in HCT116 colon cancer cells, which is entrained (reset) by the SOX inducer, menadione (MD). This reset was dependent on the expression of p53, and it doubled the sensitivity of the cells to MD. The period of oscillation was found to have a linear correlation with MD concentration, given by the equation, T, in h = 23.52 - 1.05 [MD concentration in µM]. Further, we developed a mathematical model to better understand the molecular mechanisms involved in rhythm reset. Biologically meaningful parameters were obtained through parameter estimation techniques; the model can predict experimental profiles of SOX, establish qualitative relations between interacting species in the system and serves as an important tool to understand the profiles of various species. The model was also able to successfully predict the rhythm reset in MD treated hepatoma cell line, HepG2.

Visualizing participant experiences in maternal and child nutrition studies using timeline mapping.

Iron and folic acid (IFA) supplementation is one of the most cost-effective interventions to prevent and treat anemia during pregnancy. Despite having the highest global burden of anemia among pregnant women, rates of IFA uptake in pregnancy in India are still very low, particularly in the state of Uttar Pradesh. Timeline maps were developed as a visual qualitative tool to explore the nuances of health behaviors among pregnant women with respect to antenatal care (ANC) services, including IFA consumption.  Timeline maps were used to elicit and visually document critical events pertaining to ANC services chronologically, including details on contact points with the health system and events specific to IFA distribution, consumption and counselling. The tool consists of a horizontal straight line with nine suspended boxes corresponding to each month of pregnancy, with legends on how to illustrate IFA receipt and consumption. In this instance, the woman's last menstrual period and expected date of delivery were used as a frame of reference for the duration of pregnancy.  Six research assistants (RAs) were trained on how to use timeline maps to elicit and record participant narratives. The RAs later participated in a focus group discussion to gain insight about their experiences using the tool. The timeline maps were easy-to-use and facilitated in-depth conversations with participants. RAs were able to actively engage the participants in co-creating the maps. The visual nature of the tool prompted participants' recall of key pregnancy events and reflexivity. Challenges reported with the tool/process included recollection of past events and potential misrepresentation of information. These highlight a need to restructure training processes. Our findings indicate that timeline maps have the potential to be used in a variety of other program contexts, and merit further exploration.