Zebrafish: A Model to Study and Understand the Diabetic Nephropathy and Other Microvascular Complications of Type 2 Diabetes Mellitus.

Diabetes mellitus (DM) is a complicated metabolic illness that has had a worldwide impact and placed an unsustainable load on both developed and developing countries' health care systems. According to the International Diabetes Federation, roughly 537 million individuals had diabetes in 2021, with type 2 diabetes mellitus accounting for the majority of cases (T2DM). T2DM is a chronic illness defined by insufficient insulin production from pancreatic islet cells. T2DM generates various micro and macrovascular problems, with diabetic nephropathy (DN) being one of the most serious microvascular consequences, and which can lead to end-stage renal disease. The zebrafish (Danio rerio) has set the way for its future as a disease model organism. As numerous essential developmental processes, such as glucose metabolism and reactive metabolite production pathways, have been identified in zebrafish that are comparable to those seen in humans, it is a good model for studying diabetes and its consequences. It also has many benefits over other vertebrate models, including the permeability of its embryos to small compounds, disease-driven therapeutic target selection, in vivo validation, and deconstruction of biological networks. The organism can also be utilized to investigate and understand the genetic abnormalities linked to the onset of diabetes problems. Zebrafish may be used to examine and visualize the growth, morphology, and function of organs under normal physiological and diabetic settings. The zebrafish has become one of the most useful models for studying DN, especially when combined with genetic alterations and/or mutant or transgenic fish lines. The significant advancements of CRISPR and next-generation sequencing technology for disease modelling in zebrafish, as well as developments in molecular and nano technologies, have advanced the understanding of the molecular mechanisms of several human diseases, including DN. In this review, we emphasize the physiological and pathological processes relating to microvascular problems in zebrafish, as well as the many experimental zebrafish models used to research DN, and the DN-related outcomes and mechanisms observed in zebrafish.

Deep sequencing unveils altered cardiac miRNome in congenital heart disease.

Congenital heart disease (CHD) surges from fetal cardiac dysmorphogenesis and chiefly contributes to perinatal morbidity and cardiovascular disease mortality. A continual rise in prevalence and prerequisite postoperative disease management creates need for better understanding and new strategies to control the disease. The interaction between genetic and non-genetic factors roots the multifactorial status of this disease, which remains incompletely explored. The small non-coding microRNAs (miRs, miRNAs) regulate several biological processes via post-transcriptional regulation of gene expression. Abnormal expression of miRs in developing and adult heart is associated with anomalous cardiac cell differentiation, cardiac dysfunction, and cardiovascular diseases. Here, we attempt to discover the changes in cardiac miRNA transcriptome in CHD patients over those without CHD (non-CHD) and find its role in CHD through functional annotation. This study explores the miRNome in three most commonly occurring CHD subtypes, namely atrial septal defect (ASD), ventricular septal defect (VSD), and tetralogy of fallot (TOF). We found 295 dysregulated miRNAs through high-throughput sequencing of the cardiac tissues. The bioinformatically predicted targets of these differentially expressed miRs were functionally annotated to know they were entailed in cell signal regulatory pathways, profoundly responsible for cell proliferation, survival, angiogenesis, migration and cell cycle regulation. Selective miRs (hsa-miR-221-3p, hsa-miR-218-5p, hsa-miR-873-5p) whose expression was validated by qRT-PCR, have been reported for cardiogenesis, cardiomyocyte proliferation, cardioprotection and cardiac dysfunction. These results indicate that the altered miRNome to be responsible for the disease status in CHD patients. Our data expand the existing knowledge on the epigenetic changes in CHD. In future, characterization of these cardiac-specific miRs will add huge potential to understand cardiac development, function, and molecular pathogenesis of heart diseases with a prospect of epigenetic manipulation for cardiac repair.

Neuro-protective effects of nano-formulated hesperetin in a traumatic brain injury model of Danio rerio.

Understanding the mechanism behind neuronal regeneration is critical for treating ischemic stroke and traumatic brain injury. The presence of neural stem cells in and around the sub-ventricular zone of human and also in zebrafish is evidenced. In this current study, the neuro-protective potential of nano-formulated hesperetin on injury-induced neurogenesis in zebrafish was assessed. Nanoformulation of hesperetin was prepared by anti-solvent precipitation technique using sodium dodecyl sulfate (SDS) as the stabilizing agent. The synthesized particles were characterized using SEM, DLS, XRD and FT-IR. Anti-oxidant capacity of nano hesperetin (nHST) in in vitro followed by in vivo studies in a traumatic brain injury (TBI) model of adult zebrafish (Danio rerio), catalase activity, histological analysis and gene expression studies for the genes Sox2, Nestin, Fabp7a and HuC were carried out. The synthesized particles were found to be in nanoscale and SDS had successfully integrated with hesperetin. Moreover, nHST had a significantly higher anti-oxidant capacity in vitro. Catalase levels in nHST treated group were significantly restored compared to other groups. Histological studies supported reduced tissue damage on oral administration of nano-hesperetin as compared to other groups. Gene expression studies showed that nano-hesperetin at a concentration of 10 µM when administered orally induced proliferation of neural stem cells without inducing cell death.

Isopimpinellin extends antiangiogenic effect through overexpression of miR-15b-5p and downregulating angiogenic stimulators.

BACKGROUND: Angiogenesis is the formation of new blood vessels from an existing vasculature through a series of processes such as activation, proliferation, and directed migration of endothelial cells. Angiogenesis is instrumental in the metastatic spread of tumors. Isopimpinellin, a furanocoumarin group of phytochemicals, is an anticarcinogenic agent. However, no studies have proven its antiangiogenic effects. The current study thus aimed to screen the antiangiogenic effect of isopimpinellin. METHODS AND RESULTS: Human Umblical Vein Endothelial Cell (HUVEC) as an in vitro model and zebrafish embryos as an in vivo model was used in this study. The experimental results showed that isopimpinellin effectively inhibited HUVEC proliferation, invasion, migration, and tube formation, which are the key steps in angiogenesis by markedly suppressing the expression of pro-angiogenic genes VEGF, AKT, and HIF-1α. In addition, isopimpinellin exerts its anti-angiogenic effect through the regulation of miR-15b-5p and miR-542-3p. Furthermore, in zebrafish embryos, isopimpinellin inhibited the development of intersegmental vessels (ISVs) through the significant downregulation of all pro-angiogenic genes vegf, vegfr2, survivin, angpt-1, angpt-2, and tie-2. CONCLUSION: Collectively, these experimental findings offer novel insights into the antiangiogenic nature of isopimpinellin and open new avenues for therapeutic approaches.

Naringenin alleviates 6-hydroxydopamine induced Parkinsonism in SHSY5Y cells and zebrafish model.

6-Hydroxydopamine (6-OHDA) is a neurotoxin that inhibits the mitochondrial complex I causing mitochondrial impairment, aetiology of Parkinson's. Naringenin is a flavanone predominantly present in citrus fruits. Due to its high antioxidant and anti-inflammatory potential, it has been widely studied against various disorders. In this study, the neuroprotective effect of naringenin was determined against 6-OHDA induced toxicity with Levodopa (l-DOPA) as the standard. Naringenin reduced 6-OHDA induced oxidative stress biomarker levels such as CAT, GSH, SOD, and ROS. Naringenin rescued 6-OHDA induced reduction of the mitochondrial membrane potential. Treatment with naringenin improved the locomotion of the 6-OHDA treated zebrafish larvae which showed stagnant swimming patterns. Naringenin was also found to downregulate the expression of some Parkinsonian genes such as casp9, lrrk2, and polg and upregulate pink1. These studies attribute to naringenin as a viable molecule to study further for its neuroprotective effects against 6-OHDA induced neurotoxicity and neurodegeneration.

Hesperidin downregulates kinases lrrk2 and gsk3ß in a 6-OHDA induced Parkinson's disease model.

The depletion of dopamine in the striatum region and Lewy bodies are the hallmark characteristics of Parkinson's disease. The pathology also includes the upregulation of various Parkinson's disease (PARK) genes and kinases. Two such kinases, LRRK2 and GSK-3ß have been directly implicated in the formation of tau and alpha-synuclein proteins, causing PD. Hesperidin (HES) is a flavanone glycoside that has multiple therapeutic benefits including neuroprotective effects. In this study, we examined the neuroprotective effects of HES against 6-hydroxydopamine (6-OHDA) induced-neurotoxicity in the in-vitro and in-vivo model. Hesperidin significantly protected the SH-SY5Y cells' stress against 6-OHDA induced toxicity by downregulating biomarkers of oxidative stress. Furthermore, HES downregulated the kinases lrrk2 and gsk3ß along with casp3, casp9, and polg in the zebrafish model. The treatment with HES also improved the locomotor pattern of zebrafish that was affected by 6-OHDA. This study suggests that hesperidin could be a drug of choice in targeting kinases against a 6-OHDA model of PD.

A marine sponge associated fungal metabolite monacolin X suppresses angiogenesis by down regulating VEGFR2 signaling.

Cancer is one of the leading causes of global death and there is an urgent need for the development of cancer treatment; targeting VEGFR2 could be one of the promising therapies. In the present study, previously isolated marine fungal metabolite monacolin X, suppresses in vitro angiogenic characteristics such as proliferation, migration, adhesion, invasion and tube formation of HUVECs when stimulated by VEGF, at a non-toxic concentration. Monacolin X downregulated VEGFR2, PKCα and PKCη mRNA expression. Further, monacolin X inhibited in vivo angiogenesis in CAM assay, vascular sprouting in aortic ring, decreased ISV and SIV length and diameter in Tg (Kdr:EGFP)/ko1 zebrafish embryos. Monacolin X showed reduced protein expression of pVEGFR2, pAKT1, pMAPKAPK2, pFAK and pERK1 in breast cancer lines and in DMBA induced mammary carcinoma in SD rats showed tumor regression and anti-angiogenesis ability via decrease pVEGFR2 and pAKT1 protein expression. In silico studies also revealed monacolin X ability to bind to crucial amino acid Cys 919 in the active site of VEGFR2 suggesting it to be a potent VEGFR2 inhibitor.

Novel gas producing Vibrio cholerae: a case report of gastroenteritis with acute kidney injury.

BACKGROUND: Bacterial characterization is important in clinical and epidemiological studies. We herein report the first case of gas-producing Vibrio cholera gastroenteritis with acute kidney injury. CASE PRESENTATION: A 30-year-old female presented to the emergency department with complaints of about ten episodes of watery diarrhea, four episodes of vomiting and elevated serum urea/creatinine levels. Although the bacteria were first misidentified as Vibrio furnissii by gas production on carbohydrate fermentation and triple sugar iron agar, it was later confirmed as Vibrio cholerae by 16 S rRNA gene sequencing and specific PCR. The treatment regimen was followed as for Vibrio species with intravenous fluids, ciprofloxacin and doxycycline. The patient recovered without relapse. CONCLUSIONS: Literature survey from the PubMed database shows no gas-producing Vibrio cholerae isolate being reported in the world. Further, genotype studies are warranted to look into the gas production of Vibrio cholerae .

Canonical Wnt pathway gene expression and their clinical correlation in oral squamous cell carcinoma.

AIM: The aim of this study is to explore the prognostic significance and clinicopathological correlations of the Wnt pathway genes in a cohort of surgically treated patients with oral squamous cell carcinoma (OSCC) patients. SETTINGS AND DESIGN: A prospective genetic study on patients with OSCC was carried out during the period from July 2014 to January 2016. Informed consent from patients and institutional ethical approval for the study was obtained and the guidelines were strictly followed for collection of samples. SUBJECTS AND METHODS: Clinical data and mRNA expression analysis of ten genes in the canonical Wnt pathway were evaluated and their relationships with clinical and demographic variables were studied in 58 tissue samples. Wnt-3a, ß-catenin, secreted frizzled-related proteins sFRP-1, sFRP-2, sFRP-4, sFRP-5, Wnt inhibitory factor 1, dickkopf-1, c-MYC, and cyclin-D1 from cancer (n = 29) and normal (n = 29) tissue samples were investigated using quantitative reverse transcription-polymerase chain reaction. STATISTICAL ANALYSIS: Descriptive statistics were used to summarize the sample characteristics and clinical variables. If the data were normal, then parametric tests were used; otherwise, nonparametric alternatives were used. All the analyses were carried out using SPSS version 23.0 (IBM SPSS Inc., USA). RESULTS: Expression of sFRP-1, sFRP-2, and sFRP-5 in control samples and expression of c-MYC and cyclin D1 in cancer samples showed statistical significance. Significant expression of Wnt3A was observed among patients who had recurrence and were deceased. CONCLUSION: Wnt3A, ß-catenin, and cyclin D1 are recognized as key components of Wnt/ß-catenin signaling. However, in this study, there was no significant expression of all the three genes in OSCC. The proto-oncogene c-MYC showed statistically significant upregulation in cancer tissue samples suggesting that the OSCC among South Indian population is primarily not mediated by the canonical Wnt signaling pathway.

Association between specific periodontal pathogens, Toll-like receptor-4, and nuclear factor-κB expression in placental tissues of pre-eclamptic women with periodontitis.

AIM: The aim of the present study was to determine the association between the presence of specific periodontal pathogens, Toll-like receptor-4 (TLR-4), and nuclear factor-κB (NF-κB) expression in the placental tissues of pre-eclamptic women. METHODS: Antenatal periodontal screening was performed in 25 normotensive pregnant women and 25 pre-eclamptic women. Subgingival plaque and placental tissue samples were collected from both groups and screened for the presence of Porphyromonas gingivalis (P. gingivalis), Tannerella forsythia, Aggregatibacter actinomycetemcomitans, and Prevotella intermedia (P. intermedia) using real-time polymerase chain reaction. The placental samples were also analyzed to quantify TLR-4 and NF-κB expression. RESULTS: The subgingival plaque samples of pre-eclamptic women showed significantly higher frequencies of P. intermedia. In the placental samples, P. gingivalis, P. intermedia, and the expression of TLR-4 and NF-κB were found to be at significantly higher levels compared to normotensive pregnant women. Using linear regression analysis, the expression of TLR-4 was significantly influenced by the presence of P. gingivalis (coefficient=3.176, 95% confidence interval [CI]: 367-5.986) and P. intermedia (coefficient=2.886, 95% CI: 0.77-5.696), whereas NF-κB expression was influenced only by the presence of P. intermedia (coefficient=2.220, 95% CI: 0.051-4.388) in the placental tissues of pre-eclamptic women. CONCLUSION: An association exists between P. gingivalis and P. intermedia with increased TLR-4 and NF-κB expression in the placenta of pre-eclamptic women with periodontitis.

Chitosan-based nano-formulation enhances the anticancer efficacy of hesperetin.

Cancer is one of the major causes of increased morbidity and mortality in modern society. Colorectal cancer is the third leading cause for cancer related death worldwide. Current chemotherapeutics are not very effective and have severe side effects. Hesperetin is a bioflavonoid from citrus fruits and its clinical use is restricted because of the poor water solubility. Folate receptor is overexpressed in various cancer cells. Therefore, we synthesized the chitosan folate hesperetin nanoparticle (CFH) by covalently conjugating folic acid with chitosan molecules. The size of the CFH nanoparticles is around 450nm, which is advantageous for passively targeting the cancer cell specifically due to the leaky vasculature of the tumour. Particle surface and size were observed using SEM and TEM studies. The results show that hesperetin has an IC50 value of 190µM and it induces apoptosis in HCT15 cells, however, CFH is very potent in inhibiting the proliferation with the IC50 value of 28µM. In addition, CFH inhibited colony formation and induced apoptosis by regulating the expression of proapoptotic genes expression. Therefore, the chitosan - folic acid conjugation appears to be the suitable carrier for colorectal cancer cell-specific delivery of hesperetin.

Evidence Linking the Role of Placental Expressions of Peroxisome Proliferator-Activated Receptor-γ and Nuclear Factor-Kappa B in the Pathogenesis of Preeclampsia Associated With Periodontitis.

BACKGROUND: Peroxisome proliferator-activated receptor (PPAR)-γ activation leads to suppression of production of a broad range of proinflammatory molecules. It plays a role in differentiation of trophoblasts and helps in normal placentation and formation of vascular exchange interface. Activation of nuclear factor-kappa (NF-κ) B triggers proinflammatory molecules inducing abnormal placentation and premature labor. This study aims to explore expression of PPAR-γ and NF-κB in placentas of women with periodontitis-associated preeclampsia compared with that in normotensive pregnant women. METHODS: Fifty pregnant women were included. Twenty-five were controls (normotensive pregnant women) and 25 were pregnant women with preeclampsia, including those with gestational hypertension. Demographic data, pregnancy characteristics, and periodontal parameters were recorded, including: 1) plaque index; 2) gingival index; 3) bleeding on probing (BOP); 4) probing depth; and 5) attachment loss (AL). Placental tissue samples were collected from both groups and analyzed to quantify expression of PPAR-γ and NF-κB using real-time polymerase chain reaction. RESULTS: BOP and AL were significantly higher in pregnant women with preeclampsia compared with normotensive pregnant women (P <0.05). Expression of PPAR-γ was downregulated in patients with preeclampsia compared with that of healthy normotensive patients, which was statistically significant (P <0.05), whereas NF-κB was significantly activated (P <0.05) in pregnant women with preeclampsia compared with normotensive pregnant women. CONCLUSIONS: Higher periodontal disease prevalence is found among pregnant women with preeclampsia, with increased percentage of sites with BOP and greater AL. This study provides novel information on host response to systemic inflammation induced by periodontal pathogens through mechanisms involving downregulation of PPAR-γ and increased activation of NF-κB.

Impact of occupational health hazards on serum markers of bone formation in spray painters of Chennai region in Tamil Nadu.

CONTEXT: The association between spray paint exposure and bone remodeling received little attention despite the high usage of spray paints in automobile industries, steel furniture workshops etc. AIM: The present study was aimed at investigating the level of serum markers of bone formation in spray painters. The spray painting subjects were selected from automobile body repair workshops in Chennai region of TamilNadu which constitutes 30% of India's automobile industry. SETTING AND DESIGN: All the study subjects, exposed to spray paint were working in a workshop without standard spraying room and did not wore any aerosol removing respirator. The controls were selected from random population irrespective of occupation. Data relevant to the socioeconomic features and personal history was collected using a questionnaire. The current study included 50 spray painters and 25 control subjects of same age group. MATERIALS AND METHODS: We examined the level of serum calcium, serum phosphorus, serum differentiation markers of bone such as alkaline phosphatase (bone specific) and serum osteocalcin in which these levels were found to be high in serum of spray painters. CONCLUSION: The current study concludes dysregulation in bone remodeling of spray painters exposed to chronic solvents and paint pigments.

Myocilin mutations among POAG patients from two populations of Tamil Nadu, South India, a comparative analysis.

PURPOSE: Primary open angle glaucoma (POAG) is the most common type of glaucoma. Among the POAG genes identified so far, myocilin (MYOC) is the most frequently mutated gene in POAG patients worldwide. The MYOC Gln48His mutation is unique among Indian POAG patients. This mutation has not been observed in some populations within India and in other populations worldwide. The objectives of this work were to characterize and compare the mutation spectrum among POAG patients from two places of South India and identify the occurrence and prevalence of Gln48His mutation in our study populations. METHODS: One hundred-one (101) POAG patients from Chennai, South India were recruited for the study. Earlier, 100 patients from the southernmost part of India, Kanyakumari district, were screened. MYOC was screened by polymerase chain reaction based single stand conformation polymorphism (PCR-SSCP) methodology. DNA sequencing of deviant samples was performed. Secondary structures of the proteins with amino acid sequence variations were predicted. RESULTS: The mutation frequency of MYOC among POAG patients in Chennai was 2%. Three types of mutations were observed. The MYOC Gln48His mutation was observed among 2 POAG patients from Chennai. However, absence of this mutation among patients from Kanyakumari suggests possible involvement of demographic factors in disease causation via this mutation. Two heterozygous sequence variants, Thr353Ile and Asn480Lys, in the same exon (exon III) of MYOC were observed in one POAG patient who had a severe disease phenotype. This is the first such report of a compound heterozygote individual with two mutations in the same exon of MYOC. CONCLUSIONS: The presence of mutations at a rate similar to other studies suggests the causative role of MYOC among POAG patients from Chennai. Screening of more patients and families from all parts of India is required to identify the actual frequency of the Gln48His mutation and thus highlight its importance. The compound heterozygote with a severe disease phenotype reiterates the importance of MYOC in certain POAG patients.