Modulation of APOL1-miR193a Axis Prevents Podocyte Dediffrentiation in High Glucose Milieu

The loss of podocyte (PD) molecular phenotype is an important feature of diabetic podocytopathy. We hypothesized that high glucose (HG) induces dedifferentiation in differentiated podocytes (DPDs) through alterations in the apolipoprotein (APO) L1-microRNA (miR) 193a axis. HG-induced DPD dedifferentiation manifested in the form of downregulation of Wilms’ tumor 1 (WT1) and upregulation of paired box 2 (PAX2) expression. WT1-silenced DPDs displayed enhanced expression of PAX2. Immunoprecipitation of DPD cellular lysates with anti-WT1 antibody revealed formation of WT1 repressor complexes containing Polycomb group proteins, enhancer of zeste homolog 2, menin, and DNA methyltransferase (DNMT1), whereas silencing of either WT1 or DNMT1 disrupted this complex with enhanced expression of PAX2. HG-induced DPD dedifferentiation was associated with a higher expression of miR193a, whereas inhibition of miR193a prevented DPD dedifferentiation in HG milieu. HG downregulated DPD expression of APOL1. miR193a-overexpressing DPDs displayed downregulation of APOL1 and enhanced expression of dedifferentiating markers; conversely, silencing of miR193a enhanced the expression of APOL1 and preserved DPD phenotype. Moreover, stably APOL1G0-overexpressing DPDs displayed the enhanced expression of WT1 but attenuated expression of miR193a; nonetheless, silencing of APOL1 reversed these effects. Since silencing of APOL1 enhanced miR193a expression as well as dedifferentiation in DPDs, it appears that downregulation of APOL1 contributed to dedifferentiation of DPDs through enhanced miR193a expression in HG milieu. Vitamin D receptor agonist downregulated miR193a, upregulated APOL1 expression, and prevented dedifferentiation of DPDs in HG milieu. These findings suggest that modulation of the APOL1-miR193a axis carries a potential to preserve DPD molecular phenotype in HG milieu.</jats:p

Molecular Docking Studies of a Cyclic Octapeptide-Cyclosaplin from Sandalwood

Natural products from plants, such as chemopreventive agents, attract huge attention because of their low toxicity and high specificity. The rational drug design in combination with structure-based modeling and rapid screening methods offer significant potential for identifying and developing lead anticancer molecules. Thus, the molecular docking method plays an important role in screening a large set of molecules based on their free binding energies and proposes structural hypotheses of how the molecules can inhibit the target. Several peptide-based therapeutics have been developed to combat several health disorders, including cancers, metabolic disorders, heart-related diseases, and infectious diseases. Despite the discovery of hundreds of such therapeutic peptides however, only few peptide-based drugs have made it to the market. Moreover, the in silico activities of cyclic peptides towards molecular targets, such as protein kinases, proteases, and apoptosis related proteins have not been extensively investigated. In this study, we explored the in silico kinase and protease inhibitor potentials of cyclosaplin, and studied the interactions of cyclosaplin with other apoptosis-related proteins. Previously, the structure of cyclosaplin was elucidated by molecular modeling associated with dynamics that were used in the current study as well. Docking studies showed strong affinity of cyclosaplin towards cancer-related proteins. The binding affinity closer to 10 kcal/mol indicated efficient binding. Cyclosaplin showed strong binding affinities towards protein kinases such as EGFR, VEGFR2, PKB, and p38, indicating its potential role in protein kinase inhibition. Moreover, it displayed strong binding affinity to apoptosis-related proteins and revealed the possible role of cyclosaplin in apoptotic cell death. The protein&ndash;ligand interactions using LigPlot displayed some similar interactions between cyclosaplin and peptide-based ligands, especially in case of protein kinases and a few apoptosis related proteins. Thus, the in silico analyses gave the insights of cyclosaplin being a potential apoptosis inducer and protein kinase inhibitor

Evaluation of Cyclosaplin Efficacy Using a Silk Based 3D Tumor Model

Development of novel anti-cancer peptides requires a rapid screening process which can be accelerated by using appropriate in vitro tumor models. Breast carcinoma tissue is a three-dimensional (3D) microenvironment, which contains a hypoxic center surrounded by dense proliferative tissue. Biochemical clues provided by such a 3D cell mass cannot be recapitulated in conventional 2D culture systems. In this experiment, we evaluate the efficacy of the sandalwood peptide, cyclosaplin, on an established in vitro 3D silk breast cancer model using the invasive MDA-MB-231 cell line. The anti-proliferative effect of the peptide on the 3D silk tumor model is monitored by alamarBlue assay, with conventional 2D culture as control. The proliferation rate, glucose consumed, lactate dehydrogenase (LDH), and matrix metalloproteinase 9 (MMP-9) activity of human breast cancer cells are higher in 3D constructs compared to 2D. A higher concentration of drug is required to achieve 50% cell death in 3D culture than in 2D culture. The cyclosaplin treated MDA-MB-231 cells showed a significant decrease in MMP-9 activity in 3D constructs. Microscopic analysis revealed the formation of cell clusters evenly distributed in the scaffolds. The drug treated cells were less in number, smaller and showed unusual morphology. Overall, these findings indicate the role of cyclosaplin as a promising anti-cancer therapeutic

Perinatal Outcomes in Premature Placental Calcification and the Association of a Color Doppler Study: Report from a Tertiary Care Hospital in Eastern India

Introduction: Placental calcification, identified before the 36th week of gestational age, is known as premature placental calcification (PPC). PPC could be a clue for the poor fetal outcome. However, its association with adverse perinatal outcomes is yet to be confirmed. Objective: The primary objective was to determine and compare the perinatal outcomes in pregnancies with and without documented premature placental calcification. Methodology: The present study was a prospective cohort study performed from October 2017 to September 2019. We consecutively enrolled 494 antenatal women who presented to our antenatal OPD after taking consent to participate in our study. Transabdominal sonographies were conducted between 28–36 weeks of gestation to document placental maturity. We compared maternal and fetal outcomes between those who were identified with grade III placental calcification (n = 140) and those without grade III placental calcification (n = 354). Results: The incidence of preeclampsia, at least one abnormal Doppler index, obstetrics cholestasis, placental abruption, and FGR (fetal growth restriction) pregnancies were significantly higher in the group premature placental calcification. We also found a significantly increased incidence of Low APGAR (Appearance, Pulse, Grimace, Activity, and Respiration) scores, NICU (Neonatal Intensive Care Unit) Admission, Abnormal CTG (cardiotocography), meconium-stained liquor, and low birth weight babies in those with grade III placental calcification. Conclusion: Clinicians should be aware of documenting placental grading while performing ultrasonography during 28 to 36 weeks. Ultrasonographically, the absence of PPC can define a subcategory of low-risk pregnant populations which probably need no referral to specialized centers and can be managed in these settings

Effect of APOL1 disease risk variants on APOL1 gene product

Gene sequence mutations may alter mRNA transcription, transcript stability, protein translation, protein stability and protein folding. Apolipoprotein L1 (APOL1) has two sets of sequence variants that are risk factors for kidney disease development, APOL1G1 (substitution mutation) and APOL1G2 (deletion mutation). Our present study focuses on the impact of these variants on APOL1 mRNA transcription and translation. APOL1 plasmids (EV, G0, G1 and G2) were transfected into human embryonic kidney (HEK) 293T cells. APOL1 variant expression was observed to be significantly lower than that of APOL1G0. Podocyte cell lines stably expressing APOL1 transgenes also showed lower levels of APOL1 expression of APOL1 variants (G1 and G2) compared with APOL1G0 by Western blotting and FACS analysis. The enhanced expression of GRP78 by podocytes expressing APOL1 variants would indicate endoplasmic reticulum (ER) stress. Bioinformatics evaluation using two different programs (MUPro and I-Mutant 2.0) predicted that APOL1 variants were less stable than APOL1G0. Concomitant with protein levels, APOL1 mRNA levels were also depressed following induction of APOL1 variant compared with APOL1G0 in both proliferating and differentiated podocytes. APOL1 mRNA transcript stability was tested after actinomycin D pulsing; APOL1G1 and APOL1G2 mRNAs transcript decayed 10-15% and 15-20% (within a period of 0.5-3 h) respectively. Our data suggest that down-regulated APOL1 protein expression in APOL1 variants is due to compromised transcription and decay of the APOL1 variant transcripts

Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes

<div><p>Human skin is body’s vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated <i>in vitro</i>. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC₅₀ values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H₂O₂) induced RBC haemolysis (IC₅₀ 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H₂O₂-induced damage; inhibited H₂O₂ induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells <i>in vitro</i> and can be used as a potential ingredient in skin care formulations.</p></div

Radical scavenging effect of TE on HDF cells against H2O2-induced cell damage.

<p>(<b>A</b>) HDF cells were pre-treated with various concentrations of TE for 1 h and exposed to 10 μM H2O2, and further incubated for 24 h at 37°C. (<b>B</b>) HDFs were co-treated with various concentrations of TE and 10 μM H2O2, incubated for 24 h at 37°C. Cell viability was determined by MTT assay and the results expressed as mean ± SEM (n = 3). <b>(C) Protective effect of TE on DNA damage in HDF cells by release of OH radicals from Fenton reaction.</b> The DNA were isolated from HDF cells and treated with two concentrations of TE (50 and 100μg/mL), FeSO₄ (2mM) and H₂O₂ (1 mM), incubated for 1 h at 37°C. DNA bands were resolved in 1% agarose gel stained with ethidium bromide. Densitometry analysis showing the protective effect of TE on H₂O₂-induced DNA damage. Values shown depict arbitrary units. Data is expressed as mean ± SEM (n = 3).</p

Effect of TE on NO production in LPS-stimulated RAW264.7 cells.

<p>Cells were treated with TE (50 and 100 μg/mL) and dexamethasone (Dexa) (100 μM), with/without LPS (1μg/mL) and incubated for 24 h at 37°C. After incubation, the cell supernatant was used to determine NO level by Griess reagent method. The values are expressed as mean ± SEM of three experiments (<i>p</i> ≤ 0.01, p ≤ 0.05).</p

(A) Effect of TE on H₂O₂-induced RBC hemolysis. RBC cells were treated with different concentrations of TE and ascorbic acid (AA) (25–100 μg/mL) and the cells were induced with 100 μM H₂O₂ for cell lysis. Data was expressed as percentage of control. Results are shown as mean ± SEM of three experiments. (B) Reducing power activity of TE at different concentrations (0–400 μg/ml).

<p>Absorbance was read at 700 nm and the data are represented as ascorbic acid equivalents. Results expressed as mean ± SEM (n = 3).</p