Systems biology approach: identification of hub genes, signaling pathways, and molecular docking of COL1A1 gene in cervical insufficiency.

The collagen type I alpha 1 (COL1A1, OMIM #120,150) gene, encoding the alpha-1 chain of type I collagen (UniProt #P02452), plays a key role in life-homeostasis due to its remarkable involvement in collagen synthesis. It is a promising candidate gene implicated in the pathogenesis of cervical insufficiency (CI). This study aimed to identify genetic variations within the COL1A1 gene that contribute to the development of CI. Polymerase chain reaction (PCR) and amplicon sequencing were implemented for single nucleotide polymorphisms (SNPs) detection (+ 1245G/T, SP1 rs1800012), which revealed wild-type sequence for targeted SNPs in enrolled proband indicated negative results regarding COL1A1 gene involvement for current form of CI. It allows further investigation of other closely connected genes probed in this study. Computational approaches viz. Protein-protein interaction (PPI), gene ontology (GO), and pathway participation were used to identify the crucial hub genes and signaling pathways for COL1A1 and CI. Using the Yet Another Scientific Artificial Reality Application (YASARA) software, molecular docking, and molecular dynamic (MD) simulation with the oxytocin (CID 439,302), estradiol (CID 129,728,744), progesterone (CID 5994) and hydroxyprogesterone (CID 150,788) were done. Interactive bioinformatics analysis demonstrated that the COL1A1 and more than 10 collagen sister genes had a strong connection with CI. In sum, the findings of this study provide insights into a modus operandi that can be utilized to illuminate the path toward studying sister genes and smooth diagnosis of CI. These findings have implications for understanding the foundational process of the condition and potentially developing screening, diagnostic, and therapeutic interventions.

CRISPR-based genome editing of zebrafish.

CRISPR/Cas9, once discovered as an adaptive immune system in bacteria, has emerged as a disruptive technology in the field of genetic engineering. Technological advancements in the recent past has enhanced the applicability of CRISPR/Cas9 tool for gene editing, gene therapies, developmental studies and mutational analysis in various model organisms. Zebrafish, one of the excellent animal models, is preferred for conducting CRISPR/Cas9 studies to assess the functional implication of specific genes of interest. CRISPR/Cas9 mediated gene editing techniques, such as, knock-out and knock-in approaches, provide evidences to identify the role of different genes through loss-of-function studies. Also, CRISPR/Cas9 has been proved to be an efficient tool for designing disease models for gene expression studies based on phenotypic screening. The present chapter provides an overview of CRISPR/Cas9 mechanism, different strategies for DNA modifications and gene function analysis, highlighting the translational applications for future prospects, such as screening of drug toxicity and efficacy.

Design of non-viral vector with improved regulatory features towards therapeutic application.

Viral vectors based gene therapy is often compromised by adverse immunological reactions raising safety concerns. Hence, improved design and development of non-viral vectors with strong regulatory regions is desired. We describe the design of a non-viral mammalian expression vector in which the primary transgene (a truncated dystrophin gene linked with Duchenne muscular dystrophy (DMD)) named microdystrophin delR4-R23/delCT (MD1) is under the transcriptional control of elements of desmin locus control region (DES-LCR). The designed vector, named as DES-LCR/MD1-EGFP, was constructed by cloning two fragments into the pBluescript backbone. Fragment 1 contains DES-LCR enhancer and DES-LCR promoter region while fragment 2 contains MD1 transgene and reporter EGFP (enhanced green fluorescent protein) gene separated by linker P2A (2A peptide). This vector design provides a framework for strong regulation with non-viral features. This design forms the foundation for application in conditions linked to multisystem diseases.

A Systematic Bioinformatics Approach to Motif-Based Analysis of Human Locus Control Regions.

Locus control regions (LCRs), cis-acting, noncoding regulatory elements with strong transcription-enhancing activity, are conserved in sequence and organization, and exhibit strict gene-specific expression. LCRs have been reported and studied in several mammalian gene systems, signifying that they play an important role in eukaryotic gene expression control. Their highly regulated, stable, and precise levels of expression have made them a strong candidate for use in gene therapy vectors. In this study, we attempted to determine the unique signatures of human LCRs by analyzing a data set of LCR sequences for the presence of motifs through systematic bioinformatics approach. Using web-based regulatory sequence analysis tools (RSAT), motif-based analysis was performed. Detected significant motifs were analyzed further for their identity using Tomtom tool. RSAT analysis revealed that significant motifs are existent within the LCRs. Identity analysis using Tomtom showed that detected significant motifs were comparable with known transcription factor (TF) binding sites and the top scoring motifs belong to zinc finger-containing proteins, an important group of proteins involved in a variety of cellular activities. Correspondence to segment of known motif indicates the biological relevance of the detected motifs. Motif-based analysis is valuable for analyzing the various characteristics of sequences, notably TF binding models in this study. Owning to their unique expression control abilities, LCRs form an important component of integrating vectors, therefore identification of unique signatures present within LCR sequences will be instrumental in the design of new generation of regulatory elements containing LCR sequences.

Molecular interactions of active constituents of essential oils in zwitterionic lipid bilayers.

Eugenol and its related compounds are major active constituents of essential oils and have been extensively used as food flavoring agents with significant lipid peroxidation inhibition activity, highlighting the importance of understanding detailed molecular mechanisms behind their interactions with lipid bilayer. For this, we studied antioxidant activity of essential oils rich extract of Cinnamomum tamala leaves and molecular dynamics simulations of eugenol, isoeugenol, methyleugenol, acetyleugenol and eugenol oxide in POPC and PLPC lipid bilayers. All the compounds penetrated into bilayer however, isoeugenol showed highest affinity for the pure POPC and PLPC bilayers with lowest free energy profiles, formed more H-bonds with bilayer oxygen atoms and more pronounced changes in area per lipid and thickness of the bilayer, thus more efficient for scavenging radicals coming from outside as well as centrally located lipid peroxyl radicals. These molecular interactions rationalize the difference in inhibition activities of lipid peroxidation by eugenol and its related compounds.

Mechanism of protective effect of phyllanthin against carbon tetrachloride-induced hepatotoxicity and experimental liver fibrosis in mice.

Chronic injury to liver triggers synthesis of extracellular matrix components resulting in progressive fibrosis and eventually cirrhosis. Transforming growth factor-ß1 (TGF-ß1) transduces its signal by binding to TGF-ß type 1 receptor kinase or activin like kinase (ALK5) receptor and mediates hepatic fibrosis by increasing the transcription of downstream entities such as collagen via Smad2 and Smad3. The present study was carried out to investigate the mechanism by which phyllanthin, a hepatoprotective lignin isolated from the plant Phyllanthus amarus (P. amarus) exerts its anti-fibrotic effect. The inhibitory role of phyllanthin on ALK5 was first analyzed using molecular docking experiments. Phyllanthin was found to effectively bind to serine (Ser) 280 at the active site of ALK5 by forming hydrogen bonds. The in vivo protective effect of phyllanthin against carbon tetrachloride (CCl4)-induced hepatic fibrosis was established by studying the protein expressions of TGF-ß1, ALK5 and Smad2 and 3 and by determining various biochemical and histopathological parameters. Phyllanthin was found to exert its anti-fibrotic effect by down-regulating TGF signaling pathway via ALK5 and Smad2 and 3 inhibition.

Antinephrolithiatic and antioxidative efficacy of Dolichos biflorus seeds in a lithiasic rat model.

CONTEXT: Dolichos biflorus sensu auct non L. (Fabaceae) is widely used for the treatment of kidney stones, leucorrhoea, urinary disorders, and menstrual troubles, and is known for its antioxidant activity. OBJECTIVES: To evaluate the preventive effect of hydro-alcoholic extract of Dolichos biflorus seeds (DBE) in ethylene glycol induced nephrolithiasis. MATERIALS AND METHODS: In vitro antioxidative capacity of DBE was estimated in terms of reducing power, superoxide radical, 2,2- diphenyl-1-picrylhydrazyl radical, and nitric oxide scavenging activity. A validated HPLC method was used for standardization using quercetin as a marker. Adult female Wistar rats were administered with DBE (150 and 300 mg/kg body weight/day) along with ethylene glycol (0.75%, v/v) for 28 d. The various biochemical parameters were measured in urine, serum, and kidney followed by histochemistry. RESULTS: Ethylene glycol caused a significant increase in calcium, oxalate, phosphate, and total protein in urine as well as in kidney whereas decrease in calcium, sodium, and magnesium in serum was observed (p < 0.001). Ethylene glycol also caused a significant increase in lipid peroxidation and concurrent decrease in activities of antioxidant enzymes in kidney (p < 0.001). However, the seed extract of D. biflorus caused significant restoration of all these parameters (p < 0.001). Histopathological and histochemical studies also showed the reduced calcifications in kidney of seed extract treated rats. DISCUSSION AND CONCLUSION: These results indicated that seeds of D. biflorus have significant prophylactic effect in preventing the nephrolithiasis, which might be due to the antioxidant activity of the active compounds of the plant.

Testing the efficacy of quercetin in mitigating bisphenol A toxicity in liver and kidney of mice.

Quercetin (3,5,7,3',4'-pentahydroxy flavone) is a potent antioxidant found in various fruits and vegetables. The present investigation was an attempt to evaluate the mitigatory effect of quercetin on the damage caused by bisphenol A (BPA; 2,2-bis (4-hydroxyphenyl) propane), a well-known xenoestrogen, on liver and kidney of mice. Swiss strain adult male albino mice were orally administered with 120 and 240 mg/kg body weight (bw)/day BPA with or without quercetin (60 mg/kg bw/day) for 30 days. On the completion of the treatment period, animals were killed; organs were isolated and used for the study. Results revealed that oral administration of BPA for 30 days caused significant and dose-dependent decrease in body weight. Absolute and relative organ weights, total lipid and cholesterol contents were significantly increased in liver and kidney of mice when compared with vehicle control. BPA treatment also caused, when compared with vehicle control, a statistically significant reductions in the activities of catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase and glutathione-S-transferase as well as in glutathione and total ascorbic acid contents; however, significant increase was found in malondialdehyde (MDA) levels. Histopathological studies revealed hepatocellular necrosis, cytoplasmic vacuolization and decrease in hepatocellular compactness in liver as well as distortion of the tubules, increased vacuolization, necrosis and disorganization of glomerulus in the kidney of BPA-treated mice. All these effects were dose-dependent. Co-treatment with quercetin (60 mg/kg bw) and BPA (low dose and high dose) alleviates the changes in body weight, as well as absolute and relative organ weights of mice. It also ameliorates the oxidative stress created by BPA by lowering MDA levels and by increasing enzymatic and nonenzymatic antioxidants as well as it brings back the normal histoarchitecture of liver and kidney of mice. The present results revealed that graded doses of BPA caused oxidative damage in liver and kidney of mice, which is mitigated by quercetin.

Inhibition of calcium oxalate crystallisation in vitro by an extract of Bergenia ciliata.

OBJECTIVE: To evaluate the effectiveness of an extract obtained from the rhizomes of Bergenia ciliata (Saxifragaceae) on the inhibition of calcium oxalate (CaOx) crystallisation in vitro. MATERIALS AND METHODS: A hydro-alcoholic extract (30:70, v/v) of rhizomes of B. ciliata was prepared at different concentrations (1-10 mg/mL). The crystallisation of CaOx monohydrate (COM) was induced in a synthetic urine system. The nucleation and aggregation of COM crystals were measured using spectrophotometric methods. The rates of nucleation and aggregation were evaluated by comparing the slope of the turbidity of a control system with that of one exposed to the extract. The results were compared with a parallel study conducted with a marketed poly-herbal combination, Cystone, under identical concentrations. Crystals generated in the urine were also analysed by light microscopy. Statistical differences and percentage inhibitions were calculated and assessed. RESULTS: The extract of B. ciliata was significantly more effective in inhibiting the nucleation and aggregation of COM crystals in a dose-dependent manner than was Cystone. Moreover, the extract induced more CaOx dihydrate crystals, with a significant reduction in the number and size of COM crystals. CONCLUSION: An extract of the traditional herb B. ciliata has an excellent inhibitory activity on crystalluria and therefore might be beneficial in dissolving urinary stones. However, further study in animal models of urolithiasis is needed to evaluate its potential anti-urolithiatic activity.

Protective effect of black tea infusion on aflatoxin-induced hepatotoxicity in mice.

BACKGROUND: Aflatoxins are a group of mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus and are potent inducers of hepatotoxicity. OBJECTIVE: The present study was carried out to investigate the effect of black tea infusion on aflatoxin-induced hepatotoxicity in male mice. METHODS: A 2% black tea infusion in drinking water was prepared and orally administered along with aflatoxin (750 and 1500 µg/kg body weight) for 30 days. Morphological investigation, body weight and organ weight calculations and histopathological analysis were carried out. Serum hepatic marker enzymes namely alanine aminotransferase and aspartate aminotransferase were estimated. RESULTS: The results clearly indicated that aflatoxin treatment for 30 days caused significant dose-dependent reduction in body weight and increase in liver weight. The activities of ALT and AST were found to be elevated while protein content was found to be decreased in aflatoxin-treated mice as compared to vehicle control. Histopathological analysis showed hepatocellular necrosis and cytoplasmic vacuolization along with fatty infiltration in toxin-treated animals. Results revealed significant (p < 0.05) restoration of aflatoxin-induced damages in body weight, organ weight, serum chemistry and histopathological features in aflatoxin plus black tea infusion administered mice in a dose dependant manner. CONCLUSION: It is concluded from the present study that supplementation of black tea infusion can be beneficial in positively modulating aflatoxin-induced alterations in liver.

Aflatoxin-induced biochemical changes in liver of mice and its mitigation by black tea extract.

Aflatoxin belongs to the class of naturally occurring mycotoxins, food contaminants having potent carcinogenicity. We have evaluated the ameliorative role of black tea extract on aflatoxin-induced biochemical changes in the liver of albino male mice. Adult male mice were orally administered with 750 and 1500 pg of aflatoxin in 0.2 mL olive oil/kg b.w./day for 30 days. Oral administration of aflatoxin caused, as compared with controls, significant, dose-dependent reduction in DNA, RNA, protein and glycogen contents; however, cholesterol content and phsphorylase activity were significantly increased. Black tea is one of the most potent antioxidants containing numerous bioactive phytonurtients having therapeutic applications. Aflatoxin-induced changes in the liver of mice were significantly ameliorated on co-treatment of black tea extract (2% infusion in water).

Protection against butyl p-hydroxybenzoic acid induced oxidative stress by Ocimum sanctum extract in mice liver.

Prime focus of the present investigation was to evaluate hepatoprotective potency of Ocimum sanctum (O. sanctum) aqueous extract against butyl p-hydroxybenzoic acid (butylparaben) toxicity in mice. Oral treatment of butylparaben (1320 mg/kg b.w./day) to mice for 30 days resulted in significant (p < 0.05) elevation in hepatic lipid peroxidation, which could be due to significant (p < 0.05) reduction in non-enzymatic (glutathione and total ascorbic acid) antioxidant contents and enzymatic (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione transferase) antioxidants activities. Co-treatment of O. sanctum extracts in three different doses (100, 200 and 300 mg/kg b.w./day) resulted in significant (p < 0.05) reduction in butylparaben-induced hepatic changes. Oral administration of O. sanctum with butylparaben resulted in dose-dependent and significant (p < 0.05) reduction in lipid peroxidation as compared to butylparaben alone treated group. Similarly, all three doses of O. sanctum reduced butylparaben-induced changes in non-enzymatic and enzymatic antioxidants. The effect was significant (p < 0.05) and dose-dependent. All three doses of O. sanctum ameliorated butylparaben-induced changes, showing maximum protection at 300 mg/kg b.w./day dose. Results of present study indicate that butylparaben-induced hepatotoxicity involves its ability to induce oxidative stress, whereas antihepatotoxic effect of O. sanctum was mainly due to its antioxidative potency.

Butyl p-hydroxybenzoic acid induces oxidative stress in mice liver--an in vivo study.

Present study focuses on the evaluation of butyl p-hydroxybenzoic acid (butylparaben CAS No: 94-26-8) exerted hepatotoxicity in mice. Oral administration of three different doses of butylparaben (40, 20 and 13.33 mg/0.2 mL olive oil/kg b.w./day) for 30 days has resulted in marked increase in lipid peroxidation. The effect was dose-dependent. Biochemical analysis revealed significant (p < 0.05) and dose-dependant reduction in non-enzymatic antioxidants such as glutathione and ascorbic acid content. Significant (p < 0.05) reduction in enzymatic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, glutathione transferase were also observed in butylparaben treated groups as compared to control. Our findings prove that the oxidative stress induced by butylparaben plays the central role in the toxicity.

Phyllanthin of Standardized Phyllanthus amarus Extract Attenuates Liver Oxidative Stress in Mice and Exerts Cytoprotective Activity on Human Hepatoma Cell Line.

BACKGROUND: Phyllanthus amarus, a traditional herbal liver-protecting medicine, is known to contain an active ingredient phyllanthin. Many research studies and clinical trials performed in the past using this plant have given contentious results which clearly accentuates the need for the standardization of the extracts. AIM: In this study, P. amarus extract was standardized for phyllanthin content by high performance thin layer chromatography (HPTLC) and high performance liquid chromatography (HPLC) analysis. The preventive role of a standardized extract of P. amarus against CC14-induced hepatotoxicity in vivo and in vitro using mice model and human hepatoma HepG2 cell line, respectively, was investigated. METHODS: Phyllanthin was used as a marker phytochemical for the standardization of P. amarus extract. The extracts were verified for phyllanthin content by HPTLC and HPLC. Female mice were orally administered with CCl4 either with or without standardized P. amarus extract in three different doses. Similarly, the cytoprotective role of the standardized extract in vitro was studied in HepG2 cell line. RESULTS: Oral administration of CCl4 resulted in increased oxidative stress, decreased antioxidative defense, and liver injury. Treatment with P. amarus along with CCl4 significantly mitigated the increase in activities of liver marker enzymes, lipid peroxidation, and bilirubin content. It also increased the antioxidant enzymatic and non-enzymatic defense parameter levels. The results of the in vitro study conducted in HepG2 cells indicated that the hepatotoxin lowered 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Mil) metabolism and increased the release of transaminases which were corrected with co-incubation with P. amarus. CONCLUSION: The study established a significant liver-protecting role of standardized P. amarus extract due to the presence of active ingredient phyllanthin.

Mitigation of carbon tetrachloride-induced damage by Phyllanthus amarus in liver of mice.

Liver disease has become a global concern worldwide. In absence of reliable liver protective drugs in modem medicine, a large number of medicinal preparations are recommended for the treatment of liver disorders as they are believed to be harmless based on their natural origin. The aim of the present study was to determine the hepatoprotective activity of Phyllanthus amarus plant extract against carbon tetrachloride (CCl4-induced liver damage in female mice. Carbon tetrachloride administration caused a significant increase in liver and serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and acid phosphatase (ACP), while total protein content significantly decreased as compared to vehicle control. The effect was dose-dependent. Oral administration of aqueous extract of Phyllanthus amarus along with carbon tetrachloride caused significant mitigation of CCl4-induced changes.

Ameliorative effects of ginger extract on paraben-induced lipid peroxidation in the liver of mice.

We have evaluated the ameliorative effect of ginger extract on paraben (p-hydroxybenzoic acid)-induced lipid peroxidation in the liver of mice. Adult female albino mice were orally administered with 2.25 or 4.50 mg of paraben in 0.2 mL olive/animal/day (67.5 and 135 mg/kg of body weight) for 30 days. The results revealed significantly higher (p < or = 0.05) lipid peroxidation in the liver of paraben-treated mice than that of controls. As compared with the controls, the levels of non-enzymatic antioxidants: glutathione and ascorbic acid, as well as the enzymatic antioxidants: superoxide dismutase, glutathione peroxidase and catalase were significantly (p < or = 0.05) lowered in the liver of paraben-treated mice. Oral administration of aqueous extract of Zinziber officinale (3 mg/animal/day) along with paraben for 30 days (Groups 6 and 7) caused significant (p < or = 0.05) amelioration in paraben-induced lipid peroxidation and increased significantly (p < or = 0.05) the activities of enzymatic (superoxide dismutase, glutathione peroxidase, catalase) and contents of non-enzymatic (glutathione and ascorbic acid) antioxidants in the liver of mice, as compared with those given paraben alone (Groups 4, 5). Thus, oral administration of aqueous extract of Zinziber officinale along with paraben significantly (p < or = 0.05) ameliorates paraben-induced lipid peroxidation in the liver of mice.