LncRNA MALAT1 Expression Regulates Breast Cancer Progression via PI3K/AKT/mTOR Pathway Modulation.

Breast cancer is a significant health challenge for women globally, including the Pakistani population. Numerous pathways and small molecules like noncoding ribonucleotides are implicated in breast cancer development and progression. Among these, lncRNAs, have garnered considerable attention due to their role in breast cancer tumorigenesis and metastasis. In the current study involving 52 mammary tumor samples from the Pakistani population, the expression of lncRNA MALAT1 (metastasis associated lung adenocarcinoma transcript 1) was studied via RT-PCR (Real-Time polymerase chain reaction). In addition, PI3K/AKT/mTOR pathway expression was also assessed through RT-PCR and immunohistochemistry in breast cancer patient samples. The study also investigated the cross-talk of lncRNA MALAT1 and PI3K pathway genes by inhibiting it with PI3K inhibitor (LY294002) in MDA-MB-231 cell line. Furthermore, lncRNA MALAT1 was silenced in MDA-MB-231 cells using siRNA to determine its impact on breast cancer proliferation and metastasis. The results revealed an upregulated expression of MALAT1 and PI3K/AKT/mTOR pathway genes in grade II and III breast tissue samples before chemotherapy. The proliferation, growth, and invasion of breast cancer cells were significantly reduced upon MALAT1 silencing in MDA-MB-231. Further, its downregulation substantially reduced the PI3K pathway expression levels at mRNA and protein levels. In conclusion, the current study suggests that MALAT1 could serve as a therapeutic target for breast cancer, underscoring its role in breast cancer proliferation and metastasis. Moreover, the study proposes a mechanism of action of MALAT1, demonstrating that its inhibition can reduce the expression of the PI3K/AKT/mTOR axis. These findings emphasize the potential significance of targeting MALAT1 as a therapeutic strategy for breast cancer, and further exploration of this interaction is warranted to gain deeper insight into the molecular mechanism of this lncRNA.

Prevalence of BRCA1 and BRCA2 germline variants in an unselected pancreatic cancer patient cohort in Pakistan.

BACKGROUND: BRCA1 and BRCA2 (BRCA1/2) are the most frequently investigated genes among Caucasian pancreatic cancer patients, whereas limited reports are available among Asians. We aimed to investigate the prevalence of BRCA1/2 germline variants in Pakistani pancreatic cancer patients. METHODS: One hundred and fifty unselected and prospectively enrolled pancreatic cancer patients were comprehensively screened for BRCA1/2 germline variants using denaturing high-performance liquid chromatography and high-resolution melting analyses, followed by DNA sequencing of the variant fragments. The novel variants were analyzed for their pathogenic effect using in-silico tools. Potentially functional variants were further screened in 200 cancer-free controls. RESULTS: Protein truncating variant was detected in BRCA2 only, with a prevalence of 0.7% (1/150). A frameshift BRCA2 variant (p.Asp946Ilefs*14) was identified in a 71-year-old male patient of Pathan ethnicity, with a family history of abdominal cancer. Additionally, we found a novel variant in BRCA2 (p.Glu2650Gln), two previously reported variants in BRCA1 (p.Thr293Ser) and BRCA2 (p.Ile2296Leu) and a recurrent nonsense variant in BRCA2 (p.Lys3326Ter). These variants were classified as variants of uncertain significance (VUS). It is noteworthy that none of these VUS carriers had a family history of pancreatic or other cancers. CONCLUSIONS: In this first study, BRCA1/2 pathogenic variant is identified with a low frequency in pancreatic cancer patients from Pakistan. Comprehensive multigene panel testing is recommended in the Pakistani pancreatic cancer patients to enhance genetic understanding in this population.

Anticancer genes (NOXA, PAR-4, TRAIL) are de-regulated in breast cancer patients and can be targeted by using a ribosomal inactivating plant protein (riproximin).

BACKGROUND: Anticancer genes are an endogenous defense against transformed cells as they impose antineoplastic effects upon ectopic expression. Profiling the expression of these genes is fundamental for exploring their prognostic and therapeutic relevance in cancers. Natural compounds can upregulate anticancer genes in malignant cells and thus be useful for therapeutic purposes. In this study, we identified the expression levels of anticancer genes in breast cancer clinical isolates. In addition, the purified and sequenced plant protein (riproximin) was evaluated for its potential to induce anticancer genes in two breast cancer cell lines. METHODOLOGY: Expression profiles of three anticancer genes (NOXA, PAR-4, TRAIL) were identified by immunohistochemistry in 45 breast cancer clinical isolates. Breast cancer cells were exposed to riproximin and expression of the anticancer genes was determined by microarray, real-time PCR and western blot methodologies. Lastly, a bioinformatic approach was adopted to highlight the molecular/functional significance of the anticancer genes. RESULTS: NOXA expression was evenly de-regulated among the clinical isolates, while PAR-4 was significantly down-regulated in majority of the breast cancer tissues. In contrast, TRAIL expression was increased in most of the clinical samples. Expression levels of the anticancer genes followed a distinct trend in accordance with the disease severity. Riproximin showed a substantial potential of inducing expression of the anticancer genes in breast cancer cells at transcriptomic and protein levels. The bioinformatic approach revealed involvement of anticancer genes in multiple cellular functions and signaling cascades. CONCLUSION: Anticancer genes were de-regulated and showed discrete expression patterns in breast cancer patient samples. Riproximin effectively induced the expression of selected anticancer genes in breast cancer cells.

Contribution of germline PALB2 variants to an unselected and prospectively registered pancreatic cancer patient cohort in Pakistan.

BACKGROUND: Partner and localizer of BRCA2 (PALB2) is a pancreatic cancer (PC) susceptibility gene reported in Caucasians. However, limited data are available among Asians. We investigated the contribution of PALB2 germline variants to Pakistani PC patients. METHODS: 150 unselected and prospectively enrolled PC patients were comprehensively screened for PALB2 variants, using denaturing high-performance liquid chromatography and DNA sequencing. Novel variants were investigated for their pathogenic effect using in-silico tools. Potentially functional variants were screened in 200 controls. RESULTS: Twenty-two different PALB2 variants were identified. A missense variant (p.Arg37His) was identified in a 48-years-old male patient with a family history of breast cancer. Another missense variant (p.Trp898Arg) was identified in a 48-years-old male patient with a family history of esophageal cancer. A novel 3' downstream variant (c.∗480A>G) was detected in a 34-years-old female patient with family history of lung cancer. Another novel 3' downstream variant (c.∗417A>C) was identified in a 41-years-old male patient. All these variants were absent in 200 controls. p.Arg37His and p.Trp898Arg were predicted as likely pathogenic. c.∗417A>C and c.∗480A>G were classified as variants of uncertain significance. CONCLUSION: This is the first study that suggests a minimal contribution of PALB2 variants to PC risk in Pakistani population.

pH Responsive Abelmoschus esculentus Mucilage and Administration of Methotrexate: In-Vitro Antitumor and In-Vivo Toxicity Evaluation.

The rapid progression in biomaterial nanotechnology apprehends the potential of non-toxic and potent polysaccharide delivery modules to overcome oral chemotherapeutic challenges. The present study is aimed to design, fabricate and characterize polysaccharide nanoparticles for methotrexate (MTX) delivery. The nanoparticles (NPs) were prepared by Abelmoschus esculentus mucilage (AEM) and chitosan (CS) by the modified coacervation method, followed by ultra-sonification. The NPs showed much better pharmaceutical properties with a spherical shape and smooth surface of 213.4-254.2 nm with PDI ranging between 0.279-0.485 size with entrapment efficiency varying from 42.08 ± 1.2 to 72.23 ± 2.0. The results revealed NPs to possess positive zeta potential and a low polydispersity index (PDI). The in-vitro drug release showed a sustained release of the drug up to 32 h with pH-dependence. Blank AEM -CS NPs showed no in-vivo toxicity for a time duration of 14 days, accompanied by high cytotoxic effects of optimized MTX loaded NPs against MCF-7 and MD-MBA231 cells by MTT assay. In conclusion, the findings advocated the therapeutic potential of AEM/CS NPs as an efficacious tool, offering a new perspective for pH-responsive routing of anticancer drugs with tumor cells as a target.

The Insight of In Silico and In Vitro evaluation of Beta vulgaris phytochemicals against Alzheimer's disease targeting acetylcholinesterase.

B. vulgaris extracts possess antioxidant, anti-inflammatory along with its role in improving memory disorders. Subsequently, in vitro and in silico studies of its purified phytochemicals may expand complementary and alternative Alzheimer's therapeutic option. Super activation of acetylcholinesterase enzyme is associated explicitly with Alzheimer's disease (AD) ultimately resulting in senile dementia. Hence, acetylcholinesterase enzyme inhibition is employed as a promising approach for AD treatment. Many FDA approved drugs are unable to cure the disease progression completely. The Present study was devised to explore the potential bioactive phytochemicals of B. vulgaris as alternative therapeutic agents against AD by conducting in vitro and in silico studies. To achieve this, chemical structures of phytochemicals were recruited from PubChem. Further, these compounds were analyzed for their binding affinities towards acetylcholinesterase (AChE) enzyme. Pharmacophoric ligand-based models showed major characteristics like, HBA, HBD, hydrophobicity, aromaticity and positively ionizable surface morphology for receptor binding. Virtual screening identified three hit compounds including betanin, myricetin and folic acid with least binding score compared to the reference drug, donepezil (-17 kcal/mol). Further, in vitro studies for anti-acetylcholinesterase activity of betanin and glycine betaine were performed. Dose response analysis showed 1.271 µM and 1.203 µM 50% inhibitory concentration (IC50) values for betanin and glycine betaine compounds respectively. Our findings indicate that phytoconstituents of B. vulgaris can be implicated as an alternative therapeutic drug candidate for cognitive disorders like Alzheimer's disease.

Berberis lyceum and Fumaria indica: in vitro cytotoxicity, antioxidant activity, and in silico screening of their selected phytochemicals as novel hepatitis C virus nonstructural protein 5A inhibitors.

Berberis lyceum and Fumaria indica are two Pakistani indigenous herbal medicines used to treat liver infections, including hepatitis C virus (HCV). This study aimed to evaluate the cytotoxicity, and antioxidant activity of these plant extracts and computationally screen their selected phytoconstituents as HCV NS5A inhibitors. The viability of HepG2 cells was assessed 24 h and 48 h post-treatment using colorimetric and dye exclusion methods. Antioxidant properties were examined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH), reducing power, and total antioxidant capacity assays. Seventeen known phytochemicals identified from each plant were docked into the active binding site of HCV NS5A protein. The top hit ligands were analyzed for their druglikeness properties and the indices of absorption, distribution, metabolism, elimination, and toxicity (ADMET). The results showed that both plant extracts were non-toxic (CC50 > 200 µg/ml). The IC50 values of DPPH-radical scavenging activity were 51.02 ± 0.94 and 62.91 ± 1.85 µg/ml for B. lyceum and F. indica, respectively. They also exhibited reducing power and total antioxidant capacity.The phytochemicals were identified as potent HCV NS5A inhibitors with good druglikeness and ADMET properties. Six of the docked phytochemicals exhibited higher binding scores (-17.9 to -19.2 kcal/mol) with HCV NS5A protein than the standard drug, daclatasvir (-17.2 kcal/mol). Molecular dynamics (MD) simulation confirmed the stability of two compounds, berbamine and paprafumine at 100 ns with active site of HCV NS5A protein. The identified compounds through molecular docking and MD simulation could have potential as HCV NS5A inhibitor after further validation.

Phytochemical Screening and Protective Effects of Prunus persica Seeds Extract on Carbon Tetrachloride-Induced Hepatic Injury in Rats.

BACKGROUND AND PURPOSE: Carbon tetrachloride (CCl4) is a dynamic environmental toxin released from chemical factories and its concentration in the atmosphere is accelerating at an alarming proportion. The potential presence of CCl4 in the human body causes liver injury via free radical stimulated inflammatory responses. OBJECTIVES: In this study, protective effects of hydromethanolic seeds extract of Prunus persica (PPHM) were evaluated for free radical scavenging potential in CCl4 mediated acute liver toxicity in the murine model. EXPERIMENTAL APPROACH: Followed by acute oral toxicity analysis, liver cells of Sprague-Dawley (SD) rats were treated with CCl4 and subsequently, the chemoprophylactic effect of extract (400 mg/Kg dose) was evaluated using in vivo studies including, silymarin as the positive control. Biochemical parameters, staining (hematoxylin and eosin (H & E) and Masson's Trichome) and quantitative gene expression analysis via real-time PCR were used to evaluate hepatic damage control. RESULTS: The results illustrated that PPHM extract exhibit strong anti-oxidant activity, comparable to the positive control, gallic acid. Research study results also demonstrated that the extract treatment at 400 mg/Kg concentration is highly effective in protecting liver damage due to CCl4 exposure. Mechanistic investigations indicated that the therapeutic action of PPHM was correlated with the increase in Nrf2, NQO-1 and decrease in collagen III mRNA genes expression compared to CCl4 treated group. CONCLUSIONS AND IMPLICATIONS: Accordingly, our research study indicated that PPHM alleviated CCl4-mediated oxidative stress through Nrf2/NQO-1 pathway, thereby protecting liver damage against environmental toxins. Our findings provide supportive evidence to suggest PPHM as a novel nontoxic hepatoprotective agent.

Ficus benghalensis as Potential Inhibitor of 5α-Reductase for Hair Growth Promotion: In Vitro, In Silico, and In Vivo Evaluation.

The screening of hair follicles, dermal papilla cells, and keratinocytes through in vitro, in vivo, and histology has previously been reported to combat alopecia. Ficus benghalensis has been used conventionally to cure skin and hair disorders, although its effect on 5α-reductase II is still unknown. Currently, we aim to analyze the phytotherapeutic impact of F. benghalensis leaf extracts (FBLEs) for promoting hair growth in rabbits along with in vitro inhibition of the steroid isozyme 5α-reductase II. The inhibition of 5α-reductase II by FBLEs was assessed by RP-HPLC, using the NADPH cofactor as the reaction initiator and Minoxin (5%) as a positive control. In silico studies were performed using AutoDock Vina to visualize the interaction between 5α-reductase II and the reported phytoconstituents present in FBLEs. Hair growth in female albino rabbits was investigated by applying an oral dose of the FBLE formulation and control drug to the skin once a day. The skin tissues were examined by histology to see hair follicles. Further, FAAS, FTIR, and antioxidants were performed to check the trace elements and secondary metabolites in the FBLEs. The results of RP-HPLC and the binding energies showed that FBLEs reduced the catalytic activity of 5α-reductase II and improved cell proliferation in rabbits. The statistical analysis (p < 0.05 or 0.01) and percentage inhibition (>70%) suggested that hydroalcoholic FBLE has more potential in increasing hair growth by elongating hair follicle's anagen phase. FAAS, FTIR, and antioxidant experiments revealed sufficient concentrations of Zn, Cu, K, and Fe, together with the presence of polyphenols and scavenging activity in FBLE. Overall, we found that FBLEs are potent in stimulating hair follicle maturation by reducing the 5α-reductase II action, so they may serve as a principal choice in de novo drug designing to treat hair loss.

In vitro phytochemical and anticancer activity of Misopates orontium L. and Dicliptera bupleuroides Nees.

In the present study phytochemical analysis and anticancer activity of Misopates orontium L. and Dicliptera bupleuroides Nees was carried out. Methanolic extracts of M. orontium and D. bupleuroides were selected for phytochemical analysis. The present analysis showed the presence of phytochemical such as carbohydrates, proteins, tannins, glycosides, alkaloids, saponins, phenols and flavonoids in M. orontium and D. bupleuroides. Anticancer assays including MTT, Alamar Blue (AB), Neutral Red (NR) and lactate dehydrogenase (LDH) were employed on whole herb methanolic extract and all other fractions of both plants to calculate the % age of cell viability and cell cytotoxicity. The percentage of cell viability was highly significant in all anticancer assays for all fractions. Therefore, ethyl acetate and aqueous fractions showed the excellent profile in evaluation of cytotoxicity in each assay. All above findings indicated that the whole herb of both selected plants have strong anticancer activity.

Discovery of Novel HCV NS5B polymerase inhibitor, 2-(3,4-dimethyl-5,5-dioxidobenzo[e]pyrazolo[4,3-c][1,2]thiazin-2(4H)-yl)-N-(2-fluorobenzyl)acetamide via molecular docking and experimental approach.

Hepatitis C Virus (HCV) is a viral infection posing a severe global threat that left untreated progresses to end-stage liver disease, including cirrhosis and hepatocellular carcinoma (HCC). Moreover, no prophylactic approach exists so far enabling its prevention. The NS5B polymerase holds special significance as the target of intervention against HCV infection. The current study kindles benzothiazine derivatives against HCV NS5B polymerase through in silico and experimental approaches. Following docking, the compound 2-(3,4-dimethyl-5,5-dioxidobenzo[e]pyrazolo[4,3-c][1,2]thiazin-2(4H)-yl)-N-(2-fluorobenzyl)acetamide was revealed to form effective binding interaction in the proposed site of HCV NS5B with a score of -10 kcal/mol and subsequently was deciphered through molecular dynamics (MD) simulation study which indicated interaction of residues TYR_382, VAL_381 and HIS_467 through hydrophobic interaction and two residues such as GLU_202 and LYS_209 contributed in the formation of water bridges. The subsequent in silico pharmacological analysis revealed its safe drug profile. The cytotoxicity activity of compound 6c indicated to be non-toxic in HepG2 cells at concentration ranges from 0.001-1.0 µmol/L with >80% cell viability and diminished expression of the HCV NS5B to 98% at the dose of 1.0 µmol/L and 90% at 0.5µmol/L. Thus the hit compound 6c might be a potent NS5B polymerase inhibitor required to be validated further through in vivo and preclinical studies.

GC-MS analysis, anticancer and anti-inflammatory activities of Saussurea hypoleuca spreng. Root.

Study has been premeditated to appraise the anticancer and anti-inflammatory activities of a native medicinal plant Saussurea hypoleuca Spreng root. Anticancer assays including MTT, Alamar Blue (AB), Neutral Red (NR) & LDH were employed on root methanolic extract (RME) and all fractions to calculate % age of cell viability and cell cytotoxicity. All fractions of plant root were tested for in vitro as well as in vivo anti-inflammatory assays by reported methods. GC-MS analysis of n-hexane: chloroform fractions in column chromatography has shown isopropyl myristate, hexadecanoic acid, 11-octadecenoic acid, Di-n-octyl phthalate, dioctyl ether, decanedioic acid, 1H-3a,7-Methanoazulene, 3,4-hexanedione and Tetracosapentaene. Percentage of cell viability in anticancer assays was significantly high in all fractions. However, whole results were momentous with ethyl acetate and aqueous fractions owning to excellent profile in evaluating cytotoxicity in each assay. COX-2 inhibition was calculated which was high in RME (68.69%), ethyl acetate (56.52%), aqueous (55.21%) and chloroform fraction (53.47%). Carrageenan and formalin models were developed on rats to investigate in vivo anti-inflammatory activity. RME (56.19%, 71.09%, 66.4%, 67.99%) and ethyl acetate (51.36%, 64.97%, 55.63% & 61.01%) produced significant % age inhibition in dose dependent manner at 200 and 400 mg/kg doses respectively. All above findings direct that plant root holds strong anticancer and anti-inflammatory activities.

Perception of breast cancer risk factors: Dysregulation of TGF-ß/miRNA axis in Pakistani females.

Breast cancer poses a serious health risk for women throughout the world. Among the Asian population, Pakistani women have the highest risk of developing breast cancer. One out of nine women is diagnosed with breast cancer in Pakistan. The etiology and the risk factor leading to breast cancer are largely unknown. In the current study the risk factors that are most pertinent to the Pakistani population, the etiology, molecular mechanisms of tumor progression, and therapeutic targets of breast cancer are studied. A correlative, cross-sectional, descriptive, and questionnaire-based study was designed to predict the risk factors in breast cancer patients. Invasive Ductal Carcinoma (90%) and grade-II tumor (73.2%) formation are more common in our patient's data set. Clinical parameters such as mean age of 47.5 years (SD ± 11.17), disturbed menstrual cycle (> 2), cousin marriages (repeated), and lactation period (< 0.5 Y) along with stress, dietary and environmental factors have an essential role in the development of breast cancer. In addition to this in silico analysis was performed to screen the miRNA regulating the TGF-beta pathway using TargetScanHuman, and correlation was depicted through Mindjet Manager. The information thus obtained was observed in breast cancer clinical samples both in peripheral blood mononuclear cells, and biopsy through quantitative real-time PCR. There was a significant dysregulation (**P>0.001) of the TGF-ß1 signaling pathway and the miRNAs (miR-29a, miR-140, and miR-148a) in patients' biopsy in grade and stage specifically, correlated with expression in blood samples. miRNAs (miR-29a and miR-140, miR-148a) can be an effective diagnostic and prognostic marker as they regulate SMAD4 and SMAD2 expression respectively in breast cancer blood and biopsy samples. Therefore, proactive therapeutic strategies can be devised considering negatively regulated cascade genes and amalgamated miRNAs to control breast cancer better.

Association of depression with dental caries and periodontal disease at a tertiary care hospital.

OBJECTIVE: To determine the association of depression with dental caries and periodontal disease. METHODS: The cross-sectional descriptive study was conducted at the Nishtar Institute of Dentistry, Multan, Pakistan, from May 7, 2018 to January 7, 2019, and comprised samples from subjects with dental caries and periodontal disease. Hospital Anxiety and Depression Scale was applied to screen the participants for the presence or absence of depression. Data was analysed using SPSS 21. RESULTS: Of the 296 participants, 125(42.2%) were males and 171(57.7%) were females. The overall mean age was 38.74±12.87 years. Depression was found in 195(65.8%) patients. Significant association of depression in patients of dental caries and periodontal disease was found with female gender, age <50 years, illiteracy, marital status, pre-existing hypertension, coronary artery disease, illicit substance addiction and psychotropic medication use (p<0.05). CONCLUSION: There was high frequency of depression among patients of dental caries and periodontal disease.

Tectona grandis leaf extract ameliorates hepatic fibrosis: Modulation of TGF- ß /Smad signaling pathway and upregulating MMP3/TIMP1 ratio.

ETHNOBOTANICAL RELEVANCE: Tectona grandis L.f (or syn: Jatus grandis (L.f.) Kuntze Revis), from family Lamiaceae, also known as Teak, is widely recognized in ayurvedic system of medicine and confer curative potential against inflammation, liver disorders, biliousness, diabetes, bronchitis, leprosy and dysentery. Its leaves are rich source of edible food colorant and reported nontoxic for liver and various organs. AIM OF STUDY: Hepatic injury progression to liver cirrhosis and cancer is a serious health issue across the world. Currently, anti-fibrotic therapeutic options are limited and expensive with no FDA approved direct anti-hepato-fibrotic drug validated in clinic. Thus, the aim of this study was to understand ameliorative effect of Tectona grandis L.f, leaves in early liver fibrosis. METHOD AND RESULTS: C57BL/6 mice suffering from CCl4 induced liver injury, were orally administered at three different doses (50, 100 & 200 mg/kg) of Tectona grandis L.f, leaf extract, thrice a week, up to 4 and 8 weeks. Anti-fibrotic effect was evaluated through animal body/liver weight measurements, serological tests (AST, ALT, GSH, MDA and LDH assays), tissue hydroxyproline content, and histochemical analysis (H&E, Masson trichrome, Sirius red and αSMA localization). Moreover, transcriptional and post-transcriptional expression of fibrosis associated biomarkers and TGF-ß/Smad cascade were analyzed. It was observed that 100 mg/kg dose optimally downregulated TGF-ß1/Smad2 with upregulation of Smad7 and regulated αSMA, Col 1, PDGF, TIMP1 and MMP3 expression, post 8 weeks of treatment. In addition, MMP3/TIMP1 ratio was upregulated to 0.7, 2.5 and 1.7 fold at 50 mg/kg, 100 mg/kg & 200 mg/kg treatments respectively, in comparison to untreated liver fibrosis models. The extract contains gallic acid, caffeic acid, sinapinic acid and myricetin when analyzed through high performance liquid chromatography. CONCLUSION: Tectona grandis L.f, leaves have potential to ameliorate liver fibrosis induced by CCl4 in mice via modulation of TGF-ß1/Smad pathway and upregulated MMP3/TIMP1 ratio.

Phytochemical analysis of Berberis lyceum methanolic extract and its antiviral activity through the restoration of MAPK signaling pathway modulated by HCV NS5A

Objective: To evaluate the antiviral activity and phytochemicals of selected plant extracts and their effect on the mitogen-activated protein kinase (MAPK) signaling pathway modulated by hepatitis C virus (HCV) nonstructural protein 5A (NS5A). Methods: A total of ten plant extracts were initially screened for their toxicities against HepG2 cells. The non-toxic plants were tested for their inhibitory effect on the expression of HCV NS5A at both mRNA and protein levels using real-time PCR and Western blotting assays, respectively. The differential expression of the genes associated with MAPK pathway in the presence of NS5A gene and plant extract was measured through real-time PCR. Subsequently, the identification of secondary metabolites was carried out by phytochemical and HPLC analysis. Results: The phytochemical profiling of Berberis lyceum revealed the presence of alkaloids, phenols, saponins, tannins, flavonoids, carbohydrates, terpenoids, steroids, and glycosides. Similarly, quercetin, myricetin, gallic acid, caffeic acid, and ferulic acid were identified through HPLC analysis. The methanolic extract of Berberis lyceum strongly inhibited HCV RNA replication with an IC50 of 11.44 μg/mL. RT-PCR and Western blotting assays showed that the extract reduced the expression of HCV NS5A in a dose-dependent manner. Berberis lyceum extract also attenuated NS5A-induced dysregulation of the MAPK signaling pathway. Conclusions: Our findings suggest that Berberis lyceum extract strongly inhibits HCV propagation by reducing HCV NS5A-induced perturbation of MAPK signaling.

Discovery of novel Hepatitis C virus inhibitor targeting multiple allosteric sites of NS5B polymerase.

HCV is a viral infection posing a severe global threat when left untreated progress to end-stage liver disease, including cirrhosis and HCC. The NS5B polymerase of HCV is the most potent target that harbors four allosteric binding sites that could interfere with the HCV infection. We present the discovery of a novel synthetic compound that harbors the potential of NS5B polymerase inhibition. All eight compounds belonging to the benzothiazine family of heterocycles displayed no cellular cytotoxicity in HepG2 cells at nontoxic dose concentration (200 µM). Subsequently, among eight compounds of the series, merely compound 5b exhibited significant inhibition of the expression of the HCV NS5B gene as compared to DMSO control in semi-quantitative PCR. Based on our western blot result, 5b at the range of 50, 100 and 200 µM induced 20, 40, and 70% inhibition of NS5B protein respectively. To estimate the binding potential, 5b was docked at respective allosteric sites followed by molecular dynamics (MD) simulations for a period of 20 ns. In addition, binding free energy calculation by MM-GB/PBSA method revealed a conserved interaction profile of residues lining the allosteric sites in agreement with the reported NS5B co-crystallized inhibitors. The presented results provide important information about a novel compound 5b which may facilitate the the discovery of novel inhibitors that tends to target multiple sites on NS5B polymerase.

Structural and Functional Analysis of Pullulanase Type 1 (PulA) from Geobacillus thermopakistaniensis.

Pullulanase type I (PulA) is a debranching enzyme that specifically cleaves α-1,6-glycosidic linkages in pullulan. Pullulan has not only diverse applications in food industry but also has immune-stimulatory effects on B and T cells, and found to enhance the production of various anti-inflammatory cytokines in human. Moreover, pullulan has been suggested as a possible anti-cancer drug delivery agent without adjuvant due to its unique structure. The process of pullulan degradation is unresolved due to imprecise pullulanase structural characteristics. Therefore, the present study aimed to understand the structural and functional characteristics of pullulanase enzyme from Geobacillus thermopakistaniensis MAS1 strain using various computational approaches. The physio-chemical topographies and secondary structure of GT_PulA were explored using ProPram, InterPro and SMART. Various tools like I-TASSER, ModRefiner, RAMPAGE, PROCHECK and MOE 2009.10 were used to construct and verify the 3D structural model. The structural elucidation confirmed the significant domains, i.e., CBM48, CBM2, and TIM barrel having catalytically active residues, and conserved region YNGWDP. CBM2 domain along with TIM barrel has a capacity to bind different ligands and proved favorable for multiple substrate catalyses. These structural properties can have a potential effect on enhancing enzymatic activity of GT_PulA enzyme.

Modification of miRNA Expression through plant extracts and compounds against breast cancer: Mechanism and translational significance.

BACKGROUND: Cancer is hyper-proliferative, multi-factorial and multi-step, heterogeneous group of molecular disorders. It is the second most reported disease after heart diseases. Breast carcinoma is the foremost death causing disease in female population worldwide. Cancer can be controlled by regulating the gene expression. Current therapeutic options are associated with severe side effects and are expensive for the people living in under-developed countries. Plant derived substances have potential application against different diseases like cancer, inflammation and viral infections. HYPOTHESIS: The mechanism of action of the medicinal plants is largely unknown. Targeting gene network and miRNA using medicinal plants could help in improving the therapeutic options against cancer. METHODS: The literature from 135 articles was reviewed by using PubMed, google scholar, Science direct to find out the plants and plant-based compounds against breast cancer and also the studies reporting their mechanistic route of action both at coding and noncoding RNA levels. RESULTS: Natural products act as selective inhibitors of the cancerous cells by targeting oncogenes and tumor suppressor genes or altering miRNA expression. Natural compounds like EGCG from tea, Genistein from fava beans, curcumin from turmeric, DIM found in cruciferous, Resveratrol a polyphenol and Quercetin a flavonoid is found in various plants have been studied for their anticancer activity. The EGCG was found to inhibit proliferative activity by modulating miR-16 and miR-21. Similarly, DIM was found to down regulate miR-92a which results to modulate NFkB and stops cancer development. Another plant-based compound Glyceollins found to upregulate miR-181c and miR-181d having role in tumor suppression. It also found to regulate miR-22, 29b and c, miR-30d, 34a and 195. Quercetin having anti-cancer activity induce the apoptosis through regulating miR-16, 26b, 34a, let-7g, 125a and miR-605 and reduce the miRNA expression like miR-146a/b, 503 and 194 which are involved in metastasis. CONCLUSION: Targeting miRNA expression using natural plant extracts can have a reverse effect on cell proliferation; turning on and off tumor-inducing and suppressing genes. It can be efficiently adopted as an adjuvant with the conventional form of therapies to increase their efficacy against cancer progression.

Terminalia arjuna gum/alginate in situ gel system with prolonged retention time for ophthalmic drug delivery.

Poor availability is the major barrier to accept the new smart gel system as a preferred ophthalmic solution for various eye problems. Smart gel system especially derived from natural source allows the rapid transition of ocular solution into gel form upon contact to tear solution. The present experimental scheme was intended to prepare and characterize a pH triggered in situ gelling system using moxifloxacin HCl (MOX-HCl). Gum was extracted from Terminalia arjuna bark resin and used as gelling agent in blend with sodium alginate. Sterilized formulations were developed and characterized for their physicochemical attributes. These were further investigated for microbiological testing and eye irritation studies. Drug loaded in situ gel was appeared as clear sol that converted into gel phase in presence of tear solution. Optimized formulation was stable, therapeutically efficacious, non-irritant and has a sustained release of the drug for twelve hours period. Instillation of MOX-HCl loaded in situ gel did not cause any type of irritation symptoms like redness, inflammation and excessive tear production in rabbits as compared to control. MOX-HCl loaded in situ gel can be appraised as a substitute for conventional eye drops for extended precorneal retention, improved corneal permeability along with better ocular bioavailability.