Isolation, purification and structural elucidation of Mellein from endophytic fungus Lasiodiplodia theobromae strain (SJF-1) and its broad-spectrum antimicrobial and pharmacological properties.

In an on-going investigation of bioactive metabolites producing potential endophytic fungi, the strain Lasiodiplodia theobromae (SJF-1) was isolated from a medicinal plant Syzygium cumini. The cultural, morphological and molecular identification was done with the SJF-1 strain. The obtained gene sequence was deposited in NCBI with accession number MG 938644. The methanolic extract of SJF-1 strain possessed one major bioactive fraction, and it was purified by column chromatography. Further, it was identified as Mellein by various spectroscopic studies (1 H, 13 C, DEPT-135°, FT-IR, ESI-HR-MS and 2D NMR). Biologically, Mellein showed potent anti-Xanthomonas activity with minimum inhibitory concentration (MIC) values ranging from 1·9 to 62·5 µg ml-1 against 11 Xanthomonas strains, a broad-spectrum antimicrobial activity with MIC 7·8-31·25 µg ml-1 and 1·9-31·25 µg ml-1 towards both bacterial and fungal strains, respectively. The scanning electron microscope analysis proved the antimicrobial efficacy of a Mellein by rupturing the cell walls of Xanthomonas sp. Molecular docking studies further supported that the Mellein showed good binding interactions with the proteins of Xanthomonas sp. to reduce pathogenicity. Further, in silico pharmacological studies showed that this metabolite exhibited high gastrointestinal absorption properties and promising oral drug bioavailability. We report, anti-Xanthomonas, in silico docking and pharmacological studies of Mellein from (SJF-1) strain for the first time.

Synthesis and biological evaluation of Schizandrin derivatives as tubulin polymerization inhibitors.

A series of oxime ester-derivatives were prepared by utilizing the schizandrin (1), a major compound isolated from Schisandra grandiflora, which is deployed in different traditional system of medicine. The in vitro antiproliferative activities of the synthesized compounds were assessed against a selected panel of human cancer cell lines (A549, RKO P3, DU145 and Hela) and normal cell (HEK293). Several of these derivatives were found more potent in comparison to parent compound, schizandrin (1). Particularly, 4a and 4b demonstrated potent activity against DU-145 and RKOP3 cell lines with IC50 values of 3.42 µM and 3.35 µM respectively. To characterize the molecular mechanisms involved in antitumoral activity, these two compounds, 4a and 4b were selected for further studies. Cell cycle analysis revealed that both the compounds were able to induce apoptosis and cell cycle arrest at G0/G1 phase. To know the extent of apoptosis in DU145 and RKOP3 cell lines, Annexin V-FITC were performed. Moreover, the tubulin polymerization assay indicated that 4a and 4b exhibits potent inhibitory effect on the tubulin assembly. Molecular docking studies and competitive binding assay also indicated that 4a and 4b effectively bind at the colchicine binding site of the tubulin.

Punica granatum as a salutiferous superfruit in the treatment of oral candidiasis - An in-vitro study.

CONTEXT: The rise in the incidence of clinical resistance to antifungal therapy and failure to respond in recent years underscores the need for a time-honored approach to treat the disease using natural drugs instead of synthetic drugs, which have lesser adverse effects and good patient response. Punica granatum (pomegranate) is a fruit that has admirable medicinal value. AIMS: The study aimed to evaluate the in vitro antifungal efficacy of P. granatum peel extract against oral Candida compared with clotrimazole. SETTINGS AND DESIGN: The study design involves an in-vitro study. SUBJECTS AND METHODS: Saliva from candidiasis patients was inoculated and cultured on 60 separate Sabouraud dextrose agar plates and incubated at 37°C for 48 h from which Candida species were collected. Agar well-diffusion method was followed. Different concentrations of P. granatum peel extracts, ethanol solvent (control) and standard clotrimazole were added into the wells and allowed to diffuse at room temperature for 2 h. The plates were incubated at 37°C for 48 h. The antifungal potential of test compounds was determined based on the mean diameter of the zone of inhibition around the well in millimeters. STATISTICAL ANALYSIS USED: Statistical analysis was performed using IBM software SPSS version 20 at one-way ANOVA. RESULTS: Antifungal efficacies of P. granatum peel extract and clotrimazole were statistically significant, and there was an increase in inhibitory efficacy with an increase in concentration. Minimum inhibitory concentration of P. granatum peel extract approximated with that of clotrimazole. CONCLUSIONS: The results of this study indicate that P. granatum peel extract can be used as an effective natural substitute for synthetic antifungal agents.

A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies.

Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 106. Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity.

A humanized monoclonal antibody targeting secreted anterior gradient 2 effectively inhibits the xenograft tumor growth.

Anterior Gradient 2 (AGR2) is a potential anti-tumor target and we previously reported a murine antibody 18A4 with specific binding to AGR2. However, humanization is a must to overcome immunogenicity before considering for clinical use and optimized vectors for mammalian expression are also necessary for following industrialized manufacture. Here, we describe an anti-tumor humanized antibody blocking secreted AGR2 activity. We employed the CDR grafting technique and deimmunization analysis to construct humanized antibody variants of 18A4, and 18A4Hu I was selected as the best humanization candidate, characterized by physical and chemical property comparison. Mouse xenograft study showed that 18A4Hu I could effectively inhibit the xenograft tumor growth, antibody blocking epitope analysis using AGR2 mutants indicated that the inhibition activity of 18A4Hu I is exerted probably through blocking the AGR2 functions which rely on the amino acid sites of E60-H76 and A86-E153. What's more, in this report, we also describe a pHAb-FAST vector system which is specifically designed for humanized antibody mammalian expression vector fast construction. With pHAb-FAST system, expression vector of 18A4Hu I could be quickly constructed only through twice overlapping PCR reactions. To our knowledge, AGR2-targeted 18A4Hu I is a promising humanized anti-tumor drug candidate, and pHAb-FAST system is a useful optimized mammalian expression vector construction tool. Our findings are supposed to accelerate the development of antibody-based cancer therapy.

Self healing hemophilic pseudotumor of the mandible in a 5-year-old boy, an interesting and rare finding: Case report and review.

Hemophilic pseudotumor (PT) is a very rare complication of hemophilia consisting of a chronic, encapsulated, hemorrhagic fluid collection occurring both in the soft tissues and/or bone. Radiological features of osseous hemophilic PT are nonspecific and mimic several other benign or malignant bone tumors or infectious processes. Although the diagnosis is usually made on the location of the lesion and by the knowledge of the underlying disease, the radiologist should be aware of the imaging characteristics, in order to avoid misinterpretation as a malignant tumor, as biopsy of these lesions is contraindicated.

Phytochemical investigation of sesquiterpenes from the fruits of Schisandra chinensis and their cytotoxic activity.

Phytochemical investigation of ethanolic extract from the fruits of Schisandra chinensis led to the isolation of four new sesquiterpenes (1-4); their structures were determined by a combination of NMR (1D and 2D) and MS spectroscopic techniques. In addition, all these isolates were screened for their cytotoxic activities against MCF-7, Caco-2, Hela, Lncap, Hep G2 and MDA-MB231 cancer cell lines. Results indicated that compounds 2 and 3 displayed potent cytotoxic activity against Caco2 cell lines with IC50 values of 17.10 µg/mM and 16.46 µg/mM, respectively.

Association of gene polymorphism in detoxification enzymes and urinary 8-OHdG levels in traffic policemen exposed to vehicular exhaust.

CONTEXT: With rapid economic growth and massive development of transportation, the number of automobiles has greatly increased. Traffic police are the one of the vulnerable groups predominantly exposed to vehicular exhaust during traffic control. OBJECTIVE: The present study is aimed to study the relation between occupational exposure to vehicular exhaust and oxidative stress (OS) in traffic police. We investigated the levels of 8- hydroxydeoxyguanosine (8-OHdG), one of the most sensitive biomarkers for measuring OS and the association between polymorphisms in Cytochrome P450 (CYP) and Glutathione S-Transferase (GST) genes that are known to play a significant role in the activation and detoxification of xenobiotics. MATERIALS AND METHODS: 148 non smoking male traffic policemen and 135 control subjects were selected for this study. The 8-OHdG levels were analyzed by liquid chromatography with electrochemical detection method. Gene polymorphism was detected by multiplex PCR and RFLP method. RESULTS: 8-OHdG levels were found to be increased in traffic police with increase in the years of service in traffic control (p = 0.02) when compare to the controls. The results showed a significant increase in urinary 8-OHdG levels in mutated CYP1A1m1 (p < 0.007) and null GSTM1 (p < 0.01) genotypes. However the genotype frequencies of CYP1A1 m2 and GSTT1 genes did not vary in both exposed and control groups. CONCLUSION: Our study suggests that exposure to vehicular exhaust over a period of time increases oxidative stress and subsequently induces oxidative DNA damage in traffic policemen. Preventive and therapeutic strategies may be considered for traffic policemen to minimize the adverse effects due to vehicular exposure.

Timing of surgery in aortic stenosis.

Aortic valve stenosis (AVS) usually results from three distinct processes (degenerative-calcific, rheumatic, and congenital), with a final common pathway of significant aortic outflow tract obstruction. The stenotic lesion tends to progress slowly, but once symptoms develop clinical deterioration can ensue rapidly. Chest pain, dyspnea, and syncope are the most common symptoms of significant AVS. Detection of symptoms, subtle or obvious, is critical to the management of AVS because their presence portends a worse overall prognosis and is an indication for intervention. There are several special clinical scenarios that require added consideration, including individuals with concomitant coronary artery disease, the presence of a relatively small transvalvular pressure gradient in the setting of low cardiac output (so-called low-gradient AVS), and elderly with severe AVS. Surgical aortic valve replacement (AVR) is the mainstay treatment for relief of obstruction in patients with symptomatic AVS. Percutaneous balloon valvuloplasty is reserved for the small minority of patients who are not surgical candidates and is associated with a high restenosis rate. Percutaneous AVR is a new technology that is being tested in a few select centers on patients who are not operative candidates.

Idiopathic dilated cardiomyopathy: a common but mystifying cause of heart failure.

While researchers try to elucidate the origins of idiopathic dilated cardiomyopathy, clinicians continue to face the challenges of identifying and treating the causes of this condition to improve symptoms and survival. We review classification schemes for dilated cardiomyopathy and the current range of diagnostic and therapeutic options and treatment goals.