Identification of In-Vitro Red Fluorescent Protein with Antipathogenic Activity from the Midgut of the Silkworm (Bombyx Mori L.).

BACKGROUND: The midgut of silkworm (Bombyx mori L.) plays an important role as a natural barrier and source of innate immunity. We had purified the novel red fluorescent protein (RFP) from the midgut of the silkworm Bombyx mori L. and bioassay studies confirmed RFPs possess antiviral, antifungal and antibacterial properties. N-terminal sequence of RFP analysis predicted chbp gene and it belongs to lipocalin gene family and is known to involve in anti-pathogenic activities. OBJECTIVE: The main objective of this study was to purify RFP from the midgut of Kolar Gold silkworm and confirm its antimicrobial activity. METHODS: For isolation of RFP, midgut juice was collected by brief exposure to chloroform vapours to fifth instar Kolar Gold silkworm larvae. Juice was purified by 40 % ammonium sulfate precipitation and purified by gel filtration chromatography (GFC) and fractions with fluorescence red under Ultra violet (UV) were collected. Molecular weight and purity of RFP was identified using PAGE, MALDI-TOF and HPLC. Antimicrobial property of purified RFP against BmNPV, Escherichia coli, Klebsiella pneumonia, Bacillus subtilis and Phytophthora meadii was performed. N-terminal sequencing of RFP was performed using Edman degradation method. Using ten amino acid sequence, using default parameter BLAST search was performerd. From the fifth day old fifth instar silkworm midgut mRNA was isolated and cDNA was synthesized using oligo-dt primer and amplification of ChBP gene was carried out by using cDNA as the template and ChBP gene specific primers. chbp protein sequence as a input built the homology model by using SWISS-MODEL. RESULTS: RFP was purified by 40 % ammonium sulfate precipitation and gel filtration chromatography (GFC) and fractions with fluorescence red under Ultra violet (UV) were collected and SDS - PAGE revealed a size of 40 kDa. RFP purified by GFC was further reconfirmed by HPLC with a single peak with a retention time of 8.755 min. MALDI-TOF produced a peak at a molecular mass of 40 kDa. RFP from the midgut juice showed antiviral activity against the silkworm virus BmNPV, antibacterial activity against Escherichia coli, Klebsiella pneumonia, Bacillus subtilis and Phytophthora meadii. N-terminal sequencing of RFP by Edman degradation method sequenced TQTIETDYWV amino acids and BLAST analysis predicted the Chlorophyllide-a Binding Protein (chbp) with B. mori. PCR product was sequenced and obtained 911bp nucleotides encoding 302 amino acid residues and deposited with the accession number KX186723 in NCBI. Sequence analysis revealed Chbp belongs to lipocalin gene family and known to involve in antiviral, antifungal and anti-bacterial properties. Chbp gene homology model was predicted using crystal structure of insecticyanin A from the tobacco hornworm as a template. CONCLUSION: Our results indicated RFP present in midgut juice of 5th instar larvae of kolar gold silkworm. We have purified novel RFP with molecular mass of 40 kDa and showed its antipathogenic activities. Chbp gene synthesises RFP and further it could be utilized for agriculture and pharmaceutical industry.

Detection of Quorum Sensing Molecules and Biofilm Formation in Ralstonia solanacearum.

Many bacteria use small diffusible signaling molecules to communicate each other termed as quorum sensing (QS). Most Gram-negative bacteria use acyl homoserine lactone (AHL) as QS signal molecules. Using these signaling molecules, bacteria are able to express specific genes in response to population density. This work aimed to detect the production of QS signal molecules and biofilm formation in Ralstonia solanacearum isolated from various diseased tomato plants with symptoms of bacterial wilt. A total of 30 R. solanacearum strains were investigated for the production of QS signal molecules using Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1 (pZLR4) biosensor systems. All 30 bacterial isolates from various bacterial wilt-affected tomato plants produced AHL molecules that induced the biosensor. The microtiter plate assay demonstrated that of the 30 bacterial isolates, 60 % formed biofilm, among which four isolates exhibited a higher degree of biofilm formation. The biofilm-inducing factor was purified from these four culture supernatants. The structure of the responsible molecule was solved using nuclear magnetic resonance and mass spectroscopy and was determined to be 2-hydroxy-4-((methylamino)(phenyl)methyl) cyclopentanone (HMCP), which was confirmed by chemical synthesis and NMR. The Confocal laser scanning microscopic analysis showed well-developed biofilm architecture of bacteria when treated with HMCP. The knowledge we obtained from this study will be useful for further researcher on the role of HMCP molecule in biofilm formation.

Phytochemically Functionalized Cu and Ag Nanoparticles Embedded in MWCNTs for Enhanced Antimicrobial and Anticancer Properties.

Nanomedicine is an emerging field concerned with the use of precision engineered nanomaterials, which leads to the development of novel remedial and diagnostic modalities for human use. In this study, Cu(NO3)2 and AgNO3 precursors were reduced to copper nanoparticles (CuNPs) and silver nanoparticles (AgNPs) using Terminalia arjuna bark extracts under microwave irradiation in the presence of well-dispersed multi-walled carbon nanotubes (MWCNTs) in aqueous medium. The formation of CuNPs or AgNPs and their functionalization with MWCNTs via bioactive molecules of plant extract were evidenced from UV-Vis spectra, XRD, FTIR, FESEM, EDX, and TEM images. The phytochemically functionalized Cu-MWCNTs and Ag-MWCNTs nanomaterials showed enhanced biocide activity, and the inhibitory activity for bacteria was higher than that of fungus. Furthermore, these biohybrid nanomaterials are non-toxic to normal epithelial cells (Vero), whereas they are highly toxic for tested human cancer cells of MDA-MB-231, HeLa, SiHa, and Hep-G2. The cell viability was found to decrease with the increasing dose from 10 to 50 µg mL-1, as well as incubation time from 24 to 72 h. For instance, the cell viability was found to be ~91 % for normal Vero cells and ~76 % for cancer cells for lower dose of 10 µg mL-1.

Stereoselective synthesis of oxa- and aza-angular triquinanes using tandem radical cyclization to vinylogous carbonates and carbamates.

Tandem radical cyclization to vinylogous carbonates and carbamates is developed for a new, highly stereoselective synthesis of heterocyclic angular triquinanes. The strategy is also useful to gain access to oxa- and azatriquinanes, which incorporate the spiroindoline moiety. The method is further extended to the synthesis of lactone-bearing as well as uracil-fused angular triquinanes.

Stereoselective synthesis of hexahydro-3-methyl-1-arylchromeno[3,4-b]pyrrole and its annulated heterocycles as potent antimicrobial agents for human pathogens.

Synthesis of a series of novel hexahydrochromenopyrrole analogues has been accomplished through an intramolecular 1,3-dipolar cycloaddition (1,3-DC reaction) of azomethine ylides, generated by the aldehyde induced decarboxylation of secondary amino acids. These compounds were screened for antibacterial and antifungal activities against six human pathogenic bacteria and three human pathogenic fungi and found to have good antimicrobial properties against most of the microorganisms.

Weaning success among ventilator-dependent patients in a rehabilitation facility.

OBJECTIVES: To determine outcomes of difficult-to-wean, ventilator-dependent patients transferred from intensive care units to rehabilitation hospitals and to determine predictors of weaning success in such patients. DESIGN: A retrospective cohort study. SETTING: A rehabilitation facility. PARTICIPANTS: One hundred forty-five difficult-to-wean patients (55.2% men; 83.4% white; mean age +/- standard deviation, 65.8 +/- 16.4y) transferred to a rehabilitation facility between July 1994 and June 1996. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Demographic and clinical data, including variables identified previously as predictive of weaning success among highly selected populations. RESULTS: Patients' Gillespie categories (reason for ventilator dependency) included "other medical conditions" (eg, pneumonia, neurologic) in 42.1% of the cases, postoperative in 24.8%, previous lung disease (eg, chronic obstructive pulmonary disease, interstitial lung disease) in 15.2%, trauma in 11.7%, respiratory failure with multisystem failure in 3.4%, and uncomplicated acute lung injury (acute respiratory distress syndrome) in 2.8%. Of 145 patients, 50.3% were completely weaned, 4.8% were partially weaned, and 44.8% remained ventilator dependent. In a stepwise multivariable logistic regression analysis, significant predictors of weaning success included white race (odds ratio [OR] = 3.4), serum albumin level (OR = 2.1g/dL), and blood urea nitrogen (BUN) level (OR = .97mg/dL); in addition, compared with postoperative patients, patients with "other medical conditions" (OR = .15) or previous lung disease (OR = .08) were less likely to be weaned (area under receiver operating characteristic curve = .76). Among 31 long-term survivors who were interviewed at least 6 months after discharge from the rehabilitation facility, 58.1% rated their health-related quality of life as good or better. CONCLUSIONS: Half of the patients admitted to a rehabilitation facility were weaned from their ventilators. Predictors of weaning success included race, BUN level, albumin level, and reason for ventilator dependency.