Differential proteomic analysis under pesticides stress and normal conditions in Bacillus cereus 2D

A potential pesticide degrading bacterial isolate (2D), showing maximum tolerance (450 mg∙L-1) for cypermethrin, fipronil, imidacloprid and sulfosulfuron was recovered from a pesticide contaminated agricultural field. The isolate degraded cypermethrin, imidacloprid, fipronil and sulfosulfuron in minimal salt medium with 94, 91, 89 and 86% respectively as revealed by high performance liquid chromatography (HPLC) and gas chromatography (GC) analysis after 15 days of incubation. Presence of cyclobutane, pyrrolidine, chloroacetic acid, formic acid and decyl ester as major intermediate metabolites of cypermethrin biodegradation was observed in gas chromatography mass spectrometry (GC-MS) analysis. Results based on 16S rDNA sequencing, and phylogenetic analysis showed maximum similarity of 2D with Bacillus cereus (Accession ID: MH341691). Stress responsive and catabolic/pesticide degrading proteins were over expressed in the presence of cypermethrin in bacteria. Enzymatic kinetics of laccase was deduced in the test isolate under normal and pesticide stress conditions which suggested that the production of enzyme was induced significantly in pesticide stress (163 μg.μL-1) as compare to normal conditions(29 μg.μL-1) while the Km value was decreased in pesticides stress condition (Km = 10.57 mM) and increases in normal condition (Km = 14.33 mM).Amplification of laccase gene showed a major band of 1200bp. The present study highlights on the potential of 2D bacterial strain i.e., high tolerance level of pesticide, effective biodegradation rate, and presence of laccase gene in bacterial strain 2D, could become a potential biological agent for large-scale treatment of mixture of pesticide (cypermethrin, fipronil, imidacloprid and sulfosulfuron) in natural environment (soil and water).

Differential analysis of pesticides biodegradation in soil using conventional and high-throughput technology

A potential pesticide degrading bacterial isolate (2D), showing maximum tolerance (450 ppm) for cypermethrin, fipronil, imidacloprid and sulfosulfuron was recovered from a pesticide contaminated agricultural field. The isolate degraded cypermethrin, imidacloprid, fipronil and sulfosulfuron in minimal salt medium with 94, 91, 89 and 86% respectively as revealed by HPLC and GC analysis after 15 days of incubation. Presence of cyclobutane, pyrrolidine, chloroacetic acid, formic acid and decyl ester as major intermediate metabolites of cypermethrin biodegradation was observed in GC-MS analysis. Results based on 16S rDNA sequencing, and phylogenetic analysis showed maximum similarity of 2D with Bacillus cereus (MH341691). Stress responsive and catabolic/ pesticide degrading proteins were over expressed in the presence of cypermethrin in bacteria. Enzyme kinetics of laccase was deduced in the test isolate under normal and pesticide stress conditions. Amplification of laccase gene showed a major band of 1200bp. Maximum copy number of 16S rDNA was seenin uncontaminated soil as compared to pesticide contaminated soil using qRT-PCR. The metagenome sequencing revealed reduction in the population of proteobacteria in contaminated soil as compared to uncontaminated soil but showed dominance of actinobacteria, firmicutes and bacteriodates in pesticide spiked soil. Presence of some new phyla like chloroflexi, planctomycetes, verrucomicrobia was observed followed by extinction of acidobacteria and crenarchaeota in spiked soil. The present study highlights on the potential of 2D bacterial strain i.e., high tolerance level of pesticide, effective biodegradation rate, and presence of laccase gene in bacterial strain 2D, could become a potential biological agent for large-scale treatment of mixture of pesticide (cypermethrin, fipronil, imidacloprid and sulfosulfuron) in natural environment (soil and water).

The potential of raman spectroscopy as a monitoring tool for thermal therapy

Laser Interstitial thermal Therapy (LITT) is a minimally invasive technique for treating localized solid tumors through heating with light. LITT is not routinely employed in a clincal setting due to difficulties in real-time monitoring of tissue heating. This work investigates the feasibility of Raman Spectroscopy (RS) to monitor thermal therapies. RS has the ability to detect changes in the seconcary structure of proteins, and may prove useful as an indicator of tissue coagulation in real-time during thermal therapy. Tissue equivalent albumen phantoms were heated in a water bath and bovine muscle samples where heated in a water bath and through laser photocoagulation. Raman spectra were acquired after heating and increases in the overall Raman intensity and shifts in major band locations were observed after heating. Correlations between Raman intensity and thermal dose were also observed. These results indicate that RS may be employable as a real-time monitoring tool for LITT.

The potential of raman spectroscopy as a monitoring tool for thermal therapy

Laser Interstitial thermal Therapy (LITT) is a minimally invasive technique for treating localized solid tumors through heating with light. LITT is not routinely employed in a clincal setting due to difficulties in real-time monitoring of tissue heating. This work investigates the feasibility of Raman Spectroscopy (RS) to monitor thermal therapies. RS has the ability to detect changes in the seconcary structure of proteins, and may prove useful as an indicator of tissue coagulation in real-time during thermal therapy. Tissue equivalent albumen phantoms were heated in a water bath and bovine muscle samples where heated in a water bath and through laser photocoagulation. Raman spectra were acquired after heating and increases in the overall Raman intensity and shifts in major band locations were observed after heating. Correlations between Raman intensity and thermal dose were also observed. These results indicate that RS may be employable as a real-time monitoring tool for LITT.

Domain Unknown Function DUF1668-Containing Genes in Multiple Lineages Are Responsible for F1 Pollen Sterility in Rice

Postzygotic reproductive isolation maintains species integrity and uniformity and contributes to speciation by restricting the free gene flow between divergent species. In this study we identify causal genes of two Mendelian factors S22A and S22B on rice chromosome 2 inducing F1 pollen sterility in hybrids between Oryza sativa japonica-type cultivar Taichung 65 (T65) and a wild relative of rice species Oryza glumaepatula. The causal gene of S22B in T65 encodes a protein containing DUF1668 and gametophytically expressed in the anthers, designated S22B_j. The O. glumaepatula allele S22B-g, allelic to S22B_j, possesses three non-synonymous substitutions and a 2-bp deletion, leading to a frameshifted translation at the S22B C-terminal region. Transcription level of S22B-j and/or S22B_g did not solely determine the fertility of pollen grains by genotypes at S22B. Western blotting of S22B found that one major band with approximately 46 kDa appeared only at the mature stage and was reduced on semi-sterile heterozygotes at S22B, implying that the 46 kDa band may associated in hybrid sterility. In addition, causal genes of S22A in T65 were found to be S22A_j1 and S22A_j3 encoding DUF1668-containing protein. The allele of a wild rice species Oryza meridionalis Ng at S22B, designated S22B_m, is a loss-of-function allele probably due to large deletion of the gene lacking DUF1668 domain and evolved from the different lineage of O. glumaepatula. Phylogenetic analysis of DUF1668 suggested that many gene duplications occurred before the divergence of current crops in Poaceae, and loss-of-function mutations of DUF1668-containing genes represent the candidate causal genetic events contributing to hybrid incompatibilities. The duplicated DUF1668-domain gene may provide genetic potential to induce hybrid incompatibility by consequent mutations after divergence.

An Abundant New Saponin-Polybromophenol Antibiotic (CU1) from Cassia fistula Bark Against Multi-Drug Resistant Bacteria Targeting RNA Polymerase

Spread of multidrug-resistant infections is a threat to human race and need for new drug development is great. Bark ethanol extract of Cassia fistula inhibited MDR bacteria isolated from Ganga River water, human and animal. On TLC, a gray colour major band ran fast (CU1; 6.6% of bark and ~30% of crude extract) which quickly purified on HPLC C18 column at 3 min. Chemical assays suggested a triterpene linked to polyphenol known as saponin. CHN Elements analysis (35.9% C; 5.5% H) did not identified nitrogen suggesting a polyphenol or glycoside. VU-Vis spectra gave high peak at below 200nm with a secondary peak at 275nm with minor hinge at 578nm indicating a fused ring with bromo-polyphenol. CU1 Mass (897 Daltons) with fragments of 515, 325, 269, 180 daltons and six halogen substitutions reflected by 82 molecular mass of DBr deviate six larger fragments. FT-IR suggested broad band at 3500-3000 cm−1 for −OH where as two strong peaks at 1552cm−1 for aromatic C=C and 1408cm−1 for phenol. Proton-NMR confirmed polymeric phenol at δ 4.86-4.91 ppm and tetratet at δ3.57-3.618 ppm with phenolic bromo-substituents. Carbon-NMR identified a strong peak at δ=23.7ppm for many C-Br and at 165ppm for a polybenzoid compound. CU1 inhibited the RNA Polymerase of E. coli (IC50=23μg/ml) and M. tuberculosis (IC50=34μg/ml) but not DNA polymerase. Gel shift assays demonstrated that CU1 drug interacted with enzyme and inhibited its binding to open promoter complex. Thus, CU1 phyto-chemical is an alternative safer and low cost drug against MDR-TB as well as other MDR pathogens.

Raman Spectroscopic Study of Valuable Idols from UNESCO World Heritage Sites in Kathmandu, Nepal

As an important step towards the conservation of valuable world heritage assets in Kathmandu, we performed Raman spectral studies on several valuable idols located in UNESCO World Heritage Sites for internal identification purposes. A spectrum of a stone idol in the Mohankali Chowk has a major band at 1093.5 cm-1 which may be identified as a C-O stretching vibration within the carbonate groups of CaCO3. The Raman spectra of a bull situated in the same Chowk has two major bands at the wavenumbers of 1812.7 and 3552.4 cm-1 which are assigned as combined vibrational modes of CO3 and hydroxyl stretching band, respectively. Similarly, the spectrum of a Shivalinga located at Pashupati Bankali has a major band at 467.7 cm-1. This band is formed by the movement of the oxygen atom in Si-O-Si, which is a symmetric stretching mode indicating the presence of crystalline silica (SiO2) in the Shivalinga. The Raman spectrum of Lord Changunarayan in Garuda located in the Changunarayan temple premises has a strong band at the wavenumber 462.6 cm-1, denoting the presence of quartz (SiO2).

Similar Allergenicity to Different Artemisia Species Is a Consequence of Highly Cross-Reactive Art v 1-Like Molecules

Background and objectives: Pollens of weeds are relevant elicitors of type I allergies. While many Artemisia species occur worldwide, allergy research so far has only focused on Artemisia vulgaris. We aimed to characterize other prevalent Artemisia species regarding their allergen profiles. Materials and Methods: Aqueous extracts of pollen from seven Artemisia species were characterized by gel electrophoresis and ELISA using sera from mugwort pollen-allergic patients (n = 11). The cDNA sequences of defensin–proline-linked proteins (DPLPs) were obtained, and purified proteins were tested in a competition ELISA, in rat basophil mediator release assays, and for activation of Jurkat T cells transduced with an Art v 1-specific TCR. IgE cross-reactivity to other allergens was evaluated using ImmunoCAP and ISAC. Results: The protein patterns of Artemisia spp. pollen extracts were similar in gel electrophoresis, with a major band at 24 kDa corresponding to DPLPs, like the previously identified Art v 1. Natural Art v 1 potently inhibited IgE binding to immobilized pollen extracts. Six novel Art v 1 homologs with high sequence identity and equivalent IgE reactivity were identified and termed Art ab 1, Art an 1, Art c 1, Art f 1, Art l 1, and Art t 1. All proteins triggered mediator release and cross-reacted at the T cell level. The Artemisia extracts contained additional IgE cross-reactive molecules from the nonspecific lipid transfer protein, pectate lyase, profilin, and polcalcin family. Conclusions: Our findings demonstrate that DPLPs in various Artemisia species have high allergenic potential. Therefore, related Artemisia species need to be considered to be allergen elicitors, especially due to the consideration of potential geographic expansion due to climatic changes.

Preliminary Assessment of the Antihypertensive and Antioxidative Activities of the Peptides from “Saba” Banana (Musa balbisiana Colla) Flesh

The crude proteins were isolated using 0.125 M Tris-HCl with 50 mM NaCl at pH 7.4. The protein content of the crude extract was determined using the Lowry assay and was found to be 4.28 mg/mL. The major band which corresponds to the major protein has an approximate molecular weight of 20 KDa. The isolated crude proteins were subjected to enzymatic digestion using pepsin, trypsin, chymotrypsin, and thermolysin for 3, 4, 12, and 24 hours. The 24-hour digest was found to have the highest percent anti-Angiotensin Converting Enzyme (ACE) activity (36.02%) while the 12-hour digest was found to have the highest anti-oxidative activity (33.14%) using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The 24-hour digest was subjected to Ultra-Performance Liquid Chromatography (UPLC) to determine the peptide fraction responsible for the ACE inhibitory activity. Three peptide fractions (PF1, PF2 and PF3) were chosen and PF2 exhibited the highest percent inhibitory activity (32.21) against ACE. PF2 was subjected to thin layer chromatography (TLC) and the possible identity is EK based on the Rf value traveled by glutamic acid (E) (0.43) and Lysine (K) (0.13). In silico analysis was done to correlate the results with the presence of putative peptides with antioxidative and antihypertensive activities. Results showed that antihypertensive peptides EK, GS, TY, FNE, FP, LKA, PT, PP, FAL and antioxidative peptides IR and VPW were found based on the sequence of the protein in “Saba” banana. The presence of the antihypertensive peptide EK was verified using thin layer chromatography (TLC).

Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

The main goal of the current study was cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand, by using two sets of specific primers contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E.coli DH5α competent cells. pPic9k –hpi, which was propagated in the positive transformant E. coli cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115 using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human insulin with the theoretical molecular weight (5.8 KDa).