Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part 5: Pulse radiolysis studies on the antioxidant action of 7,8-diacetoxy-4-methylcoumarin.

7,8-Dihydroxy-4-methylcoumarin (1, DHMC) and 7,8-diacetoxy-4-methylcoumarin (2, DAMC) were shown to possess radical scavenging property and strongly inhibit membrane lipid peroxidation. Although free polyphenolic compounds are known to be antioxidants, the antioxidant action of the acetoxy compound DAMC was intriguing. Hence, pulse radiolysis studies were undertaken to explain the antioxidant action of DAMC. Accordingly, DAMC and DHMC were separately reacted with the system generating azide radicals and the resulting transient spectra were recorded. The spectra so obtained in both the cases demonstrated peak at 410 nm, characteristic of phenoxyl radical. The rate constants for the formation of phenoxyl radical from DHMC and DAMC were 34 x 10(8) M(-1) s(-1) and 6.2 x 10(8) M(-1) s(-1), respectively. We propose that the free radical mediated oxidation of DAMC initially produces a radical cation that loses an acetyl carbocation to yield the phenoxyl radical. It is possible to conclude that the mechanism of the antioxidant action of DAMC follows the pathway similar to that of DHMC involving the formation of a stable phenoxyl radical.     

Development and Evaluation of Simple Dot–Blot Assays for Rapid Detection of Staphylococcal Enterotoxin-A in Food

The present study was aimed to develop and evaluate dot–blot assays for rapid detection of staphylococcal enterotoxin-A (SEA) in food. Dot blots were developed in two formats, indirect and sandwich utilizing mouse monoclonal anti-SEA and rabbit polyclonal anti-SEA antibodies. In indirect dot–blot format, recombinant SEA was directly coated on NCM dot–blot strip and detection was carried out by anti-SEA antibodies. In sandwich dot–blot format, SEA was trapped between anti-SEA capture and detection antibodies. Both the dot–blot assays exhibited a sensitivity of ~48 ng ml^?1 when tested in different food matrices. The developed assays were highly specific as no cross-reactivity was detected with other classical staphylococcal enterotoxins, toxigenic bacteria and foodborne pathogens. Sensitivity and specificity of developed indirect and sandwich dot–blot assays with respect to PCR was found to be 100 and 99%, respectively. The results shows that the developed dot–blot assays can be used as rapid preliminary screening tests for detection of SEA in food or determining the toxigenic potential of staphylococci, especially in resource-limited settings.     

Diversity analysis,pathogen load and gene expression studies of mango cultivars infected by mango malformation disease

Mango (Mangifera indica L.) is known as “king of fruits” in India. More than 1000 mango varieties are currently cultivated in Indian Sub-continent. However most of the orchards of mango are infected with mango malformation disease (MMD), which every year leads to huge losses in yield of mango in range of 40 to 80?% in India. Till date there is no effective control measure against MMD. Floral Malformation, in contrast to vegetative one, is very virulent and can cause the loss of the entire crop. In the present study, six mango cultivars commonly grown in Gujarat, and all infected with various degrees of MMD were taken for studying their molecular relatedness, pathogen load and defense responsiveness via gene expression to rate whether hybrids or landrace among mango cultivars are better equipped to fight MMD. Genetic diversity analysis was performed using 30 SSR markers in order to bring out clustering pattern among the six cultivars belonging to orchards of Balisana and Prantij, Gujarat. The diversity analysis gave clues to the existence of wide genetic base among the six cultivars. Fungal load studies using Real Time PCR lead to the ranking of cultivars based on maximum and minimum infection load of pathogen. Absolute quantitation studies found that cultivars like Totapuri, Neelam and Amrapali were more resistant to MMD than highly popular cultivars like Kesar. The six mango cultivars were further quantified for pathogen responsiveness with 21 defense responsive genes using Real Time PCR. Among the 21 genes selected for the study, 11 genes were directly part of defense responsive pathways like Phenyl propanoid pathway and jasmonic acid pathway. Gene expression studies aided in ranking mango hybrid like Amrapali having better systemic acquired resistance response as 11 defense responsive genes were found upregulated in this cultivar followed by landrace Neelam which is in fact a parental line of Amrapali. If MMD remains unchecked it may lead to evolution of more virulent strains of Fusarium; propelling devastating consequences in mango cultivation. Hence mango hybrids developed via molecular and expressional screening will fasten process of establishment of resistant mango cultivars.     

Chemodiversity of Epicuticular n‐Alkanes and Morphological Traits of Natural Populations of Satureja subspicata Bartl. ex Vis. along Dinaric Alps – Ecological and Taxonomic Aspects

Morphological characters and the composition of epicuticular leaf n ‐alkanes of two Satureja subspicata Bartl . ex Vis . subspecies (subsp. liburnica ?ili? and subsp. subspicata ) from nine natural populations along Dinaric Alps range were studied. Morphological characters were chosen based on ?ili? ?s subspecies separation. Seventeen n ‐alkane homologues (C₁₉ – C₃₅) were identified using gas chromatography/mass spectrometry (GC/MS) and GC/flame ionisation detector (FID). The most abundant n ‐alkane in all populations was n ‐nonacosane (C₂₉), followed by n ‐hentriacontane (C₃₁), with the exception of Diva?a population where these two alkanes were co‐dominant. Diversity and variability of n ‐alkane patterns and morphological characters and their relation to different geographic and bioclimatic parameters, including exposure, were analysed by several statistical multivariate methods (PCA, HCA, Discriminant Analysis, Mantel test). These tests showed clear separation of subsp. liburnica from subsp. subspicata , even though population Velebit showed separation from other subsp. liburnica populations based on phytochemical characters. Mantel test showed high correlation with geographical distribution in both investigated data sets. High correlation between morphological and phytochemical characters was also established. However, exposure can influence n ‐alkane profile, suggesting precaution while taking samples from natural habitats.     

AZD1775 induces toxicity through double-stranded DNA breaks independently of chemotherapeutic agents in p53-mutated colorectal cancer cells

AZD1775 is a small molecule WEE1 inhibitor used in combination with DNA-damaging agents to cause premature mitosis and cell death in p53-mutated cancer cells. Here we sought to determine the mechanism of action of AZD1775 in combination with chemotherapeutic agents in light of recent findings that AZD1775 can cause double-stranded DNA (DS-DNA) breaks. AZD1775 significantly improved the cytotoxicity of 5-FU in a p53-mutated colorectal cancer cell line (HT29 cells), decreasing the IC₅₀ from 9.3 μM to 3.5 μM. Flow cytometry showed a significant increase in the mitotic marker pHH3 (3.4% vs. 56.2%) and DS-DNA break marker γH2AX (5.1% vs. 50.7%) for combination therapy compared with 5-FU alone. Combination therapy also increased the amount of caspase-3 dependent apoptosis compared with 5-FU alone (4% vs. 13%). The addition of exogenous nucleosides to combination therapy significantly rescued the increased DS-DNA breaks and caspase-3 dependent apoptosis almost to the levels of 5-FU monotherapy. In conclusion, AZD1775 enhances 5-FU cytotoxicity through increased DS-DNA breaks, not premature mitosis, in p53-mutated colorectal cancer cells. This finding is important for designers of future clinical trials when considering the optimal timing and duration of AZD1775 treatment.     

Development of LSPR-Based Optical Fiber Dopamine Sensor Using L-Tyrosine-Capped Silver Nanoparticles and Its Nonlinear Optical Properties

Facile synthesis of L-tyrosine-capped silver nanoparticles (Tyr-AgNPs) was carried out, and its linear and nonlinear optical properties were investigated. Further, the sensing properties of Tyr-AgNPs toward dopamine were explored. Tyr-AgNPs exhibit a decrease in fluorescence intensity while a linear increase in absorption spectrum against increase in dopamine (DA) concentration (0–50 μM) at room temperature. Tyr-AgNPs are used as the sensing material for the fabrication of fiber optic dopamine sensor. Sensitivity, selectivity, and limit of detection of the sensor are evaluated. This proposed fiber optic sensor may offer sensitive and low-cost strategy for DA detection.

     

High Yield Preparation of Functionally Active Catalytic-Translocation Domain Module of Botulinum Neurotoxin Type A That Exhibits Uniquely Different Enzyme Kinetics

Botulinum neurotoxins (BoNTs) are the most toxic proteins known to cause flaccid muscle paralysis as a result of inhibition of neurotransmitter release from peripheral cholinergic synapses. BoNT type A (BoNT/A) is a 150 kDa protein consisting of two major subunits: light chain (LC) and heavy chain (HC). The LC is required for the catalytic activity of neurotoxin, whereas the C and N terminal domains of the HC are required for cell binding, and translocation of LC across the endosome membranes, respectively. To better understand the structural and functional aspects of BoNT/A intoxication we report here the development of high yield Escherichia coli expression system (2–20-fold higher yield than the value reported in the literature) for the production of recombinant light chain-translocation domain (rLC-TD/A) module of BoNT/A which is catalytically active and translocation competent. The open reading frame of rLC-TD/A was PCR amplified from deactivated recombinant BoNT/A gene (a non-select agent reagent), and was cloned using pET45b (+) vector to express in E. coli cells. The purification procedure included a sequential order of affinity chromatography, trypsinization, and anion exchange column chromatography. We were able to purify?>?95% pure, catalytically active and structurally well-folded protein. Comparison of enzyme kinetics of purified LC-TD/A to full-length toxin and recombinant light chain A suggest that the affinity for the substrate is in between endopeptidase domain and botulinum toxin. The potential application of the purified protein has been discussed in toxicity and translocation assays.     

Development and application of anticancer fluorescent CdS nanoparticles enriched Lactobacillus bacteria as therapeutic microbots for human breast carcinoma

Applied Microbiology and Biotechnology - Applications of probiotic bacteria and nanoparticles (NPs) as therapeutic agents have great importance. This study demonstrates a combinatorial approach of...