Purification and characterization of a new cold active lipase, EnL A from Emericella nidulans NFCCI 3643

A mesophilic fungi producing an extracellular cold-active lipase was isolated from the soil samples of palm oil mill effluent dump sites, Pedavegi, West Godavari Dist, A.P. India and was identified as Emericella nidulans. The enzyme was purified by ammonium sulfate fractionation followed by hydrophobic interaction chromatography using phenyl sepharose. The enzyme was 35 fold pure compared to crude with a specific activity of 1494.51 U/mg. SDS PAGE analysis revealed that the protein is monomeric with a MW of ˜54 kDa and zymogram analysis showed that the purified protein was active. Characterization studies revealed that the temperature optimum was at 30°C and an optimum pH of 5. The Km and Vmax values were found to be 0.61 mM and 322.58 mM/min.mg, respectively. Sequencing of the purified protein by MALDI TOF-MS analysis followed by BLAST P analysis indicated that the protein is a putative secretary lipase from E. nidulans. Search of lipase engineering data base (LED) revealed that this protein belongs to a newly introduced super family of Candida antarctica lipase A like and to the homologous family of Aspergillus lipase like.Key words: Cold active lipase, Emericella nidulans, hydrophobic interaction chromatography, Candida antarctica lipase A like

The Molecular Switch of Telomere Phages: High Binding Specificity of the PY54 Cro Lytic Repressor to a Single Operator Site

Temperate bacteriophages possess a molecular switch, which regulates the lytic and lysogenic growth. The genomes of the temperate telomere phages N15, PY54 and ɸKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator. The roles of these products are thought to be similar to those of the lambda proteins CI, Cro and Q, respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ɸKO2 are also reminiscent of lambda-like phages. By contrast, in silico analyses revealed the presence of only one operator (O$_{\rm{R}}$3) in PY54. The purified PY54 Cro protein was used for EMSA studies demonstrating that it exclusively binds to a 16-bp palindromic site (O$_{\rm{R}}$3) upstream of the prophage repressor gene. The O$_{\rm{R}}$3 operator sequences of PY54 and ɸKO2/N15 only differ by their peripheral base pairs, which are responsible for Cro specificity. PY54 cI and cro transcription is regulated by highly active promoters initiating the synthesis of a homogenious species of leaderless mRNA. The location of the PY54 Cro binding site and of the identified promoters suggests that the lytic repressor suppresses cI transcription but not its own synthesis. The results indicate an unexpected diversity of the growth regulation mechanisms in lambda-related phages

Effects of chlorantraniliprole and thiamethoxam rice seed treatments on egg numbers and first instar survival of Lissorhoptrus oryzophilus (Coleoptera: Curculionidae)

Effects of treatment of rice seeds with an anthranilic diamide, chlorantraniliprole, and a neonicotinoid, thiamethoxam, on egg laying and first instar survival in rice water weevil, Lissorhoptrus oryzophilus Kuschel, were examined under greenhouse conditions. Exposure of adult weevils to rice (6-7 leaf stage) grown from seeds treated with chlorantraniliprole and thiamethoxam resulted in reduction in numbers of eggs and first instars. The low egg numbers by adults exposed to chlorantraniliprole-treated plants was confirmed as a sublethal effect on adults: adult survival was not impacted after 4 d of feeding on foliage from chlorantraniliprole-treated plants but the number of eggs laid by these weevils was reduced when released on untreated plants. Furthermore, a comparison of first instar emergence from chlorantraniliprole-treated plants and from untreated plants infested with weevils previously exposed to this chemical suggested that chlorantraniliprole was also reducing egg or first instar survival. In contrast, adults that fed on foliage from thiamethoxam-treated plants showed increased mortality. Possible sublethal effects of thiamethoxam on the number of eggs laid by adults were investigated by infesting untreated plants with weevils that survived exposure to thiamethoxam via foliar feeding (7 μg active ingredient / seed). Prior exposure to thiamethoxam through adult feeding reduced egg numbers. However, potential larvicidal or ovicidal effects of thiamethoxam seed treatments could not be detected in this study because of low first instar emergence from both thiamethoxam-treated plants and from untreated plants infested with weevils previously exposed to this chemical. These experiments revealed that the two seed treatments accomplish weevil control in different ways. © 2013 Entomological Society of America

Optical characterization of porcine tissues from various organs in the 650-1100 nm range using time-domain diffuse spectroscopy

We present a systematic characterization of the optical properties (µa and µs') of nine representative ex vivo porcine tissues over a broadband spectrum (650-1100 nm). We applied time-resolved diffuse optical spectroscopy measurements for recovering the optical properties of porcine tissues depicting a realistic representation of the tissue heterogeneity and morphology likely to be found in different ex vivo tissues. The results demonstrate a large spectral and inter-tissue variation of optical properties. The data can be exploited for planning or simulating ex vivo experiments with various biophotonics techniques, or even to construct artificial structures mimicking specific pathologies exploiting the wide assortment in optical properties.This article is freely available via Open Access. Click on the Publisher URL to access it via the publisher's site.Horizon 2020 Framework Programme10.13039/100010661 (654148;, 675332); Engineering and Physical Sciences Research Council10.13039/501100000266 (EP/R020965/1).published version, accepted versio

Unsaturated fatty acids are inhibitors of bacterial conjugation

This report describes a high-throughput assay to identify substances that reduce the frequency of conjugation in Gram-negative bacteria. Bacterial conjugation is largely responsible for the spread of multiple antibiotic resistances in human pathogens. Conjugation inhibitors may provide a means to control the spread of antibiotic resistance. An automated conjugation assay was developed that used plasmid R388 and a laboratory strain of Escherichia coli as a model system, and bioluminescence as a reporter for conjugation activity. Frequencies of conjugation could be measured continuously in real time by the amount of light produced, and thus the effects of inhibitory compounds could be determined quantitatively. A control assay, run in parallel, allowed elimination of compounds affecting cell growth, plasmid stability or gene expression. The automated conjugation assay was used to screen a database of more than 12 000 microbial extracts known to contain a wide variety of bioactive compounds (the NatChem library). The initial hit rate was 1·4 %. From these, 48 extracts containing active compounds and representing a variety of organisms and extraction conditions were subjected to fractionation (24 fractions per extract). The 52 most active fractions were subjected to a secondary analysis to determine the range of plasmid inhibition. Plasmids R388, R1 and RP4 were used as representatives of a variety of plasmid transfer systems. Only one fraction (of complex composition) affected transfer of all three plasmids, while four other fractions were active against two of them. Two separate compounds were identified from these fractions: linoleic acid and dehydrocrepenynic acid. Downstream analysis showed that the chemical class of unsaturated fatty acids act as true inhibitors of conjugation

Non-invasive investigation of adipose tissue by time domain diffuse optical spectroscopy

The human abdominal region is very heterogeneous and stratified with subcutaneous adipose tissue (SAT) being one of the primary layers. Monitoring this tissue is crucial for diagnostic purposes and to estimate the effects of interventions like caloric restriction or bariatric surgery. However, the layered nature of the abdomen poses a major problem in monitoring the SAT in a non-invasive way by diffuse optics. In this work, we examine the possibility of using multi-distance broadband time domain diffuse optical spectroscopy to assess the human abdomen non-invasively. Broadband absorption and reduced scattering spectra from 600 to 1100 nm were acquired at 1, 2 and 3 cm source-detector distances on ten healthy adult male volunteers, and then analyzed using a homogeneous model as an initial step to understand the origin of the detected signal and how tissue should be modeled to derive quantitative information. The results exhibit a clear influence of the layered nature on the estimated optical properties. Clearly, the underlying muscle makes a relevant contribution in the spectra measured at the largest source-detector distance for thinner subjects related to blood and water absorption. More unexpectedly, also the thin superficial skin layer yields a direct contamination, leading to higher water content and steeper reduced scattering spectra at the shortest distance, as confirmed also by simulations. In conclusion, provided that data analysis properly accounts for the complex tissue structure, diffuse optics may offer great potential for the continuous non-invasive monitoring of abdominal fat

Exploitation of Wild Cicer reticulatum Germplasm for Resistance to Helicoverpa armigera

In the absence of high levels of resistance to Helicoverpa armigera (Hübner) in the cultivated germplasm of chickpea, we evaluated accessions of Cicer spp. mostly Cicer reticulatum Ladzinsky, for resistance to this important pest. Under multichoice conditions in the field, 10 accessions showed lower leaf damage and lower numbers of eggs, larvae, or both of H. armigera. Of these, IG 69960, IG 72934, and IG 72936 showed significantly lower leaf feeding than the cultivated genotypes or other accessions at the vegetative and reproductive stages. Larval weight was lower or comparable with that on C. bijugum (IG 70019) and C. judaicum (IG 70032) in C. reticulatum accessions IG 72933, IG 72934, IG 72936, and IG 72953 at the seedling stage and on IG 69960 and IG 72934 at the flowering stage. The accessions showing resistance to H. armigera in the field and laboratory conditions were placed in different groups, indicating the presence of diversity in C. reticulatum accessions for resistance to this pest. Less than seven larvae survived on IG 70020, IG 72940, IG 72948, and IG 72949, and IG 72964 compared with 12 on ICC 506. Larval and total developmental periods were prolonged by 6-15 and 3-8 d, respectively, on C. reticultatum accessions compared with those on ICCC 37. Less than five larvae pupated on the C. reticulatum accessions (except IG 72958 and ICC 17163) compared with 11 in ICCC 37. Accessions showing lower leaf feeding and adverse effects on the survival and development can be used in increasing the levels and diversifying the basis of resistance to H. armigera in chickpea

Antibiosis mechanism of resistance to pod borer, Helicoverpa armigera in wild relatives of chickpea.

The pod borer, Helicoverpa armigera, is one of the major constraints to chickpea production worldwide. The levels of resistance to pod borer in the cultivated chickpea germplasm are moderate, and therefore, we studied the reaction of 32 accessions of wild relatives of chickpea for resistance to H. armigera under greenhouse conditions. Accessions ICC 17257, IG 70002, IG 70003, IG 70012, (Cicer bijugum), IG 69948 (C. pinnatifidum), IG 69979 (C. cuneatum), IG 70032, IG 70033, IG 70038, and IG 72931 (C. judaicum) showed lower leaf feeding, a drastic reduction in larval weight, and poor host suitability index at the vegetative and/or flowering stages of crop growth as compared to the cultivated chickpeas. Based on percentage pods damaged by 5th day (<52% pods damaged compared to 90% pods damaged in Annigeri), and percentage weight gain by the larvae (<35% weight gain compared to 366% weight gain on ICCV 2); accessions IG 69979 (C. cuneatum), IG 70003, IG 70022, IG 70016, IG 70013, IG 70012, IG 70010, IG 70001, IG 70018, and IG 70002 (C. bijugum), and IG 72953 (C. reticulatum) showed high levels of resistance to H. armigera. Larvae of H. armigera weighed <50 mg when reared on C. pinnatifidum (IG6 9948 and IG 70039), and C. judaicum (IG 72931) compared to 301.95 mg on C. arietinum (ICCC 37 - the cultivated chickpea). Larval weights on many accessions of the wild relatives of chickpea were much lower than those on the cultivated chickpeas, indicating the existence of different mechanisms of resistance to H. armigera. There was no pupation and adult emergence when the larvae were reared on accessions of C. pinnatifidum (IG 69948 and IG 70039), and C. judaicum (IG 69980, IG 70032, IG 70033 and IG 72931). The wild relatives of chickpea showing high levels of antibiosis to H. armigera can be used to introgress diverse resistance genes into cultivated chickpea to increase the levels and diversify the basis of resistance to this insec

Time-domain NIRS system based on supercontinuum light source and multi-wavelength detection: Validation for tissue oxygenation studies

We present and validate a multi-wavelength time-domain near-infrared spectroscopy (TD-NIRS) system that avoids switching wavelengths and instead exploits the full capability of a supercontinuum light source by emitting and acquiring signals for the whole chosen range of wavelengths. The system was designed for muscle and brain oxygenation monitoring in a clinical environment. A pulsed supercontinuum laser emits broadband light and each of two detection modules acquires the distributions of times of flight of photons (DTOFs) for 16 spectral channels (used width 12.5 nm / channel), providing a total of 32 DTOFs at up to 3 Hz. Two emitting fibers and two detection fiber bundles allow simultaneous measurements at two positions on the tissue or at two source-detector separations. Three established protocols (BIP, MEDPHOT, and nEUROPt) were used to quantitatively assess the system's performance, including linearity, coupling, accuracy, and depth sensitivity. Measurements were performed on 32 homogeneous phantoms and two inhomogeneous phantoms (solid and liquid). Furthermore, measurements on two blood-lipid phantoms with a varied amount of blood and Intralipid provide the strongest validation for accurate tissue oximetry. The retrieved hemoglobin concentrations and oxygen saturation match well with the reference values that were obtained using a commercially available NIRS system (OxiplexTS) and a blood gas analyzer (ABL90 FLEX), except a discrepancy occurs for the lowest amount of Intralipid. In-vivo measurements on the forearm of three healthy volunteers during arterial (250 mmHg) and venous (60 mmHg) cuff occlusions provide an example of tissue monitoring during the expected hemodynamic changes that follow previously well-described physiologies. All results, including quantitative parameters, can be compared to other systems that report similar tests. Overall, the presented TD-NIRS system has an exemplary performance evaluated with state-of-the-art performance assessment methods

Evaluation of Wild Relatives of Chickpea (Cicer spp.) for Resistance to Pod Borer, Helicoverpa armigera (Hubner)

The legume pod borer, Helicoverpa armigera, is a major constraint to chickpea production worldwide. The levels of resistance in the cultivated chickpea germplasm are moderate, and therefore, we evaluated 93 accessions of annual wild relatives of chickpea in the field, and 141 accessions under greenhouse conditions for resistance to H. armigera. Under field conditions, 24 accessions showed a leaf feeding score of <2.0 compared to 6.0 to 6.5 of the Cicer reticulatum accession IG 69975. These accessions also had less than 2 eggs and/or larvae of H. armigera and <2 larvae of Spodoptera exigua per plant at the flowering/podding stages. Based on leaf feeding, larval survival, and larval weights in the detached leaf assay, 41 accessions showed low leaf feeding, reduced larval weights, and/or low larval survival. Accessions IG 69947, IG 70002, IG 70003, IG 70009, IG 70019, IG 70022, ICC 17125, IG 69979 ICC 17122, ICC 17156, IG 70006, and ICC 17187 (C. bijugum), IG 69995 and IG 70030 (C. judaicum), and IG 69988, IG 69999 IG 70021, IG 70025, and IG 70028 (C. pinnatifidum) showed low leaf feeding, low larval weights, and low host suitability index. These accessions can be exploited for introgressing resistance genes from the wild relatives into the cultivated chickpeas to increase the levels and diversify the basis of resistance to H. armigera