Simple fluorescence-based high throughput cell viability assay for filamentous fungi.

UNLABELLED: Filamentous fungi are important model organisms to understand the eukaryotic process and have been frequently exploited in research and industry. These fungi are also causative agents of serious diseases in plants and humans. Disease management strategies include in vitro susceptibility testing of the fungal pathogens to environmental conditions and antifungal agents. Conventional methods used for antifungal susceptibilities are cumbersome, time-consuming and are not suitable for a large-scale analysis. Here, we report a rapid, high throughput microplate-based fluorescence method for investigating the toxicity of antifungal and stress (osmotic, salt and oxidative) agents on Magnaporthe oryzae and compared it with agar dilution method. This bioassay is optimized for the resazurin reduction to fluorescent resorufin by the fungal hyphae. Resazurin bioassay showed inhibitory rates and IC50 values comparable to the agar dilution method and to previously reported IC50 or MICs for M. oryzae and other fungi. The present method can screen range of test agents from different chemical classes with different modes of action for antifungal activities in a simple, sensitive, time and cost effective manner. SIGNIFICANCE AND IMPACT OF THE STUDY: A simple fluorescence-based high throughput method is developed to test the effects of stress and antifungal agents on viability of filamentous fungus Magnaporthe oryzae. This resazurin fluorescence assay can detect inhibitory effects comparable to those obtained using the growth inhibition assay with added advantages of simplicity, time and cost effectiveness. This high throughput viability assay has a great potential in large-scale screening of the chemical libraries of antifungal agents, for evaluating the effects of environmental conditions and hyphal kinetic studies in mutant and natural populations of filamentous fungi.

Identification of different species of Bacillus isolated from Nisargruna Biogas Plant by FTIR, UV-Vis and NIR spectroscopy.

Definitive identification of microorganisms, including pathogenic and non-pathogenic bacteria, is extremely important for a wide variety of applications including food safety, environmental studies, bio-terrorism threats, microbial forensics, criminal investigations and above all disease diagnosis. Although extremely powerful techniques such as those based on PCR and microarrays exist, they require sophisticated laboratory facilities along with elaborate sample preparation by trained researchers. Among different spectroscopic techniques, FTIR was used in the 1980s and 90s for bacterial identification. In the present study five species of Bacillus were isolated from the aerobic predigester chamber of Nisargruna Biogas Plant (NBP) and were identified to the species level by biochemical and molecular biological (16S ribosomal DNA sequence) methods. Those organisms were further checked by solid state spectroscopic absorbance measurements using a wide range of electromagnetic radiation (wavelength 200 nm to 25,000 nm) encompassing UV, visible, near Infrared and Infrared regions. UV-Vis and NIR spectroscopy was performed on dried bacterial cell suspension on silicon wafer in specular mode while FTIR was performed on KBr pellets containing the bacterial cells. Consistent and reproducible species specific spectra were obtained and sensitivity up to a level of 1000 cells was observed in FTIR with a DTGS detector. This clearly shows the potential of solid state spectroscopic techniques for simple, easy to implement, reliable and sensitive detection of bacteria from environmental samples.

Synthesis of uniform gold nanoparticles using non-pathogenic bio-control agent: evolution of morphology from nano-spheres to triangular nanoprisms.

Green synthesis of gold nanospheres with uniform diameter and triangular nanoprisms with optically flat surface was carried out using a non-pathogenic bio-control agent Trichoderma asperellum for reduction of HAuCl(4). Kinetics of the reaction was monitored by UV-Vis absorption spectroscopy. No additional capping/complexing agent was used for stabilizing the gold nanoparticles. Evolution of morphology from pseudospherical nanoparticles to triangular nanoprisms was studied by transmission electron microscopy (TEM). It revealed that three or more pseudospheres fused to form nanoprisms of different shapes and sizes. Slow rate of reduction of HAuCl(4) by constituents of cell-free fungal extract was instrumental in producing such exotic morphologies. Isolation of gold nanotriangles from the reacting masses was achieved by differential centrifugation.

Does dementia increase risk of thrombolysis?: a case-control study.

BACKGROUND: Dementia represents a potential challenge when thrombolysis is a treatment option. In this study, we assess the impact of dementia on the rate of intracerebral hemorrhage (ICH) and hospital mortality associated with acute ischemic stroke (AIS) in patients treated with thrombolysis. METHODS: A cohort of patients with AIS was identified from the National Inpatient Sample database for the years 2000 to 2007. Vascular and degenerative types of dementia were identified by the International Classification of Diseases-9-CM codes. A matched random sample without dementia was selected from a pool of those with AIS and treated with thrombolysis. RESULTS: In this analysis, 35,557 patients with diagnosis of dementia were included; 207 (0.56%) received thrombolysis. In-hospital mortality (17.48% vs 8.63%) and ICH (5.80% vs 0.38%) were higher in the thrombolysis group (p < 0.0001) compared to those who did not receive thrombolysis. Multivariate analysis showed that thrombolysis was associated with increased hospital mortality (odds ratio [OR] 16.15; 95% confidence interval [CI] 8.54-30.53) and ICH (OR 2.80; 95% CI 1.82-4.32). Compared to a matched population of patients without dementia treated with thrombolysis (n = 621), those who had dementia and were treated with thrombolysis had similar risks of ICH (5.80% vs 4.51%, p = 0.45) and mortality (17.39% vs 14.49%, p = 0.31) rates. With thrombolysis, ICH remained a predictor of mortality for both dementia and control groups (OR 2.25; 95% CI 1.02-4.99). CONCLUSION: The administration of thrombolysis for AIS in patients with dementia was not associated with increased risk of ICH or death compared to the counterparts without dementia. ICH remained as predictor of mortality.

Chemical Characterization, Crossfeeding and Uptake Studies on Hydroxamate Siderophore of Alcaligenes faecalis.

We report the production of two types of siderophores namely catecholate and hydroxamate in modified succinic acid medium (SM) from Alcaligenes faecalis. Two fractions of siderophores were purified on amberlite XAD, major fraction was hydroxamate type having a λ(max) at 224 nm and minor fraction appeared as catecholate with a λ(max) of 264 nm. The recovery yield obtained from major and minor fractions was 297 and 50 mg ml(-1) respectively. The IEF pattern of XAD-4 purified siderophore suggested the pI value of 6.5. Cross feeding studies revealed that A. faecalis accepts heterologous as well as self (hydroxamate) siderophore in both free and iron complexed forms however; the rate of siderophore uptake was more in case of siderophores complexed to iron. Siderophore iron uptake studies indicated the differences between hydroxamate siderophore of A. faecalis and Alc E, a siderophore of Alcaligenes eutrophus.

Green synthesis of highly stabilized nanocrystalline silver particles by a non-pathogenic and agriculturally important fungus T. asperellum.

A controlled and up-scalable biosynthetic route to nanocrystalline silver particles with well-defined morphology using cell-free aqueous filtrate of a non-pathogenic and commercially viable biocontrol agent Trichoderma asperellum is being reported for the first time. A transparent solution of the cell-free filtrate of Trichoderma asperellum containing 1 mM AgNO(3) turns progressively dark brown within 5 d of incubation at 25 °C. The kinetics of the reaction was studied using UV-vis spectroscopy. An intense surface plasmon resonance band at ∼410 nm in the UV-vis spectrum clearly reveals the formation of silver nanoparticles. The size of the silver particles using TEM and XRD studies is found to be in the range 13-18 nm. These nanoparticles are found to be highly stable and even after prolonged storage for over 6 months they do not show significant aggregation. A plausible mechanism behind the formation of silver nanoparticles and their stabilization via capping has been investigated using FTIR and surface-enhanced resonance Raman spectroscopy.

Analysis of aflatoxin regulatory factors in serial transfer-induced non-aflatoxigenic Aspergillus parasiticus.

Aflatoxins (AFs) are carcinogenic secondary metabolites of Aspergillus parasiticus. In previous studies, non-toxigenic A. parasiticus sec- (for secondary metabolism negative) variants were generated through serial transfer of mycelia from their toxigenic sec+ (for secondary metabolism positive) parents for genetic and physiological analysis for understanding regulation of AF biosynthesis. Previous studies have shown no difference in the DNA sequence of aflR, a positive regulator of AF production, in the sec+ and sec- strains. In this study, AflJ, another positive regulator of AF production, laeA, a global regulator of secondary metabolism, and the intergenic region between aflR and aflJ, were analysed to determine if they play a role in establishment of the sec- phenotype. The study showed that while this sequence identity extended to the aflJ as well as the aflJ-aflR intergenic region, expression of aflR in the sec- strain was several fold lower than that observed in the sec+ strain, while aflJ expression was barely detectable in the sec- strain. Western blot analysis indicated that despite AflR protein being present in the sec- strain, no toxin production resulted. Introduction of a second copy of aflR into the sec- strain increased aflR expression, but did not restore AF production. Lastly, reverse transcription-PCR analysis revealed that laeA was expressed in both sec+ and sec- strains. These results suggest that although aflR, aflJ and laeA are necessary for AF production, they are not sufficient. We propose that the aflR and aflJ expression may be regulated by element(s) downstream from laeA or from pathways not influenced by laeA.

Adsorption-desorption of HCH and endosulfan on a soil.

Sorption - desorption of HCH (1,2,3,4,5,6 - Hexachlorocyclohexane) and Endosulfan (1,2,3,4,7,7 - Hexachlorobicyclo [2,2,1] -2 - heptene - 5,6 - bisoxy methylene sulfite) on soil was studied using the batch equilibration technique with initial concentrations for the two chemicals ranging from 0.03 to 340 microg ml(-1), which corresponds to a field application rate of 0.1 2.0 kg m(-2). Calculated slopes of the Freundlich sorption isotherms were near to 1. The Kf, Kd, and Koc values determined for endosulfan were at least an order of magnitude greater than that determined for HCH. Kf values were 209 and 8035 for HCH and Endosulfan respectively. Hysteresis was observed for desorption of both the chemicals. Studies showed that Endosulfan and HCH binding to soil was strong and desorption cannot be predicted from adsorption isotherms only.

Correct anatomical location of entry point for antegrade femoral nailing.

Closed intramedullary nailing is a well-accepted method of treatment for femoral shaft fractures. The issue of the correct entry point for antegrade nailing remains a matter of controversy, and the literature is confusing. We reviewed the opinions of 100 orthopaedic surgeons by means of questionnaires. Only four surgeons were able to identify and label their respective entry points for femoral nailing correctly, possibly because of incorrect illustration in publications or errors in terminology. Although the piriformis fossa appears to be the ideal entry point, the importance of exact localisation in the sagittal plane, centered over the axis of medullary canal, cannot be overlooked.

Regulatory elements in aflatoxin biosynthesis.

This review provides a synopsis of factors involved in the regulation of aflatoxin inAspergillus species at the molecular level. Much of the knowledge available today on the regulation of secondary metabolite production in fungi has been gleaned from studies of the aflatoxin gene cluster inA. flavus andA. parasiticus and the sterigmatocystin gene cluster inA. nidulans. Regulation of these two gene clusters is under the control of both pathway-specific transcription factors such as AflR and AflJ and global or broad-domain transcription factors such as AreA and PacC. Study of secondary metabolite (sec-) mutants inA. parasiticus first identified an association between mycotoxin production and fungal development. This linkage has been extended at the molecular level by the characterization of a G-protein/cAMP/Protein kinase A signaling pathway that regulates sporulation via the transcription factor BrlA and aflatoxin/sterigmatocystin production via AflR. Another global regulator of mycotoxin production, VeA, mediates a developmental light-response inA. nidulans andA. flavus. Though not similar to any known fungal transcriptional regulators, VeA controls aflatoxin/sterigmatocystin production via transcriptional control of AflR and it also regulates development of sexual structures such as cleistothecia inA. nidulans and sclerotia inA. flavus.

Metabolism of 14C-naphthalene in marine sediments.

Metabolism of 14C-naphthalene was studied in aerobic and anaerobic marine sediments from the Mumbai coast, India using a continuous flow-through system for 5 weeks. There was no volatilisation of naphthalene from anaerobic sediment. Naphthalene underwent more extensive mineralization in aerobic sediment (31.6% of the applied activity) than in anaerobic sediment (5% of the applied activity). No metabolite of naphthalene was present in sediments at the end of the incubation period.

Uptake of 14C-chlorpyrifos by clams.

The uptake of 14C-chlorpyrifos by clams (Katalysia opima) was studied to determine the bioaccumulation potential over a period of five days. Chlorpyrifos was applied to a model ecosystem in beakers at the rate of 3 mg l(-1) of seawater. Clams showed a maximum uptake of 14C-chlorpyrifos in the first 8 hours of exposure. Subsequently these residues decreased significantly and at the end of 5 days about 1.5% of the applied activity could be recovered from the clam samples. The half-life of chlorpyrifos in this marine water system was about a day. However, after 5 days about 28% of the applied 14C-activity was present in water. This may be significant and could possibly play a role in finding the residue of this insecticide in water bodies. Clams brought about rapid degradation of chlorpyrifos in the first 48 hours. The stabilised residues in water were reflected later in clams.

Degradation of 14C-carbofuran in soil using a continuous flow system.

14C-carbofuran underwent considerable mineralization (approximately 30% of the applied activity) in Vertisol soil under moist and flooded conditions during 60 days incubation. Bound residues were formed under both the conditions, the extent being more in moist soils (approximately 55% of the applied activity) than under flooded conditions (approximately 41% of the applied activity). 3-Keto carbofuran was the only significant metabolite observed under flooded conditions.

Monitoring of organochlorine pesticide residues in the Indian marine environment.

Organochlorine pesticide residues in sediment and fish samples collected from the east and west coasts of India are presented. HCH isomers and DDT and its metabolites are the predominantly identified compounds in most of the samples. Despite the higher quantity of consumption, HCH and DDT levels in fish in India were lower than those in temperate countries suggesting a lower accumulation in tropical fish, which could be due to rapid volatilization and degradation of these insecticides in the tropical environment. The predominance of alpha- and beta-HCH reflect the use of technical grade HCH in India. The high temperature in the tropics also enhances the elimination rate of chemicals in fish, as the biological half-lives of semivolatile compounds such as DDT are short at high temperature.

Bioremediation of DDT in soil by genetically improved strains of soil fungus Fusarium solani.

Bioremediation of DDT in soil by genetically improved recombinants of the soil fungus Fusarium solani was studied. The parent strains were isolated from soil enriched with DDD or DDE (immediate anaerobic and aerobic degradation products of DDT), as further degradation of these products are slow processes compared to the parent compound. These naturally occurring strains isolated from soil, however, are poor degraders of DDT and differed in their capability to degrade its metabolites such as DDD, DDE, DDOH and DBP and other organochlorine pesticides viz. kelthane and lindane. Synergistic effect was shown by some of these strains, when grown together in the medium containing DDD and kelthane under mixed culture condition. No synergism in DDE degradation was observed with the strains isolated from enriched soil. DDD-induced proteins extracted from individual culture filtrate (exo-enzyme) when subjected to SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) showed complementary polypeptide bands in these strains i.e., each strain produced distinct DDD degrading polypeptide bands and the recombinant or hybrid strains produced all of the bands of the two parents and degraded DDD better than the parental strains. Recombinant hybrid strains with improved dehalogenase activity were raised by parasexual hybridisation of two such complementary isolates viz. isolate 1(P-1) and 4(P-2) showing highest complementation and are compatible for hyphal fusion inducing heterokaryosis. These strains are genetically characterised as Kel+Ben(R)DBP-Lin- and Kel-Ben(r)DBP+Lin+ respectively. Recombinants with mixed genotype, i.e., Kel+Ben(R)DBP+Lin+ showing superior degradation quality for DDT were selected for bioremediation study. Recombination was confirmed by polypeptide band analysis of DDD induced exo-proteins from culture filtrate using SDS-Polyacrylamide Gel Electrophoresis (PAGE) and RAPD (Random Amplified Polymorphic DNA) of genomic DNA using PCR (Polymerase Chain Reaction) technique. SDS-PAGE showed combination of DDD induced polypeptide bands characteristic of both the parents in the recombinants or the hybrids. PCR study showed the parent specific bands in the recombinant strains confirming gene transformation.

Studies on degradation of 14C-DDT in the marine environment.

Degradation of 14C-DDT was studied in a marine ecosystem for 60 days and in marine sediments under moist and flooded conditions using a continuous flow system for a period of 130 days. 14C-DDT residues were recovered in sediments of the marine ecosystem at uniform level of 60-65% of the applied 14C-activity throughout the incubation period. DDD was a major metabolite in sediments while DDMU was a major metabolite in clams. Clams brought about substantial degradation of DDT. However, 14C-residues recovered form clams are not suggestive of significant bioaccumulation. In the continuous flow experiment, under both moist and flooded conditions, DDT underwent degradation and about 22% of the applied 14C-activity was recovered as volatiles under both conditions. In sediments, extractable 14C-residues accounted for about 30 and 19% under moist and flooded conditions, respectively. DDT was the major compound in extractable residues as identified by TLC-autoradiographic procedures. More bound residues were formed under flooded than under moist conditions.

Studies on degradation of 14C-chlorpyrifos in the marine environment.

Degradation of 14C-chlorpyrifos was studied in a marine ecosystem for 60 days and in marine sediment under moist and flooded conditions using a continuous flow system allowing a total 14C-mass balance for a period of 40 days. In the marine ecosystem, 14C-chlorpyrifos underwent rapid degradation and very little (1-2%) 14C-residues of the applied activity were detected after two months in sediments. Clams were major component of the ecosystem and played a significant role in degradation of the insecticide. In the continuous flow system chlorpyrifos did not undergo substantial mineralization. Volatilization accounted for 0.8-1% loss during first ten days of application. The amounts of extractable 14C-activity were higher in flooded sediments than in moist sediment. More bound residues were formed under moist conditions. TCP (3,5,6-trichloro-2-pyridinol) was the major degradation product formed under both moist and flooded conditions, its formation being higher in the latter conditions. These studies underline the role of clams in degradation of chlorpyrifos and lack of microbial degradation. In absence of clams, chlorpyrifos underwent abiotic degradation in marine sediment with formation of bound residues.

Caffeine protects mice against whole-body lethal dose of gamma-irradiation.

Administration of caffeine (1,3,7-trimethylxanthine), a major component of coffee, to Swiss mice at doses of 80 or 100 mg/kg body weight 60 min prior to whole-body lethal dose of gamma-irradiation (7.5 Gy) resulted in the survival of 70 and 63% of animals, respectively, at the above doses in contrast to absolutely no survivors (LD-100/25 days) in the group exposed to radiation alone. Pre-treatment with a lower concentration of caffeine (50 mg/kg) did not confer any radioprotection. The protection exerted by caffeine (80 mg/kg), however, was reduced from 70 to 50% if administered 30 min prior to irradiation. The trend statistics reveal that a dose of 80 mg/kg administered 60 min before whole-body exposure to 7.5 Gy is optimal for maximal radioprotection. However, caffeine (80 mg/kg) administered within 3 min after irradiation offered no protection. While there is documentation in the literature that caffeine is an antioxidant and radioprotector against the oxic pathway of radiation damage in a wide range of cells and organisms, this is the first report demonstrating unequivocally its potent radioprotective action in terms of survival of lethally whole-body irradiated mice.

Characterization of experimentally induced, nonaflatoxigenic variant strains of Aspergillus parasiticus.

Six previously isolated, nonaflatoxigenic variants of Aspergillus parasiticus, designated sec mutants, were characterized morphologically by electron microscopy, biochemically by biotransformation studies with an aflatoxin precursor, and genetically by Northern (RNA) hybridization analysis of aflatoxin biosynthetic gene transcripts. Scanning electron micrographs clearly demonstrated that compared with the parental sec+ forms, the variant sec forms had an abundance of vegetative mycelia, orders of magnitude reduced number of conidiophores and conidia, and abnormal metulae. Conidiospores were detected in sec cultures only at higher magnifications (x 500), in contrast to the sec+ (wild-type) strain, in which abundant conidiospores (masking the vegetative mycelia) were observed at even lower magnifications (x 300). All sec+ forms, but none of the sec forms, showed bioconversion of sterigmatocystin to aflatoxins. Northern blots probed with pathway genes demonstrated lack of expression of both the aflatoxin biosynthetic pathway structural (nor-1 and omtA) and regulatory (aflR) genes in the sec forms; PCR and Southern hybridization analysis confirmed the presence of the genes in the sec genomes. Thus, the loss of aflatoxigenic capabilities in the sec form is correlated with alterations in the conidial morphology of the fungus, suggesting that the regulation of aflatoxin synthesis and conidiogenesis may be interlinked.