Chronopathological aspects of disease incidence in rice (Oryza sativa L.)

Rice seedlings maintained under uncontrolled glasshouse conditions were inoculated with conidial suspensions of a fungal pathogen, Helminthosporium oryzae, at various times during the 24 h. Significant increase in the percent germination and germ tube length of conidia were observed in the rice samples inoculated at 02:00 and 06:00h. The 24 h temporal variation in leaf temperature was positively correlated with variation in stomatal movements. The results indicate a 24 h rhythm in the behavior of the fungal pathogen on the host in relation to the conditions of the growing environment. In all the inoculated seedlings, the appearance of a large number of brown leaf spots was confined to the light span. Among the plants inoculated, earlier initiation of brown leaf spot appearance, maximum number of leaf spots, and highest disease severity were observed when plants were inoculated at 02:00h. There was a positive correlation between disease severity of the host and in vivo values of percent germination of conidia and germ tube length of the pathogen in plants inoculated between 02:00 and 06:00h. The findings of this study implicate that light intensity and temperature could play a predominant role in controlling disease susceptibility rhythms in plants.     

Measurement and comparison of head scatter factor for 7 MV unflattened (FFF) and 6 MV flattened photon beam using indigenously designed columnar mini phantom

Aim

To measure and compare the head scatter factor for 7 MV unflattened and 6 MV flattened photon beam using a home-made designed mini phantom.

Background

The head scatter factor (Sc) is one of the important parameters for MU calculation. There are multiple factors that influence the Sc values, like accelerator head, flattening filter, primary and secondary collimators.

Materials and methods

A columnar mini phantom was designed as recommended by AAPM Task Group 74 with high and low atomic number material for measurement of head scatter factors at 10 cm and d_max dose water equivalent thickness.

Results

The Sc values measured with high-Z are higher than the low-Z mini phantoms observed for both 6MV-FB and 7MV-UFB photon energies. Sc values of 7MV-UFB photon beams were smaller than those of the 6MV-FB photon beams (0.6–2.2% (Primus), 0.2–1.4% (Artiste) and 0.6–3.7% (Clinac iX (2300CD))) for field sizes ranging from 10 cm × 10 cm to 40 cm × 40 cm. The SSD had no influence on head scatter for both flattened and unflattened beams. The presence of wedge filters influences the Sc values. The collimator exchange effects showed that the opening of the upper jaw increases Sc irrespective of FF and FFF.

Conclusions

There were significant differences in Sc values measured for 6MV-FB and unflattened 7MV-UFB photon beams over the range of field sizes from 10 cm × 10 cm to 40 cm × 04 cm. Different results were obtained for measurements performed with low-Z and high-Z mini phantoms.     

The Role of ARF6 in Biliary Atresia

Background & Aims

Altered extrahepatic bile ducts, gut, and cardiovascular anomalies constitute the variable phenotype of biliary atresia (BA).

Methods

To identify potential susceptibility loci, Caucasian children, normal (controls) and with BA (cases) at two US centers were compared at >550000 SNP loci. Systems biology analysis was carried out on the data. In order to validate a key gene identified in the analysis, biliary morphogenesis was evaluated in 2-5-day post-fertilization zebrafish embryos after morpholino-antisense oligonucleotide knockdown of the candidate gene ADP ribosylation factor-6 (ARF6, Mo-arf6).

Results

Among 39 and 24 cases at centers 1 and 2, respectively, and 1907 controls, which clustered together on principal component analysis, the SNPs rs3126184 and rs10140366 in a 3’ flanking enhancer region for ARF6 demonstrated higher minor allele frequencies (MAF) in each cohort, and 63 combined cases, compared with controls (0.286 vs. 0.131, P = 5.94x10^-7, OR 2.66; 0.286 vs. 0.13, P = 5.57x10^-7, OR 2.66). Significance was enhanced in 77 total cases, which included 14 additional BA genotyped at rs3126184 only (p = 1.58x10^-2, OR = 2.66). Pathway analysis of the 1000 top-ranked SNPs in CHP cases revealed enrichment of genes for EGF regulators (p<1 x10^-7), ERK/MAPK and CREB canonical pathways (p<1 x10^-34), and functional networks for cellular development and proliferation (p<1 x10^-45), further supporting the role of EGFR-ARF6 signaling in BA. In zebrafish embryos, Mo-arf6 injection resulted in a sparse intrahepatic biliary network, several biliary epithelial cell defects, and poor bile excretion to the gall bladder compared with uninjected embryos. Biliary defects were reproduced with the EGFR-blocker AG1478 alone or with Mo-arf6 at lower doses of each agent and rescued with arf6 mRNA.

Conclusions

The BA-associated SNPs identify a chromosome 14q21.3 susceptibility locus encompassing the ARF6 gene. arf6 knockdown in zebrafish implicates early biliary dysgenesis as a basis for BA, and also suggests a role for EGFR signaling in BA pathogenesis.     

Effect of N-benzoyl-D-phenylalanine and metformin on insulin receptors in neonatal streptozotocin-induced diabetic rats: studies on insulin binding to erythrocytes

In the present study, we focused on the insulin-receptor binding in circulating erythrocytes of N-benzoyl-D-phenylalanine (NBDP) and metformin in neonatal streptozotocin (nSTZ)-induced male Wistar rats. We measured blood levels of glucose and plasma insulin and the binding of insulin to cell-membrane ER receptors in NBDP and metformin-treated diabetic rats. The mean specific binding of insulin to ER was significantly lower in diabetic control rats (DC) (53.0 +/- 3.1%) than in NBDP (62.0 +/- 3.1%), metformin (66.0 +/- 3.3%) and NBDP and metformin combination-treated (72.0 +/- 4.2%) diabetic rats, resulting in a significant decrease in plasma insulin. Scatchard plot analysis demonstrated that the decrease in insulin binding was accounted for by a lower number of insulin receptor sites per cell in DC rats when compared with NBDP and metformin-treated rats. High-affinity (Kd1), low-affinity (Kd2), and kinetic analysis revealed an increase in the average receptor affinity in ER from NBDP and metformin-treated diabetic rats having NBDP 2.0 +/- 0.10 x 10(-10) M(-1) (Kd1); 12.0 +/- 0.85 x 10(-8) M(-1) (Kd2), Metformin 2.1 +/- 0.15 x 10(-10) M(-1) (Kd1); 15.0 +/- 0.80 x 10(-8) M(-1) (Kd2), NBDP and metformin 2.7 +/- 0.10 x 10(-10) M(-1) (Kd1); 20.0 +/- 1.2 x 10(-8) M(-1) (Kd2) compared with 0.9 +/- 0.06 x 10(-10) M(-1) (Kd1); 6.0 +/- 0.30 x 10(-8) M(-1) (Kd2) in DC rats. The results suggest an acute alteration in the number of insulin receptors on ER membranes in nSTZ induced diabetic control rats. Treatment with NBDP along with metformin significantly improved specific insulin binding, with receptor number and affinity binding reaching almost normal non-diabetic levels. The data presented here show that NBDP along with metformin increase total ER membrane insulin binding sites with a concomitant significant increase in plasma insulin.     

Molecular properties of lysozyme-microbubbles: towards the protein and nucleic acid delivery

Microbubbles (MBs) have specific acoustic properties that make them useful as contrast agents in ultrasound imaging. The use of the MBs in clinical practice led to the development of more sensitive imaging techniques both in cardiology and radiology. Protein-MBs are typically obtained by dispersing a gas phase in the protein solution and the protein deposited/cross-linked on the gas-liquid interface stabilizes the gas core. Innovative applications of protein-MBs prompt the investigation on the properties of MBs obtained using different proteins that are able to confer them specific properties and functionality. Recently, we have synthesized stable air-filled lysozyme-MBs (LysMBs) using high-intensity ultrasound-induced emulsification of a partly reduced lysozyme in aqueous solutions. The stability of LysMBs suspension allows for post-synthetic modification of MBs surface. In the present work, the protein folded state and the biodegradability property of LysMBs were investigated by limited proteolysis. Moreover, LysMBs were coated and functionalized with a number of biomacromolecules (proteins, polysaccharides, nucleic acids). Remarkably, LysMBs show a high DNA-binding ability and protective effects of the nucleic acids from nucleases and, further, the ability to transform the bacteria cells. These results highlight on the possibility of using LysMBs for delivery of proteins and nucleic acids in prophylactic and therapeutic applications.     

Molecular characterization of <Emphasis Type="Italic">bmyC</Emphasis> gene of the mosquito pupicidal bacteria, <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> (VCRC B483) and in silico analysis of bacillomycin D synthetase C protein

A strain of Bacillus amyloliquefaciens (VCRC B483) exhibiting mosquito pupicidal, keratinase and antimicrobial activities was isolated from mangrove forest ecosystem of Andaman and Nicobar Islands. Molecular characterization of the strain showed the presence of lipopeptide encoding bmyC gene. Phylogenetic tree based on protein sequence of this gene exhibited homology with mycosubtilin synthetase of Bacilus atropheus and Iturin synthetase of Bacillus subtilis and B. amyloliquefaciens. This is the first report on the evolutionary conservation of amino acids concerned with the function and structure of bmyC protein of B. amyloliquefaciens. The presence of valine at the 1197th position in our strain was found to be unique and different from the existing strains of B. subtilis and B. amyloliquefaciens. Molecular modelling studies revealed significant changes in the structure of epimerization domain of the bmyC protein with A1197V variation. Crude metabolite of this strain exhibited antifungal activity against Fusarium sp. and Carvularia sp.     

Effect of Long-term Fluoride Exposure on Growth, Nutrient Utilization and Fluoride Kinetics of Calves Fed Graded Levels of Dietary Protein

In order to assess the influence of dietary protein levels on the fluoride (F) bioavailability, 30 crossbred calves (6-8 months; approximately 104 kg BW) initially exposed to different dietary protein levels were allotted into six groups in a 3?×?2 factorial design. The factors included three different levels of protein viz. normal (100%; NP), low (75%; LP), and high (125%; HP) as per Kearl recommendations besides two levels of supplemental fluorine (as sodium fluoride) at 0 or 200 mg/kg diet. The animals were fed on the respective concentrate mixture and wheat straw for 210 days. A metabolism trial was conducted at 200 days post-feeding to study digestibility, plane of nutrition, and nutrient balances. The final body weight at the end of 210 days was lower (p?<?0.01) in animals fed 200 mg/kg F (164.2?±?8.92 kg) compared to those fed no F (200.7?±?8.05 kg). Calves on LP diets attained lower (p?<?0.05) average daily gain in comparison to NP or HP fed calves. The F-supplemented calves exhibited lower (p?<?0.01) voluntary feed intake than their non-supplemented control. The digestibility of proximate nutrients other than ether extract exhibited higher (p?<?0.01) values in F-fed calves attributable chiefly to reduced consumption of dry matter. The calves fed extra F retained lower mean daily nitrogen; calcium, and phosphorus compared to the calves fed no F. The mean daily intake, excretion, and retention of F were higher (p?<?0.01) in the F-supplemented calves. A significant (p?<?0.01) interaction between protein levels and F was evident in the urinary excretion of F; calves on LP diet exhibiting lower urinary excretion. Consequently, the bioavailability of F tended to be higher on LP than NP or HP diets. From the results, it is concluded that protein levels in the diet do not impart significant influence on susceptibility to fluorosis in crossbred calves. However, the bioavailability of F tended to increase on diets low in protein.     

Beta-fructofuranosidase production by 2-deoxyglucose resistant mutants of aspergillus niger in submerged and solid-state fermentation

Aspergillus niger produces extracellular beta-fructofuranosidase under submerged (SmF) and solid state fermentation (SSF) conditions. After UV mutagenesis of conidiospores of A. niger, 2-deoxyglucose (10 g/l) resistant mutants were isolated on Czapek's minimal medium containing glycerol as a carbon source and the mutants were examined for improved production of beta-fructofuranosidase in SmF and SSF conditions. One of such mutant DGRA-1 overproduced beta-fructofuranosidase in both SmF and SSF conditions. In SmF, the mutant DGRA-1 showed higher beta-fructofuranosidase productivity (110.8 U/l/hr) than the wild type (48.3 U/l/hr). While in SSF the same strain produced 322 U/l/hr of beta-fructofuranosidase, 2 times higher than that of wild type (154.2 U/l/hr). In SmF, both wild type and mutants produced relatively low level of beta-fructofuranosidase in medium containing sucrose with glucose than from the sucrose medium. However in SSF, the DGRA-1 mutant grown in sucrose and sucrose+ glucose did not show any difference with respect to beta-fructofuranosidase production. These results indicate that the catabolite repression of beta-fructofuranosidase synthesis is observed in SmF whereas in SSF such regulation was not prominent.     

Potential risk modifications of GSTT1, GSTM1 and GSTP1 (glutathione-S-transferases) variants and their association to CAD in patients with type-2 diabetes

Background

Type-2 diabetes mellitus (T2DM) is a major risk factor for coronary artery disease (CAD) resulting in high morbidity and mortality. Glutathione S-transferases (GSTM1, GSTT1 and GSTP1) are known for their broad range of detoxification and in the metabolism of xenobiotics. Recent studies revealed the relationship of GSTs variants with T2DM and CAD. In this case-control study we ascertained the association of GSTs variants in association with the development of CAD in patients with T2DM.

Methods

From the Southern part of India, we enrolled 222 T2DM patients, 290 T2DM patients with CAD and 270 healthy controls matched for age, sex and origin. Serum lipid profiles were measured and DNA was extracted from the blood samples. Multiplex PCR for GSTM1/T1 (null polymorphism) and PCR-RFLP for GSTP1 (105 A > G), were performed for genotyping of study participants. Gene frequency and lipid profiles were statistically analyzed for disease association.

Results

Regression analysis showed that, GSTM1-null genotype is associated with a 2-fold increase (OR = 2.925; 95% CI = 2.078–4.119; P < 0.0001) and GSTT1-null genotype is associated with a 3-fold increase (OR = 3.114; 95% CI = 2.176–4.456; P < 0.0001) to T2DM development. Ile/Val and Val/Val genotypes of GSTP1 also showed a significant risk for T2DM (OR = 1.423, CI = 1.041–1.946; P = 0.027 and OR = 1.829, CI = 1.064–3.142; P = 0.029). Increased odds ratio showed that GSTT1-null genotype had a moderately higher occurrence in T2DM–CAD patients (OR = 1.918, 95% CI = 1.144–3.214; P = 0.014) than T2DM patients without CAD. The level of HDL has significantly decreased in GSTT1-present than in GSTT1-null genotype (43.50 ± 4.10 vs. 45.20 ± 3.90; P = 0.004) when compared with control and T2DM patients. However, LDL level showed a significant increase in GSTT1-null than GSTT1-present genotype (108.70 ± 16.90 vs. 102.20 ± 12.60; P = 0.005). Although the GSTM1-null polymorphism showed no correlation with lipid profiles among T2DM and T2DM with CAD patients, GSTT1-null polymorphism attained a statistical significance for the level of LDL (127 ± 28.20 vs. 134 ± 29.10; P = 0.039) and triglycerides in T2DM with CAD patients (182.10 ± 21.10 vs. 191.20 ± 24.10; P = 0.018).

Conclusion

Our work concludes that GSTM1, GSTT1 and GSTP1 variants might contribute to the development of T2DM and GSTT1 variant alone is involved in the development of T2DM associated CAD complications in the South Indian population.