The efficacy of antisense-based construct for inducing resistance against Croton yellow vein mosaic virus in Nicotiana tabacum.

Begomoviruses have increased pathogenicity because of their adaptation to a wide host range; consequently, these viruses cause a major loss to agroeconomic crops worldwide. In this study, we designed a gene construct representing an antisense coat protein gene. We also analyzed the efficacy of the induced resistance against Croton yellow vein mosaic virus (CrYVMV) affecting papaya in Nicotiana tabacum plants. Positive control plants developed typical leaf curl symptoms, whereas transgenic plants were symptomless. Moreover, the key component (i.e., short interfering RNA) of the antisense pathway was upregulated in transgenic plants. This finding demonstrates the activation of the gene silencing mechanism in transgenic plants. Thus, these results confirm that our construct is functional and effectively induces transient resistance against CrYVMV infections.

Transcriptome Analysis of Aspergillus flavus Reveals veA-Dependent Regulation of Secondary Metabolite Gene Clusters, Including the Novel Aflavarin Cluster.

The global regulatory veA gene governs development and secondary metabolism in numerous fungal species, including Aspergillus flavus. This is especially relevant since A. flavus infects crops of agricultural importance worldwide, contaminating them with potent mycotoxins. The most well-known are aflatoxins, which are cytotoxic and carcinogenic polyketide compounds. The production of aflatoxins and the expression of genes implicated in the production of these mycotoxins are veA dependent. The genes responsible for the synthesis of aflatoxins are clustered, a signature common for genes involved in fungal secondary metabolism. Studies of the A. flavus genome revealed many gene clusters possibly connected to the synthesis of secondary metabolites. Many of these metabolites are still unknown, or the association between a known metabolite and a particular gene cluster has not yet been established. In the present transcriptome study, we show that veA is necessary for the expression of a large number of genes. Twenty-eight out of the predicted 56 secondary metabolite gene clusters include at least one gene that is differentially expressed depending on presence or absence of veA. One of the clusters under the influence of veA is cluster 39. The absence of veA results in a downregulation of the five genes found within this cluster. Interestingly, our results indicate that the cluster is expressed mainly in sclerotia. Chemical analysis of sclerotial extracts revealed that cluster 39 is responsible for the production of aflavarin.

"BABY, BABY I'VE GOT HEADACHE".

A 26-year-old woman with no prior medical history presented post-partum with altered mental status. She had no complications during pregnancy and had a spontaneous vaginal delivery at term one week prior. On post-partum day five, she began complaining of headaches, initially responsive to ibuprofen but eventually worsened with no relief. On the evening of admission, her boyfriend noted strange behavior and movements consistent with a tonic-clonic seizure. On the way to the hospital, she had two more similar seizures witnessed by emergency medical serevices (EMS). EMS reported her blood pressures in route to be 200/100s. She was given 5 mg of magnesium by EMS due to concern for postpartum eclampsia. Upon arrival at the emergency room, she was somnolent but arousable although unable to answer any questions. She was mildly tachycardic at 106 beats per minute and had a temperature of 38.2°C. Her blood pressure was elevated at 165/95 mm Hg. On exam, dried blood was noted on her lips and her tongue was swollen. On auscultation, she was tachycardic with clear lung sounds. Her abdomen was soft and non-tender and there was no vaginal bleeding or other discharge. Laboratory values revealed a sodium of 142, potassium of 3.3, chloride of 110, bicarbonate of 16, creatinine of 1.1, magnesium of 3.9, and white blood cell count of 12.3 x103/mm3 with 88% neutrophils and no bands. A toxicology panel was negative for opiates, benzodiazepines, or other illicit drugs. Urine was remarkable for large blood, 448 red blood cells, protein, moderate leukocyte esterase, and 73 white blood cells. Chest x-ray and CT scan of the head were both normal. She was admitted to the medical intensive care unit for close monitoring, neurological checks, and continued magnesium administration. By hospital day two, her mental status had improved significantly.

Blue light (470 nm) effectively inhibits bacterial and fungal growth.

UNLABELLED: Blue light (470 nm) LED antimicrobial properties were studied alone against bacteria and with or without the food grade photosensitizer, erythrosine (ERY) against filamentous fungi. Leuconostoc mesenteroides (LM), Bacillus atrophaeus (BA) or Pseudomonas aeruginosa (PA) aliquots were exposed on nutrient agar plates to Array 1 (AR1, 0·2 mW cm(-2)) or Array 2 (AR2, 80 mW cm(-2)), which emitted impure or pure blue light (0-300 J cm(-2)), respectively. Inoculated control (room light only) plates were incubated (48 h) and colonies enumerated. The antifungal properties of blue light combined with ERY (11·4 and 22·8 µmol l(-1)) on Penicillium digitatum (PD) and Fusarium graminearum (FG) conidia were determined. Conidial controls consisted of: no light, room light-treated conidia and ERY plus room light. Light-treated (ERY + blue light) conidial samples were exposed only to AR2 (0-100 J cm(-2)), aliquots spread on potato dextrose agar plates, incubated (48 h, 30°C) and colonies counted. Blue light alone significantly reduced bacterial and FG viability. Combined with ERY, it significantly reduced PD viability. Blue light is lethal to bacteria and filamentous fungi although effectiveness is dependent on light purity, energy levels and microbial genus. SIGNIFICANCE AND IMPACT OF THE STUDY: Light from two arrays of different blue LEDs significantly reduced bacterial (Leuconostoc mesenteroides, Bacillus atrophaeus and Pseudomonas aeruginosa) viabilities. Significant in vitro viability loss was observed for the filamentous fungi, Penicillium digitatum and Fusarium graminearum when exposed to pure blue light only plus a photosensitizer. F. graminearum viability was significantly reduced by blue light alone. Results suggest that (i) the amount of significant loss in bacterial viability observed for blue light that is pure or with traces of other wavelengths is genus dependent and (ii) depending on fungal genera, pure blue light is fungicidal with or without a photosensitizer.

Volatile trans-2-hexenal, a soybean aldehyde, inhibits Aspergillus flavus growth and aflatoxin production in corn.

UNLABELLED: Trans-2-hexenal, a volatile aldehyde, is produced by soybean (Glycine max [L.] Merr) and other plants via the lipoxygenase pathway. In vitro tests showed it significantly (P < 0.001) reduced Aspergillus flavus germinating conidial viability at 10 µM, with approximately 95% viability reduction observed at 20 µM. The viability of nongerminated conidia was not reduced. To test the effectiveness of this volatile to prevent fungal growth in stored corn, trans-2-hexenal was pumped intermittently into glass jars containing corn. Experiments were performed to determine the ability of 2 different pump cycle time-courses to prevent A. flavus growth on sterile corn (23% moisture). Intermittently (30-min pumping period) over 7 d, this volatile was pumped through 350 g of corn kernels inoculated with 1 mL of 3 × 104 conidia of A. flavus. Controls consisted of (1) sterile corn, (2) corn inoculated with A. flavus with no pumped air, and (3) corn inoculated with A. flavus with intermittently pumped air. Aflatoxin B1 (AFB1), viability counts, and aldehyde concentration in the headspace were performed in each experiment. To determine whether an increased time period between volatile pumping would prevent A. flavus growth, a 2nd series of experiments were performed that were similar to the 1st series except that trans-2-hexenal (only) was pumped for a 30-min period every 12 h. Experiments were performed 3 times for each time course. Both experiments showed that intermittent pumping of volatile trans-2-hexenal significantly (P < 0.001) prevented A. flavus growth and aflatoxin B1 production over a 7-d period. PRACTICAL APPLICATION: Results from this study indicate that intermittent pumping of volatile trans-2-hexenal could be used to protect stored corn from A. flavus growth and aflatoxin contamination.

Study of betasatellite molecule from leaf curl disease of sunn hemp (Crotalaria juncea) in India.

Leaves of sunn hemp (Crotalaria juncea) showing geminiviral symptoms were collected from Lucknow, India during rainy season in 2008. DNA template isolated from the symptomatic leaf tissues were subjected to polymerase chain reaction (PCR) using specific primers to amplify coat protein (CP) gene of DNA-A as well as betasatellite DNA associated with the leaf curl disease. CP gene showed 97% sequence identity with that of Cotton leaf curl Burewala virus (CLCuBwV). Further, the betasatellite DNA molecule revealed sequence similarity with previously characterized betasatellite DNA of begomoviruses affecting malvaceous crops from different regions of India and Pakistan. Maximum similarity (>90%) of betasatellite DNA under study was observed with Cotton leaf curl Multan betasatellite (CLCuMB-[Pak: Mul17:08) and other betasatellite DNA from Pakistan thus confirming possible infection of C. juncea with begomovirus. A complementary sense open reading frame (ORF) ßC1 is present at nucleotide position 194-550. Sequence comparison of this ORF with other members of begomoviruses further confirmed association of a begomovirus with C. juncea. The betasatellite DNA when expressed under the control of CaMV35S promoter Nicotiana tabacum, showed leaf deformities. Our results demonstrated that a malvaceous betasatellite is adapted by a nonmalvaceous host and causes similar disease symptoms.

Correlation and classification of single kernel fluorescence hyperspectral data with aflatoxin concentration in corn kernels inoculated with Aspergillus flavus spores.

The objective of this study was to examine the relationship between fluorescence emissions of corn kernels inoculated with Aspergillus flavus and aflatoxin contamination levels within the kernels. Aflatoxin contamination in corn has been a long-standing problem plaguing the grain industry with potentially devastating consequences to corn growers. In this study, aflatoxin-contaminated corn kernels were produced through artificial inoculation of corn ears in the field with toxigenic A. flavus spores. The kernel fluorescence emission data were taken with a fluorescence hyperspectral imaging system when corn kernels were excited with ultraviolet light. Raw fluorescence image data were preprocessed and regions of interest in each image were created for all kernels. The regions of interest were used to extract spectral signatures and statistical information. The aflatoxin contamination level of single corn kernels was then chemically measured using affinity column chromatography. A fluorescence peak shift phenomenon was noted among different groups of kernels with different aflatoxin contamination levels. The fluorescence peak shift was found to move more toward the longer wavelength in the blue region for the highly contaminated kernels and toward the shorter wavelengths for the clean kernels. Highly contaminated kernels were also found to have a lower fluorescence peak magnitude compared with the less contaminated kernels. It was also noted that a general negative correlation exists between measured aflatoxin and the fluorescence image bands in the blue and green regions. The correlation coefficients of determination, r(2), was 0.72 for the multiple linear regression model. The multivariate analysis of variance found that the fluorescence means of four aflatoxin groups, <1, 1-20, 20-100, and >or=100 ng g(-1) (parts per billion), were significantly different from each other at the 0.01 level of alpha. Classification accuracy under a two-class schema ranged from 0.84 to 0.91 when a threshold of either 20 or 100 ng g(-1) was used. Overall, the results indicate that fluorescence hyperspectral imaging may be applicable in estimating aflatoxin content in individual corn kernels.

Triglycerides and small dense low density lipoprotein in the discrimination of coronary heart disease risk in South Asian populations.

INTRODUCTION: Coronary heart disease (CHD) is exceptionally prevalent amongst globally dispersed migrant groups originating from the Indian subcontinent, but the contribution of dyslipidaemia to their increased risk remains poorly defined. METHODS: Fasting lipids and lipoproteins, apolipoproteins (Apo), low density lipoprotein (LDL) diameter and oxidised LDL were measured amongst rural Indians in India (n=294) and their migrant contemporaries in the UK (n=242). The performance of qualitative and quantitative measures of lipid metabolism were compared in the discrimination of WHO defined metabolic risk and raised Framingham CHD risk scores (>15%) using Receiver Operating Characteristic (ROC) curves. RESULTS: LDL diameter was correlated with triglycerides (R(2)=0.12, P<0.001) and with high density lipoprotein (HDL) cholesterol levels (R(2)=0.15, P<0.001) in both groups. Migrants had less small dense LDL (95% CI: 12.5-14.2%) vs. rural Indians (15.7-17.2, P<0.05). On ROC analysis, triglycerides were the only consistent discriminators of metabolic and CHD risk scores (all P< or =0.001). Apo B was also a strong indicator of raised CHD risk scores. Irrespective of site, individuals with raised triglycerides also had higher total cholesterol and Apo B, denser LDL, lower HDL and more oxidised LDL (all P< or =0.01). DISCUSSION: Fasting triglycerides reflect both qualitative and quantitative aspects of lipid metabolism, and are a comprehensive discriminator of CHD risk in this South Asian population.

Expression of E-Cadherin in breast carcinomas and its association with other biological markers - a prospective study.

CONTEXT: E-cadherin (E-CD) is an important cell adhesion molecule in normal epithelial cells and has been shown to be an invasion tumor suppressor gene. MATERIALS AND METHODS: Various clinicopathological parameters like age, family history, tumor stage, histological grade, lymph node status and other biological markers were also analyzed. Present study reveals E-CD expression in 65 cases of breast cancer including 41 (63%) cases of pure infiltrating ductal carcinoma (IDC), 11 (16.9%) of pure infiltrating lobular carcinoma (ILC); another 10 (15.3%) of mixed ductal/lobular type, and remaining 3 (4.6%) miscellaneous types. RESULTS: Negative E-CD expression was noticed more in advancing age groups (P = 0.01). About 59.2% cases showing negative E-CD expression had family history of breast and/or other cancers. E-CD expression was found significantly higher in cases of pure IDC (55.5%) than in pure ILC cases (18.1%) (P = 0.04). Eleven (68.7%) of the total 16 high-grade IDC cases, revealed negative expression. Both cases of comedo carcinoma revealed negative expression. Three (30%) out of 10 mixed cases revealed negative expression in both ductal and lobular areas, while in remaining 7 cases, positvity was seen in ductal areas only. Invasive cribriform and medullary carcinoma revealed a stronger expression, while negative staining was observed in sweat gland carcinoma. E-CD re-expression was noticed in lymph node tumor deposits. A direct association of E-CD expression with ER expression and an inverse association with that of p53 were also observed (P = 0.001), (P = 0.04). CONCLUSIONS: E-CD expression is useful, but limited, in differentiating IDCs from ILCS. Its negative expression correlates with certain poor prognostic parameters reflecting its use as a marker for invasive cancers. It re-expresses at metastatic sites.

Vitamin D receptor gene polymorphism(s) and breast cancer risk in north Indians.

BACKGROUND: Vitamin D (1,25-dihydroxyVitamin D3) has shown experimentally anticarcinogenic effects and is thought to protect against breast cancer. The actions of Vitamin D are mediated via the Vitamin D receptor (VDR), and the polymorphisms at 3'UTR region of this gene are associated with the risk and progression of breast carcinoma. The current study is an attempt to examine the association of these variations with breast cancer risk in north Indians. METHODS: A total of 160 cases and 140 control subjects were studied for the polymorphisms at 3' end of the VDR gene. A polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) method and fragment analysis was performed to determine ApaI and TaqI polymorphisms and variable length poly-A microsatellite repeats. Linkage disequilibrium (LD) was calculated for each pair of polymorphisms. Unadjusted and adjusted odds ratios for breast cancer with genotypes comprising the polymorphic sites were calculated to understand their role towards breast cancer susceptibility. RESULTS: Patient's with long poly-A repeat showed a significant association with disease (chi 2 = 9.52, df = 2, P

Family tracing to identify patients with familial hypercholesterolaemia: the second audit of the Department of Health Familial Hypercholesterolaemia Cascade Testing Project.

BACKGROUND: Family tracing is a method recognized to find new patients with familial hypercholesterolaemia (FH). We have implemented family tracing led by FH Nurses and have determined acceptability to patients, feasibility and costs. METHODS: Nurses were located at five National Health Service (NHS) Trusts; they identified FH patients and offered them family tracing. Responses and test results were recorded on a database and summarized on a family pedigree. RESULTS: The majority ( approximately 70%) of index cases participated; the proportion was lower when patients had been discharged from the clinics and in metropolitan areas. On average, 34% (range 13-50%) of relatives lived outside the catchment area of the clinics and could not attend the nurse-led FH clinics. Of the previously untested relatives, 76% who lived in the catchment area of the clinic came forward to be tested. One-third of the relatives who came forward for testing were children

Apolipoprotein E polymorphism and its relation to plasma lipids in coronary heart disease.

BACKGROUND: The present investigation is aimed at examining the Apolipoprotein E (APOE) genotypic influence on coronary heart disease (CHD) risk in northwest India (Punjab), where this disease is emerging as a major threat to public-health care system. MATERIALS AND METHODS: The present study comprised of angiographically diagnosed coronary heart disease patients (n = 193) and controls (n = 150) of Punjab. Genetic polymorphism of APOE gene was investigated by polymerase chain reaction (PCR), and its association with lipid levels was evaluated. RESULTS: The allele frequencies of epsilon2, epsilon3, and epsilon4 were 0.054, 0.795, 0.151; and 0.077, 0.856, 0.067 in patients and controls respectively. The bearers of E3/E4 genotype had threefold higher propensity of developing CHD in this population (OR, 3.04; CI, 1.55-6.25; P < 0.001), which exacerbated (OR, 4.18; CI, 2.03-9.27; P < 0.001) after correcting for age, sex, BMI, and lipid-lowering drugs. Lower HDL-C levels and higher LDL-C levels were found to be correlated with E3/E4 genotype (P < 0.01). Other concomitants like body mass index (BMI), total cholesterol (TC), and triglyceride (TG) levels did not show up as genetic determinants in this part of the region. CONCLUSIONS: A significant association (P = 0.016) of epsilon4 allele, especially E3/E4 genotype, with CHD was observed, along with HDL-C and LDL-C concentrations, in the population of northwest India.

Are patients with familial hypercholesterolaemia well managed in lipid clinics? An audit of eleven clinics from the Department of Health Familial Hypercholesterolaemia Cascade Testing project.

BACKGROUND: Familial hypercholesterolaemia (FH) is an autosomal co-dominant disorder which is relatively common, leads to high levels of LDL-cholesterol and if untreated to early coronary heart disease. An audit of current practice at National Health Service Trusts in England was undertaken to determine whether FH patients meet the diagnostic criteria for FH; are being offered appropriate advice and treatment; and to what extent their families are contacted and offered testing for the disorder. METHODS: Medical records of known FH patients (over 18 years of age and diagnosed before 31 December 2003) were accessed to obtain information on diagnosis, treatment and family tracing. RESULTS: The records of 733 FH patients were examined, 79% met the UK 'Simon Broome' register criteria for the diagnosis of definite or possible FH. Analyses showed that patients were usually offered appropriate advice and treatment, with 89% being on a statin. However, the audit indicated a high variability in family tracing between the sites, with significant differences in the frequency of inclusion of a family pedigree in the notes (range 1-71%, mean 35%); the general practitioner (GP) being advised that first-degree relatives should be tested (range 4-52%, mean 27%); and the proportion of relatives contacted and tested (range 6-50%, mean 32%). CONCLUSION: FH patients are well cared for in lipid clinics in England, are being given appropriate lifestyle advice and medication, but an increase in recording of LDL-cholesterol levels may lead to improvements in their management. Practice in family tracing appears to vary widely between clinics.

Role of enteric fever in ileal perforations: an overstated problem in tropics?

PURPOSE: To determine the role of enteric fever in ileal perforations. METHODS: A prospective cohort of 47 patients of ileal perforation was subjected to clinical examination and investigations for APACHE II scoring. Blood, ulcer edge biopsy, mesenteric lymph node and peritoneal aspirate were subjected to culture to determine the predominant aerobic bacterial isolate and its antibiogram. RESULTS: Seven patients (14.9%) required intensive care and seven (14.9%) developed septicaemia. Mortality was 17%. Highest isolation rate was seen in ulcer edge (70.2%) followed by lymph node (66%) culture. The bacterial spectrum was Escherichia coli (23.4%), Enterococcus faecalis (21.3%), Salmonella enterica serovar Typhi (6.3%), Salmonella enterica serovar Paratyphi A (4.2%), etc. CONCLUSIONS: Enteric fever organisms are not the predominant causative agents of ileal perforations. Culture of ulcer edge biopsy, lymph node is crucial for aetiological diagnosis. The use of APACHE II triaging and prescription of antimicrobials based on the local pattern of susceptibility profile of the aetiological agent is recommended.

Aflatoxin formation and gene expression in response to carbon source media shift in Aspergillus parasiticus.

Aflatoxins are toxic and carcinogenic polyketide metabolites produced by fungal species, including Aspergillus flavus and A. parasiticus. The biosynthesis of aflatoxins is modulated by many environmental factors, including the availability of a carbon source. The gene expression profile of A. parasiticus was evaluated during a shift from a medium with low concentration of simple sugars, yeast extract (YE), to a similar medium with sucrose, yeast extract sucrose (YES). Gene expression and aflatoxins (B1, B2, G1, and G2) were quantified from fungal mycelia harvested pre- and post-shifting. When compared with YE media, YES caused temporary reduction of the aflatoxin levels detected at 3-h post-shifting and they remained low well past 12 h post-shift. Aflatoxin levels did not exceed the levels in YE until 24 h post-shift, at which time point a tenfold increase was observed over YE. Microarray analysis comparing the RNA samples from the 48-h YE culture to the YES samples identified a total of 2120 genes that were expressed across all experiments, including most of the aflatoxin biosynthesis genes. One-way analysis of variance (ANOVA) identified 56 genes that were expressed with significant variation across all time points. Three genes responsible for converting norsolorinic acid to averantin were identified among these significantly expressed genes. The potential involvement of these genes in the regulation of aflatoxin biosynthesis is discussed.

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.

Migration is associated with lower total, but not free testosterone levels in South Asian men.

OBJECTIVE: Serum testosterone measurement is an integral part of the endocrine assessment of men. Little is known about its variation in relation to migration. We examined within a South Asian group the effect of migration to the UK on androgen levels. DESIGN: Circulating testosterone and SHBG concentrations were measured in 97 Gujarati men resident in India and in 79 men from the same villages of origin living in Birmingham, UK. Free testosterone was calculated by Vermeulen's method. Insulin sensitivity (HOMA-S) was determined from paired fasting plasma intact insulin and glucose values. RESULTS: Circulating testosterone was significantly lower in UK Gujarati men (17.2 nmol/l [15.7-18.7]) vs. Indian Gujarati men (21.7 [20.0-23.5]) (P = 0.0002) (age-adjusted median [95% CI]). There was no difference by migration status in circulating free testosterone. Sex hormone binding globulin (SHBG) levels were lower in UK migrants (16.8 nmol/l [15.5-18.1]) than in nonmigrants (21.9 nmol/l [20.5-23.3]) (P < 0.0001). Testosterone level correlated positively with insulin sensitivity (HOMA-S) (rho 0.16, P = 0.04). In multivariate analysis, total testosterone was independently and positively associated with logSHBG (normalized beta (beta) = 0.29, P = 0.002) and independently and negatively with waist circumference (beta = -0.19, P = 0.04), in a model also including height, age, migration status, leptin and fasting insulin. CONCLUSION: Lower circulating testosterone in UK Gujarati men and its association with markers of insulin sensitivity suggest a profound influence of body composition change with migration on testosterone levels. The lower SHBG in this group restores parity in free testosterone. Account should be taken of SHBG in interpreting testosterone levels in men, as well as in women.