Efficient extraction of proteins from recalcitrant plant tissue for subsequent analysis by two-dimensional gel electrophoresis.

Protein extraction for two-dimensional electrophoresis from tissues of recalcitrant species is quite problematic and challenging due to the low protein content and high abundance of contaminants. Proteomics in Shorea robusta is scarcely conducted due to the lack of a suitable protein preparation procedure. To establish an effective protein extraction protocol suitable for two-dimensional electrophoresis in Shorea robusta, four procedures (borate buffer/trichloroacetic acid extraction, organic solvent/trichloroacetic acid precipitation, sucrose/Tris/phenol, and organic solvent/phenol/sodium dodecyl sulfate) were evaluated. Following these, proteins were isolated from mature leaves and were analyzed for proteomics, and also for potential contaminants, widely reported to hinder proteomics. The borate buffer/trichloroacetic acid extraction had the lowest protein yield and did not result in any banding even in one-dimensional electrophoresis. In contrast, organic solvent/phenol/sodium dodecyl sulfate extraction allowed the highest protein yield. Moreover, during proteomics, organic solvent/phenol/sodium dodecyl sulfate extracted protein resolved the maximum number (144) of spots. Further, when proteins were evaluated for contaminants, significant (77-95%) reductions in the nucleic acids, phenol, and sugars were discernible with refinement in extraction procedure. Accumulated data suggested that the organic solvent/phenol/sodium dodecyl sulfate extraction was the most effective protocol for protein isolation for proteomics of Shorea robusta and can be used for plants that have a similar set of contaminants.

Socio-economic and ethnic inequalities in diabetes retinal screening.

OBJECTIVE: We aimed to quantify socio-economic and ethnic inequalities in diabetes retinal screening. METHODS: Data were analysed for the retinal screening programme for three South London boroughs for the 18-month period to February 2009. Sight-threatening diabetic retinopathy (STDR) was defined as the occurrence of diabetic maculopathy, severe non-proliferative or proliferative diabetic retinopathy. Odds ratios were adjusted for sex, age group, duration and type of diabetes, self-reported ethnicity and deprivation quintile by participant postal code. RESULTS: There were 76 351 records obtained but, after excluding duplicate and ineligible records, data were analysed for 59 495 records from 31 484 subjects. There were 7026 (22%) subjects called for appointments who were not screened in the period, with 24 458 (78%) having one or more screening episodes. Non-attendance for screening was highest in young adults aged 18-34 years (32%) and in those aged 85 years or greater (28%). In the most deprived quintile, non-attendance was 23% compared with 21% in the least deprived quintile [odds ratio (OR) 1.37, 95% confidence interval (CI) 1.16-1.61, P < 0.001]. There were 2819 (11.5%) participants with STDR, including 10.8% in the least deprived quintile and 12.2% in the most deprived quintile (OR 1.10, 95% CI 0.95-1.16, P = 0.196). Compared with white Europeans (9.4%), STDR was higher in Africans (15.2%) and African Caribbeans (14.7%), resulting from a higher frequency of diabetic maculopathy. CONCLUSION: Socio-economic inequality in diabetes retinal screening may be smaller than reported in earlier studies. This study suggested an increased frequency of diabetic maculopathy among participants of African origins.

Multiple morbidity is associated with increased problems of food access in hospital: a cross-sectional survey utilising the Cumulative Illness Rating Scale.

BACKGROUND: Problems of undernutrition are common in hospital in-patients. Multiple morbidity increases with age and may contribute to nutritional risks. This research aimed to determine whether increased burden of long-term conditions is associated with patients' difficulties gaining access to food in hospital. METHODS: A survey was implemented in 29 wards at four hospitals using a questionnaire measure of patients' experiences of food access and the Cumulative Illness Rating Scale (CIRS) to evaluate the burden of long-term illness in each patient. Experiences of food access were evaluated in relation to CIRS score category using random effects logistic regression to adjust for age group, sex and clustering by ward. RESULTS: Data were analysed for 764/1154 (66%) eligible participants, including 384 women. The median age was 60 years (range 18-96 years). CIRS scores were analysed using the categories 0 (104 patients), 1-3 (197), 4-6 (285), 7-9 (144) and ≥10 (34). When the CIRS was zero, 10% of patients experienced physical problems with food access, whereas, when the CIRS was ≥10, 41% experienced physical barriers to food access, adjusted odds ratio 3.65 (1.14-11.7, P = 0.029). Problems with food quality were experienced by 13% with CIRS = 0 and 32% with CIRS ≥ 10 (adjusted odds ratio 3.97, 1.35-11.6, P = 0.012). Participants with greater morbidity were more likely to report that depression, breathing difficulties or chewing and swallowing difficulties affected the amount of food that they ate at mealtimes. CONCLUSIONS: Patients with multiple morbidities are more vulnerable to experiencing physical barriers to accessing food and increased concerns with food quality. Assessing barriers to food access is particularly important in multiple morbidity.

Towards an open grapevine information system.

Viticulture, like other fields of agriculture, is currently facing important challenges that will be addressed only through sustained, dedicated and coordinated research. Although the methods used in biology have evolved tremendously in recent years and now involve the routine production of large data sets of varied nature, in many domains of study, including grapevine research, there is a need to improve the findability, accessibility, interoperability and reusability (FAIR-ness) of these data. Considering the heterogeneous nature of the data produced, the transnational nature of the scientific community and the experience gained elsewhere, we have formed an open working group, in the framework of the International Grapevine Genome Program (www.vitaceae.org), to construct a coordinated federation of information systems holding grapevine data distributed around the world, providing an integrated set of interfaces supporting advanced data modeling, rich semantic integration and the next generation of data mining tools. To achieve this goal, it will be critical to develop, implement and adopt appropriate standards for data annotation and formatting. The development of this system, the GrapeIS, linking genotypes to phenotypes, and scientific research to agronomical and oeneological data, should provide new insights into grape biology, and allow the development of new varieties to meet the challenges of biotic and abiotic stress, environmental change, and consumer demand.

Targeted inactivation of the psaK1, psaK2 and psaM genes encoding subunits of Photosystem I in the cyanobacterium Synechocystis sp. PCC 6803.

PsaK and PsaM are small, integral membrane proteins, which are associated with the Photosystem I complexes of cyanobacteria. The complete genome sequence of Synechocystis sp. PCC 6803 has revealed the presence of two unlinked psaK genes: psaK1 (ssr0390) and psaK2 (sll0629). To investigate structural and functional roles of the PsaK1, PsaK2 and PsaM polypeptides in Synechocystis sp. PCC 6803, we generated targeted mutants that lack the functional psaK1, psaK2 or psaM genes. Inactivation of psaK1, psaK2 or psaM did not affect photoautotrophic growth, photosynthetic activity and accumulation of other subunits of the Photosystem I complex. The psaK1 (-), psaK2 (-) and psaK1 (-) psaK2 (-) mutants showed normal levels of Photosystem I trimers, whereas the lack of PsaM resulted in a 75% reduction in the recovery of trimers compared to the wild type. A 6.2 kDa polypeptide was observed in the Photosystem I preparations from the wild type, but not from the psaK2 (-) strain, suggesting the presence of PsaK2 in the Photosystem I complexes. Using reverse-transcription and polymerase chain reaction, we confirmed the expression of the psaK2 gene in Synechocystis sp. PCC 6803. To conclude, both psaK1 and psaK2 are expressed in Synechocystis sp. PCC 6803 and the absence of both proteins results in only a small reduction in Photosystem I electron transport. The PsaM subunit is required for the formation of stable Photosystem I trimers.

Genetic susceptibility of indigenous chicks to subgroup A Rous sarcoma virus inoculated via the chorioallantoic membrane.

An investigation was made using chicks of two Indian indigenous breeds of fowl, Kadaknath and Aseel, to ascertain genetic resistance to infection by Rous sarcoma virus of subgroup A. A standard inoculation dose of 0.2 ml virus containing 1000 pock forming units ml-1 was injected via the chorioallantoic membrane (CAM) into the 11-day-old embryos that were subsequently hatched. The sensitivity of the two indigenous breeds was compared with the highly susceptible exotic White Leghorn (WL) strain maintained in the laboratory. The Kadaknath breed was about three-fold and Assel, about six-fold less sensitive than the WL strain, indicating superiority of the indigenous breeds over the exotic breed of fowl. Most of the CAM-susceptible chicks died of liver tumour (LT) and most of the CAM-resistant chicks survived. However, conversely associated tumour phenotype subclass chicks, i.e. CAM-susceptible LT-negative chicks that survived and CAM-resistant LT-positive chicks that died, occurred consistently in the three breeds of fowl. Nevertheless, the overall survival potential of Kadaknath chicks measured up to 8 weeks post-hatching was greater than that of Aseel chicks. Neither transformation of embryonic tissue prior to hatching nor the visceral metastasis including liver conformed with the degree of CAM-infection as measured by number of pocks on CAMs.

Cellular immunity in chicks expressing fibrosarcomatous liver tumor following embryo infection with subgroup A Rous sarcoma virus.

Bryan standard strain of Rous sarcoma virus (BS-RSV) of subgroup A was inoculated into heavy and light breeds of chicken embryos via chorioallantoic membrane (CAM) to ascertain cell-mediated immune response, as measured by a leukocyte migration inhibition (LMI) test. Chicks hatched from eggs with pock-positive CAMs were more likely to develop liver tumors than those hatched from eggs with pock-negative CAMs. Chicks that developed tumors usually had a positive cell-mediated immune response, and those that were negative for liver tumor were negative, based on the LMI test.

Genetics of post-hatching survival potential of Australorp chicks infected as embryos by subgroup A Rous sarcoma virus: further support to 4-allele genetic model.

Embryos (II day-old) of Australorp breed were inoculated via chorioallantoic membrane (CAM) with subgroup A Rous sarcoma virus, and hatched subsequently. The post-hatch survival period in chicks was recorded upto the last chick that died by virus-induced liver tumour, which had a range from 3 to 50 days with an average of 13 +/- 8.7 days. The survival potential of progency tested Australorp parents selected on the basis of negative CAM-infection and those selected on uninoculated embryos, differed significantly (P less than 0.01) while maintaining an inverse relationship between liver tumour mortality and degrees of infection of CAMs. The homozygous susceptibles lacking either ar1 or ar2 or both alleles of the tva (tumour virus a) locus died within 7 days of post-hatching, supporting thereby 4-allele genetic model of tva locus recently proposed for the control of LT- and CAM-infection phenotypes.

Induced liver tumour deaths by subgroup A Rous sarcoma virus in chicks inoculated via chorioallantoic membrane, a genetic marker.

A study was made using two strains of light breed (White Leghorn strains, A and B) and four heavy beeds (Rhode Island Red, New Hampshire, Australorp, Columbian) to evaluate the breed difference in survival potential of chicks that were infected as 11-day-old embryos via chorioallantoic membranes (CAMs) with a subgroup A Rous sarcoma virus. Of the 1185 chicks hatched over multiple hatch-replicates, 845 chicks died rapidly of a fibrosarcomatous liver tumour (LT) with a peak mortality about 74% attained by the second week, post-hatch, in the heavy breeds and more than 90% by the second week in the light breed. The breeds did not differ in induced LT mortality when the chicks hatched from eggs that had at least 25 pock counts on CAMs, apparently genetically susceptible, i.e. 25 biologically active virus particles were enough to induce an unpreventable fatal LT. However, low pock-count on CAMs did not act as a pointer for predicting genetic resistance to infection because about 23% of chicks developed from eggs that had no pocks on CAMs, apparently genetically resistant, also died of LT, requiring further studies.

Subgroup-A Rous sarcoma virus-induced growth stimulation of chick embryos infected via the chorioallantoic membrane.

Chicks that hatch from eggs containing group specific antigen (gs antigen) of lymphoid leukosis virus (LLV) subgroups, grow poorly. In our laboratory for more precise identification of LLV-of subgroup A (LLV-A) resistant and susceptible genotypes by progeny testing, the chorioallantoic membrane (CAM) assay in complemented by liver tumour (LT) assay, wherein Rous sarcoma virus (RSV) of subgroup A (homologous to LLV-A) was used. The present study was conducted in a light breed (White Leghorn) and also in a heavy breed (Rhode Island Red) to ascertain the effect of infection on embryonic growth by RSV subgroup A. Mean relative body weight (rbw) of infected LT negative chicks of either breed exceeded the control highly significantly (P < 0.01) by 2%. However, neither the dose of virus inoculated per embryo, nor egg size influenced the relative body weight of day old chicks (P > 0.05). No difference in relative body weight of LT positive and control chicks was observed.

Ascorbic acid metabolism in ageing recalcitrant sal (Shorea robusta Gaertn. f.) seeds.

Changes in ascorbate content and its enzymatic utilization pattern were studied in embryonic axes and cotyledons of sal seeds undergoing rapid loss of viability, at ambient conditions. Ascorbate levels were significantly higher initially in the embryonic axes (0.32 mg/g fresh weight) and cotyledons (0.21 mg/g fresh weight) of freshly mature, relatively hydrated (42.2% moisture content) and 100% viable sal seeds. It declined sharply as the tissues; embryonic axes and cotyledons, desiccated with absolutely no detectable amount in non-viable seeds (21% moisture content). Significantly strong correlation was obtained between desiccation of embryonic axes (r = 0.96) and cotyledon (r = 0.97) with loss of ascorbate levels and loss of germinability. Higher rates of ascorbic acid utilization (AAU) recorded in the embryonic axes of 100% viable seed declined sharply as the seed viability reduced due to desiccation below 36.8% moisture content. AAU was not detected in the cotyledons.

Expression of tumour phenotypes, hatchability and liver tumour mortality in two routes of inoculation in fowl.

A total of 350 and 200 eleven-day-olc embryos (pooled breeds) of twelve hatch replicates were inoculated with pseudotype of Bryan high titre, RSV(RAV-49) of subgroup C viz CAM (chorioallantoic membrane) and YS (yolk sac) route, respectively. An increase in hatchability (about 16%) and decrease in the incidence of CAM(+) [71%] and LT(+) [47%] phenotypes was noticed when inoculation was done via YS route as compared to the inoculation via CAM routes. A delay in LT(+) mortality was also recorded in YS route of infection. Chi-square analysis within a route basis indicated highly significant contingency (P < 0.01) in association of CAM infection phenotypes and LT incidence phenotypes for CAM route of infection in contrast to the YS route of infection.

Genetic control of cellular infection by subgroups A and C RNA tumour viruses in guinea fowl.

An investigation was carried out in guinea fowl to determine their susceptibility to infection by Rous sarcoma viruses of subgroups A and C. A standard dose of each subgroup virus was inoculated into 14-day-old embryos via the chorioallantoic membrane (CAM). On the 10th day after inoculation, 50% of the embryonic chorioallantoic membranes were harvested to assess their infection status (CAM(+) or (-)), while the rest were allowed to hatch. The hatchabilities of the embryos inoculated with subgroups A and C were about 50% and 57%, respectively. The relative sensitivities of guinea fowl to infection by viruses of subgroups A and C were observed to be 0.220 and 0.003, respectively, as compared to chickens (1.00). Mortality due to subgroup A virus-induced liver tumours (LT) was 54% and four phenotypic subclasses, namely CAM(+) LT(+), CAM(+) LT(-), CAM(-) LT(+) and CAM(-) LT(-), were observed in guinea fowl as in chickens. However, a higher incidence (31%) of conversely associated phenotypes, i.e. CAM(+) LT(-) and CAM(-) LT(+), were observed in guinea fowl. Mortality caused by subgroup A virus-induced liver tumours was first observed in inoculated guinea fowl keets during the 3rd week after hatching, and 93% of the mortality occurred within 6 weeks. The peak mortality occurred in the 4th week after hatching. The target organs for transformation were considered to be the liver and spleen because of the equal incidence of tumours in these organs. Males and females were equally likely to die from liver tumours.(ABSTRACT TRUNCATED AT 250 WORDS)

The genetic control of susceptibility to subgroup D RNA tumour virus infection in commercial breeds of chickens.

The genetic control of resistance to RSV (RAV-50) in chick embryos inoculated via the chorioallantoic membrane route was studied in three breeds of chickens. The relative susceptibility to subgroup D infection was 0.01, 0.02 and 0.003 in broilers, White Leghorns and Bantam x Australorp crosses, respectively. The distribution pattern of putative dr gene frequencies were 0.80, 0.68 and 0.90, respectively, in the same three breeds. Liver tumours resulting from the virus infection were observed in a very few birds about 28 days after hatching.

Diurnal regulation of plastid genes in Populus deltoides.

Light regulates leaf and chloroplast development, together with overall chloroplast gene expression at various levels. Plants respond to diurnal and seasonal changes in light by changing expression of photosynthesis genes and metabolism. In Populus deltoides, a deciduous tree species, leaf development begins in the month of March and leaf maturation is attained by summer, which is subsequently followed by autumnal senescence and fall. In the present study, diurnal changes in the steady state transcript levels of plastid genes were examined in the fully developed leaves during summer season. Our results show that steady state level of the psaA/B, psbA, psbEFLJ and petA transcripts showed differential accumulation during diurnal cycle in summer. However, there was no significant change in the pigment composition during the day/night cycle. Our studies suggest that the diurnal regulation of steady state mRNA accumulation may play a crucial role during daily adjustments in plants life with rapidly changing light irradiance and temperature.

ROS production and lipid catabolism in desiccating Shorea robusta seeds during aging.

Reactive oxygen species (ROS) and lipid peroxidation products appear to correlate strongly with the desiccation induced loss of viability in recalcitrant sal seeds. The 100% germination in fresh sal seeds declined with dehydration under natural storage conditions (26 ± 1 °C, relative humidity 52 ± 2%). Seeds became non-viable within 8 days. Desiccation induced disturbances in the metabolic activity of seeds resulted in generation of enormous amounts of ROS that are responsible for cellular damage and viability loss. Oxidative stress in the dehydrating aging sal seeds was further aggravated by inducing lipid peroxidation as the amounts of free fatty acid, conjugated diene, lipid hydroperoxide and secondary free radicals; malondialdehyde and 4-hydroxy-2-nonenal, were also promoted. In addition, significant rise in lipid degrading enzymes; lipase (EC 3.1.1.3) and lipoxygenase (LOX, EC 1.13.11.12) were detected in dehydrating sal seeds. Our results indicated multiple pathways (ROS, lipid peroxidation & lipase and LOX) that operate in the dehydrating recalcitrant sal seeds finally contributing to loss of viability.

Inheritance of fibrosarcomatous liver tumours in White Leghorn chicks inoculated vu the chorioallantoic membrane with subgroup A Rous sarcoma virus: A four-allele genetic model.

An investigation was made using White Leghorn fowl to study the genetic control of subgroup A Rous sarcoma virus-induced fibrosarcomatous liver tumours (LT) in chicks inoculated via the chorioallantoic membrane (CAM). A total of 723 CAM-inoculated embryos were hatched in three experiments. In Expt 1, CAM-susceptibility and LT-mortality were examined on a within sire family basis to ascertain the inter-relationship between the two parameters. In Expt 2, sires that had records of high or low LT(+) deaths in the families were selected to produce progeny within high and low incidence lines in the next generation to ascertain the amenability of the trait (LT death) to selection pressure. Survivors of LT-assay (Expt 1) were mated inter se in Expt 3 to study the inheritance of the two traits according to known or proposed genetic models. It was shown that LT mortality is a genetic trait because of its amenability to selection, with a high realised heritability (h(2)(R)= 1.16). In the three experiments, most CAM-susceptibles (S) died of LT(+), and most CAM-resistants(R) survived, but there were some conversely associated phenotypes i.e. S(LT-) and R(LT+). The conventional 2-allele model of the tva (tumour virus a) locus with pleiotropic effect, or a 2-locus model with linkage, were considered inadequate to explain the occurrence of conversely associated phenotypes on a within family basis. However, a 4-allele model of the tva locus showed a good fit to the results of this study.

Hatchability and mortality following infection of chick embryos with subgroup a Rous sarcoma virus.

An investigation, using Rhode Island Red, New Hampshire, Columbian and Australorp, was conducted to study the correlation between chorioallantoic membrane (CAM)-infection phenotype to challenge with Bryan strain of Rous sarcoma virus (BS-RSV) of subgroup A and subsequent mortality, following hatching. A total of 806 CAMs and 438 chicks over nine hatch-replicates in three experiments provided evidence that infection via an inoculation of the CAM induced rapid and high mortality, about 72%, averaged over the four breeds, from a fibrosarcomatous liver tumour (LT) in CAM-susceptible chicks. In contrast, only 14% of chicks inoculated via the yolk sac died from liver tumours. Infection via the CAM reduced hatchability by about 28% (P<0.01) as compared to the hatchability of embryos infected via the yolk sac. About 21% of chicks which developed no pocks on the CAM after RSV inoculation developed liver tumours and died.

Characterization of the orf31-petG gene cluster from the plastid genome of Populus deltoides.

The orf31-petG gene cluster is located approximately 1.2 kb away from the psbEFLJ operon in the chloroplast genome of Populus deltoides. The orf31 (ycf7) encodes an unidentified polypeptide while the petG gene encodes subunit V of an important component, cytochrome b6/f complex, involved in photosynthetic electron transport. We have determined the nucleotide sequence of the orf31-petG gene cluster from the plastid genome of a tree, Populus deltoides. Our sequence analysis suggests that these genes possess high homology with the published sequences of these genes from other plants. Northern analysis suggests development dependent transcription of the orf31-petG cluster in leaves.