Identification and characterisation of the Arabidopsis thaliana cell wall proteome: unravelling novel cell wall proteins and new potential functions of the plant extracellular matrix

The application of the proteomic approach has facilitated efforts directed toward the mapping of the entire Arabidopsis cell wall proteome. Proteins were sequentially extracted from purified cell walls using 0.2 M CaC1(_2) followed by a urea buffer. The extracts were resolved via large format two dimensional polyacrylamide gel electrophoresis (2-D PAGE) and were visualised via Coomassie brilliant blue staining. The aim was to identify and characterise as many cell wall proteins as possible, with the hope of identifying novel cell wall proteins. Out of 325 spots visualised on the 2-D polyacrylamide gel, 144 gave a positive protein identification representing 104 different proteins. The identified proteins were divided into 3 categories. The first category included proteins that have been previously identified as plant cell wall proteins. The second category was designated to include novel cell wall proteins (hypothetical proteins) and the third category was made up of proteins, which had recognised functions, but had never hitherto been known to be secreted to the extracellular matrix. Among the identified novel cell wall proteins there were several that shared high homology with protein kinases. These proteins possessed all the characteristics of secreted polypeptides, such as the cleavable N-terminal signal peptide, and were found to lack both the transmembrane domain and the endoplasmic reticulum retention tetrapeptides (HDEL and KDEL). These observations suggested that, as in animal cells, plant cells had extracellular protein kinase activity (phosphorylation). This was supported by the recent discovery that plant cells secrete ATP to the extracellular matrix (Thomas et al., 2000). Verification of the occurrence of extracellular protein kinase activity was further strengthened by the identification of phosphorylated bona fide cell wall proteins and stress responses caused by the depletion extracellular ATP

Identification and profiling of salinity stress-responsive proteins in Sorghum bicolor seedlings

Sorghum bicolor, a drought tolerant cereal crop, is not only an important food source in the semi arid/arid regions but also a potential model for studying and gaining a better understanding of the molecular mechanisms of drought and salt stress tolerance in cereals. In this study, seeds of a sweet sorghumvariety, MN1618, were planted and grown on solid MS growth medium with or without 100mM NaCl. Heat shock protein expression immunoblotting assays demonstrated that this salt treatment induced stress within natural physiological parameters for our experimental material. 2D PAGE in combination with MS/MS proteomics techniques were used to separate, visualise and identify salinity stress responsive proteins in young sorghum leaves. Out of 281 Coomassie stainable spots, 118 showed statistically significant responses (p<0.05) to salt stress treatments. Of the 118 spots, 79 were selected for tandem mass spectrometric identification, owing to their good resolution and abundance levels, and of these, 55 were positively identified. Identified proteins were divided into six functional categories including both known and novel/putative stress responsive proteins. Molecular and physiological functions of some of our proteins of interest are currently under investigation via bioinformatic and molecular biology approaches.Web of Scienc

Molecular adaptation mechanisms employed by ethanologenic bacteria in response to lignocellulose-derived inhibitory compounds

Current international interest in finding alternative sources of energy to the diminishing supplies of fossil fuels has encouraged research efforts in improving biofuel production technologies. In countries which lack sufficient food, the use of sustainable lignocellulosic feedstocks, for the production of bioethanol, is an attractive option. In the pre-treatment of lignocellulosic feedstocks for ethanol production, various chemicals and/or enzymatic processes are employed. These methods generally result in a range of fermentable sugars, which are subjected to microbial fermentation and distillation to produce bioethanol. However, these methods also produce compounds that are inhibitory to the microbial fermentation process. These compounds include products of sugar dehydration and lignin depolymerisation, such as organic acids, derivatised furaldehydes and phenolic acids. These compounds are known to have a severe negative impact on the ethanologenic microorganisms involved in the fermentation process by compromising the integrity of their cell membranes, inhibiting essential enzymes and negatively interact with their DNA/RNA. It is therefore important to understand the molecular mechanisms of these inhibitions, and the mechanisms by which these microorganisms show increased adaptation to such inhibitors. Presented here is a concise overview of the molecular adaptation mechanisms of ethanologenic bacteria in response to lignocellulose-derived inhibitory compounds. These include general stress response and tolerance mechanisms, which are typically those that maintain intracellular pH homeostasis and cell membrane integrity, activation/regulation of global stress responses and inhibitor substrate-specific degradation pathways. We anticipate that understanding these adaptation responses will be essential in the design of ‘intelligent’ metabolic engineering strategies for the generation of hyper-tolerant fermentation bacteria strains.IS

Mapping and characterisation of the sorghum cell suspension culture secretome

Here we reported the first secretomic study of sorghum (Sorghum bicolor), a naturally drought tolerant cereal crop. In this study, we used a gel-based proteomic approach in combination with mass spectrometry to separate and identify proteins secreted into the culture medium of sorghum cell suspensions, a first step towards understanding their functions during plant growth and development. Proteins secreted into the culture medium of 10-day old sorghum cell suspension cultures termed culture filtrate (CF) proteins were separated by two dimensional gel electrophoresis (2DE) and visualised using Coomassie brilliant blue (CBB) R-250 staining. Of the 25 visualised CBB stainable spots, 15 abundant, well resolved and reproducible spots amongst the three biological replicates used were selected for identification using matrix assisted laser desorption/ionisation-time of flight/time of flight tandem mass spectrometry (MALDI-TOF-TOF MS). Of these spots, 14 were positively identified, representing four different protein classes: Peroxidases, germin proteins, oxalate oxidases and α-galactosidases. All the identified proteins are known secretory proteins, with predicted signal peptides, which target proteins for the secretory pathway. The identified proteins have known functions in signalling processes, defence mechanisms and cell wall metabolism which is consistent with their location outside the cell. Western blotting analysis of the CF protein extracts using an antibody against β-tubulin, a cytoplasmic protein, indicated that our CF protein preparations are free from any detectable amounts of this marker protein. Therefore, our sorghum cell culture system is ideal for use in the proteomic analysis of secreted proteins. The findings of this study are a step in the process of bridging the gap that currently exists in sorghum proteomics and also provides a foundation for future studies on understanding the roles played by secreted proteins during plant growth and development of the same crop.Keywords: Sorghum, cell suspension cultures, culture filtrate, secreted proteins, proteomics analysis, two-dimensional gel electrophoresis, β-tubuli

Mapping and characterisation of the sorghum cell suspension culture secretome

Here we reported the first secretomic study of sorghum (Sorghum bicolor), a naturally drought tolerant cereal crop. In this study, we used a gel-based proteomic approach in combination with mass spectrometry to separate and identify proteins secreted into the culture medium of sorghum cell suspensions, a first step towards understanding their functions during plant growth and development. Proteins secreted into the culture medium of 10-day old sorghum cell suspension cultures termed culture filtrate (CF) proteins were separated by two-dimensional gel electrophoresis (2DE) and visualised using Coomassie brilliant blue (CBB) R-250 staining. Of the 25 visualised CBB stainable spots, 15 abundant, well-resolved and reproducible spots amongst the three biological replicates used were selected for identification using matrix assisted laser desorption/ionisation-time of flight/time of flight tandem mass spectrometry (MALDI-TOF-TOF MS). Of these spots, 14 were positively identified, representing four different protein classes: Peroxidases, germin proteins, oxalate oxidases and ?-galactosidases. All the identified proteins are known secretory proteins, with predicted signal peptides, which target proteins for the secretory pathway. The identified proteins have known functions in signalling processes, defence mechanisms and cell wall metabolism which is consistent with their location outside the cell. Western blotting analysis of the CF protein extracts using an antibody against ?-tubulin, a cytoplasmic protein, indicated that our CF protein preparations are free from any detectable amounts of this marker protein. Therefore, our sorghum cell culture system is ideal for use in the proteomic analysis of secreted proteins. The findings of this study are a step in the process of bridging the gap that currently exists in sorghum proteomics and also provides a foundation for future studies on understanding the roles played by secreted proteins during plant growth and development of the same crop

Characterisation of Thiol-releasing and Lower Volatile Acidityforming Intra-genus Hybrid Yeast Strains for Sauvignon blanc Wine

A single Saccharomyces cerevisiae wine yeast strain produces a range of aroma and flavour metabolites (e.g. volatile thiols), as well as unfavourable metabolites (e.g. volatile acidity [VA]) during the alcoholic fermentation of white wine, especially Sauvignon blanc. The former contributes to the organoleptic quality of the final wine. Previous research showed that yeast derived enzymes (proteins) are involved in the release of wine quality enhancing or reducing metabolites during fermentation. Small-scale winemaking trials were initiated to evaluate protein expression and metabolite release of tropical fruit aroma wine producing S. cerevisiae hybrid yeasts. Commercial ‘thiol-releasing’ wine yeasts (TRWY) were included in winemaking trials as references. Improved hybrids were identified which showed enhanced thiol-releasing, specifically 3-mercaptohexanol (3MH), and lower VA formation during the production of Sauvignon blanc wines compared to some commercial TRWY references. It is noteworthy that the hybrid NH 56 produced wines with the second highest 3MH levels after hybrid NH 84, and lowest acetic acid of all strains included in this study. This yeast was also the only strain to have down-regulated proteins linked to amino acid biosynthesis, pentose phosphate pathway, glycolysis and fructose and galactose metabolism during the lag phase. Furthermore, differences in protein expression were reflected in the variation of metabolite release by different strains, thereby confirming that enzymes (proteins) are the final effectors for metabolite release. 

Establishment of sorghum cell suspension culture system for proteomics studies

This study describes the establishment of sorghum cell suspension culture system for use in proteomics studies. Friable sorghum callus was initiated from young shoots under completely dark conditions on MS medium supplemented with 3 mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 2.5 mg/L 1-naphthaleneacetic acid (NAA). Additionally, sorghum cell suspension cultures have been initiated from the friable callus masses in liquid medium with the same composition as the callusinitiation medium. Total soluble proteins (TSP) and culture filtrate (CF) proteins were extracted from the cell culture system and solubilised in urea buffer (9 M urea, 2 M thiourea and 4% CHAPS). Both onedimensional (1D) and two-dimensional (2D) gel analysis of these two proteomes show that the TSP and CF proteomes have different protein expression profiles. The sorghum TSP proteome, which is highlycomplex, is best resolved when separated on large format, 18 cm, pH 4 - 7 isoelectric focusing (IEF) immobilised pH gradient (IPG) strips. On the other hand, the sorghum CF proteome (secretome) is lesscomplex with most proteins being resolved on mini format, 7 cm, pH 3 - 10 IPG strips. Furthermore, narrowing down the pH range from 3 - 10 to 4 - 7 for the CF proteome resulted in improved protein spotresolution

A Comparative Study of Selected Physical and Biochemical Traits of Wild-Type and Transgenic Sorghum to Reveal Differences Relevant to Grain Quality

Transgenic sorghum featuring RNAi suppression of certain kafirins was developed recently, to address the problem of poor protein digestibility in the grain. However, it was not firmly established if other important quality parameters were adversely affected by this genetic intervention. In the present study several quality parameters were investigated by surveying several important physical and biochemical grain traits. Important differences in grain weight, density and endosperm texture were found that serve to differentiate the transgenic grains from their wild-type counterpart. In addition, ultrastructural analysis of the protein bodies revealed a changed morphology that is indicative of the effect of suppressed kafirins. Importantly, lysine was found to be significantly increased in one of the transgenic lines in comparison to wild-type; while no significant changes in anti-nutritional factors could be detected. The results have been insightful for demonstrating some of the corollary changes in transgenic sorghum grain, that emerge from imposed kafirin suppression

FINANCEMENT DES SOINS PAR APROCHE MUTUELLE DE SANTE : AVIS DES ENSEIGNANTS D’EPST-RD CONGO SUR LA QUALITE DE SERVICES DE LA MESP.

The problem of access to health care poses a problem in our country [Amuli jiwe,2014]. Primary, secondary, and vocational chalk professionals are not excluded. To avoid the uncontrolled ou flow of funds, the means being limited, although the need is enormous, the teachers are affiliated to the mutual health insurance, while it is noted that 9 out of 10 Teachers who attend medical training in partnership with the said mutual health insurance are really disappointed with the quality of care and the management of the mutual, which is why we asked ourselves the question; what are the factors associated with the dissatisfaction of teachers at EPST about their mutual insurance we started from a hypothesis according to the dissatisfaction of MESP members would be linked to the quality of care, which would be less effective. Our scientific study was carried out in the city province of Kinshasa with the MESP. We used the survey method and the structured interview technique guided by the survey questionnaire, which served as an interview of 384 participants obtained using the inclusion and exclusion criteria and Fisher statistical formula [Bura P.2014]. After analysis and interpretation of the data, we found the following results:205 respondents, 53,3% say that members' money is more allocated for the functioning of the administration(salaries of agents and workers of the mutual fund) than health care is members followed by 84 or 21,8%  who say that the money for health is not only implanted in some four cities out of the  26 provinces of the country. On the other hand, 123members or 32,1%  blame the health facilities for the less satisfactory quality of relational care, followed by 103 or 26,8% who attest that the number of health facilities is insufficient for the best care of patients who are members of the mutual insurance company, 13or 3,4 who would say that the hygiene of hospitals is defective.  The problem of access to health care poses a problem in our country [Amuli jiwe,201]. Primary, secondary, and vocational chalk professionals are not excluded. To avoid the uncontrolled ou flow of funds, the means being limited, although the need is enormous, the teachers are affiliated to the mutual health insurance, while it is noted that 9 out of 10 Teachers who attend medical training in partnership with the said mutual health insurance are really disappointed with the quality of care and the management of the mutual, which is why we asked ourselves the question; what are the factors associated with the dissatisfaction of teachers at EPST about their mutual insurance we started from a hypothesis according to the dissatisfaction of MESP members would be linked to the quality of care, which would be less effective. Our scientific study was carried out in the city province of Kinshasa with the MESP. We used the survey method and the structured interview technique guided by the survey questionnaire, which served as an interview of 384 participants obtained using the inclusion and exclusion criteria and Fisher statistical formula [Bura P.2014]. After analysis and interpretation of the data, we found the following results:205 respondents, 53,3% say that members' money is more allocated for the functioning of the administration(salaries of agents and workers of the mutual fund) than health care is members followed by 84 or 21,8% &nbsp;who say that the money for health is not only implanted in some four cities out of the &nbsp;26 provinces of the country. On the other hand, 123members or 32,1% &nbsp;blame the health facilities for the less satisfactory quality of relational care, followed by 103 or 26,8% who attest that the number of health facilities is insufficient for the best care of patients who are members of the mutual insurance company, 13or 3,4 who would say that the hygiene of hospitals is defective. &nbsp

INPPO actions and recognition as a driving force for progress in plant proteomics: Change of guard, INPPO update, and upcoming activities

The International Plant Proteomics Organization (INPPO) is a non-profit organization whose members are scientists involved or interested in plant proteomics. Since the publication of the first INPPO highlights in 2012, continued progress on many of the organization’s mandates/goals has been achieved. Two major events are emphasized in this second INPPO highlights. First, the change of guard at the top, passing of the baton from Dominique Job, INPPO founding President to Ganesh Kumar Agrawal as the incoming President. Ganesh K. Agrawal, along with Dominique Job and Randeep Rakwal initiated the INPPO. Second, the most recent INPPO achievements and future targets, mainly the organization of first the INPPO World Congress in 2014, tentatively planned for Hamburg (Germany), are mentioned.Web of Scienc