V2: Integrated management of rainwater for crop-livestock agroecosystems

With mixed crop-livestock systems projected to remain the main providers of food in the coming decades, opportunities exist for smallholders to participate and benefit from emerging crop and livestock markets in the Volta Basin. This project intends to identify, evaluate, adapt, and disseminate best-fit integrated rainwater management strategies (RMS), targeted to different biophysical and socio-economic domains. The integrated RMS are comprised of technological solutions, directed at different components of the agroecosystems, underpinned by enabling institutional and policy environments and linked to market incentives that can drive adoptio

The toxicological intersection between allergen and toxin: A structural comparison of the cat dander allergenic protein Fel d1 and the slow loris brachial gland secretion protein

Slow lorises are enigmatic animal that represent the only venomous primate lineage. Their defensive secretions have received little attention. In this study we determined the full length sequence of the protein secreted by their unique brachial glands. The full length sequences displayed homology to the main allergenic protein present in cat dander. We thus compared the molecular features of the slow loris brachial gland protein and the cat dander allergen protein, showing remarkable similarities between them. Thus we postulate that allergenic proteins play a role in the slow loris defensive arsenal. These results shed light on these neglected, novel animals

Antifungal benzo[b]thiophene 1,1-dioxide IMPDH inhibitors exhibit pan-assay interference (PAINS) profiles

Fungi cause serious life-threatening infections in immunocompromised individuals and current treatments are now complicated by toxicity issues and the emergence of drug resistant strains. Consequently, there is a need for development of new antifungal drugs. Inosine monophosphate dehydrogenase (IMPDH), a key component of the de novo purine biosynthetic pathway, is essential for growth and virulence of fungi and is a potential drug target. In this study, a high-throughput screen of 114,000 drug-like compounds against Cryptococcus neoformans IMPDH was performed. We identified three 3-((5-substituted)-1,3,4-oxadiazol-2-yl)thio benzo[b]thiophene 1,1-dioxides that inhibited Cryptococcus IMPDH and also possessed whole cell antifungal activity. Analogs were synthesized to explore the SAR of these hits. Modification of the fifth substituent on the 1,3,4-oxadiazole ring yielded compounds with nanomolar in vitro activity, but with associated cytotoxicity. In contrast, two analogs generated by substituting the 1,3,4-oxadiazole ring with imidazole and 1,2,4-triazole gave reduced IMPDH inhibition in vitro, but were not cytotoxic. During enzyme kinetic studies in the presence of DTT, nucleophilic attack of a free thiol occurred with the benzo[b]thiophene 1,1-dioxide. Two representative compounds with substitution at the 5 position of the 1,3,4-oxadiazole ring, showed mixed inhibition in the absence of DTT. Incubation of these compounds with Cryptococcus IMPDH followed by mass spectrometry analysis showed non-specific and covalent binding with IMPDH at multiple cysteine residues. These results support recent reports that the benzo[b]thiophene 1,1-dioxides moiety as PAINS (pan-assay interference compounds) contributor

Direct involvement of the TEN domain at the active site of human telomerase

Telomerase is a ribonucleoprotein that adds DNA to the ends of chromosomes. The catalytic protein subunit of telomerase (TERT) contains an N-terminal domain (TEN) that is important for activity and processivity. Here we describe a mutation in the TEN domain of human TERT that results in a greatly increased primer Kd, supporting a role for the TEN domain in DNA affinity. Measurement of enzyme kinetic parameters has revealed that this mutant enzyme is also defective in dNTP polymerization, particularly while copying position 51 of the RNA template. The catalytic defect is independent of the presence of binding interactions at the 5′-region of the DNA primer, and is not a defect in translocation rate. These data suggest that the TEN domain is involved in conformational changes required to position the 3′-end of the primer in the active site during nucleotide addition, a function which is distinct from the role of the TEN domain in providing DNA binding affinity

The molybdoproteome of Starkeya novella - insights into the diversity and functions of molybdenum containing proteins in response to changing growth conditions

Molybdenum enzymes are known to underpin key reactions in the biological carbon, nitrogen and sulfur cycles, however, the diversity of these enzymes and the reactions they catalyze especially in bacteria is much greater than currently known. We have analysed the molybdoproteome of the soil bacterium Starkeya novella as a function of growth mode and identified a complete pathway for Mo-PPT synthesis, a Mo transporter and 18 gene loci encoding mononuclear Mo-enzymes of the Xanthine Oxidase, Sulfite Oxidase and DMSO Reductase enzyme families. This relatively high number of Mo enzymes may be a specific property of the taxonomic group (Xanthobacteraceae) to which S. novella belongs. About 70% of S. novella Mo enzymes have no characterized close relatives, and two thirds of them are expressed under the conditions analysed, which included heterotrophy, methylotrophy, chemolithotrophy and mixotrophy. Many enzymes were clearly regulated in response to either the type of carbon source present in the growth medium or the presence of thiosulfate, and two, in particular, including an uncharacterized enzyme of the XO family (Snov_3370) were highly abundant under all growth conditions tested. We also uncovered novel enzymes with links to growth in the presence of thiosulfate, such as a PaoABC-type aldehyde oxidoreductase and an uncharacterized group 1 sulfite oxidase family enzyme, although the function of these enzymes during sulfur oxidation is unclear at present. Clearly, further work is needed to uncover the significance of these enzymes for cell metabolism

Metabolic adaptation and trophic strategies of soil bacteria--C1- metabolism and sulfur chemolithotrophy in Starkeya novella

The highly diverse and metabolically versatile microbial communities found in soil environments are major contributors to the global carbon, nitrogen, and sulfur cycles. We have used a combination of genome based pathway analysis with proteomics and gene expression studies to investigate metabolic adaptation in a representative of these bacteria, Starkeya novella, which was originally isolated from agricultural soil. This bacterium was the first facultative sulfur chemolithoautotroph that was isolated and it is also able to grow with methanol and on over 39 substrates as a heterotroph. However, using glucose, fructose, methanol, thiosulfate as well as combinations of the carbon compounds with thiosulfate as growth substrates we have demonstrated here that contrary to the previous classification, S. novella is not a facultative sulfur chemolitho- and methylotroph, as the enzyme systems required for these two growth modes are always expressed at high levels. This is typical for key metabolic pathways. In addition enzymes for various pathways of carbon dioxide fixation were always expressed at high levels, even during heterotrophic growth on glucose or fructose, which suggests a role for these pathways beyond the generation of reduced carbon units for cell growth, possibly in redox balancing of metabolism. Our results then indicate that S. novella, a representative of the Xanthobacteraceae family of methylotrophic soil and freshwater dwelling bacteria, employs a mixotrophic growth strategy under all conditions tested here. As a result the contribution of this bacterium to either carbon sequestration or the release of climate active substances could vary very quickly, which has direct implications for the modeling of such processes if mixotrophy proves to be the main growth strategy for large populations of soil bacteria

Proteome analysis of outer membrane and extracellular proteins from Pseudomonas aeruginosa for vaccine discovery

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The synaptic proteome in Alzheimer's disease

Background: Synaptic dysfunction occurs early in Alzheimer's disease (AD) and is recognized to be a primary pathological target for treatment. Synapse degeneration or dysfunction contributes to clinical signs of dementia through altered neuronal communication; the degree of synaptic loss correlates strongly with cognitive impairment. The molecular mechanisms underlying synaptic degeneration are still unclear, and identifying abnormally expressed synaptic proteins in AD brain will help to elucidate such mechanisms and to identify therapeutic targets that might slow AD progression. Methods: Synaptosomal fractions from human autopsy brain tissue from subjects with AD (n = 6) and without AD (n = 6) were compared using two-dimensional differential in-gel electrophoresis. AD pathology is region specific; human subjects can be highly variable in age, medication, and other factors. To counter these factors, two vulnerable areas (the hippocampus and the temporal cortex) were compared with two relatively spared areas (the motor and occipital cortices) within each group. Proteins exhibiting significant changes in expression were identified (≥20% change, Newman-Keuls P valu

SWATH analysis of the synaptic proteome in Alzheimer's disease

Brain tissue from Alzheimer's disease patients exhibits synaptic degeneration in selected regions. Synaptic dysfunction occurs early in the disease and is a primary pathological target for treatment. The molecular mechanisms underlying this degeneration remain unknown. Quantifying the synaptic proteome in autopsy brain and comparing tissue from Alzheimer's disease cases and subjects with normal aging are critical to understanding the molecular mechanisms associated with Alzheimer pathology. We isolated synaptosomes from hippocampus and motor cortex so as to reduce sample complexity relative to whole-tissue homogenates. Synaptosomal extracts were subjected to strong cation exchange (SCX) fractionation to further partition sample complexity; each fraction received SWATH-based information-dependent acquisition to generate a comprehensive peptide-ion library. The expression of synaptic proteins from AD hippocampus and motor cortex was then compared between groups. A total of 2077 unique proteins were identified at a critical local false discovery rat

Targeted quantitative analysis of synaptic proteins in Alzheimer's disease brain

Brain tissue from Alzheimer's disease (AD) patients shows significant loss of synapses in selected regions. Synaptic degeneration is the best predictor for loss of cognitive functions ante mortem. The molecular mechanisms underlying this degeneration remain unknown. Our previous two-dimensional gel-electrophoresis proteomics study found that 26 synaptic proteins are differentially expressed in Alzheimer's brain. It is difficult to quantify global protein expression using this technique because (a) several proteins can migrate together and (b) isoforms of the same protein can migrate to different places. The present study estimated global synaptic protein levels by label-free multiple reaction monitoring. Multiple reaction monitoring is a powerful and sensitive mass spectrometry technique that specifically targets multiple protein of interests. The severely AD-affected hippocampus was compared with motor cortex, a relatively spared region. We targeted ten proteins in autopsy brain based on the earlier study. Analytes separated by high performance liquid-chromatography were monitored on a hybrid triple quadrupole linear ion trap mass spectrometer in multiple reaction monitoring mode. With the use of an internal standard protein, linear and highly reproducible (CV < 9%) label-free assays were achieved. Data were contrasted with the gel-based study to highlight differences and similarities. Significantly higher expression levels of peroxiredoxin-1 (may provide antioxidant protection) and dihydropyrimidinase-related protein-1 (associated with cytoskeletal remodeling) were found in AD hippocampus. Significantly lower levels of peroxiredoxin-1 and the energy-related enzymes creatine kinase B and fructose-bisphosphate aldolase C were found in non-AD hippocampus. Our previously reported difference in synaptotagmin expression is probably isoform-specific. These findings suggest potential roles of key proteins in synaptic loss in AD, and/or a protective mechanism in non-AD brain tissue