NMR metabolomics reveals effects of Cryptosporidium infections on host cell metabolome

Background: Cryptosporidium is an important gut microbe whose contributions towards infant and immunocompromise patient mortality rates are steadily increasing. Over the last decade, we have seen the development of various tools and methods for studying Cryptosporidium infection and its interactions with their hosts. One area that is sorely overlooked is the effect infection has on host metabolic processes. Results: Using a 1H nuclear magnetic resonance approach to metabolomics, we have explored the nature of the mouse gut metabolome as well as providing the first insight into the metabolome of an infected cell line. Statistical analysis and predictive modelling demonstrated new understandings of the effects of a Cryptosporidium infection, while verifying the presence of known metabolic changes. Of note is the potential contribution of host derived taurine to the diarrhoeal aspects of the disease previously attributed to a solely parasite-based alteration of the gut environment, in addition to other metabolites involved with host cell catabolism. Conclusion: This approach will spearhead our understanding of the Cryptosporidium-host metabolic exchange and provide novel targets for tackling this deadly parasite

A redox-active switch in fructosamine-3-kinases expands the regulatory repertoire of the protein kinase superfamily

Aberrant regulation of metabolic kinases by altered redox homeostasis substantially contributes to aging and various diseases, such as diabetes. We found that the catalytic activity of a conserved family of fructosamine-3-kinases (FN3Ks), which are evolutionarily related to eukaryotic protein kinases, is regulated by redox-sensitive cysteine residues in the kinase domain. The crystal structure of the FN3K homolog from Arabidopsis thaliana revealed that it forms an unexpected strand-exchange dimer in which the ATP-binding P-loop and adjoining β strands are swapped between two chains in the dimer. This dimeric configuration is characterized by strained interchain disulfide bonds that stabilize the P-loop in an extended conformation. Mutational analysis and solution studies confirmed that the strained disulfides function as redox “switches” to reversibly regulate the activity and dimerization of FN3K. Human FN3K, which contains an equivalent P-loop Cys, was also redox sensitive, whereas ancestral bacterial FN3K homologs, which lack a P-loop Cys, were not. Furthermore, CRISPR-mediated knockout of FN3K in human liver cancer cells altered the abundance of redox metabolites, including an increase in glutathione. We propose that redox regulation evolved in FN3K homologs in response to changing cellular redox conditions. Our findings provide insights into the origin and evolution of redox regulation in the protein kinase superfamily and may open new avenues for targeting human FN3K in diabetic complications

Structural characterisation of marine glycosaminoglycans and their interactions with proteins.

Glycosaminoglycans (GAGs) are a group of structurally related, naturally occurring polysaccharides, found as the carbohydrate moieties of proteoglycans and sometimes as free polysaccharides. GAGs are expressed ubiquitously on animal cell surfaces and within extracellular matrices. All GAGs are sulfated to various degrees, except hyaluronan, which is always non-sulfated. In this project, GAGs and other sulfated carbohydrates were purified from different marine sources and an algae species living in soil, and their structures were characterised, mainly by NMR spectroscopy. Oversulfated dermatan sulfate (DS), fucosylated chondroitin sulfate (fCS) with interesting anti-metastatic properties, a sulfated fucosylated GlcNAc polymer, naturally-occurring cellulose sulfate and a polysaccharide with a repeating disaccharide unit of IdoA-Gal, were some of the more unusual carbohydrates identified. Common GAGs like hyaluronan, chondroitin sulfate A and C, DS and heparin/heparin sulfate-like polysaccharides were also purified from lumpsuckers and ragoworms. Different depolymerisation techniques were also investigated. The effects of Fentontype reaction and photochemical free radical depolymerisation on DS were studied. Elimination of the reducing end IdoA in the case of Fenton-type produced oligosaccharides was established. The oxidisation of reducing end GalNAc to Nacetylgalactosaminic acid, as a result of both depolymerisation techniques, was also identified. In addition, the effect of mild acid hydrolysis on fCS polysaccharides were studied and this technique was deemed unfitting for the depolymerisation of the molecule, as it causes defucosylation and partial desulfation. Finally, human beta-defensin 2 (HBD2) was expressed recombinantly, as a fusion protein with pE-Sumo, in an attempt to design a high yield expression system capable of producing correctly folded protein. The significance of NMR in the identification of the state of the protein and the need for an expression system capable of carrying out correctly the post-translational modifications was demonstrated

Metabolic changes of the host-pathogen environment in a Cryptosporidium infection

Cryptosporidium is an important gut microbe whose contributions towards infant and immunocompromise patient mortality rates are steadily increasing. Current techniques for diagnosing, curing or simply understanding the biology of the parasite are few and far between, relying on a combination of in-silico predictions modelled on a varied and unique group of organisms and medical reports. The development of an in-vitro culture system, using COLO-680N cells, has provided the Cryptosporidium community with the opportunity to expand its toolkit for investigating this disease. One area in particular that is sorely overlooked is the metabolic alterations upon infection. Existing research is extremely limited and has already shown that significant variation can be found between the metabolome of different infected host species. Using a 1H Nuclear Magnetic Resonance approach to metabolomics, we have explored the nature of the mouse gut metabolome as well as providing the first insight into the metabolome of an infected cell line. Through a combination of Partial Least Squares Discriminant Analysis and predictive modelling, we exhibit new and potentially game changing insights into the effects of a Cryptosporidium parvum infection, while verifying the presence of known metabolic changes. Of particular note is the potential contribution of host derived taurine to the diuretic aspects of the disease previously attributed to a solely parasite based alteration of the gut environment. This practical and informative approach can spearhead our understanding of the Cryptosporidium-host metabolic exchange and thus provide novel targets for tackling this deadly parasite

Digestive activity and organic compounds of Nezara viridula watery saliva induce defensive soybean seed responses

The stink bug Nezara viridula is one of the most threatening pests for agriculture in North and South America, and its oral secretion may be responsible for the damage it causes in soybean (Glycine max) crop. The high level of injury to seeds caused by pentatomids is related to their feeding behavior, morphology of mouth parts, and saliva, though information on the specific composition of the oral secretion is scarce. Field studies were conducted to evaluate the biochemical damage produced by herbivory to developing soybean seeds. We measured metabolites and proteins to profile the insect saliva in order to understand the dynamics of soybean-herbivore interactions. We describe the mouth parts of N. viridula and the presence of metabolites, proteins and active enzymes in the watery saliva that could be involved in seed cell wall modification, thus triggering plant defenses against herbivory. We did not detect proteins from bacteria, yeasts, or soybean in the oral secretion after feeding. These results suggest that the digestive activity and organic compounds of watery saliva may elicit a plant self-protection response. This study adds to our understanding of stink bug saliva plasticity and its role in the struggle against soybean defenses.Fil: Giacometti, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; Argentina. Universidad de Buenos Aires. Facultad de Agronomía; ArgentinaFil: Jacobi, Vanesa Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; ArgentinaFil: Kronberg, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; Argentina. Universidad de Buenos Aires. Facultad de Agronomía; ArgentinaFil: Panagos, Charalampos. University of Georgia; Estados UnidosFil: Edison, Arthur Scott. University of Georgia; Estados UnidosFil: Zavala, Jorge Alberto. Universidad de Buenos Aires; Argentina. Instituto de Investigaciones En Biociencias Agrícolas y Ambientales; Argentin

Fucosylated chondroitin sulfates from the body wall of the sea cucumberholothuria forskali

Fucosylated chondroitin sulfate (fCS) extracted from the sea cucumber Holothuria forskali is composed of the following repeating trisaccharide unit: -> 3)GalNAc beta 4,6S(1 -> 4) [Fuc alpha X(1 -> 3)]GlcA beta(1 ->, where X stands for different sulfation patterns of fucose (X = 3,4S (46%), 2,4S (39%), and 4S (15%)). As revealed by NMR and molecular dynamics simulations, the fCS repeating unit adopts a conformation similar to that of the Le(x) blood group determinant, bringing several sulfate groups into close proximity and creating large negative patches distributed along the helical skeleton of the CS backbone. This may explain the high affinity of fCS oligosaccharides for L-and P-selectins as determined by microarray binding of fCS oligosaccharides prepared by Cu2+-catalyzed Fenton-type and photochemical depolymerization. No binding to E-selectin was observed. fCS poly- and oligosaccharides display low cytotoxicity in vitro, inhibit human neutrophil elastase activity, and inhibit the migration of neutrophils through an endothelial cell layer in vitro. Although the polysaccharide showed some anti-coagulant activity, small oligosaccharide fCS fragments had much reduced anticoagulant properties, with activity mainly via heparin cofactor II. The fCS polysaccharides showed prekallikrein activation comparable with dextran sulfate, whereas the fCS oligosaccharides caused almost no effect. The H. forskali fCS oligosaccharides were also tested in a mouse peritoneal inflammation model, where they caused a reduction in neutrophil infiltration. Overall, the data presented support the action of fCS as an inhibitor of selectin interactions, which play vital roles in inflammation and metastasis progression. Future studies of fCS-selectin interaction using fCS fragments or their mimetics may open new avenues for therapeutic intervention

A redox-active switch in fructosamine-3-kinases expands the regulatory repertoire of the protein kinase superfamily

Aberrant regulation of metabolic kinases by altered redox homeostasis substantially contributes to aging and various diseases, such as diabetes. We found that the catalytic activity of a conserved family of fructosamine-3-kinases (FN3Ks), which are evolutionarily related to eukaryotic protein kinases, is regulated by redox-sensitive cysteine residues in the kinase domain. The crystal structure of the FN3K homolog from Arabidopsis thaliana revealed that it forms an unexpected strand-exchange dimer in which the ATP-binding P-loop and adjoining β strands are swapped between two chains in the dimer. This dimeric configuration is characterized by strained interchain disulfide bonds that stabilize the P-loop in an extended conformation. Mutational analysis and solution studies confirmed that the strained disulfides function as redox "switches" to reversibly regulate the activity and dimerization of FN3K. Human FN3K, which contains an equivalent P-loop Cys, was also redox sensitive, whereas ancestral bacterial FN3K homologs, which lack a P-loop Cys, were not. Furthermore, CRISPR-mediated knockout of FN3K in human liver cancer cells altered the abundance of redox metabolites, including an increase in glutathione. We propose that redox regulation evolved in FN3K homologs in response to changing cellular redox conditions. Our findings provide insights into the origin and evolution of redox regulation in the protein kinase superfamily and may open new avenues for targeting human FN3K in diabetic complications

Use of Modis Satellite Images for Detailed Lake Morphometry: Application to Basins with Large Water Level Fluctuations

Lake morphometry is essential for managing water resources and limnetic ecosystems. For reservoirs that receive high sediment loads, frequent morphometric mapping is necessary to define both the effective life of the reservoir and its water storage capacity for irrigation, power generation, flood control and domestic water supply. The current study presents a methodology for updating the digital depth model (DDM) of lakes and reservoirs with wide intra and interannual fluctuations of water levels using satellite remote sensing. A time series of Terra MODIS satellite images was used to map shorelines formed during the annual water level change cycle, and were validated with concurrent Landsat ETM+ satellite images. The shorelines were connected with in-situ observation of water levels and were treated as elevation contours to produce the DDM using spatial interpolation. The accuracy of the digitized shorelines is within the mapping accuracy of the satellite images, while the resulting DDM is validated using in-situ elevation measurements. Two versions of the DDM were produced to assess the influence of seasonal water fluctuation. Finally, the methodology was applied to Lake Kerkini (Greece) to produce an updated DDM, which was compared with the last available bathymetric survey (1991) and revealed changes in sediment distribution within the lake