Nano-biosensor development for bacterial detection during human kidney infection: Use of glycoconjugate-specific antibody-bound gold NanoWire arrays (GNWA)

Infectious disease, commonly caused by bacterial pathogens, is now the worlds leading cause of premature death and third overall cause behind cardiovascular disease and cancer. Urinary Tract Infection (UTI), caused by E. coli bacteria, is a very common bacterial infection, a majority in women (85%) and may result in severe kidney failure if not detected quickly. Among hundreds of strains the bacteria, E. coli 0157:H7, is emerging as the most aggressive one because of its capability to produce a toxin causing hemolytic uremic syndrome (HUS) resulting in death, especially in children. In the present study, a project has been undertaken for developing a rapid method for UTI detection in very low bacteria concentration, applying current knowledge of nano-technology. Experiments have been designed for the development of biosensors using nano-fabricated structures coated with elements such as gold that have affinity for biomolecules. A biosensor is a device in which a biological sensing element is either intimately connected to or integrated within a transducer. The basic principle for the detection procedure of the infection is partly based on the enzyme-linked immunosorbent assay system. Anti-E. coli antibody-bound Gold Nanowire Arrays (GNWA) prepared on anodized porous alumina template is used for the primary step followed by binding of the bacteria containing specimen. An alkaline phosphatase-conjugated second antibody is then added to the system and the resultant binding determined by both electrochemical and optical measurements. Various kinds of GNWA templates were used in order to determine the one with the best affinity for antibody binding. In addition, an efficient method for enhanced antibody binding has been developed with the covalent immobilization of an organic linker Dithiobissuccinimidylundecanoate (DSU) on the GNWA surface. Studies have also been conducted to optimize the antibody-binding conditions to the linker-attached GNWA surfaces for their ability to detect bacteria in clinical concentrations. Published in 2004.     

Population genetics and history of the introduced fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), in Australia

Abstract  The red imported fire ant, Solenopsis invicta , a damaging invasive pest, was discovered in February 2001 in Brisbane, Australia at two sites, Fisherman Islands and suburban Richlands-Wacol. Using four microsatellite loci and the protein marker Gp-9 , we compared the two infestations with each other, and with potential source populations in North and South America to better understand the history of their introduction to Brisbane. Based on an analysis of molecular variance, as well as a maximum likelihood tree of colonies from the two Australian sites, we found that the two sites were genetically distinct and were almost certainly introduced separately. All of the colonies at Fisherman Islands were monogynous, headed by a single queen, while the Richlands-Wacol site had a mixture of single-queen monogynous and multiple-queen polygynous colonies. However, the monogynous and polygynous colonies at the Richlands-Wacol site were not genetically distinct from each other, and probably constitute a single, mixed introduction. Based on allele frequencies at the microsatellite loci, and Gp-9 , both Australian infestations were more similar to North American populations than to South American, though the Fisherman Islands infestation was intermediate, making it difficult to assign. Thus, there has been one introduction from either a North or South American monogynous population at Fisherman Islands, and one introduction from a mixed monogynous/polygynous North American population at Richlands-Wacol. These findings have implications for the control of the current infestations, as well as for the quarantine regulations necessary to prevent additional introductions to Australia.     

The family 6 carbohydrate binding module CmCBM6-2 contains two ligand-binding sites with distinct specificities

The microbial degradation of the plant cell wall is an important biological process, representing a major component of the carbon cycle. Enzymes that mediate the hydrolysis of this composite structure are modular proteins that contain non-catalytic carbohydrate binding modules (CBMs) that enhance catalytic activity. CBMs are grouped into sequence-based families, and in a previous study we showed that a family 6 CBM (CBM6) that interacts with xylan contains two potential ligand binding clefts, designated cleft A and cleft B. Mutagenesis and NMR studies showed that only cleft A in this protein binds to xylan. Family 6 CBMs bind to a range of polysaccharides, and it was proposed that the variation in ligand specificity observed in these proteins reflects the specific cleft that interacts with the target carbohydrate. Here the biochemical properties of the C-terminal cellulose binding CBM6 (CmCBM6-2) from Cellvibrio mixtus endoglucanase 5A were investigated. The CBM binds to the beta1,4-beta1,3-mixed linked glucans lichenan and barley beta-glucan, cello-oligosaccharides, insoluble forms of cellulose, the beta1,3-glucan laminarin, and xylooligosaccharides. Mutagenesis studies, informed by the crystal structure of the protein (presented in the accompanying paper, Pires, V. M. R., Henshaw, J. L., Prates, J. A. M., Bolam, D., Ferreira, L. M. A. Fontes, C. M. G. A., Henrissat, B., Planas, A., Gilbert, H. J., Czjzek, M. (2004) J. Biol. Chem. 279, 21560-21568), show that both cleft A and B can accommodate cello-oligosaccharides and laminarin displays a preference for cleft A, whereas xylooligosaccharides exhibit absolute specificity for this site, and the beta1,4,-beta1,3-mixed linked glucans interact only with cleft B. The binding of CmCBM6-2 to insoluble cellulose involves synergistic interactions between cleft A and cleft B. These data show that CmCBM6-2 contains two binding sites that display differences in ligand specificity, supporting the view that distinct binding clefts with different specificities can contribute to the variation in ligand recognition displayed by family 6 CBMs. This is in sharp contrast to other CBM families, where variation in ligand binding is a result of changes in the topology of a single carbohydrate-binding site.     

金翅夜蛾亚科八新种及一些种类的订正

金翅夜蛾亚科的种类,有的在外形上非常相似,因之过去文献记载常有混淆的地方和鉴定上的错误。本文讨论了一些种类的区别,纠正某些错误的记载,并报道8个新种。 所有新种的模式标本都暂在西北农学院昆虫标本室保存。     

Mating system and population structure of the primitively eusocial wasp Ropalidia revolutionalis: a model system for the evolution of complex societies

Mating systems are important determinants of genetic structure in cooperative groups, and their effects can influence profoundly the interactions of group members. The primitively eusocial wasp, Ropalidia revolutionalis, has an interesting genetic and social structure that makes it an excellent model system for examining the evolution of more complex societies. In particular, its colonies sometimes have multiple queens, a key characteristic of more advanced wasp societies. In this study, we have characterized the mating system of the social wasp Ropalidia revolutionalis to understand better its colony genetic structure. R. revolutionalis females nearly always mate singly and they are unrelated to their mates. However, different females in the same colony do mate with males, on average, who are related as cousins. Single mating will help to maintain high relatedness, which should be important for continued cooperation in multiple queen societies, but it creates potential conflicts in single queen colonies over the production of males as well as over the timing of male production. We have also characterized the population structure of R. revolutionalis from Townsville, in tropical north Queensland, to Brisbane in the subtropics. Even at such a large scale, the population is remarkably unstructured with an average F(ST) of 0.0546. There is weak isolation by distance, and evidence for subtle differentiation between a southern region with no dry season, which extends as far north as Rockhampton, and a northern region with a severe to moderate dry season. This may reflect historical effects of extreme aridity on the population structure.     

Escherichia coli LipA is a lipoyl synthase: in vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein

The Escherichia coli lipA gene product has been genetically linked to carbon-sulfur bond formation in lipoic acid biosynthesis [Vanden Boom, T. J., Reed, K. E., and Cronan, J. E., Jr. (1991) J. Bacteriol. 173, 6411-6420], although in vitro lipoate biosynthesis with LipA has never been observed. In this study, the lipA gene and a hexahistidine tagged lipA construct (LipA-His) were overexpressed in E. coli as soluble proteins. The proteins were purified as a mixture of monomeric and dimeric species that contain approximately four iron atoms per LipA polypeptide and a similar amount of acid-labile sulfide. Electron paramagnetic resonance and electronic absorbance spectroscopy indicate that the proteins contain a mixture of [3Fe-4S] and [4Fe-4S] cluster states. Reduction with sodium dithionite results in small quantities of an S = 1/2 [4Fe-4S](1+) cluster with the majority of the protein containing a species consistent with an S = 0 [4Fe-4S](2+) cluster. LipA was assayed for lipoate or lipoyl-ACP formation using E. coli lipoate-protein ligase A (LplA) or lipoyl-[acyl-carrier-protein]-protein-N-lipoyltransferase (LipB), respectively, to lipoylate apo-pyruvate dehydrogenase complex (apo-PDC) [Jordan, S. W., and Cronan, J. E. (1997) Methods Enzymol. 279, 176-183]. When sodium dithionite-reduced LipA was incubated with octanoyl-ACP, LipB, apo-PDC, and S-adenosyl methionine (AdoMet), lipoylated PDC was formed. As shown by this assay, octanoic acid is not a substrate for LipA. Confirmation that LipA catalyzes formation of lipoyl groups from octanoyl-ACP was obtained by MALDI mass spectrometry of a recombinant PDC lipoyl-binding domain that had been lipoylated in a LipA reaction. These results provide information about the mechanism of LipA catalysis and place LipA within the family of iron-sulfur proteins that utilize AdoMet for radical-based chemistry.     

Pyruvate formate-lyase-activating enzyme: strictly anaerobic isolation yields active enzyme containing a [3Fe-4S](+) cluster

Pyruvate formate-lyase-activating enzyme (PFL-AE) from Escherichia coli (E. coli) catalyzes the stereospecific abstraction of a hydrogen atom from Gly734 of pyruvate formate-lyase (PFL) in a reaction that is strictly dependent on the cosubstrate S-adenosyl-l-methionine (AdoMet). Although PFL-AE is an iron-dependent enzyme, isolation of the enzyme with its metal center intact has proven difficult due to the oxygen sensitivity and lability of the metal center. We report here the first isolation of PFL-AE under nondenaturing, strictly anaerobic conditions. Iron and sulfide analysis as well as UV-visible, EPR, and resonance Raman data support the presence of a [3Fe-4S](+) cluster in the purified enzyme. The isolated native enzyme, but not apo-enzyme, exhibits a high specific activity (31 U/mg) in the absence of added iron, indicating that the native cluster is necessary and sufficient for enzymatic activity.