Multiple oligomeric structures of a bacterial small heat shock protein

Small heat shock proteins are ubiquitous molecular chaperones that form the first line of defence against the detrimental effects of cellular stress. Under conditions of stress they undergo drastic conformational rearrangements in order to bind to misfolded substrate proteins and prevent cellular protein aggregation. Owing to the dynamic nature of small heat shock protein oligomers, elucidating the structural basis of chaperone action and oligomerization still remains a challenge. In order to understand the organization of sHSP oligomers, we have determined crystal structures of a small heat shock protein from Salmonella typhimurium in a dimeric form and two higher oligomeric forms: an 18-mer and a 24-mer. Though the core dimer structure is conserved in all the forms, structural heterogeneity arises due to variation in the terminal regions.

Modelling within-host spatiotemporal dynamics of invasive bacterial disease

Mechanistic determinants of bacterial growth, death, and spread within mammalian hosts cannot be fully resolved studying a single bacterial population. They are also currently poorly understood. Here, we report on the application of sophisticated experimental approaches to map spatiotemporal population dynamics of bacteria during an infection. We analyzed heterogeneous traits of simultaneous infections with tagged Salmonella enterica populations (wild-type isogenic tagged strains [WITS]) in wild-type and gene-targeted mice. WITS are phenotypically identical but can be distinguished and enumerated by quantitative PCR, making it possible, using probabilistic models, to estimate bacterial death rate based on the disappearance of strains through time. This multidisciplinary approach allowed us to establish the timing, relative occurrence, and immune control of key infection parameters in a true host-pathogen combination. Our analyses support a model in which shortly after infection, concomitant death and rapid bacterial replication lead to the establishment of independent bacterial subpopulations in different organs, a process controlled by host antimicrobial mechanisms. Later, decreased microbial mortality leads to an exponential increase in the number of bacteria that spread locally, with subsequent mixing of bacteria between organs via bacteraemia and further stochastic selection. This approach provides us with an unprecedented outlook on the pathogenesis of S. enterica infections, illustrating the complex spatial and stochastic effects that drive an infectious disease. The application of the novel method that we present in appropriate and diverse host-pathogen combinations, together with modelling of the data that result, will facilitate a comprehensive view of the spatial and stochastic nature of within-host dynamics. © 2008 Grant et al.

Spread of Salmonella enterica in the body during systemic infection: unravelling host and pathogen determinants.

Salmonella enterica causes a range of life-threatening diseases in humans and animals worldwide. Current treatments for S. enterica infections are not sufficiently effective, and there is a need to develop new vaccines and therapeutics. An understanding of how S. enterica spreads in tissues has very important implications for targeting bacteria with vaccine-induced immune responses and antimicrobial drugs. Development of new control strategies would benefit from a more sophisticated evaluation of bacterial location, spatiotemporal patterns of spread and distribution in the tissues, and sites of microbial persistence. We review here recent studies of S. enterica serovar Typhimurium (S. Typhimurium) infections in mice, an established model of systemic typhoid fever in humans, which suggest that continuous bacterial spread to new infection foci and host phagocytes is an essential trait in the virulence of S. enterica during systemic infections. We further highlight how infections within host tissues are truly heterogeneous processes despite the fact that they are caused by the expansion of a genetically homogeneous microbial population. We conclude by discussing how understanding the within-host quantitative, spatial and temporal dynamics of S. enterica infections might aid the development of novel targeted preventative measures and drug regimens.

Probing biomolecular interaction forces using an anharmonic acoustic technique for selective detection of bacterial spores.

Receptor-based detection of pathogens often suffers from non-specific interactions, and as most detection techniques cannot distinguish between affinities of interactions, false positive responses remain a plaguing reality. Here, we report an anharmonic acoustic based method of detection that addresses the inherent weakness of current ligand dependant assays. Spores of Bacillus subtilis (Bacillus anthracis simulant) were immobilized on a thickness-shear mode AT-cut quartz crystal functionalized with anti-spore antibody and the sensor was driven by a pure sinusoidal oscillation at increasing amplitude. Biomolecular interaction forces between the coupled spores and the accelerating surface caused a nonlinear modulation of the acoustic response of the crystal. In particular, the deviation in the third harmonic of the transduced electrical response versus oscillation amplitude of the sensor (signal) was found to be significant. Signals from the specifically-bound spores were clearly distinguishable in shape from those of the physisorbed streptavidin-coated polystyrene microbeads. The analytical model presented here enables estimation of the biomolecular interaction forces from the measured response. Thus, probing biomolecular interaction forces using the described technique can quantitatively detect pathogens and distinguish specific from non-specific interactions, with potential applicability to rapid point-of-care detection. This also serves as a potential tool for rapid force-spectroscopy, affinity-based biomolecular screening and mapping of molecular interaction networks.

Identificación molecular de Burkholderia glumae, causante del añublo bacterial, en cinco zonas arroceras de Nicaragua

De octubre-diciembre 2012 se realizó el diagnóstico molecular de la bacteria causante del añublo bacterial (Burkholderia glumae) en cinco zonas arroceras de Nicaragua: Sébaco, Malacatoya, Boaco, León y Chinandega. Se colectaron 133 muestras en las zonas de estudio, con el fin de aislar e identificar las cepas de Burkholderia glumae. Todas las muestras fueron procesadas en el Centro de Biología Molecular de la Universidad Centroamericana. Se estandarizó y optimizó la prueba de PCR, para la identificación de la bacteria se amplificó un fragmento de 282 pares de bases de la región 16S de ADN ribosomal. 74% de las muestras resultaron positivas. La bacteria está presente en las zonas de estudio. Se realizaron aislamientos de colonias para su posterior identificación mediante la técnica de secuenciación. La cepa BGR1 está presente en las zonas evaluadas. Se requiere monitorear Burkholderia glumae en ambas épocas de siembra para llevar un registro de daños y la identificación de diferentes cepas.

Increased expression of cystine/glutamate antiporter in multiple sclerosis

Background: Glutamate excitotoxicity contributes to oligodendrocyte and tissue damage in multiple sclerosis (MS). Intriguingly, glutamate level in plasma and cerebrospinal fluid of MS patients is elevated, a feature which may be related to the pathophysiology of this disease. In addition to glutamate transporters, levels of extracellular glutamate are controlled by cystine/glutamate antiporter x(c)(-), an exchanger that provides intracellular cystine for production of glutathione, the major cellular antioxidant. The objective of this study was to analyze the role of the system x(c)(-) in glutamate homeostasis alterations in MS pathology. -- Methods: Primary cultures of human monocytes and the cell line U-937 were used to investigate the mechanism of glutamate release. Expression of cystine glutamate exchanger (xCT) was quantified by quantitative PCR, Western blot, flow cytometry and immunohistochemistry in monocytes in vitro, in animals with experimental autoimmune encephalomyelitis (EAE), the animal model of MS, and in samples of MS patients. -- Results and discussion: We show here that human activated monocytes release glutamate through cystine/glutamate antiporter x(c)(-) and that the expression of the catalytic subunit xCT is upregulated as a consequence of monocyte activation. In addition, xCT expression is also increased in EAE and in the disease proper. In the later, high expression of xCT occurs both in the central nervous system (CNS) and in peripheral blood cells. In particular, cells from monocyte-macrophage-microglia lineage have higher xCT expression in MS and in EAE, indicating that immune activation upregulates xCT levels, which may result in higher glutamate release and contribution to excitotoxic damage to oligodendrocytes. -- Conclusions: Together, these results reveal that increased expression of the cystine/glutamate antiporter system x(c)(-) in MS provides a link between inflammation and excitotoxicity in demyelinating diseases.

Recognizing and managing common fish diseases

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Control of bacterial disease in small scale fresh-water aquaculture: Project R0754. Project completion report December 1998 - May 1999

The work presented here represents an 18-month study to examine the relationship between environmental conditions, bacterial load in the water and bacteria levels in tissue macrophages of a range of clinically healthy freshwater fish species, farmed in a range of culture systems in Thailand and Vietnam. Preliminary assessment was made of the clinical significance of the macrophage bacterial load. The aim of this work was to improve production in fresh-water aquaculture through the control of clinical bacterial disease and subclinical infection, and to identify management practices most effective in promoting fish health. [PDF contains 37 pages]

Biocompatible Bacterial Cellulose-Poly(2-hydroxyethyl methacrylate) Nanocomposite Films

A series of bacterial cellulose-poly(2-hydroxyethyl methacrylate) nanocomposite films was prepared by in situ radical polymerization of 2-hydroxyethyl methacrylate (HEMA), using variable amounts of poly(ethylene glycol) diacrylate (PEGDA) as crosslinker. Thin films were obtained, and their physical, chemical, thermal, and mechanical properties were evaluated. The films showed improved translucency compared to BC and enhanced thermal stability and mechanical performance when compared to poly(2-hydroxyethyl methacrylate) (PHEMA). Finally, BC/PHEMA nanocomposites proved to be nontoxic to human adipose-derived mesenchymal stem cells (ADSCs) and thus are pointed as potential dry dressings for biomedical applications.

Bacterial flora of the gut of the African snakehead, Channa obscura (Pisces: Channidae)

A qualitative and quantitative investigation of the bacterial flora of the gut of the African snakehead, Channa obscura was undertaken. The types of bacteria isolated from the different parts of the gut of C. obscura include Pseudomonas, Streptococcus, Citrobacter and Proteus. The coliform (Escherichia coli, Enterobacter) and some other Enterobacteriaceae such as Salmonella were also present. The stomach and intestine were found to have a preponderance of Pseudomonas and Vibrio species. Klebsiella sp. and Bacillus sp. (only in the pyloric caeca) were also isolated. On the whole, the correlation coefficients of the two incubation temperatures showed a high statistical significance. Thus the bacterial load of the gut of C. obscura has been shown as a function of temperature

Watershed monitoring and the TMDL modeling to assess bacterial loading in estuarine environments to improve management

Shellfish bed closures along the North Carolina coast have increased over the years seemingly concurrent with increases in population (Mallin 2000). More and faster flowing storm water has come to mean more bacteria, and fecal indicator bacterial (FIB) standards for shellfish harvesting are often exceeded when no source of contamination is readily apparent (Kator and Rhodes, 1994). Could management reduce bacterial loads if the source of the bacteria where known? Several potentially useful methods for differentiating human versus animal pollution sources have emerged including Ribotyping and Multiple Antibiotic Resistance (MAR) (US EPA, 2005). Total Maximum Daily Load (TMDL) studies on bacterial sources have been conducted for streams in NC mountain and Piedmont areas (U.S. EPA, 1991 and 2005) and are likely to be mandated for coastal waters. TMDL analysis estimates allowable pollutant loads and allocates them to known sources so management actions may be taken to restore water to its intended uses (U.S. EPA, 1991 and 2005). This project sought first to quantify and compare fecal contamination levels for three different types of land use on the coast, and second, to apply MAR and ribotyping techniques and assess their effectiveness for indentifying bacterial sources. Third, results from these studies would be applied to one watershed to develop a case study coastal TMDL. All three watershed study areas are within Carteret County, North Carolina. Jumping Run Creek and Pettiford Creek are within the White Oak River Basin management unit whereas the South River falls within the Neuse River Basin. Jumping Run Creek watershed encompasses approximately 320 ha. Its watershed was a dense, coastal pocosin on sandy, relic dune ridges, but current land uses are primarily medium density residential. Pettiford Creek is in the Croatan National Forest, is 1133 ha. and is basically undeveloped. The third study area is on Open Grounds Farm in the South River watershed. Half of the 630 ha. watershed is under cultivation with most under active water control (flashboard risers). The remaining portion is forested silviculture.(PDF contains 4 pages)

Revealing, illuminating, and modifying proteins in human diseases using noncanonical amino acids

To better understand human diseases, much recent work has focused on proteins to either identify disease targets through proteomics or produce therapeutics via protein engineering. Noncanonical amino acids (ncAAs) are tools for altering the chemical and physical properties of proteins, providing a facile strategy not only to label proteins but also to engineer proteins with novel properties. My thesis research has focused on the development and applications of noncanonical amino acids in identifying, imaging, and engineering proteins for studying human diseases. Chapter 1 introduces the concept of ncAAs and reveals insights to how I chose my thesis projects.

ncAAs have been incorporated to tag and enrich newly synthesized proteins for mass spectrometry through a method termed BONCAT, or bioorthogonal noncanonical amino acid tagging. Chapter 2 describes the investigation of the proteomic response of human breast cancer cells to induced expression of tumor suppressor microRNA miR-126 by combining BONCAT with another proteomic method, SILAC or stable isotope labeling by amino acids in cell culture. This proteomic analysis led to the discovery of a direct target of miR-126, shedding new light on its role in suppressing cancer metastasis.

In addition to mass spectrometry, ncAAs can also be utilized to fluorescently label proteins. Chapter 3 details the synthesis of a set of cell-permeant cyclooctyne probes and demonstration of selective labeling of newly synthesized proteins in live mammalian cells using azidohomoalanine. Similar to live cell imaging, the ability to selectively label a particular cell type within a mixed cell population is important to interrogating many biological systems, such as tumor microenvironments. By taking advantage of the metabolic differences between cancer and normal cells, Chapter 5 discusses efforts to develop selective labeling of cancer cells using a glutamine analogue.

Furthermore, Chapter 4 describes the first demonstration of global replacement at polar amino acid positions and its application in developing an alternative PEGylation strategy for therapeutic proteins. Polar amino acids typically occupy solvent-exposed positions on the protein surface, and incorporation of noncanonical amino acids at these positions should allow easier modification and cause less perturbation compared to replacements at the interior positions of proteins.

Gut microbiota promote hematopoiesis to control bacterial infection

The commensal microbiota impacts specific immune cell populations and their functions at peripheral sites, such as gut mucosal tissues. However, it remains unknown whether gut microbiota control immunity through regulation of hematopoiesis at primary immune sites. We reveal that germ-free mice display reduced proportions and differentiation potential of specific myeloid cell progenitors of both yolk sac and bone marrow origin. Homeostatic innate immune defects may lead to impaired early responses to pathogens. Indeed, following systemic infection with Listeria monocytogenes, germ-free and oral antibiotic-treated mice display increased pathogen burden and acute death. Recolonization of germ-free mice with a complex microbiota restores defects in myelopoiesis and resistance to Listeria. These findings reveal that gut bacteria direct innate immune cell development via promoting hematopoiesis, contributing to our appreciation of the deep evolutionary connection between mammals and their microbiota.

Determination of optimum stocking density of Macrobrachium rosenbergii larvae using multiple feed in a commercial hatchery at Cox’s Bazar, Bangladesh

Experimental stocking density of Macrobrachium rosenbergii in larval rearing was conducted in A.G. Aqua Hatchery, Chakaria, Bangladesh to study the effect of different stocking densities on growth, survival rate and diseases stress under hatchery condition. The research work was conducted using six cemented rectangular tanks having 3m3 capacity (1.5mX2mX1m) each. Stocking density were maintained in three experimental setup as 200, 150 and 100ind/L of the T1, T2 and T3 respectively with one replicate each. The larvae were fed with Artemia nauplii, Custard, Maxima and brine shrimp flakes. Water quality was maintained by exchanging 20-30% (12ppt saline water) daily. During the study period, temperature, pH, DO, salinity, nitrite-nitrogen, ammonia and alkalinity were maintained from 28.5-31.5ºC, 7.5-7.8, 5.8-5.9mg/L, 12-13ppt, 0.14-0.2 mg/L, 0.22-0.3mg/L, and 140-160mg/L respectively. The growth rates of larvae at 11th stage were recorded in terms of body length 0.115, 0.136, and 0.169 mm/day whereas body weight were observed 0.000115, 0.000180, and 0.000240g/day. The survival rate of larvae were found 21.8%, 30.4% and 51.3% in treatments T1, T2 and T3 respectively. PL was obtained as 43, 45, and 51PL/L and days required of 41, 38 and 34 days in stocking density of 200, 150, and 100ind/L respectively. It was found that the minimum of 34 days was required to attain the PL (12th stage) using the stocking density of 100 individuals/L. Cannibalism, Zoothamnium, Exuvia Entrapment Disease (EED), and Bacterial Necrosis (BN) were found to be the threat to the commercial hatchery operation that might responsible for potential larval damages which can be reduced by lowering the stocking densities in larval rearing tank that also increased the survival and growth rate.