31P-NMR spectroscopy of perifused rat hepatocytes immobilized in agarose threads: application to chemical-induced hepatotoxicity.

A system consisting of isolated rat hepatocytes immobilized in agarose threads continuously perifused with oxygenated Krebs-Henseleit (KH) solution has been found to maintain cell viability with excellent metabolic activity for more than 6 h. The hepatocytes were monitored by phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy at 4.7 Tesla, by measurement of oxygen consumption and by the leakage of lactate dehydrogenase (LD) and alanine aminotransferase (ALT). The data obtained were comparable to those found for an isolated perfused whole liver in vitro. The effects of allyl alcohol (AA), ethanol, and 4-acetaminophenol (AP) were examined. A solution of 225 microM AA perifused for 90 min caused the disappearance of the beta-phosphate resonance of adenosine triphosphate (ATP) in the 31P-NMR spectra, a 7-fold increase in LD leakage and a 70% reduction in oxygen consumption. Ethanol (1.0 M) perifused for 90 min reduced the beta-ATP signal intensity ratio by 20%, the phosphomonoester (PME) signal by 50% and inorganic phosphate (Pi) by 33% (P less than 0.05). AP (10 mM) caused only mild liver-cell damage. The results demonstrate that perifused immobilized hepatocytes can be used as a liver model to assess the effects of a wide range of chemicals and other xenobiotics by NMR spectroscopy.     

Natural feeding influences protein expression in the dogfish shark rectal gland: A proteomic analysis

The rectal gland is the principal salt-secreting organ in elasmobranchs, yet its functional response to normal physiological variation (e.g., due to feeding, stress) has only recently been examined. To complement studies on acid-base, digestive, and osmoregulatory physiology in response to natural feeding, we investigated protein-level responses in the rectal gland of spiny dogfish (Squalus acanthias) 6 h, 20 h, and 5 days (reference control) after a meal. Our objective was to identify proteins involved in regulation of osmoregulatory and metabolic processes in response to feeding. Proteins were separated by two-dimensional gel electrophoresis, and protein spots that were significantly up- or down-regulated > 2 fold (i.e., abundance increased more than 100% or decreased more than 50%) were detected using gel image analysis software. Of 684 proteins analyzed on 2D gels, 16 proteins changed significantly 6 h after feeding vs. 5 day controls (5 decreased; 11 increased), and 12 proteins changed > 2 fold 20 h after feeding vs. 5 day controls (2 decreased; 10 increased). Thirteen of these proteins were identified using mass spectrometry and classified into functional pathways using the PANTHER bioinformatics database. Rectal gland proteins that were regulated following feeding fell into three main categories: cytoskeletal/muscular (e.g., tropomyosin alpha chain, transgelin), energy metabolism (e.g., malate dehydrogenase, ATP synthase), and nucleotide metabolism (nucleoside diphosphate kinase). The data also revealed that previously documented increases in the activity of isocitrate dehydrogenase after feeding are at least partially due to increased abundance of a cytosolic, NADP-dependent isoform of this enzyme. One of the primary components of the rectal gland's response to feeding appears to be maintenance of the cellular supply of energy, which would be necessary to fuel increased activities of enzymes involved in salt secretion and oxidative metabolism in the rectal gland following a meal.     

Pyrosequencing study of fecal microflora of autistic and control children

There is evidence of genetic predisposition to autism, but the percent of autistic subjects with this background is unknown. It is clear that other factors, such as environmental influences, may play a role in this disease. In the present study, we have examined the fecal microbial flora of 33 subjects with various severities of autism with gastrointestinal symptoms, 7 siblings not showing autistic symptoms (sibling controls) and eight non-sibling control subjects, using the bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) procedure. The results provide us with information on the microflora of stools of young children and a compelling picture of unique fecal microflora of children with autism with gastrointestinal symptomatology. Differences based upon maximum observed and maximum predicted operational taxonomic units were statistically significant when comparing autistic and control subjects with p-values ranging from <0.001 to 0.009 using both parametric and non-parametric estimators. At the phylum level, Bacteroidetes and Firmicutes showed the most difference between groups of varying severities of autism. Bacteroidetes was found at high levels in the severely autistic group, while Firmicutes were more predominant in the control group. Smaller, but significant, differences also occurred in the Actinobacterium and Proteobacterium phyla. Desulfovibrio species and Bacteroides vulgatus are present in significantly higher numbers in stools of severely autistic children than in controls. If the unique microbial flora is found to be a causative or consequent factor in this type of autism, it may have implications with regard to a specific diagnostic test, its epidemiology, and for treatment and prevention.     

Microarray based comparison of two Escherichia coli O157:H7 lineages

Background  

Previous research has identified the potential for the existence of two separate lineages of Escherichia coli O157:H7. Clinical isolates tended to cluster primarily within one of these two lineages. To determine if there are virulence related genes differentially expressed between the two lineages we chose to utilize microarray technology to perform an initial screening.     

Heterologous expression of bacteriocin E-760 in Chlamydomonas reinhardtii and functional analysis

The use of antimicrobial peptides (AMPs) synthesized by bacteria (bacteriocins) is an alternative for combating multidrug resistant bacterial strains and their production by recombinant route is a viable option for their mass production. The bacteriocin E-760 isolated from the genus Enterococcus sp. has been shown to possess inhibitory activity against Gram-negative and Gram-positive bacteria. In this study, the expression of a chimeric protein coding for E-760 in the nucleus of C. reinhardtii was evaluated, as well as, its antibacterial activity. The synthetic gene E-760S was inserted into the genome of C. reinhardtii using Agrobacterium tumefaciens. A transgenic line was identified in TAP medium with hygromycin and also by PCR. The increment in the culture medium temperature of the transgenic strain at 35 °C for 10 minutes, increased the production level of the recombinant protein from 0.14 (Noninduced culture, NIC) to 0.36% (Induced culture, IC) of total soluble proteins (TSP); this was quantified by an ELISA assay. Recombinant E-760 possesses activity against Staphylococcus aureus in 0.34 U log, Streptococcus agalactiae in 0.48 U log, Enterococcus faecium in 0.36 U log, Pseudomonas aeruginosa in 2 U log and for Klebsiella pneumoniae, the activity was 0.07 U log. These results demonstrate that the nucleus transformation of C. reinhardtii can function as a stable expression platform for the production of the synthetic gene E-760 and it can potentially be used as an antibacterial agent.     

Polymicrobial nature of chronic diabetic foot ulcer biofilm infections determined using bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP)

Background

Diabetic extremity ulcers are associated with chronic infections. Such ulcer infections are too often followed by amputation because there is little or no understanding of the ecology of such infections or how to control or eliminate this type of chronic infection. A primary impediment to the healing of chronic wounds is biofilm phenotype infections. Diabetic foot ulcers are the most common, disabling, and costly complications of diabetes. Here we seek to derive a better understanding of the polymicrobial nature of chronic diabetic extremity ulcer infections.

Methods and Findings

Using a new bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) approach we have evaluated the bacterial diversity of 40 chronic diabetic foot ulcers from different patients. The most prevalent bacterial genus associated with diabetic chronic wounds was Corynebacterium spp. Findings also show that obligate anaerobes including Bacteroides, Peptoniphilus, Fingoldia, Anaerococcus, and Peptostreptococcus spp. are ubiquitous in diabetic ulcers, comprising a significant portion of the wound biofilm communities. Other major components of the bacterial communities included commonly cultured genera such as Streptococcus, Serratia, Staphylococcus and Enterococcus spp.

Conclusions

In this article, we highlight the patterns of population diversity observed in the samples and introduce preliminary evidence to support the concept of functional equivalent pathogroups (FEP). Here we introduce FEP as consortia of genotypically distinct bacteria that symbiotically produce a pathogenic community. According to this hypothesis, individual members of these communities when they occur alone may not cause disease but when they coaggregate or consort together into a FEP the synergistic effect provides the functional equivalence of well-known pathogens, such as Staphylococcus aureus, giving the biofilm community the factors necessary to maintain chronic biofilm infections. Further work is definitely warranted and needed in order to prove whether the FEPs concept is a viable hypothesis. The findings here also suggest that traditional culturing methods may be extremely biased as a diagnostic tool as they select for easily cultured organisms such as Staphylococcus aureus and against difficult to culture bacteria such as anaerobes. While PCR methods also have bias, further work is now needed in comparing traditional culture results to high-resolution molecular diagnostic methods such as bTEFAP.     

Contributions of the hippocampus and medial prefrontal cortex to energy and body weight regulation

The effects of selective ibotenate lesions of the complete hippocampus (CHip), the hippocampal ventral pole (VP), or the medial prefrontal cortex (mPFC) in male rats were assessed on several measures related to energy regulation (i.e., body weight gain, food intake, body adiposity, metabolic activity, general behavioral activity, conditioned appetitive responding). The testing conditions were designed to minimize the nonspecific debilitating effects of these surgeries on intake and body weight. Rats with CHip and VP lesions exhibited significantly greater weight gain and food intake compared with controls. Furthermore, CHip-lesioned rats, but not rats with VP lesions, showed elevated metabolic activity, general activity in the dark phase of the light-dark cycle, and greater conditioned appetitive behavior, compared with control rats without these brain lesions. In contrast, rats with mPFC lesions were not different from controls on any of these measures. These results indicate that hippocampal damage interferes with energy and body weight regulation, perhaps by disrupting higher-order learning and memory processes that contribute to the control of appetitive and consummatory behavior.     

From receptor recognition mechanisms to bioinspired mimetic antagonists in HIV-1/cell docking

Understanding the ways in which two or more proteins interact may give insight into underlying binding and activation mechanisms in biology, methods for protein separation and structure-based antagonism. This review describes ways in which protein recognition has been explored in our laboratory for the HIV-1/cell entry process. Initial contact between an HIV-1 virion particle and a human cell occurs between gp120 (an HIV-1 envelope protein) and CD4 (a human extracellular signaling protein). This interaction leads to a sequence of events which includes a conformational change in gp120, fusion of the HIV-1 and cellular membranes and eventual infection of the cell. Using an optical biosensor and a reporter antibody, we have been able to measure the conformational change in gp120 that occurs upon CD4 binding. We also have used this biosensor system to characterize CD4 mimetics, obtained by peptide synthesis in miniprotein scaffolds. Phage display techniques have been employed to identify novel miniprotein sequences. The combination of biosensor interaction kinetics analysis and phage display provides a useful approach for understanding the recognition mechanisms involved in the HIV/cell docking process. This approach may also be useful in investigating other protein complexes of importance in health and disease.     

Multiple scale analysis of factors influencing the distribution of an invasive aquatic grass

With the expected increase in the spread of invasive species, examination of factors controlling distributions at multiple spatial scales and ecological modelling of their potential distributions are important analyses for informed decision-making. The scale-dependence of mechanisms influencing invasion by non-native species has been shown previously, indicating that studies of key factors affecting invasive species distributions at multiple spatial scales are critical for successful management. Freshwater systems are particularly vulnerable to invasive species, yet few studies have examined the environmental factors influencing distributions of invasive species at multiple spatial scales. We examined the effect of environmental variables on the predicted distribution of the invasive aquatic grass Glyceria maxima over continental, regional and local scales in Australia. We undertook an initial critical evaluation of which predictor variables were most appropriate to use at each scale, largely considering prior knowledge. On a continental scale, climatic, topographic and hydrological variables predicted well the potential distribution of G. maxima, identifying temperate regions as most susceptible to invasion. The regional analysis found that dense, woody, riparian vegetation has a strong negative impact on the occurrence of G. maxima, especially at intermediate elevations. The invasive grass was found less often on biotite granite and on fluvial geology. At a local scale, occurrence of G. maxima was related positively to soil phosphorus and nitrogen, and negatively related to soil organic carbon. The identification of key factors affecting invasive species distributions at multiple spatial scales will inform prevention schemes, assist targeted field sampling for the development of monitoring programs, and allow prioritization of control methods.