The Hydrogenase Chip: a tiling oligonucleotide DNA microarray technique for characterizing hydrogen-producing and -consuming microbes in microbial communities

We developed a broad-ranging method for identifying key hydrogen-producing and consuming microorganisms through analysis of hydrogenase gene content and expression in complex anaerobic microbial communities. The method is based on a tiling hydrogenase gene oligonucleotide DNA microarray (Hydrogenase Chip), which implements a high number of probes per gene by tiling probe sequences across genes of interest at 1.67 × –2 × coverage. This design favors the avoidance of false positive gene identification in samples of DNA or RNA extracted from complex microbial communities. We applied this technique to interrogate interspecies hydrogen transfer in complex communities in (i) lab-scale reductive dehalogenating microcosms enabling us to delineate key H₂-consuming microorganisms, and (ii) hydrogen-generating microbial mats where we found evidence for significant H₂ production by cyanobacteria. Independent quantitative PCR analysis on selected hydrogenase genes showed that this Hydrogenase Chip technique is semiquantitative. We also determined that as microbial community complexity increases, specificity must be traded for sensitivity in analyzing data from tiling DNA microarrays.     

Kinetic characterization of glutathione reductase from the malarial parasite Plasmodium falciparum. Comparison with the human enzyme

The homodimeric flavoenzyme glutathione reductase (GR) maintains high intracellular concentrations of the antioxidant glutathione (GSSG + NADPH + H(+) <--> 2 GSH + NADP(+)). Due to its central function in cellular redox metabolism, inhibition of GR from the malarial parasite Plasmodium falciparum represents an important approach to antimalarial drug development; therefore, the catalytic mechanism of GR from P. falciparum has been analyzed and compared with the human host enzyme. The reductive half-reaction is similar to the analogous reaction with GR from other species. The oxidative half-reaction is biphasic, reflecting formation and breakdown of a mixed disulfide between the interchange thiol and GSH. The equilibrium between the E(ox)-EH(2) and GSSG-GSH couples has been modeled showing that the Michaelis complex, mixed disulfide-GSH, is the predominant enzyme form as the oxidative half-reaction progresses; rate constants used in modeling allow calculation of an K(eq) from the Haldane relationship, 0.075, very similar to the K(eq) of the same reaction for the yeast enzyme (0.085) (Arscott, L. D., Veine, D. M., and Williams, C. H., Jr. (2000) Biochemistry 39, 4711-4721). Enzyme-monitored turnover indicates that E(FADH(-))(S-S). NADP(+) and E(FAD)(SH)(2).NADPH are dominant enzyme species in turnover. Since the individual forms of the enzyme differ in their susceptibility to inhibitors, the prevailing states of GR in the cell are of practical relevance.     

Association of PARL rs3732581 genetic variant with insulin levels, metabolic syndrome and coronary artery disease

PARL (presenilin-associated rhomboid-like) is a mitochondrial protein involved in mitochondrial membrane remodelling, and maps to a quantitative trait locus (3q27) associated with metabolic traits. Recently the rs3732581 (Leu262Val) variant was found to be associated with increased levels of plasma insulin, a finding not replicated in a larger cohort. The aim of the current study was to investigate the associations between rs3732581 and levels of plasma insulin, metabolic syndrome (MetS) and its components, and cardiovascular disease. The CUPID population consisted of 556 subjects with angiographically proven CAD and the CUDAS cohort consisted of 1,109 randomly selected individuals from Perth, Western Australia. Samples were genotyped using mutation-specific PCR. No significant associations were observed between rs3732581 and levels of plasma insulin, glucose, BMI or MetS in either population. However, carriers of the minor allele had significantly lower mean intima-media thickness (IMT) [0.69 mm, 95% CI (0.69, 0.70 mm); P = 0.004], compared with major allele homozygotes [mean IMT = 0.71 mm, 95% CI (0.70, 0.72 mm)] in the CUDAS population. Further analysis using a recessive model showed homozygous carriers of the minor allele were predisposed to CAD [OR 1.55, 95% CI (1.11, 2.16); P = 0.01]. Despite the functional evidence for a role of PARL in regulating insulin levels, no association with rs3732581 was found in the current study. Additionally, there were no associations with glucose levels, BMI or MetS. There were significant effects of the variant on mean IMT and risk of CAD. A role for PARL in metabolic conditions cannot be excluded and more comprehensive genetic studies are warranted.     

Degeneration of the intervertebral disc

The intervertebral disc is a cartilaginous structure that resembles articular cartilage in its biochemistry, but morphologically it is clearly different. It shows degenerative and ageing changes earlier than does any other connective tissue in the body. It is believed to be important clinically because there is an association of disc degeneration with back pain. Current treatments are predominantly conservative or, less commonly, surgical; in many cases there is no clear diagnosis and therapy is considered inadequate. New developments, such as genetic and biological approaches, may allow better diagnosis and treatments in the future.     

High pressures and asymmetrical stresses in the scoliotic disc in the absence of muscle loading

Background

Loads acting on scoliotic spines are thought to be asymmetric and involved in progression of the scoliotic deformity; abnormal loading patterns lead to changes in bone and disc cell activity and hence to vertebral body and disc wedging. At present however there are no direct measurements of intradiscal stresses or pressures in scoliotic spines. The aim of this study was to obtain quantitative measurements of the intradiscal stress environment in scoliotic intervertebral discs and to determine if loads acting across the scoliotic spine are asymmetric. We performed in vivo measurements of stresses across the intervertebral disc in patients with scoliosis, both parallel (termed horizontal) and perpendicular (termed vertical) to the end plate, using a side mounted pressure transducer (stress profilometry)

Methods

Stress profilometry was used to measure horizontal and vertical stresses at 5 mm intervals across 25 intervertebral discs of 7 scoliotic patients during anterior reconstructive surgery. A state of hydrostatic pressure was defined by identical horizontal and vertical stresses for at least two consecutive readings. Results were compared with similar stress profiles measured during surgery across 10 discs of 4 spines with no lateral curvature and with data from the literature.

Results

Profiles across scoliotic discs were very different from those of normal, young, healthy discs of equivalent age previously presented in the literature. Hydrostatic pressure regions were only seen in 14/25 discs, extended only over a short distance. Non-scoliotic discs of equivalent age would be expected to show large centrally placed hydrostatic nuclear regions in all discs. Mean pressures were significantly greater (0.25 MPa) than those measured in other anaesthetised patients (<0.07 MPa). A stress peak was seen in the concave annulus in 13/25 discs. Stresses in the concave annulus were greater than in the convex annulus indicating asymmetric loading in these anaesthetised, recumbent patients.

Conclusion

Intradiscal pressures and stresses in scoliotic discs are abnormal, asymmetrical and high in magnitude even in the absence of significant applied muscle loading. The origin of these abnormal stresses is unclear.     

Histopathological diagnosis of myocarditis in a dengue outbreak in Sri Lanka, 2009

Background

In 2009, an outbreak of dengue caused high fatality in Sri Lanka. We conducted 5 autopsies of clinically suspected myocarditis cases at the General Hospital, Peradeniya to describe the histopathology of the heart and other organs.

Methods

The diagnosis of dengue was confirmed with specific IgM and IgG ELISA, HAI and RT-PCR techniques. The histology was done in tissue sections stained with hematoxylin and eosin.

Results

Of the 319 cases of dengue fever, 166(52%) had severe infection. Of them, 149 patients (90%) had secondary dengue infection and in 5 patients, DEN-1 was identified as the causative serotype. The clinical diagnosis of myocarditis was considered in 45(27%) patients. The autopsies were done in 5 patients who succumbed to shock (3 females and 2 males) aged 13- 31 years. All had pleural effusions, ascites, bleeding patches in tissue planes and histological evidence of myocarditis. The main histological findings of the heart were interstitial oedema with inflammatory cell infiltration and necrosis of myocardial fibers. One patient had pericarditis. The concurrent pulmonary abnormalities were septal congestion, pulmonary haemorrhage and diffuse alveolar damage; one case showed massive necrosis of liver.

Conclusions

The histology supports occurrence of myocarditis in dengue infection.

     

Production of neosolaniol monoacetate by an undescribedFusarium species resemblingF camptoceras

Cultures of 12 South African isolates of an undescribedFusarium species resembling but distinct fromF camptoceras were analysed for the presence of diacetoxyscirpenol (DAS), neosolaniol monoacetate (NMA), and T-2 toxin, by capillary gas chromatography utilizing electron capture detection. No DAS or T-2 toxin could be detected in any of the cultures of the isolates. NMA was, however, detected in 10 of the 12 isolates at levels ranging from 310 to 2060 ng/g. The method used, was primarily developed for the determination of DAS and T-2 toxin in fungal cultures and grain samples but was found to be suitable for the coextraction of NMA at an average recovery of 80.8%, with a detection limit in the order of 100 ng/g. Supportive evidence for the presence of the NMA was obtained by capillary gas chromatography / mass spectrometry. Regarded as a relatively rare trichothecene, NMA has never been reported to occur naturally and has previously been shown to be produced by only a fewFusarium strains.