Rapid development of application-specific flexible MRI receive coils.

Over the last 30 years, there have been dramatic changes in phased array coil technology leading to increasing channel density and parallel imaging functionality. Current receiver array coils are rigid and often mismatched to patient's size. Recently there has been a move towards flexible coil technology, which is more conformal to the human anatomy. Despite the advances of so-called flexible surface coil arrays, these coils are still relatively rigid and limited in terms of design conformability, compromising signal-to-noise ratio (SNR) for flexibility, and are not designed for optimum parallel imaging performance. The purpose of this study is to report on the development and characterization of a 15-channel flexible foot and ankle coil, rapidly designed and constructed using highly decoupled radio-frequency (RF) coil elements. Coil performance was evaluated by performing SNR and g-factor measurements. In vivo testing was performed in a healthy volunteer using both the 15-channel coil and a commercially available 8-channel foot coil. The highly decoupled elements used in this design allow for extremely rapid development and prototyping of application-specific coils for different patient sizes (adult vs child) with minimal additional design consideration in terms of coil overlap and geometry. Image quality was comparable to a commercially available RF coil.

From 'OPAT' to 'COpAT': implications of the OVIVA study for ambulatory management of bone and joint infection.

Bone and joint infection contributes significantly to clinical activity within outpatient parenteral antimicrobial therapy (OPAT) services. The OVIVA (oral versus intravenous antibiotics for bone and joint infection) randomized study has challenged the practice of prolonged intravenous therapy, because non-inferiority of oral antibiotic therapy was demonstrated, thereby implying that early transition to oral therapy is an appropriate alternative to prolonged intravenous therapy. We examine the caveats to the study and discuss the implications for OPAT practice, highlighting the importance of careful oral antibiotic selection with attention to bioavailability, bone penetration, drug interactions, compliance and toxicity monitoring. We emphasize that ambulatory antibiotic therapy (whether intravenous or oral) in this patient group requires expert multidisciplinary management, monitoring and follow-up, and ideally should be undertaken within existing OPAT or, more accurately, complex outpatient antibiotic therapy (COpAT) services.

Legacy introductions and climatic variation explain spatiotemporal patterns of invasive hybridization in a native trout.

Hybridization between invasive and native species, a significant threat to worldwide biodiversity, is predicted to increase due to climate-induced expansions of invasive species. Long-term research and monitoring are crucial for understanding the ecological and evolutionary processes that modulate the effects of invasive species. Using a large, multidecade genetics dataset (N = 582 sites, 12,878 individuals) with high-resolution climate predictions and extensive stocking records, we evaluate the spatiotemporal dynamics of hybridization between native cutthroat trout and invasive rainbow trout, the world's most widely introduced invasive fish, across the Northern Rocky Mountains of the United States. Historical effects of stocking and contemporary patterns of climatic variation were strongly related to the spread of hybridization across space and time. The probability of occurrence, extent of, and temporal changes in hybridization increased at sites in close proximity to historical stocking locations with greater rainbow trout propagule pressure, warmer water temperatures, and lower spring precipitation. Although locations with warmer water temperatures were more prone to hybridization, cold sites were not protected from invasion; 58% of hybridized sites had cold mean summer water temperatures (<11°C). Despite cessation of stocking over 40 years ago, hybridization increased over time at half (50%) of the locations with long-term data, the vast majority of which (74%) were initially nonhybridized, emphasizing the chronic, negative impacts of human-mediated hybridization. These results show that effects of climate change on biodiversity must be analyzed in the context of historical human impacts that set ecological and evolutionary trajectories.

MEMS switch integrated radio frequency coils and arrays for magnetic resonance imaging.

Surface coils are widely used in magnetic resonance imaging and spectroscopy. While smaller diameter coils produce higher signal to noise ratio (SNR) closer to the coil, imaging larger fields of view or greater distance into the sample requires a larger overall size array or, in the case of a channel count limited system, larger diameter coils. In this work, we consider reconfiguring the geometry of coils and coil arrays such that the same coil or coil array may be used in multiple field of view imaging. A custom designed microelectromechanical systems switch, compatible with magnetic resonance imaging, is used to switch in/out conductive sections and components to reconfigure coils. The switch does not degrade the SNR and can be opened/closed in 10 µs, leading to rapid reconfiguration. Results from a single coil, configurable between small/large configurations, and a two-coil phased array, configurable between spine/torso modes, are presented.

Restrictive antibiotic stewardship associated with reduced hospital mortality in gram-negative infection.

INTRODUCTION: : Antimicrobial stewardship has an important role in the control of Clostridium difficile infection (CDI) and antibiotic resistance. An important component of UK stewardship interventions is the restriction of broad-spectrum beta-lactam antibiotics and promotion of agents associated with a lower risk of CDI such as gentamicin. While the introduction of restrictive antibiotic guidance has been associated with improvements in CDI and antimicrobial resistance, evidence of the effect on outcome following severe infection is lacking. METHODS: : In 2008, Glasgow hospitals introduced a restrictive antibiotic guideline. A retrospective before/after study assessed outcome following Gram-negative bacteraemia in the 2-year period around implementation. RESULTS: : Introduction of restrictive antibiotic guidelines was associated with a reduction in utilization of ceftriaxone and co-amoxiclav and an increase in amoxicillin and gentamicin. Approximately 1593 episodes of bacteremia were included in the study. The mortality over 1-year following Gram-negative bacteraemia was lower in the period following guideline implementation (RR 0.852, P = 0.045). There was no evidence of a difference in secondary outcomes including ITU admission, length of stay, readmission, recurrence of bacteraemia and need for renal replacement therapy. There was a fall in CDI (RR 0.571, P = 0.014) and a reduction in bacterial resistance to ceftriaxone and co-amoxiclav but no evidence of an increase in gentamicin resistance after guideline implementation. CONCLUSION: : Restrictive antibiotic guidelines were associated with a reduction in CDI and bacterial resistance but no evidence of adverse outcomes following Gram-negative bacteraemia. There was a small reduction in one year mortality.

Analysis of three genomes within the thermophilic bacterial species Caldanaerobacter subterraneus with a focus on carbon monoxide dehydrogenase evolution and hydrolase diversity.

BACKGROUND: The Caldanaerobacter subterraneus species includes thermophilic fermentative bacteria able to grow on carbohydrates substrates with acetate and L-alanine as the main products. In this study, comprehensive analysis of three genomes of C. subterraneus subspecies was carried in order to identify genes encoding key metabolic enzymes and to document the genomic basis for the evolution of these organisms. METHODS: Average nucleotide identity and in silico DNA relatedness were estimated for the studied C. subterraneus genomes. Genome synteny was evaluated using R2CAT software. Protein conservation was analyzed using mGenome Subtractor. Horizontal gene transfer was predicted through the GOHTAM pipeline (using tetranucleotide composition) and phylogenetic analyses (by maximum likelihood). Hydrolases were identified through the MEROPS and CAZy platforms. RESULTS: The three genomes of C. subterraneus showed high similarity, although there are substantial differences in their gene composition and organization. Each subspecies possesses a gene cluster encoding a carbon monoxide dehydrogenase (CODH) and an energy converting hydrogenase (ECH). The CODH gene is associated with an operon that resembles the Escherichia coli hydrogenase hyc/hyf operons, a novel genetic context distinct from that found in archetypical hydrogenogenic carboxydotrophs. Apart from the CODH-associated hydrogenase, these bacteria also contain other hydrogenases, encoded by ech and hyd genes. An Mbx ferredoxin:NADP oxidoreductase homolog similar to that originally described in the archaeon Pyrococcus furiosus was uniquely encoded in the C. subterraneus subsp. yonseiensis genome. Compositional analysis demonstrated that some genes of the CODH-ECH and mbx operons present distinct sequence patterns in relation to the majority of the other genes of each genome. Phylogenetic reconstructions of the genes from these operons and those from the ech operon are incongruent to the species tree. Notably, the cooS gene of C. subterraneus subsp. pacificus and its homologs in C. subterraneus subsp. tengcongensis and C. subterraneus subsp. yonseiensis form distinct clades. The strains have diverse hydrolytic enzymes and they appear to be proteolytic and glycolytic. Divergent glycosidases from 14 families, among them amylases, chitinases, alpha-glucosidases, beta-glucosidases, and cellulases, were identified. Each of the three genomes also contains around 100 proteases from 50 subfamilies, as well about ten different esterases. CONCLUSIONS: Genomic information suggests that multiple horizontal gene transfers conferred the adaptation of C. subterraneus subspecies to extreme niches throughout the carbon monoxide utilization and hydrogen production. The variety of hydrolases found in their genomes indicate the versatility of the species in obtaining energy and carbon from diverse substrates, therefore these organisms constitute a remarkable resource of enzymes with biotechnological potential.

Y chromosome phylogeny for cutthroat trout (Oncorhynchus clarkii) subspecies is generally concordant with those of other markers.

Sequence divergence was evaluated in the non-recombining, male-specific OmyY1 region of the Y chromosome among the subspecies of cutthroat trout (Oncorhynchus clarkii) in the western United States. This evaluation identified subspecies-discriminating OmyY1-haplotypes within a ∼1200bp region of the OmyY1 locus and localized the region to the end of the Y chromosome by FISH analysis. OmyY1 sequences were aligned and used to reconstruct a phylogeny of the cutthroat trout subspecies and related species via maximum-parsimony and Bayesian analyses. In the Y-haplotype phylogeny, clade distributions generally corresponded to the geographic distributions of the recognized subspecies. This phylogeny generally corresponded to a mitochondrial tree obtained for these subspecies in a previous study. Both support a clade of trout vs. Pacific salmon, of rainbow trout, and of a Yellowstone cutthroat group within the cutthroat trout. In our OmyY1 tree, however, the cutthroat "clade", although present topologically, was not statistically significant. Some key differences were found between trees obtained from the paternally-inherited OmyY1 vs. maternally-inherited mitochondrial haplotypes in cutthroat trout compared to rainbow trout. Other findings are: The trout OmyY1 region evolves between 3 and 13 times slower than the trout mitochondrial regions that have been studied. The Lahontan cutthroat trout had a fixed OmyY1 sequence throughout ten separate populations, suggesting this subspecies underwent a severe population bottleneck prior to its current dispersal throughout the Great Basin during the pluvial phase of the last ice age. The Yellowstone group is the most derived among the cutthroat trout and consists of the Yellowstone, Bonneville, Colorado, Rio Grande and greenback subspecies. Identification of subspecies and sex with this Y-chromosome marker may prove useful in conservation efforts.

RAD sequencing yields a high success rate for westslope cutthroat and rainbow trout species-diagnostic SNP assays.

Hybridization with introduced rainbow trout threatens most native westslope cutthroat trout populations. Understanding the genetic effects of hybridization and introgression requires a large set of high-throughput, diagnostic genetic markers to inform conservation and management. Recently, we identified several thousand candidate single-nucleotide polymorphism (SNP) markers based on RAD sequencing of 11 westslope cutthroat trout and 13 rainbow trout individuals. Here, we used flanking sequence for 56 of these candidate SNP markers to design high-throughput genotyping assays. We validated the assays on a total of 92 individuals from 22 populations and seven hatchery strains. Forty-six assays (82%) amplified consistently and allowed easy identification of westslope cutthroat and rainbow trout alleles as well as heterozygote controls. The 46 SNPs will provide high power for early detection of population admixture and improved identification of hybrid and nonhybridized individuals. This technique shows promise as a very low-cost, reliable and relatively rapid method for developing and testing SNP markers for nonmodel organisms with limited genomic resources.

Hybridization rapidly reduces fitness of a native trout in the wild.

Human-mediated hybridization is a leading cause of biodiversity loss worldwide. How hybridization affects fitness and what level of hybridization is permissible pose difficult conservation questions with little empirical information to guide policy and management decisions. This is particularly true for salmonids, where widespread introgression among non-native and native taxa has often created hybrid swarms over extensive geographical areas resulting in genomic extinction. Here, we used parentage analysis with multilocus microsatellite markers to measure how varying levels of genetic introgression with non-native rainbow trout (Oncorhynchus mykiss) affect reproductive success (number of offspring per adult) of native westslope cutthroat trout (Oncorhynchus clarkii lewisi) in the wild. Small amounts of hybridization markedly reduced fitness of male and female trout, with reproductive success sharply declining by approximately 50 per cent, with only 20 per cent admixture. Despite apparent fitness costs, our data suggest that hybridization may spread due to relatively high reproductive success of first-generation hybrids and high reproductive success of a few males with high levels of admixture. This outbreeding depression suggests that even low levels of admixture may have negative effects on fitness in the wild and that policies protecting hybridized populations may need reconsideration.

Counterselection of prokaryotic ribosomal RNA during reverse transcription using non-random hexameric oligonucleotides.

Ribosomal RNA (rRNA) is the major component in total RNA extracts, interfering with the synthesis of cDNA corresponding to messenger RNA (mRNA). In this study, we present a novel strategy for selectively discriminating against rRNA and favoring mRNA from prokaryotes during synthesis of cDNA by reverse transcriptase. Our technique is based on the fact that rRNA sequences, in many species, are G+C rich relative to the genome at large, and highly conserved among prokaryotes. The sequence TTTT is therefore rarely found in rRNA sequences. However, TTTT priming sites are found at a much higher frequency in protein-encoding gene sequences. We designed specific hexamers (HD/DHTTTT) to prime reverse transcription reactions resulting in a selective synthesis of cDNA corresponding to mRNA from prokaryotic total RNA extractions.

[Characterization of membrane-bound Fe(III)-EDTA reductase activities of the thermophilic gram-positive dissimilatory iron-reducing bacterium Thermoterrabacterium ferrireducens].

Whole-cell suspensions of T. ferrireducens reduced Fe(III) citrate, Fe(III)-EDTA, and ferrihydrite with glycerol as an electron donor. After cell disruption, the highest activity was registered with Fe(III)-EDTA as the electron acceptor and NADH or NAD(P)H as electron donors. About 80% of the NAD(P)H-dependent Fe(III)-EDTA reductase activities were associated with the membrane fraction of the cells. Treatment of the membranes with lauryl maltoside led to complete solubilization of the NADH-dependent and 70% solubilization of the NADPH-dependent Fe(III)-EDTA reductase activities. After purification by ion-exchange chromatography, the NADH-dependent activity was concentrated 8-fold, and the NADPH-dependent activity was concentrated 11-fold, with a yield of about 10% for both activities. The Fe(III)-EDTA-reducing enzyme complex included c-type cytochromes and a protein with a molecular mass of ca. 115 k Da, consisting of two polypeptides. This is the first description of membrane-bound Fe(III)-reducing oxidoreductase activities from a gram-positive dissimilatory Fe(III)-reducing bacterium.

Montelukast protects against nasal lysine-aspirin challenge in patients with aspirin-induced asthma.

Aspirin-induced asthma (AIA) is associated with increased production of cysteinyl leukotrienes (CysLT). Although leukotriene CysLT1-receptor antagonists improve lower airway outcomes in AIA, their effects and dose-response in the upper airway is less well documented. The present study evaluated the dose-response for montelukast (ML) against nasal lysine-aspirin challenge in patients with AIA. A total of 12 patients with a clear-cut history of AIA were randomised in double-blind cross-over fashion to receive single doses of ML 10 mg, ML 40 mg, or placebo (PL), with nasal lysine-aspirin challenge performed 12 h after dosing. Measurements of peak nasal inspiratory flow (PNIF), nasal blockage visual analogue scale (VAS) and forced expiratory volume in one second (FEV1) were made over 120 min after nasal lysine-aspirin challenge. Prechallenge values for mean+/-SEM PNIF (L x min(-1)) were not significantly different comparing all groups: ML 10 mg (132+/-10), ML 40 mg (125+/-12) and PL (132+/-11). There was no significant difference comparing the maximum % PNIF fall from baseline between screening (46+/-6) and PL (45+/-6). The maximum % PNIF fall from baseline was significantly greater with PL (45+/-6) compared to either ML 10 mg (34+/-6) or ML 40 mg (32+/-5). There was also a significantly greater mean % PNIF response over 120 min after lysine-aspirin challenge for PL (26+/-7) compared to either ML 10 mg (14+/-6) or ML 40 mg (17+/-6). There were no significant differences for the maximum or mean % PNIF fall from baseline comparing ML 10 mg and ML 40 mg. A significant increase in nasal blockage VAS score was observed between baseline and 60 min or 120 min with PL but not with ML 10 mg or ML 40 mg. There were no significant differences for either the maximum or mean % FEV1 over 120 min as change from baseline comparing all groups. A single 10 mg dose of montelukast partially protected against the local effects of nasal lysine-aspirin challenge, with no further benefit at 40 mg. Nasal lysine-aspirin challenge appeared to be a reproducible and safe method in assessing patients with aspirin-induced asthma.

Development of two PCR-based techniques for detecting helical and coccoid forms of Helicobacter pylori.

The primary mode of transmission of Helicobacter pylori, a human pathogen carried by more than half the population worldwide, is still unresolved. Some epidemiological data suggest water as a possible transmission route. H. pylori in the environment transforms into a nonculturable, coccoid form, which frequently results in the failure to detect this bacterium in environmental samples by conventional culture techniques. To overcome limitations associated with culturing, molecular approaches based on DNA amplification by PCR have been developed and used for the detection of H. pylori in clinical and environmental samples. Our results showed the glmM gene as the most promising target for detection of H. pylori by PCR amplification. Under optimal amplification conditions, glmM-specific primers generated PCR-amplified products that were specific for H. pylori and some other Helicobacter species. Genome sequence analysis revealed the existence of a conserved region linked to a hypervariable region upstream of the 16S rRNA gene of H. pylori. Selective PCR primer sets targeting this sequence were evaluated for the specific detection of H. pylori. One primer set, Cluster2 and B1J99, were shown to be highly specific for H. pylori strains and did not produce any PCR products when other Helicobacter species and other bacterial species were analyzed. In tests with 32 strains of H. pylori, 6 strains of other Helicobacter species, 8 strains of Campylobacter jejuni, and 21 strains belonging to different genera, the primers for glmM were selective for the Helicobacter genus and the primers containing the region flanking the 16S rRNA gene were selective for H. pylori species only. The combination of two sensitive PCR-based methods, one targeting the glmM gene and the other targeting a hypervariable flanking region upstream of the 16S rRNA gene, are complementary to each other. Whereas the glmM-specific primers provide a rapid, sensitive presumptive assay for the presence of H. pylori and closely related Helicobacter spp., the primers for sequences flanking the 16S rRNA gene can confirm the presence of H. pylori and locate the potential source of this bacterium.

A placebo-controlled evaluation of butterbur and fexofenadine on objective and subjective outcomes in perennial allergic rhinitis.

BACKGROUND: There are presently no placebo-controlled data regarding the effects of butterbur (BB) on subjective and objective outcomes in patients with perennial allergic rhinitis. OBJECTIVE: We performed a placebo-controlled evaluation of the effects of BB and fexofenadine (FEX) on subjective and objective outcomes in patients with perennial allergic rhinitis. METHODS: Sixteen patients with perennial allergic rhinitis and house dust mite sensitization were randomized in double-blind cross-over fashion to receive for 1 week either BB 50 mg twice daily, FEX 180 mg once daily and placebo (PL) once daily, or PL twice daily. The peak nasal inspiratory flow (PNIF) response to adenosine monophosphate (AMP) challenge administered as a single 400 mg/mL dose was measured over a 60-min period after challenge, and domiciliary total nasal symptom score was recorded. RESULTS: Pre-challenge values for mean+/-SEM PNIF (L/min) were not significantly different comparing all groups; BB (138+/-8), FEX (140+/-9), and PL (138+/-8). The maximum % PNIF fall from baseline after nasal AMP challenge was significantly attenuated (P<0.05) compared to PL (46+/-3), with BB (34+/-3) and FEX (39+/-3). The area under the 60-min time-response curve (%.min) was also significantly attenuated (P<0.05) compared to PL (1734+/-156), with BB (1052+/-258) and FEX (1194+/-161). There was also a significant reduction (P<0.05) in total nasal symptom score with BB (1.8+/-0.4) and FEX (1.8+/-0.4), compared to PL (2.8+/-0.5). There were no significant differences between BB and FEX for any outcomes. CONCLUSION: BB and FEX, in comparison to PL, were equally effective in attenuating the nasal response to AMP and in improving nasal symptoms, highlighting a potential role for BB in the treatment of allergic rhinitis.

Butterbur, a herbal remedy, confers complementary anti-inflammatory activity in asthmatic patients receiving inhaled corticosteroids.

BACKGROUND: The effects of butterbur (BB), a herbal remedy, as add-on therapy to inhaled corticosteroids in patients with atopic asthma is currently unknown. OBJECTIVE: We evaluated the effects of BB, given as add-on therapy to asthmatic patients maintained on inhaled corticosteroids, assessing adenosine monophosphate (AMP) bronchoprovocation (primary outcome variable) along with other surrogate inflammatory markers such as exhaled nitric oxide, serum eosinophil cationic protein and peripheral blood eosinophil count. METHODS: Sixteen atopic asthmatic patients with mean (standard error of mean) forced expiratory volume in 1 s (FEV1) of 78 (4)% predicted, maintained on their constant dose of inhaled corticosteroids throughout the study, received twice daily for 1 week either BB 25 mg or placebo (PL), in a double-blind, cross-over fashion, with a 1-week washout period prior to each randomized treatment. Measurements were made at baselines prior to each randomized treatment and following the randomized treatment period. RESULTS: Baseline values for the primary and secondary outcomes were not significantly different prior to BB and PL. AMP provocative concentration causing a 20% reduction from baseline FEV1 (PC20) as doubling dilution change from baseline, significantly improved (P<0.05) with BB, 0.6 (0.2), compared with PL, -0.1 (0.3); a 0.7 doubling dilution difference. Exhaled nitric oxide as change from baseline was significantly reduced (P<0.05) with BB, -1.2 (0.8) p.p.b., compared with PL, 0.5 (0.4) p.p.b. Both serum eosinophil cationic protein and peripheral blood eosinophil count as change from baseline were also significantly suppressed (P<0.05) with BB, -3.9 (3.3) microg/L, -31 (28)x106/L compared with PL, 3.3 (2.5) microg/L, 38 (16)x106/L, respectively. CONCLUSION: Chronic dosing with BB conferred complementary anti-inflammatory activity in atopic asthmatic patients maintained on inhaled corticosteroids. Further studies are now required to assess the potential role for BB as either monotherapy in milder patients or add-on therapy in more severe asthmatics.

Multi-subunit assembly of the Pyrococcus furiosus small heat shock protein is essential for cellular protection at high temperature.

The hyperthermophilic archaeon, Pyrococcus furiosus, expresses a small, alpha-crystallin-like protein in response to exposure to extreme temperatures, above 103 degrees C. The P. furiosus small heat shock protein (Pfu-sHSP) forms large oligomeric complexes. Based on the available crystal structures of the Methanocaldococcus jannaschii and wheat sHSPs, the protruding carboxy terminal domain is probably involved in subunit interactions. We constructed Pfu-sHSP mutants to analyze chaperone function and to study multi-subunit assembly. The results confirmed that the carboxy terminus of Pfu-sHSP is involved in inter-dimer interactions, whereas the amino terminal deletion mutant still exhibited the wild-type assembly characteristics. The ability to form oligomeric complexes via the carboxy terminal domain was shown to be necessary for thermotolerance of Escherichia coli overexpressing Pfu-sHSP. The amino terminal domain was not required for inter-species thermotolerance.

Firefly flashing is controlled by gating oxygen to light-emitting cells.

Although many aspects of firefly bioluminescence are understood, the mechanism by which adult fireflies produce light as discrete rapid flashes is not. Here we examine the most postulated theory, that flashing is controlled by gating oxygen access to the light-emitting cells (photocytes). According to this theory, the dark state represents repression of bioluminescence by limiting oxygen, which is required for bioluminescence; relief from this repression by transiently allowing oxygen access to the photocytes allows the flash. We show that normobaric hyperoxia releases the repression of light emission in the dark state of both spontaneously flashing and non-flashing fireflies, causing continual glowing, and we measure the kinetics of this process. Secondly, we determine the length of the barriers to oxygen diffusion to the photocytes in the aqueous and gas phases. Thirdly, we provide constraints upon the distance between any gas-phase gating structure(s) and the photocytes. We conclude from these data that the flash of the adult firefly is controlled by gating of oxygen to the photocytes, and demonstrate that this control mechanism is likely to act by modulating the levels of fluid in the tracheoles supplying photocytes, providing a variable barrier to oxygen diffusion.

Mechanism of pressure-induced thermostabilization of proteins: studies of glutamate dehydrogenases from the hyperthermophile Thermococcus litoralis.

In this study, we investigated the effect of pressure on protein structure and stability at high temperature. Thermoinactivation experiments at 5 and 500 atm were performed using the wild-type (WT) enzyme and two single mutants (D167T and T138E) of the glutamate dehydrogenase (GDH) from the hyperthermophile Thermococcus litoralis. All three GDHs were stabilized, although to different degrees, by the application of 500 atm. Interestingly, the degree of pressure stabilization correlated with GDH stability as well as the magnitude of electrostatic repulsion created by residues at positions 138 and 167. Thermoinactivation experiments also were performed in the presence of trehalose. Addition of the sugar stabilized all three GDHs; the degree of sugar-induced thermostabilization followed the same order as pressure stabilization. Previous studies suggested a mechanism whereby the enzyme adopts a more compact and rigid structure and volume fluctuations away from the native state are diminished under pressure. The present results on the three GDHs allowed us to further confirm and refine the proposed mechanism for pressure-induced thermostabilization. In particular, we propose that pressure stabilizes against thermoinactivation by shifting the equilibrium between conformational substates of the GDH hexamer, thus inhibiting irreversible aggregation.

Regulation and mechanism of action of the small heat shock protein from the hyperthermophilic archaeon Pyrococcus furiosus.

The small heat shock protein (sHSP) from the hyperthermophile Pyrococcus furiosus was specifically induced at the level of transcription by heat shock at 105 degrees C. The gene encoding this protein was cloned and overexpressed in Escherichia coli. The recombinant sHSP prevented the majority of E. coli proteins from aggregating in vitro for up to 40 min at 105 degrees C. The sHSP also prevented bovine glutamate dehydrogenase from aggregating at 56 degrees C. Survivability of E. coli overexpressing the sHSP was enhanced approximately sixfold during exposure to 50 degrees C for 2 h compared with the control culture, which did not express the sHSP. Apparently, the sHSP confers a survival advantage on mesophilic bacteria by preventing protein aggregation at supraoptimal temperatures.