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1.  Conversion of 4-Hydroxybutyrate to Acetyl Coenzyme A and Its Anapleurosis in the Metallosphaera sedula 3-Hydroxypropionate/4-Hydroxybutyrate Carbon Fixation Pathway 
The extremely thermoacidophilic archaeon Metallosphaera sedula (optimum growth temperature, 73°C, pH 2.0) grows chemolithoautotrophically on metal sulfides or molecular hydrogen by employing the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) carbon fixation cycle. This cycle adds two CO2 molecules to acetyl coenzyme A (acetyl-CoA) to generate 4HB, which is then rearranged and cleaved to form two acetyl-CoA molecules. Previous metabolic flux analysis showed that two-thirds of central carbon precursor molecules are derived from succinyl-CoA, which is oxidized to malate and oxaloacetate. The remaining one-third is apparently derived from acetyl-CoA. As such, the steps beyond succinyl-CoA are essential for completing the carbon fixation cycle and for anapleurosis of acetyl-CoA. Here, the final four enzymes of the 3HP/4HB cycle, 4-hydroxybutyrate-CoA ligase (AMP forming) (Msed_0406), 4-hydroxybutyryl-CoA dehydratase (Msed_1321), crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (Msed_0399), and acetoacetyl-CoA β-ketothiolase (Msed_0656), were produced recombinantly in Escherichia coli, combined in vitro, and shown to convert 4HB to acetyl-CoA. Metabolic pathways connecting CO2 fixation and central metabolism were examined using a gas-intensive bioreactor system in which M. sedula was grown under autotrophic (CO2-limited) and heterotrophic conditions. Transcriptomic analysis revealed the importance of the 3HP/4HB pathway in supplying acetyl-CoA to anabolic pathways generating intermediates in M. sedula metabolism. The results indicated that flux between the succinate and acetyl-CoA branches in the 3HP/4HB pathway is governed by 4-hydroxybutyrate-CoA ligase, possibly regulated posttranslationally by the protein acetyltransferase (Pat)/Sir2-dependent system. Taken together, this work confirms the final four steps of the 3HP/4HB pathway, thereby providing the framework for examining connections between CO2 fixation and central metabolism in M. sedula.
PMCID: PMC3993168  PMID: 24532060
2.  Initiation of health behavior discussions during primary care outpatient visits 
Patient education and counseling  2008;75(2):214-219.
Despite the importance of health promotion, rates of health behavior advice remain low and little is known about how advice is integrated into routine primary care. This study examines how health behavior topics of diet, physical activity and smoking are initiated during outpatient visits.
Audio recording of 187 adults visit to five purposefully selected physicians. An iterative analysis involved listening to and discussing cases to identify emergent patterns of initiation of health behavior talk and advice that followed.
Physicians initiated 65% of discussions and used two overarching strategies (1) Structured: a routine to ask about health behavior and (2) Opportunistic: use of a trigger to make a transition to talk about health behavior. Opportunistic strategies identified a greater proportion of patients at risk (50% vs. 34%) and led to a greater rate of advice (100% vs. 75%). Patients initiated one-third of health behavior discussions and were more likely to receive advice if they explicitly indicated readiness to change.
Opportunistic strategies show promise for a higher yield of identifying patients at risk and leading to advice.
Practice Implications
Encouraging patients to be explicit about their readiness to change is likely to increase physician advice and assistance.
PMCID: PMC4166517  PMID: 19013742
Clinician–patient communication; Health behavior change; Primary care; Practice-based research
3.  Stationary Phase and Nutrient Levels Trigger Transcription of a Genomic Locus Containing a Novel Peptide (TM1316) in the Hyperthermophilic Bacterium Thermotoga maritima 
Applied and Environmental Microbiology  2013;79(21):6637-6646.
The genome of the hyperthermophilic bacterium Thermotoga maritima encodes numerous putative peptides/proteins of 100 amino acids or less. While most of these open reading frames (ORFs) are transcribed during growth, their corresponding physiological roles are largely unknown. The onset of stationary phase in T. maritima was accompanied by significant morphological changes and upregulation of several ORFs located in the TM1298-TM1336 genome locus. This region contains putative HicAB toxin-antitoxin pairs, hypothetical proteins, radical S-adenosylmethionine (SAM) enzymes, and ABC transporters. Of particular note was the TM1315-TM1319 operon, which includes a putative 31-amino-acid peptide (TM1316) that was the most highly transcribed gene in the transcriptome during stationary phase. Antibodies directed against a synthetic version of TM1316 were used to track its production, which correlated closely with transcriptomic data. Immunofluorescence microscopy revealed that TM1316 was localized to the cell envelope and prominent in cell aggregates formed during stationary phase. The only functionally characterized locus with an organization similar to that of TM1315-TM1319 is in Bacillus subtilis, which contains subtilosin A, a cyclic peptide with Cys–to–α-carbon linkages that functions as an antilisterial bacteriocin. While the organization of TM1316 resembled that of the Bacillus peptide (e.g., in its number of amino acids and spacing of Cys residues), preparations containing high levels of TM1316 affected the growth of neither Thermotoga species nor Pyrococcus furiosus, a hyperthermophilic archaeon isolated from the same locale as T. maritima. Several other putative Cys-rich peptides could be identified in the TM1298-TM1336 locus, and while their roles are also unclear, they merit examination as potential antimicrobial agents in hyperthermophilic biotopes.
PMCID: PMC3811502  PMID: 23974142
4.  Extreme Thermophiles: Moving beyond single-enzyme biocatalysis 
Extremely thermophilic microorganisms have been sources of thermostable and thermoactive enzymes for over 30 years. However, information and insights gained from genome sequences, in conjunction with new tools for molecular genetics, have opened up exciting new possibilities for biotechnological opportunities based on extreme thermophiles that go beyond single-step biotransformations. Although the pace for discovering novel microorganisms has slowed over the past two decades, genome sequence data have provided clues to novel biomolecules and metabolic pathways, which can be mined for a range of new applications. Furthermore, recent advances in molecular genetics for extreme thermophiles have made metabolic engineering for high temperature applications a reality.
PMCID: PMC3568776  PMID: 23413412
5.  Life in hot acid: Pathway analyses in extremely thermoacidophilic archaea 
Current opinion in biotechnology  2008;19(5):445-453.
The extremely thermoacidophilic archaea are a particularly intriguing group of microorganisms that must simultaneously cope with biologically extreme pHs (≤ 4) and temperatures (Topt ≥ 60°C) in their natural environments. Their expandi ng biotechnological significance relates to their role in biomining of base and precious metals and their unique mechanisms of survival in hot acid, at both the cellular and biomolecular levels. Recent developments, such as advances in understanding of heavy metal tolerance mechanisms, implementation of a genetic system, and discovery of a new carbon fixation pathway, have been facilitated by availability of genome sequence data and molecular genetic systems. As a result, new insights into the metabolic pathways and physiological features that define extreme thermoacidophily have been obtained, in some cases suggesting prospects for biotechnological opportunities.
PMCID: PMC3771398  PMID: 18760359
extreme thermoacidophile; bioleaching; CO2 fixation; archaeal genetics
6.  The genus Thermotoga: Recent developments 
Environmental technology  2010;31(10):1169-1181.
The genus Thermotoga comprises extremely thermophilic (Topt ≥ 70°C) and hyperthermophilic (Topt ≥ 80°C) bacteria that have been extensively studied for insights into the basis for life at elevated temperatures and for biotechnological opportunities (e.g., biohydrogen production, biocatalysis). Over the past decade, genome sequences have become available for a number of Thermotoga species, leading to functional genomics efforts to understand growth physiology as well as genomics-based identification and characterization of novel high temperature biocatalysts. Discussed here are recent developments along these lines for this novel group of microorganisms.
PMCID: PMC3752655  PMID: 20718299
Thermotoga; extreme thermophiles; genomics; microbial physiology
7.  Part II: defining and quantifying individual and co-cultured intracellular proteomes of two thermophilic microorganisms by GeLC-MS2 and spectral counting 
Probing the intracellular proteome of Thermotoga maritima and Caldicellulosiruptor saccharolyticus in pure and co-culture affords a global investigation into the machinery and mechanisms enduring inside the bacterial thermophilic cell at the time of harvest. The second of a two part study, employing GeLC-MS2 a variety of proteins were confidently identified with <1% false discovery rate, and spectral counts for label-free relative quantification afforded indication of the dynamic proteome as a function of environmental stimuli. Almost 25% of the T. maritima proteome and 10% of the C. saccharolyticus proteome were identified. Through comparison of growth temperatures for T. maritima, a protein associated with chemotaxis was uniquely present in the sample cultivated at the non-optimal growth temperature. It is suspected that movement was induced due to the non-optimal condition as the organism may need to migrate in the culture to locate more nutrients. The inventory of C. saccharolyticus proteins identified in these studies and attributed to spectral counting, demonstrated that two CRISPR-associated proteins had increased expression in the pure culture versus the co-culture. Further focusing on this relationship, a C. saccharolyticus phage-shock protein was identified in the co-culture expanding a scenario that the co-culture had decreased antiviral resistance and accordingly an infection-related protein was present. Alterations in growth conditions of these bacterial thermophilic microorganisms offer a glimpse into the intricacy of microbial behavior and interaction.
PMCID: PMC3727164  PMID: 20582400
Thermotoga maritima; Caldicellulosiruptor saccharolyticus; GeLC-MS2; Co-cultivation; Intracellular proteome; Spectral counting
8.  Part I: Characterization of the Extracellular Proteome of the Extreme Thermophile Caldicellulosiruptor saccharolyticus by GeLC-MS2 
The proteome of extremely thermophilic microorganisms affords a glimpse into the dynamics of microbial ecology of high temperature environments. The secretome, or extracellular proteome of these microorganisms no doubt harbors technologically important enzymes and other thermostable biomolecules that to date have been characterized only to a limited extent. In the first of a two part study on selected thermophiles, defining the secretome requires a sample preparation method that has no negative impact on all downstream experiments. Following efficient secretome purification, GeLC-MS2 analysis and prediction servers suggest probable protein secretion to complement experimental data. In an effort to define the extracellular proteome of the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus, several techniques were considered regarding sample processing to achieve the most in-depth analysis of secreted proteins. Order of operation experiments all including the C18 bead technique demonstrated that two levels of sample purification were necessary to effectively de-salt the sample and provide sufficient protein identifications. Five sample preparation combinations yielded 71 proteins and the majority described as enzymatic and putative uncharacterized proteins anticipating consolidated bioprocessing applications. Nineteen proteins were predicted by Phobius, SignalP, SecretomeP, or TatP for extracellular secretion and 11 contain transmembrane domain stretches suggested by Phobius and TMHMM. The sample preparation technique demonstrating the most effective outcome for C. saccharolyticus secreted proteins in this study involves acetone precipitation followed by the C18 bead method in which 2.4% (63 proteins) of the predicted proteome was indentified including proteins suggested to have secretion and transmembrane moieties.
PMCID: PMC3727170  PMID: 20623222
Secretome; Caldicellulosiruptor saccharolyticus; GeLC-MS2; Thermophile; Sample Preparation
9.  Fgf10 dosage is critical for the amplification of epithelial cell progenitors and for the formation of multiple mesenchymal lineages during lung development 
Developmental biology  2007;307(2):237-247.
The key role played by of Fgf10 during early lung development is clearly illustrated in Fgf10 knockout mice, which exhibit complete lung agenesis. However, Fgf10 is continuously expressed throughout lung development suggesting extended as well as additional roles for FGF10 at later stages of lung organogenesis. We previously reported that the enhancer trap Mlcv1v-nLacZ-24 transgenic mouse strain functions as a reporter for Fgf10 expression and displays decreased endogenous Fgf10 expression (Mailleux et al., 2005). In this paper, we have generated an allelic series to determine the impact of Fgf10 dosage on lung development. We report that 80% of the newborn Fgf10 hypomorphic mice die within 24 hours of respiratory failure. These mutant lungs display severe hypoplasia, dilation of the distal airways and large hemorrhagic areas. Epithelial differentiation and proliferation studies indicate a specific decrease in the percentile of TTF1 and SP-B expressing cells correlating with reduced epithelial cell proliferation and associated with a decrease in activation of the canonical Wnt signaling in the epithelium. Analysis of vascular development shows a reduction in PECAM expression at E14.5, which is associated with a simplification of the vascular tree at E18.5. We also show a decrease in α-SMA expression in the respiratory airway suggesting defective formation of the alveolar smooth muscle cells. At the molecular level, these defects are associated with a decrease in Vegfa and Pdgfa expression likely resulting from the decrease of the epithelium/mesenchymal ratio in the Fgf10 hypomorphic lungs. Thus, our results indicate that FGF10 plays a pivotal role in maintaining epithelial progenitor cell proliferation as well as coordinating alveolar smooth muscle cell formation and vascular development.
PMCID: PMC3714306  PMID: 17560563
Fgf10 hypomorph; mesenchymal differentiation; smooth muscle cells; lung emphysema; vascularization
10.  N-terminal fusion of a hyperthermophilic chitin-binding domain to xylose isomerase from Thermotoga neapolitana enhances kinetics and thermostability of both free and immobilized enzymes 
Biotechnology progress  2010;26(4):993-1000.
Immobilization of a thermostable D-xylose isomerase (EC from Thermotoga neapolitana 5068 (TNXI) on chitin beads was accomplished via a N-terminal fusion with a chitin-binding domain (CBD) from a hyperthermophilic chitinase produced by Pyrococcus furiosus (PF1233) to create a fusion protein (CBD-TNXI). The turnover numbers for glucose to fructose conversion for both unbound and immobilized CBD-TNXI were greater than the wild-type enzyme: kcat (min−1) was approximately 1000, 3800, and 5800 at 80°C compared to 1140, 10350, and 7000 at 90°C, for the wild-type, unbound, and immobilized enzymes, respectively. These kcat values for the glucose to fructose isomerization measured are the highest reported to date for any XI at any temperature. Enzyme kinetic inactivation at 100°C, as determined from a bi-phasic inactivation model, showed that the CBD-TNXI bound to chitin had a half-life approximately three times longer than the soluble wild-type TNXI (19.9 hours vs. 6.8 hours, respectively). Surprisingly, the unbound soluble CBD-TNXI had a significantly longer half-life (56.5 hours) than the immobilized enzyme. Molecular modeling results suggest that the N-terminal fusion impacted subunit interactions, thereby contributing to the enhanced thermostability of both the unbound and immobilized CBD-TNXI. These interactions likely also played a role in modifying active site structure, thereby diminishing substrate-binding affinities and generating higher turnover rates in the unbound fusion protein.
PMCID: PMC3711014  PMID: 20730758
xylose isomerase; hyperthermophile; Thermotoga neapolitana; immobilization; chitin-binding domain
11.  Semantic Organizational Strategy Predicts Verbal Memory and Remission Rate of Geriatric Depression 
This study tests the hypothesis that the use of a semantic organizational strategy, during the free recall phase of a verbal memory task predicts remission of geriatric depression.
65 elderly patients with major depression participated in a 12-week escitalopram treatment trial. Neuropsychological performance was assessed at baseline after a 2-week drug washout period. The Hopkins Verbal Learning Test-Revised (HVLT-R)(Brandt 2001) was used to assess verbal learning and memory. Remission was defined as a Hamilton Depression Rating Scale Score (HDRS) less than or equal to 7 for two consecutive weeks and no longer meeting DSM-IV-TR criteria for major depression. The association between the number of clusters used at the final learning trial (Trial 3) and remission was examined using Cox’s proportional hazards survival analysis. The relationship between the number of clusters utilized in the final learning trial and words recalled after a 25 minute delay was examined in a regression with age and education as covariates.
Greater number of clusters utilized predicted remission rate (Hazard ratio (95% CI) = 1.26 (1.04–1.54), χ2 =4.23, df=3, p=0.04). There was a positive relationship between the total number of clusters used by the end of the third learning trial and the total number of words recalled at the delayed recall trial (F(3,58)=7.93;p<.001)
Effective semantic strategy use at baseline on a verbal list learning task by elderly depressed patients was associated with greater rate of remission with antidepressant treatment. This result provides support for previous findings indicating that measures of executive functioning at baseline are useful in predicting antidepressant response.
PMCID: PMC3188360  PMID: 21618287
Executive function; geriatric; depression; remission; semantic strategy; Hopkins Verbal Learning Test-Revised (HVLT)
12.  Executive Function and Remission of Geriatric Depression: The Role of Semantic Strategy 
This study tested the hypothesis that use of semantic organizational strategy in approaching the Mattis Dementia Rating Scale (MDRS) Complex Verbal Initiation Perseveration (I/P) task, a test of semantic fluency, is the function specifically associated with remission of late-life depression.
70 elders with major depression participated in a 12-week escitalopram treatment trial. Neuropsychological performance was assessed at baseline after a 2-week drug washout period. Patients with a Hamilton Depression Rating Scale Score less than or equal to 7 for two consecutive weeks and who no longer met DSM-IV criteria were considered to be remitted. Cox proportional hazards survival analysis was used to examine the relationship between subtests of the I/P, other neuropsychological domains and remission rate. Participants’ performance on the CV I/P was coded for perseverations, and use of semantic strategy.
The relationship of performance on the Complex Verbal I/P and remission rate was significant. No other subtest of the MDRS I/P evidenced this association. There was no significant relationship of speed, confrontation naming, verbal memory or perseveration with remission rate. Remitters’ use of verbal strategy was significantly greater than non-remitters.
Geriatric depressed patients who showed decrements in performance on a semantic fluency task showed poorer remission rates than those who showed adequate performance on this measure. Executive impairment in verbal strategy explained performance. This finding supports the concept that executive functioning exerts a “top down” effect on other basic cognitive processes, perhaps as a result of frontostriatal network dysfunction implicated in geriatric depression.
PMCID: PMC3628617  PMID: 20808124
Executive function; geriatric; depression; remission; semantic strategy; Mattis Dementia Rating Scale
13.  Identification of a Tbx1/Tbx2/Tbx3 genetic pathway governing pharyngeal and arterial pole morphogenesis 
Human Molecular Genetics  2011;21(6):1217-1229.
The 22q11.2 deletion syndrome (22q11.2DS) is the most common microdeletion disorder and is characterized by abnormal development of the pharyngeal apparatus and heart. Cardiovascular malformations (CVMs) affecting the outflow tract (OFT) are frequently observed in 22q11.2DS and are among the most commonly occurring heart defects. The gene encoding T-box transcription factor 1 (Tbx1) has been identified as a major candidate for 22q11.2DS. However, CVMs are generally considered to have a multigenic basis and single-gene mutations underlying these malformations are rare. The T-box family members Tbx2 and Tbx3 are individually required in regulating aspects of OFT and pharyngeal development. Here, using expression and three-dimensional reconstruction analysis, we show that Tbx1 and Tbx2/Tbx3 are largely uniquely expressed but overlap in the caudal pharyngeal mesoderm during OFT development, suggesting potential combinatorial requirements. Cross-regulation between Tbx1 and Tbx2/Tbx3 was analyzed using mouse genetics and revealed that Tbx1 deficiency affects Tbx2 and Tbx3 expression in neural crest-derived cells and pharyngeal mesoderm, whereas Tbx2 and Tbx3 function redundantly upstream of Tbx1 and Hh ligand expression in pharyngeal endoderm and bone morphogenetic protein- and fibroblast growth factor-signaling in cardiac progenitors. Moreover, in vivo, we show that loss of two of the three genes results in severe pharyngeal hypoplasia and heart tube extension defects. These findings reveal an indispensable T-box gene network governing pharyngeal and OFT development and identify TBX2 and TBX3 as potential modifier genes of the cardiopharyngeal phenotypes found in TBX1-haploinsufficient 22q11.2DS patients.
PMCID: PMC3491916  PMID: 22116936
14.  Epimerase (Msed_0639) and Mutase (Msed_0638 and Msed_2055) Convert (S)-Methylmalonyl-Coenzyme A (CoA) to Succinyl-CoA in the Metallosphaera sedula 3-Hydroxypropionate/4-Hydroxybutyrate Cycle 
Applied and Environmental Microbiology  2012;78(17):6194-6202.
Crenarchaeotal genomes encode the 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle for carbon dioxide fixation. Of the 13 enzymes putatively comprising the cycle, several of them, including methylmalonyl-coenzyme A (CoA) epimerase (MCE) and methylmalonyl-CoA mutase (MCM), which convert (S)-methylmalonyl-CoA to succinyl-CoA, have not been confirmed and characterized biochemically. In the genome of Metallosphaera sedula (optimal temperature [Topt], 73°C), the gene encoding MCE (Msed_0639) is adjacent to that encoding the catalytic subunit of MCM-α (Msed_0638), while the gene for the coenzyme B12-binding subunit of MCM (MCM-β) is located remotely (Msed_2055). The expression of all three genes was significantly upregulated under autotrophic compared to heterotrophic growth conditions, implying a role in CO2 fixation. Recombinant forms of MCE and MCM were produced in Escherichia coli; soluble, active MCM was produced only if MCM-α and MCM-β were coexpressed. MCE is a homodimer and MCM is a heterotetramer (α2β2) with specific activities of 218 and 2.2 μmol/min/mg, respectively, at 75°C. The heterotetrameric MCM differs from the homo- or heterodimeric orthologs in other organisms. MCE was activated by divalent cations (Ni2+, Co2+, and Mg2+), and the predicted metal binding/active sites were identified through sequence alignments with less-thermophilic MCEs. The conserved coenzyme B12-binding motif (DXHXXG-SXL-GG) was identified in M. sedula MCM-β. The two enzymes together catalyzed the two-step conversion of (S)-methylmalonyl-CoA to succinyl-CoA, consistent with their proposed role in the 3-HP/4-HB cycle. Based on the highly conserved occurrence of single copies of MCE and MCM in Sulfolobaceae genomes, the M. sedula enzymes are likely to be representatives of these enzymes in the 3-HP/4-HB cycle in crenarchaeal thermoacidophiles.
PMCID: PMC3416614  PMID: 22752162
15.  Caldicellulosiruptor Core and Pangenomes Reveal Determinants for Noncellulosomal Thermophilic Deconstruction of Plant Biomass 
Journal of Bacteriology  2012;194(15):4015-4028.
Extremely thermophilic bacteria of the genus Caldicellulosiruptor utilize carbohydrate components of plant cell walls, including cellulose and hemicellulose, facilitated by a diverse set of glycoside hydrolases (GHs). From a biofuel perspective, this capability is crucial for deconstruction of plant biomass into fermentable sugars. While all species from the genus grow on xylan and acid-pretreated switchgrass, growth on crystalline cellulose is variable. The basis for this variability was examined using microbiological, genomic, and proteomic analyses of eight globally diverse Caldicellulosiruptor species. The open Caldicellulosiruptor pangenome (4,009 open reading frames [ORFs]) encodes 106 GHs, representing 43 GH families, but only 26 GHs from 17 families are included in the core (noncellulosic) genome (1,543 ORFs). Differentiating the strongly cellulolytic Caldicellulosiruptor species from the others is a specific genomic locus that encodes multidomain cellulases from GH families 9 and 48, which are associated with cellulose-binding modules. This locus also encodes a novel adhesin associated with type IV pili, which was identified in the exoproteome bound to crystalline cellulose. Taking into account the core genomes, pangenomes, and individual genomes, the ancestral Caldicellulosiruptor was likely cellulolytic and evolved, in some cases, into species that lost the ability to degrade crystalline cellulose while maintaining the capacity to hydrolyze amorphous cellulose and hemicellulose.
PMCID: PMC3416521  PMID: 22636774
16.  Keratin-based Wound Care Products for Treatment of Resistant Vascular Wounds 
Use of new keratin-based wound dressings represent a novel approach to wound management. The authors present three patients with recalcitrant, venous and mixed venous, and arterial leg ulcers treated with these dressings. Improvement in each case was observed.
PMCID: PMC3533319  PMID: 23277802
17.  Ligand-Induced Asymmetry in Histidine Sensor Kinase Complex Regulates Quorum Sensing 
Cell  2006;126(6):1095-1108.
Bacteria sense their environment using receptors of the histidine sensor kinase family, but how kinase activity is regulated by ligand binding is not well understood. Autoinducer-2 (AI-2), a secreted signaling molecule originally identified in studies of the marine bacterium Vibrio harveyi, regulates quorum-sensing responses and allows communication between different bacterial species. AI-2 signal transduction in V. harveyi requires the integral membrane receptor LuxPQ, comprised of periplasmic binding protein (LuxP) and histidine sensor kinase (LuxQ) subunits. Combined X-ray crystallographic and functional studies show that AI-2 binding causes a major conformational change within LuxP, which in turn stabilizes a quaternary arrangement in which two LuxPQ monomers are asymmetrically associated. We propose that formation of this asymmetric quaternary structure is responsible for repressing the kinase activity of both LuxQ subunits and triggering the transition of V. harveyi into quorum-sensing mode.
PMCID: PMC3468944  PMID: 16990134
18.  Hyperthermophilic Thermotoga Species Differ with Respect to Specific Carbohydrate Transporters and Glycoside Hydrolases 
Four hyperthermophilic members of the bacterial genus Thermotoga (T. maritima, T. neapolitana, T. petrophila, and Thermotoga sp. strain RQ2) share a core genome of 1,470 open reading frames (ORFs), or about 75% of their genomes. Nonetheless, each species exhibited certain distinguishing features during growth on simple and complex carbohydrates that correlated with genomic inventories of specific ABC sugar transporters and glycoside hydrolases. These differences were consistent with transcriptomic analysis based on a multispecies cDNA microarray. Growth on a mixture of six pentoses and hexoses showed no significant utilization of galactose or mannose by any of the four species. T. maritima and T. neapolitana exhibited similar monosaccharide utilization profiles, with a strong preference for glucose and xylose over fructose and arabinose. Thermotoga sp. strain RQ2 also used glucose and xylose, but was the only species to utilize fructose to any extent, consistent with a phosphotransferase system (PTS) specific for this sugar encoded in its genome. T. petrophila used glucose to a significantly lesser extent than the other species. In fact, the XylR regulon was triggered by growth on glucose for T. petrophila, which was attributed to the absence of a glucose transporter (XylE2F2K2), otherwise present in the other Thermotoga species. This suggested that T. petrophila acquires glucose through the XylE1F1K1 transporter, which primarily serves to transport xylose in the other three Thermotoga species. The results here show that subtle differences exist among the hyperthermophilic Thermotogales with respect to carbohydrate utilization, which supports their designation as separate species.
PMCID: PMC3298158  PMID: 22247137
19.  S-Layer Homology Domain Proteins Csac_0678 and Csac_2722 Are Implicated in Plant Polysaccharide Deconstruction by the Extremely Thermophilic Bacterium Caldicellulosiruptor saccharolyticus 
The genus Caldicellulosiruptor contains extremely thermophilic bacteria that grow on plant polysaccharides. The genomes of Caldicellulosiruptor species reveal certain surface layer homology (SLH) domain proteins that have distinguishing features, pointing to a role in lignocellulose deconstruction. Two of these proteins in Caldicellulosiruptor saccharolyticus (Csac_0678 and Csac_2722) were examined from this perspective. In addition to three contiguous SLH domains, the Csac_0678 gene encodes a glycoside hydrolase family 5 (GH5) catalytic domain and a family 28 carbohydrate-binding module (CBM); orthologs to Csac_0678 could be identified in all genome-sequenced Caldicellulosiruptor species. Recombinant Csac_0678 was optimally active at 75°C and pH 5.0, exhibiting both endoglucanase and xylanase activities. SLH domain removal did not impact Csac_0678 GH activity, but deletion of the CBM28 domain eliminated binding to crystalline cellulose and rendered the enzyme inactive on this substrate. Csac_2722 is the largest open reading frame (ORF) in the C. saccharolyticus genome (predicted molecular mass of 286,516 kDa) and contains two putative sugar-binding domains, two Big4 domains (bacterial domains with an immunoglobulin [Ig]-like fold), and a cadherin-like (Cd) domain. Recombinant Csac_2722, lacking the SLH and Cd domains, bound to cellulose and had detectable carboxymethylcellulose (CMC) hydrolytic activity. Antibodies directed against Csac_0678 and Csac_2722 confirmed that these proteins bound to the C. saccharolyticus S-layer. Their cellular localization and functional biochemical properties indicate roles for Csac_0678 and Csac_2722 in recruitment and hydrolysis of complex polysaccharides and the deconstruction of lignocellulosic biomass. Furthermore, these results suggest that related SLH domain proteins in other Caldicellulosiruptor genomes may also be important contributors to plant biomass utilization.
PMCID: PMC3264102  PMID: 22138994
20.  Visual inspection of independent components: Defining a procedure for artifact removal from fMRI data 
Journal of Neuroscience Methods  2010;189(2):233-245.
Artifacts in fMRI data, primarily those related to motion and physiological sources, negatively impact the functional signal-to-noise ratio in fMRI studies, even after conventional fMRI preprocessing. Independent component analysis’ demonstrated capacity to separate sources of neural signal, structured noise, and random noise into separate components might be utilized in improved procedures to remove artifacts from fMRI data. Such procedures require a method for labeling independent components (ICs) as representing artifacts to be removed or neural signals of interest to be spared. Visual inspection is often considered an accurate method for such labeling as well as a standard to which automated labeling methods are compared. However, detailed descriptions of methods for visual inspection of ICs are lacking in the literature. Here we describe the details of, and the rationale for, an operationalized fMRI data denoising procedure that involves visual inspection of ICs (96% inter-rater agreement). We estimate that dozens of subjects/sessions can be processed within a few hours using the described method of visual inspection. Our hope is that continued scientific discussion of and testing of visual inspection methods will lead to the development of improved, cost-effective fMRI denoising procedures.
PMCID: PMC3299198  PMID: 20381530
fMRI; independent component analysis (ICA); denoising; visual inspection; artifacts; structured noise; independent component (IC) labeling
21.  Medical Transport of Children with Complex Chronic Conditions 
One of the most notable trends in child health has been the increase in the number of children with special health care needs, including those with complex chronic conditions. Care of these children accounts for a growing fraction of health care resources. We examine recent developments in health care, especially with regard to medical transport and prehospital care, that have emerged to adapt to this remarkable demographic trend. One such development is the focus on care coordination, including the dissemination of the patient-centered medical home concept. In the prehospital setting, the need for greater coordination has catalyzed the development of the emergency information form. Training programs for prehospital providers now incorporate specific modules for children with complex conditions. Another notable trend is the shift to a family-centered model of care. We explore efforts toward regionalization of care, including the development of specialized pediatric transport teams, and conclude with recommendations for a research agenda.
PMCID: PMC3270524  PMID: 22315689
22.  Group XV phospholipase A2, a lysosomal phospholipase A2 
Progress in lipid research  2011;50(1):1-13.
A phospholipase A2 was identified from MDCK cell homogenates with broad specificity toward glycerophospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. The phospholipase has the unique ability to transacylate short chain ceramides. This phospholipase is calcium-independent, localized to lysosomes, and has an acidic pH optimum. The enzyme was purified from bovine brain and found to be a water-soluble glycoprotein consisting of a single peptide chain with a molecular weight of 45 kDa. The primary structure deduced from the DNA sequences is highly conserved between chordates. The enzyme was named lysosomal phospholipase A2 (LPLA2) and subsequently designated group XV phospholipase A2. LPLA2 has 49 percent of amino acid sequence identity to lecithin cholesterol acyltransferase and is a member of the αβ-hydrolase superfamily. LPLA2 is highly expressed in alveolar macrophages. A marked accumulation of glycerophospholipids and extensive lamellar inclusion bodies, a hallmark of cellular phospholipidosis, is observed in alveolar macrophages in LPLA2−/− mice. This defect can also be reproduced in macrophages that are exposed to cationic amphiphilic drugs such as amiodarone. In addition, older LPLA2−/− mice develop a phenotype similar to human autoimmune disease. These observations indicate that LPLA2 may play a primary role in phospholipid homeostasis, drug toxicity, and host defense.
PMCID: PMC3039127  PMID: 21074554
Lysosomal phospholipase A2 (LPLA2); 1-O-acylceramide synthase; phospholipase A2; transacylase; amiodarone; cationic amphiphilic drugs; lecithin-cholesterol acyltransferase (LCAT); LCAT like lysophospholipase (LLPL); alveolar macrophages (AMs); pulmonary surfactant; phospholipidosis
23.  Hippocampal Volumes and the BDNF val66met Polymorphism in Geriatric Major Depression 
Structural abnormalities in the hippocampus have been implicated in the pathophysiology of major depressive disorder (MDD). The brain derived neurotrophic factor (BDNF) val66met polymorphism may contribute to these abnormalities and therefore confer vulnerability to MDD. This study examined whether there is a relationship among BDNF genotype, hippocampal volumes, and MDD in older adults.
Thirty-three older adults with MDD and 23 psychiatrically normal comparison subjects were studied. Structural MRI analysis was used to quantify hippocampal volumes. A repeated measures ANCOVA examined the relationships among BDNF val66met (val/val, met carrier), diagnosis (depressed, non-depressed), and hippocampal volumes (right, left). Age, gender, education, and whole brain volume were included as covariates.
Elderly MDD BDNF val/val homozygotes had significantly higher right hippocampal volumes compared with non-depressed val/val subjects. However, there was no difference between the depressed and healthy non-depressed met carriers. Additionally, depressed met carriers had an earlier age of onset of depressive illness than val/val homozygotes but age of onset did not moderate the relationship between hippocampal volumes and MDD diagnosis.
These results provide preliminary evidence of a neuroprotective role of the val/val genotype, suggesting neurotrophic factor production protects against pathophysiological processes triggered by depression in older adults with later age of onset of MDD. The BDNF val66met polymorphism may play a salient role in structural alterations of the hippocampus in older adults with MDD.
PMCID: PMC3058412  PMID: 21218562
Geriatric depression; BDNF val66met; hippocampus; older adults
25.  Complete Genome Sequences for the Anaerobic, Extremely Thermophilic Plant Biomass-Degrading Bacteria Caldicellulosiruptor hydrothermalis, Caldicellulosiruptor kristjanssonii, Caldicellulosiruptor kronotskyensis, Caldicellulosiruptor owensensis, and Caldicellulosiruptor lactoaceticus▿  
Journal of Bacteriology  2011;193(6):1483-1484.
The genus Caldicellulosiruptor contains the most thermophilic, plant biomass-degrading bacteria isolated to date. Previously, genome sequences from three cellulolytic members of this genus were reported (C. saccharolyticus, C. bescii, and C. obsidiansis). To further explore the physiological and biochemical basis for polysaccharide degradation within this genus, five additional genomes were sequenced: C. hydrothermalis, C. kristjanssonii, C. kronotskyensis, C. lactoaceticus, and C. owensensis. Taken together, the seven completed and one draft-phase Caldicellulosiruptor genomes suggest that, while central metabolism is highly conserved, significant differences in glycoside hydrolase inventories and numbers of carbohydrate transporters exist, a finding which likely relates to variability observed in plant biomass degradation capacity.
PMCID: PMC3067630  PMID: 21216991

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