Publications by authors named "Ashley Garner"

23 Publications

  • Page 1 of 1

Growth effects of N-acylethanolamines on gut bacteria reflect altered bacterial abundances in inflammatory bowel disease.

Nat Microbiol 2020 03 20;5(3):486-497. Epub 2020 Jan 20.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Inflammatory bowel diseases (IBD) are associated with alterations in gut microbial abundances and lumenal metabolite concentrations, but the effects of specific metabolites on the gut microbiota in health and disease remain largely unknown. Here, we analysed the influences of metabolites that are differentially abundant in IBD on the growth and physiology of gut bacteria that are also differentially abundant in IBD. We found that N-acylethanolamines (NAEs), a class of endogenously produced signalling lipids elevated in the stool of IBD patients and a T-cell transfer model of colitis, stimulated growth of species over-represented in IBD and inhibited that of species depleted in IBD in vitro. Using metagenomic sequencing, we recapitulated the effects of NAEs in complex microbial communities ex vivo, with Proteobacteria blooming and Bacteroidetes declining in the presence of NAEs. Metatranscriptomic analysis of the same communities identified components of the respiratory chain as important for the metabolism of NAEs, and this was verified using a mutant deficient for respiratory complex I. In this study, we identified NAEs as a class of metabolites that are elevated in IBD and have the potential to shift gut microbiota towards an IBD-like composition.
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http://dx.doi.org/10.1038/s41564-019-0655-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7047597PMC
March 2020

Microbial genes and pathways in inflammatory bowel disease.

Nat Rev Microbiol 2019 08;17(8):497-511

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Perturbations in the intestinal microbiome are implicated in inflammatory bowel disease (IBD). Studies of treatment-naive patients have identified microbial taxa associated with disease course and treatment efficacy. To gain a mechanistic understanding of how the microbiome affects gastrointestinal health, we need to move from census to function. Bacteria, including those that adhere to epithelial cells as well as several Clostridium species, can alter differentiation of T helper 17 cells and regulatory T cells. Similarly, microbial products such as short-chain fatty acids and sphingolipids also influence immune responses. Metagenomics and culturomics have identified strains of Ruminococcus gnavus and adherent invasive Escherichia coli that are linked to IBD and gut inflammation. Integrated analysis of multiomics data, including metagenomics, metatranscriptomics and metabolomics, with measurements of host response and culturomics, have great potential in understanding the role of the microbiome in IBD. In this Review, we highlight current knowledge of gut microbial factors linked to IBD pathogenesis and discuss how multiomics data from large-scale population studies in health and disease have been used to identify specific microbial strains, transcriptional changes and metabolic alterations associated with IBD.
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http://dx.doi.org/10.1038/s41579-019-0213-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6759048PMC
August 2019

CarD contributes to diverse gene expression outcomes throughout the genome of .

Proc Natl Acad Sci U S A 2019 07 19;116(27):13573-13581. Epub 2019 Jun 19.

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110;

The ability to regulate gene expression through transcription initiation underlies the adaptability and survival of all bacteria. Recent work has revealed that the transcription machinery in many bacteria diverges from the paradigm that has been established in () encodes the RNA polymerase (RNAP)-binding protein CarD, which is absent in but is required to form stable RNAP-promoter open complexes (RP) and is essential for viability in The stabilization of RP by CarD has been proposed to result in activation of gene expression; however, CarD has only been examined on limited promoters that do not represent the typical promoter structure in In this study, we investigate the outcome of CarD activity on gene expression from promoters genome-wide by performing RNA sequencing on a panel of mutants that differentially affect CarD's ability to stabilize RP In all CarD mutants, the majority of protein encoding transcripts were differentially expressed, demonstrating that CarD had a global effect on gene expression. Contrary to the expected role of CarD as a transcriptional activator, mutation of CarD led to both up- and down-regulation of gene expression, suggesting that CarD can also act as a transcriptional repressor. Furthermore, we present evidence that stabilization of RP by CarD could lead to transcriptional repression by inhibiting promoter escape, and the outcome of CarD activity is dependent on the intrinsic kinetic properties of a given promoter region. Collectively, our data support CarD's genome-wide role of regulating diverse transcription outcomes.
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http://dx.doi.org/10.1073/pnas.1900176116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6613185PMC
July 2019

Comparing glomerular filtration rate equations and the impact of different creatinine assays on the assessment of renal function in cancer patients.

Ann Clin Biochem 2019 03 21;56(2):266-274. Epub 2019 Feb 21.

1 Department of Blood Sciences, Leeds General Infirmary, Old Medical School, Leeds, UK.

Background: Equations to estimate glomerular filtration rate based on serum creatinine are commonly used in cancer patients to assess renal function. However, there is uncertainty regarding which equation is most appropriate for this population and the impact of different creatinine assays.

Methods: Measured isotopic glomerular filtration rate results from 120 oncology patients were used to evaluate and compare all four versions of the Wright equation, Cockcroft and Gault, Modification of Diet in Renal Disease (MDRD), Chronic Kidney Disease Epidemiology Collaboration and the Janowitz and Williams formula; using eight different creatinine assays (five Jaffe, three enzymatic).

Results: The enzymatic version of the Wright equation without creatine kinase performed better than the other versions for all eight creatinine assays. However, MDRD and Janowitz and Williams gave the best overall performance in this patient population. Performance was highly dependent on the creatinine assay used, for example, the percentage of results within 30% of the isotopic glomerular filtration rate (P30) ranged from 90.8% to 60.8% for MDRD.

Conclusion: The performance of any equation to estimate glomerular filtration rate is highly dependent on the creatinine assay used. Oncology units should assess the performance of glomerular filtration rate equations using their laboratory creatinine assay to determine whether they can be used safely and effectively in cancer patients.
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http://dx.doi.org/10.1177/0004563218822667DOI Listing
March 2019

Assessing the impact of inadequate hydration on isotope-GFR measurement.

Scand J Clin Lab Invest 2019 Feb - Apr;79(1-2):86-90. Epub 2019 Jan 7.

a Departments of Medical Physics and Engineering, Nuclear Medicine and Blood Sciences , The Leeds Teaching Hospitals NHS Trust , Leeds , UK.

Guidelines state that patients undergoing isotope glomerular filtration rate (GFR) tests should maintain adequate hydration, but pragmatically these tests can coincide with procedures requiring the patient not to eat or drink ('nil-by-mouth') for up to 12 hours beforehand. This study investigated the impact of a 12-hour nil-by-mouth regime on GFR measurement. Twelve healthy volunteers were recruited from our institution. Exclusion criteria included diabetes mellitus, being under 18 years of age and pregnancy. Isotope GFR measurements were carried out on these volunteers twice. One of the tests adhered strictly to the British Nuclear Medicine Society (BNMS) guidelines for GFR measurement and the other test was carried out after the volunteers had refrained from eating or drinking anything for 12 hours. The order of these tests was randomly assigned. The results show that after a nil-by-mouth regime, participants' average absolute GFR fell from 108 ml/min to 97 ml/min (p < .01), while normalised GFR fell from 97 ml/min/1.73 m to 88 ml/min/1.73m (p < .01). Serum creatinine rose from 68 mmol/L to 73 mmol/L (p < .05). There were no changes in blood pressure, serum hydration markers or bio-impedance measured fluid status. Urine analysis showed statistically significant increases in urea, creatinine and osmolality levels after the nil-by-mouth regime. The results highlight the importance of following current guidelines recommending fluid intake during the procedure. Practitioners should consider what other outpatient appointments are being scheduled concurrently with a GFR test.
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http://dx.doi.org/10.1080/00365513.2018.1555859DOI Listing
August 2019

Domains within RbpA Serve Specific Functional Roles That Regulate the Expression of Distinct Mycobacterial Gene Subsets.

J Bacteriol 2018 07 11;200(13). Epub 2018 Jun 11.

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA

The RNA polymerase (RNAP) binding protein A (RbpA) contributes to the formation of stable RNAP-promoter open complexes (RP) and is essential for viability in mycobacteria. Four domains have been identified in the RbpA protein, i.e., an N-terminal tail (NTT) that interacts with RNAP β' and σ subunits, a core domain (CD) that contacts the RNAP β' subunit, a basic linker (BL) that binds DNA, and a σ-interaction domain (SID) that binds group I and group II σ factors. Limited studies have been performed in mycobacteria, however, and how individual structural domains of RbpA contribute to RbpA function and mycobacterial gene expression remains mostly unknown. We investigated the roles of the RbpA structural domains in mycobacteria using a panel of mutants that target individual RbpA domains. The function of each RbpA domain was required for viability and optimal growth in We determined that the RbpA SID is both necessary and sufficient for RbpA interaction with the RNAP, indicating that the primary functions of the NTT and CD are not solely association with the RNAP. We show that the RbpA BL and SID are required for RP stabilization , while the NTT and CD antagonize this activity. Finally, RNA-sequencing analyses suggest that the NTT and CD broadly activate gene expression, whereas the BL and SID activate or repress gene expression in a gene-dependent manner for a subset of mycobacterial genes. Our findings highlight specific outcomes for the activities of the individual functional domains in RbpA. is the causative agent of tuberculosis and continues to be the most lethal infectious disease worldwide. Improved molecular understanding of the essential proteins involved in transcription, such as RbpA, could provide targets for much needed future therapeutic agents aimed at combatting this pathogen. In this study, we expand our understanding of RbpA by identifying the RbpA structural domains responsible for the interaction of RbpA with the RNAP and the effects of RbpA on transcription initiation and gene expression. These experiments expand our knowledge of RbpA while also broadening our understanding of bacterial transcription in general.
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http://dx.doi.org/10.1128/JB.00690-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5996690PMC
July 2018

A novel Ruminococcus gnavus clade enriched in inflammatory bowel disease patients.

Genome Med 2017 Nov 28;9(1):103. Epub 2017 Nov 28.

Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA.

Background: Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gastrointestinal tract that is associated with changes in the gut microbiome. Here, we sought to identify strain-specific functional correlates with IBD outcomes.

Methods: We performed metagenomic sequencing of monthly stool samples from 20 IBD patients and 12 controls (266 total samples). These were taxonomically profiled with MetaPhlAn2 and functionally profiled using HUMAnN2. Differentially abundant species were identified using MaAsLin and strain-specific pangenome haplotypes were analyzed using PanPhlAn.

Results: We found a significantly higher abundance in patients of facultative anaerobes that can tolerate the increased oxidative stress of the IBD gut. We also detected dramatic, yet transient, blooms of Ruminococcus gnavus in IBD patients, often co-occurring with increased disease activity. We identified two distinct clades of R. gnavus strains, one of which is enriched in IBD patients. To study functional differences between these two clades, we augmented the R. gnavus pangenome by sequencing nine isolates from IBD patients. We identified 199 IBD-specific, strain-specific genes involved in oxidative stress responses, adhesion, iron-acquisition, and mucus utilization, potentially conferring an adaptive advantage for this R. gnavus clade in the IBD gut.

Conclusions: This study adds further evidence to the hypothesis that increased oxidative stress may be a major factor shaping the dysbiosis of the microbiome observed in IBD and suggests that R. gnavus may be an important member of the altered gut community in IBD.
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http://dx.doi.org/10.1186/s13073-017-0490-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704459PMC
November 2017

Indoleacrylic Acid Produced by Commensal Peptostreptococcus Species Suppresses Inflammation.

Cell Host Microbe 2017 Jul;22(1):25-37.e6

The Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA. Electronic address:

Host factors in the intestine help select for bacteria that promote health. Certain commensals can utilize mucins as an energy source, thus promoting their colonization. However, health conditions such as inflammatory bowel disease (IBD) are associated with a reduced mucus layer, potentially leading to dysbiosis associated with this disease. We characterize the capability of commensal species to cleave and transport mucin-associated monosaccharides and identify several Clostridiales members that utilize intestinal mucins. One such mucin utilizer, Peptostreptococcus russellii, reduces susceptibility to epithelial injury in mice. Several Peptostreptococcus species contain a gene cluster enabling production of the tryptophan metabolite indoleacrylic acid (IA), which promotes intestinal epithelial barrier function and mitigates inflammatory responses. Furthermore, metagenomic analysis of human stool samples reveals that the genetic capability of microbes to utilize mucins and metabolize tryptophan is diminished in IBD patients. Our data suggest that stimulating IA production could promote anti-inflammatory responses and have therapeutic benefits.
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http://dx.doi.org/10.1016/j.chom.2017.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5672633PMC
July 2017

Effects of Increasing the Affinity of CarD for RNA Polymerase on Mycobacterium tuberculosis Growth, rRNA Transcription, and Virulence.

J Bacteriol 2017 02 30;199(4). Epub 2017 Jan 30.

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA

CarD is an essential RNA polymerase (RNAP) interacting protein in Mycobacterium tuberculosis that stimulates formation of RNAP-promoter open complexes. CarD plays a complex role in M. tuberculosis growth and virulence that is not fully understood. Therefore, to gain further insight into the role of CarD in M. tuberculosis growth and virulence, we determined the effect of increasing the affinity of CarD for RNAP. Using site-directed mutagenesis guided by crystal structures of CarD bound to RNAP, we identified amino acid substitutions that increase the affinity of CarD for RNAP. Using these substitutions, we show that increasing the affinity of CarD for RNAP increases the stability of the CarD protein in M. tuberculosis In addition, we show that increasing the affinity of CarD for RNAP increases the growth rate in M. tuberculosis without affecting 16S rRNA levels. We further show that increasing the affinity of CarD for RNAP reduces M. tuberculosis virulence in a mouse model of infection despite the improved growth rate in vitro Our findings suggest that the CarD-RNAP interaction protects CarD from proteolytic degradation in M. tuberculosis, establish that growth rate and rRNA levels can be uncoupled in M. tuberculosis and demonstrate that the strength of the CarD-RNAP interaction has been finely tuned to optimize virulence.

Importance: Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major global health problem. In order to develop new strategies to battle this pathogen, we must gain a better understanding of the molecular processes involved in its survival and pathogenesis. We have previously identified CarD as an essential transcriptional regulator in mycobacteria. In this study, we detail the effects of increasing the affinity of CarD for RNAP on transcriptional regulation, CarD protein stability, and virulence. These studies expand our understanding of the global transcription regulator CarD, provide insight into how CarD activity is regulated, and broaden our understanding of prokaryotic transcription.
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http://dx.doi.org/10.1128/JB.00698-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5287406PMC
February 2017

Cooperative stabilization of Mycobacterium tuberculosis rrnAP3 promoter open complexes by RbpA and CarD.

Nucleic Acids Res 2016 09 24;44(15):7304-13. Epub 2016 Jun 24.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA

The essential mycobacterial transcriptional regulators RbpA and CarD act to modulate transcription by associating to the initiation complex and increasing the flux of transcript production. Each of these factors interacts directly with the promoter DNA template and with RNA polymerase (RNAP) holoenzyme. We recently reported on the energetics of CarD-mediated open complex stabilization on the Mycobacterium tuberculosis rrnAP3 ribosomal promoter using a stopped-flow fluorescence assay. Here, we apply this approach to RbpA and show that RbpA stabilizes RNAP-promoter open complexes (RPo) via a distinct mechanism from that of CarD. Furthermore, concentration-dependent stopped-flow experiments with both factors reveal positive linkage (cooperativity) between RbpA and CarD with regard to their ability to stabilize RPo The observation of positive linkage between RbpA and CarD demonstrates that the two factors can act on the same transcription initiation complex simultaneously. Lastly, with both factors present, the kinetics of open complex formation is significantly faster than in the presence of either factor alone and approaches that of E. coli RNAP on the same promoter. This work provides a quantitative framework for the molecular mechanisms of these two essential transcription factors and the critical roles they play in the biology and pathology of mycobacteria.
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http://dx.doi.org/10.1093/nar/gkw577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009747PMC
September 2016

Mycobacterium tuberculosis Transcription Machinery: Ready To Respond to Host Attacks.

J Bacteriol 2016 May 14;198(9):1360-73. Epub 2016 Apr 14.

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA

Regulating responses to stress is critical for all bacteria, whether they are environmental, commensal, or pathogenic species. For pathogenic bacteria, successful colonization and survival in the host are dependent on adaptation to diverse conditions imposed by the host tissue architecture and the immune response. Once the bacterium senses a hostile environment, it must enact a change in physiology that contributes to the organism's survival strategy. Inappropriate responses have consequences; hence, the execution of the appropriate response is essential for survival of the bacterium in its niche. Stress responses are most often regulated at the level of gene expression and, more specifically, transcription. This minireview focuses on mechanisms of regulating transcription initiation that are required by Mycobacterium tuberculosis to respond to the arsenal of defenses imposed by the host during infection. In particular, we highlight how certain features of M. tuberculosis physiology allow this pathogen to respond swiftly and effectively to host defenses. By enacting highly integrated and coordinated gene expression changes in response to stress,M. tuberculosis is prepared for battle against the host defense and able to persist within the human population.
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http://dx.doi.org/10.1128/JB.00935-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836228PMC
May 2016

CarD stabilizes mycobacterial open complexes via a two-tiered kinetic mechanism.

Nucleic Acids Res 2015 Mar 19;43(6):3272-85. Epub 2015 Feb 19.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA

CarD is an essential and global transcriptional regulator in mycobacteria. While its biological role is unclear, CarD functions by interacting directly with RNA polymerase (RNAP) holoenzyme promoter complexes. Here, using a fluorescent reporter of open complex, we quantitate RPo formation in real time and show that Mycobacterium tuberculosis CarD has a dramatic effect on the energetics of RNAP bound complexes on the M. tuberculosis rrnAP3 ribosomal RNA promoter. The data reveal that Mycobacterium bovis RNAP exhibits an unstable RPo that is stabilized by CarD and suggest that CarD uses a two-tiered, concentration-dependent mechanism by associating with open and closed complexes with different affinities. Specifically, the kinetics of open-complex formation can be explained by a model where, at saturating concentrations of CarD, the rate of bubble collapse is slowed and the rate of opening is accelerated. The kinetics and open-complex stabilities of CarD mutants further clarify the roles played by the key residues W85, K90 and R25 previously shown to affect CarD-dependent gene regulation in vivo. In contrast to M. bovis RNAP, Escherichia coli RNAP efficiently forms RPo on rrnAP3, suggesting an important difference between the polymerases themselves and highlighting how transcriptional machinery can vary across bacterial genera.
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http://dx.doi.org/10.1093/nar/gkv078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381055PMC
March 2015

Social Support and Grandparent Caregiver Health: One-Year Longitudinal Findings for Grandparents Raising Their Grandchildren.

J Gerontol B Psychol Sci Soc Sci 2015 Sep 3;70(5):804-12. Epub 2014 Dec 3.

Department of Psychology, University of North Texas, Denton.

Objectives: The role of social support in predicting health among grandparents raising grandchildren was explored among 86 grandparent caregivers assessed twice over a 1-year timeframe.

Method: Relationships between social support and health were ascertained via cross-lagged analyses. Regression analyses explored the mitigating role of social support in influencing both health and depression among grandparent caregivers.

Results: Cross-lagged findings suggested that social support predicted health over time rather than vice versa. Regression analyses found that this relationship held when adjusting for multiple covariates as well as previous levels of health, depression, and parental stress. Additionally, the interaction of overall health and social support at Time 1 predicted Time 2 depression. For those who lacked social support, overall health was negatively related to self-reported depression symptoms 1 year later; this was not the case among those reporting greater social support. In addition, parental stress moderated the effects of social support on depression, and social support moderated the effects of parental stress on depression.

Discussion: Greater social support may lay the groundwork for better health, and such support, in concert with better health as well as lessened parental stress may prevent the development of depression among grandparent caregivers.
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http://dx.doi.org/10.1093/geronb/gbu165DOI Listing
September 2015

CarD integrates three functional modules to promote efficient transcription, antibiotic tolerance, and pathogenesis in mycobacteria.

Mol Microbiol 2014 Aug 16;93(4):682-97. Epub 2014 Jul 16.

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, 63110, USA.

Although the basic mechanisms of prokaryotic transcription are conserved, it has become evident that some bacteria require additional factors to allow for efficient gene transcription. CarD is an RNA polymerase (RNAP)-binding protein conserved in numerous bacterial species and essential in mycobacteria. Despite the importance of CarD, its function at transcription complexes remains unclear. We have generated a panel of mutations that individually target three independent functional modules of CarD: the RNAP interaction domain, the DNA-binding domain, and a conserved tryptophan residue. We have dissected the roles of each functional module in CarD activity and built a model where each module contributes to stabilizing RNAP-promoter complexes. Our work highlights the requirement of all three modules of CarD in the obligate pathogen Mycobacterium tuberculosis, but not in Mycobacterium smegmatis. We also report divergent use of the CarD functional modules in resisting oxidative stress and pigmentation. These studies provide new information regarding the functional domains involved in transcriptional regulation by CarD while also improving understanding of the physiology of M. tuberculosis.
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http://dx.doi.org/10.1111/mmi.12681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4127138PMC
August 2014

Health and Grandparent-Grandchild Well-Being: One-Year Longitudinal Findings for Custodial Grandfamilies.

J Aging Health 2014 Jun 28;26(4):559-582. Epub 2014 Mar 28.

University of North Texas, Denton, USA.

Objective: Comparatively little longitudinal data exist focusing on grandparent caregiving, to say nothing of health's impact over time on grandparent and grandchild well-being. Accordingly, the present study explored relationships among grandparent caregiver physical health, well-being, and adjustment, as well as with grandchild well-being across a 1-year period.

Method: Participants were 79 grandparents who had full-time responsibility for their grandchildren. Measures of grandparent physical health, well-being, and grandchild well-being were completed across two assessments, 1 year apart.

Results: Cross-lagged analyses exploring potential causality over time suggested that with one exception, the relationships between health and well-being appeared to be bidirectional.

Discussion: In general, these longitudinal data indicate that better perceived health may provide an adaptive advantage for both grandparent caregivers and their grandchildren, yet also underscore to the potentially causal role that proactivity in the face of adversity plays in the maintenance and improvement of grandparent caregiver health over time.
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http://dx.doi.org/10.1177/0898264314525664DOI Listing
June 2014

Structure and function of CarD, an essential mycobacterial transcription factor.

Proc Natl Acad Sci U S A 2013 Jul 15;110(31):12619-24. Epub 2013 Jul 15.

Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065, USA.

CarD, an essential transcription regulator in Mycobacterium tuberculosis, directly interacts with the RNA polymerase (RNAP). We used a combination of in vivo and in vitro approaches to establish that CarD is a global regulator that stimulates the formation of RNAP-holoenzyme open promoter (RPo) complexes. We determined the X-ray crystal structure of Thermus thermophilus CarD, allowing us to generate a structural model of the CarD/RPo complex. On the basis of our structural and functional analyses, we propose that CarD functions by forming protein/protein and protein/DNA interactions that bridge the RNAP to the promoter DNA. CarD appears poised to interact with a DNA structure uniquely presented by the RPo: the splayed minor groove at the double-stranded/single-stranded DNA junction at the upstream edge of the transcription bubble. Thus, CarD uses an unusual mechanism for regulating transcription, sensing the DNA conformation where transcription bubble formation initiates.
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http://dx.doi.org/10.1073/pnas.1308270110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3732983PMC
July 2013

Cloning, expression, and characterization of a cellobiose dehydrogenase from Thielavia terrestris induced under cellulose growth conditions.

Biochim Biophys Acta 2012 Jun 30;1824(6):802-12. Epub 2012 Mar 30.

Novozymes, Inc., Davis, CA 95618, USA.

The enzyme cellobiose dehydrogenase (CDH) is of considerable interest, not only for its biotechnological applications, but also its potential biological role in lignocellulosic biomass breakdown. The enzyme catalyzes the oxidation of cellobiose and other cellodextrins, utilizing a variety of one- and two-electron acceptors, although the electron acceptor employed in nature is still unknown. In this study we show that a CDH is present in the secretome of the thermophilic ascomycete Thielavia terrestris when grown with cellulose, along with a mixture of cellulases and hemicellulases capable of breaking down lignocellulosic biomass. We report the cloning of this T. terrestris CDH gene (cbdA), its recombinant expression in Aspergillus oryzae, and purification and characterization of the T. terrestris CDH protein (TtCDH). The TtCDH shows spectral properties and enzyme activity similar to other characterized CDH enzymes. Substrate specificity was determined for a number of carbohydrate electron donors in the presence of the two-electron acceptor 2,6-dichlorophenol-indophenol. The TtCDH also shows dramatic synergy with Thermoascus aurantiacus glycoside hydrolase family 61A protein in the presence of a β-glucosidase for the cleavage of cellulose.
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http://dx.doi.org/10.1016/j.bbapap.2012.03.009DOI Listing
June 2012

Detection of patients with acute kidney injury by the clinical laboratory using rises in serum creatinine: comparison of proposed definitions and a laboratory delta check.

Ann Clin Biochem 2012 Jan 30;49(Pt 1):59-62. Epub 2011 Nov 30.

Department of Clinical Biochemistry, Leeds General Infirmary, Great George Street, Leeds LS1 3EX, UK.

Background: Timely detection of acute kidney injury (AKI) in hospital patients has been hampered by the multiple definitions of AKI and difficulties applying their criteria. A laboratory delta check may provide an effective means of detecting patients developing AKI. This study compared three of the proposed AKI definitions and a delta check to detect AKI using serum creatinine results of hospital inpatients.

Methods: Serum creatinine results for 2822 inpatients were gathered retrospectively from the clinical biochemistry database. All serum creatinine results within 30 d of admission were included for each patient and assessed for AKI according to four criteria: Risk, Injury, Failure (RIFLE), Acute Kidney Injury Network (AKIN), Waikar & Bonventre or a delta check (increase of >26 μmol/L between two successive values).

Results: A total of 149 (11.3%) patients were defined as having AKI by at least one of the four criteria. Different populations of patients were identified by each criterion. The number of patients identified and the incidence of AKI were as follows: RIFLE 94 (7.1%), AKIN 125 (9.5%), Waikar & Bonventre 100 (7.6%) and delta check 146 (11.1%). The delta check detected 132 (98%) of all 135 cases detected by the other three criteria. A further 14 patients were detected solely by the delta check.

Conclusions: The different definitions proposed for AKI detect different populations of patients. A laboratory delta check detected 98% of all the patients identified by AKIN, RIFLE and Waikar & Bonventre combined and could therefore provide a practical way of detecting AKI patients.
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http://dx.doi.org/10.1258/acb.2011.011125DOI Listing
January 2012

Membrane raft actin deficiency and altered Ca2+-induced vesiculation in stomatin-deficient overhydrated hereditary stomatocytosis.

Biochim Biophys Acta 2008 Jan 29;1778(1):125-32. Epub 2007 Sep 29.

Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.

In overhydrated hereditary stomatocytosis (OHSt), the membrane raft-associated stomatin is deficient from the erythrocyte membrane. We have investigated two aspects of raft structure and function in OHSt erythrocytes. First, we have studied the distribution of other membrane and cytoskeletal proteins in rafts by analysis of detergent-resistant membranes (DRMs). In normal erythrocytes, 29% of the actin was DRM-associated, whereas in two unrelated OHSt patients the DRM-associated actin was reduced to <10%. In addition, there was a reduction in the amount of the actin-associated protein tropomodulin in DRMs from these OHSt cells. When stomatin was expressed in Madin-Darby canine kidney cells, actin association with the membrane was increased. Second, we have studied Ca2+-dependent exovesiculation from the erythrocyte membrane. Using atomic force microscopy and proteomics analysis, exovesicles derived from OHSt cells were found to be increased in number and abnormal in size, and contained greatly increased amounts of the raft proteins flotillin-1 and -2 and the calcium binding proteins annexin VII, sorcin and copine 1, while the concentrations of stomatin and annexin V were diminished. Together these observations imply that the stomatin-actin association is important in maintaining the structure and in modulating the function of stomatin-containing membrane rafts in red cells.
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http://dx.doi.org/10.1016/j.bbamem.2007.09.016DOI Listing
January 2008

Visualization of detergent solubilization of membranes: implications for the isolation of rafts.

Biophys J 2008 Feb 12;94(4):1326-40. Epub 2007 Oct 12.

Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.

Although different detergents can give rise to detergent-resistant membranes of different composition, it is unclear whether this represents domain heterogeneity in the original membrane. We compared the mechanism of action of five detergents on supported lipid bilayers composed of equimolar sphingomyelin, cholesterol, and dioleoylphosphatidylcholine imaged by atomic force microscopy, and on raft and nonraft marker proteins in live cells imaged by confocal microscopy. There was a marked correlation between the detergent solubilization of the cell membrane and that of the supported lipid bilayers. In both systems Triton X-100 and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) distinguished between the nonraft liquid-disordered (l(d)) and raft liquid ordered (l(o)) lipid phases by selectively solubilizing the l(d) phase. A higher concentration of Lubrol was required, and not all the l(d) phase was solubilized. The solubilization by Brij 96 occurred by a two-stage mechanism that initially resulted in the solubilization of some l(d) phase and then progressed to the solubilization of both l(d) and l(o) phases simultaneously. Octyl glucoside simultaneously solubilized both l(o) and l(d) phases. These data show that the mechanism of membrane solubilization is unique to an individual detergent. Our observations have significant implications for using different detergents to isolate membrane rafts from biological systems.
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http://dx.doi.org/10.1529/biophysj.107.114108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2212709PMC
February 2008

Sphingomyelin chain length influences the distribution of GPI-anchored proteins in rafts in supported lipid bilayers.

Mol Membr Biol 2007 May-Jun;24(3):233-42

Proteolysis Research Group, Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, and Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds, UK.

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are enriched in cholesterol- and sphingolipid-rich lipid rafts within the membrane. Rafts are known to have roles in cellular organization and function, but little is understood about the factors controlling the distribution of proteins in rafts. We have used atomic force microscopy to directly visualize proteins in supported lipid bilayers composed of equimolar sphingomyelin, dioleoyl-sn-glycero-3-phosphocholine and cholesterol. The transmembrane anchored angiotensin converting enzyme (TM-ACE) was excluded from the liquid ordered raft domains. Replacement of the transmembrane and cytoplasmic domains of TM-ACE with a GPI anchor (GPI-ACE) promoted the association of the protein with rafts in the bilayers formed with brain sphingomyelin (mainly C18:0). Association with the rafts did not occur if the shorter chain egg sphingomyelin (mainly C16:0) was used. The distribution of GPI-anchored proteins in supported lipid bilayers was investigated further using membrane dipeptidase (MDP) whose GPI anchor contains distearoyl phosphatidylinositol. MDP was also excluded from rafts when egg sphingomyelin was used but associated with raft domains formed using brain sphingomyelin. The effect of sphingomyelin chain length on the distribution of GPI-anchored proteins in rafts was verified using synthetic palmitoyl or stearoyl sphingomyelin. Both GPI-ACE and MDP only associated with the longer chain stearoyl sphingomyelin rafts. These data obtained using supported lipid bilayers provide the first direct evidence that the nature of the membrane-anchoring domain influences the association of a protein with lipid rafts and that acyl chain length hydrophobic mismatch influences the distribution of GPI-anchored proteins in rafts.
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http://dx.doi.org/10.1080/09687860601127770DOI Listing
December 2007

Bicyclic heteroarylpiperazines as selective brain penetrant 5-HT6 receptor antagonists.

Bioorg Med Chem Lett 2005 Nov;15(21):4867-71

Neurology and GI Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK.

Starting from the potent and selective but poorly brain penetrant 5-HT6 receptor antagonist SB-271046, a successful strategy for improving brain penetration was adopted involving conformational constraint with concomitant reduction in hydrogen bond count. This provided a series of bicyclic heteroarylpiperazines with high 5-HT6 receptor affinity. 5-Chloroindole 699929 combined high 5-HT6 receptor affinity with excellent brain penetration and also had good oral bioavailability in both rat and dog.
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http://dx.doi.org/10.1016/j.bmcl.2005.06.107DOI Listing
November 2005

SB-656104-A: a novel 5-HT(7) receptor antagonist with improved in vivo properties.

Bioorg Med Chem Lett 2002 Nov;12(22):3341-4

GlaxoSmithKline, New Frontiers Science Park, Harlow, Essex, UK.

A focused SAR study around the previously reported selective 5-HT(7) receptor antagonist, SB-269970-A has resulted in the identification of a structurally related analogue having an improved pharmacokinetic profile. Replacement of the phenolic group in SB-269970-A with an indole moiety, and replacement of the piperidinyl 4-methyl group with a heterocyclic ring system proved to be the key changes leading to the identification of SB-656104-A.
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http://dx.doi.org/10.1016/s0960-894x(02)00690-xDOI Listing
November 2002