CiteULike: jyuh's Roth

The complete genome of an individual by massively parallel DNA sequencing

David Wheeler — 2008-04-16T19:24:49-00:00

Nature, Vol. 452, No. 7189. (17 April 2008), pp. 872-876.

Isoform discovery by targeted cloning, 'deep-well' pooling and parallel sequencing

Kourosh Salehi-Ashtiani — 2008-06-28T14:29:01-00:00

Nat Meth, Vol. 5, No. 7. (July 2008), pp. 597-600.

An inducible and reversible mouse genetic rescue system.

H Zeng — 2008-07-02T06:49:30-00:00

PLoS genetics, Vol. 4, No. 5. (May 2008)

Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and cost-effective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications.

Challenges in Translating Plasma Proteomics from Bench to Bedside: Update from the NHLBI Clinical Proteomics Programs.

Robert E Gerszten — 2008-05-20T04:30:27-00:00

American journal of physiology. Lung cellular and molecular physiology (2 May 2008)

The emerging scientific field of proteomics encompasses the identification, characterization and quantification of the protein content or proteome of whole cells, tissues or body fluids. The potential for proteomic technologies to identify and quantify novel proteins in the plasma that can function as biomarkers of the presence or severity of clinical disease states holds great promise for clinical use. However, there are many challenges in translating plasma proteomics from bench to bedside and relatively few plasma biomarkers have successfully transitioned from proteomic discovery to routine clinical use. Key barriers to this translation include the need for "orthogonal" biomarkers (ie., uncorrelated with existing markers), the complexity of the proteome in biological samples, the presence of high abundance proteins such as albumin in biological samples that hinder detection of low abundance proteins, false positive associations that occur with analysis of high dimensional datasets and the limited understanding of the effects of growth, development and age on the normal plasma proteome. Strategies to overcome these challenges are discussed. Key words: proteomics, biomarkers, multiplex assays.

Berson, Yalow, and the JCI: the agony and the ecstasy.

CR Kahn — 2008-05-14T06:35:19-00:00

The Journal of clinical investigation, Vol. 114, No. 8. (October 2004), pp. 1051-1054.

The isolation of insulin in 1921 by Banting, Best, Collip, and Macleod stands as one of the most dramatic stories in modern medical investigation. Only two years passed between the initial experiments in dogs to widespread human application to the awarding of the Nobel Prize in 1923. Insulin-related research has also served as a focus, at least in part, for the work of three other Nobel Prize recipients: determination of the chemical structure of insulin by Frederick Sanger in 1958; determination of the three-dimensional structures of insulin and vitamin B12 by Dorothy Hodgkin in 1964; and finally, the development of immunoassay by Solomon Berson and Rosalyn Yalow in 1959-1960, which led to a Nobel Prize for Yalow in 1977 (five years after the untimely death of Berson). The history of Yalow and Berson's discovery and its impact on the field is an illustration of the adage that every story has two sides.

Proteomic identification of palmitoylated proteins.

AF Roth — 2008-05-11T14:44:32-00:00

Methods (San Diego, Calif.), Vol. 40, No. 2. (October 2006), pp. 135-142.

A proteomic method that purifies and identifies palmitoylated proteins from complex protein extracts is described. Using the fatty acid exchange labeling chemistry (described in the preceding report), palmitoyl modifications are exchanged for biotinylated compounds, allowing the subset of palmitoyl-proteins to be affinity-purified and then identified by mass spectroscopic protein identification technologies. The advantages and pitfalls of this new technology are discussed within the context of the recent application of this method in the yeast Saccharomyces cerevisiae.

PIASy-deficient mice display modest defects in IFN and Wnt signaling.

W Roth — 2008-04-19T14:09:26-00:00

Journal of immunology (Baltimore, Md. : 1950), Vol. 173, No. 10. (15 November 2004), pp. 6189-6199.

Protein inhibitors of activated STATs (PIAS) represent a small family of nuclear proteins that modulate the activity of many transcription factors and act as E3 ligases for covalent modification of proteins with the small ubiquitin-like modifier (SUMO). In particular, PIASy has been shown to inhibit the activation of gene expression by the IFN-responsive transcription factor STAT1 and the Wnt-responsive transcription factor LEF1. To assess the function of PIASy in vivo, we generated and analyzed mice carrying a targeted mutation of the Piasy gene. We find that homozygous mutant mice have no obvious morphological defects and have a normal distribution of lymphocyte populations. Molecular analysis of signaling in response to IFN-gamma and Wnt agonists revealed a modest reduction in the activation of endogenous and transfected target genes. Two-dimensional analysis of total proteins and SUMO-modified proteins in transformed pre-B cells showed no significant differences between wild-type mice and homozygous mutant mice. Taken together, our data indicate that PIASy has a modest effect on cytokine and Wnt signaling, suggesting a redundancy with other members of the family of PIAS proteins.

A multiplex tissue immunoblotting assay for proteomic profiling: a pilot study of the normal to tumor transition of esophageal squamous cell carcinoma.

JY Chung — 2008-03-10T10:02:54-00:00

Cancer Epidemiol Biomarkers Prev, Vol. 15, No. 7. (July 2006), pp. 1403-1408.

Esophageal cancer remains a highly lethal malignancy for which the genetic and proteomic events are poorly understood. Studies have reported dysregulated proteins in esophageal carcinoma; however, the magnitude of these changes remains largely uncharacterized. Little is known about alterations early in the neoplastic pathway. Using multiplex tissue immunoblotting, we quantified the expression of seven proteins in esophageal carcinogenesis. Regions of normal, dysplasia, and invasive carcinoma of the squamous esophagus in six patients were characterized. Pan-cytokeratin (CK) was essentially unchanged across the transition (0.96 in dysplasia and 0.69 in tumor). Expression levels of annexin 1, CK-4, and CK-14 were all decreased in dysplasia and tumor compared with normal (reference, 1.00): annexin 1, 0.30 in dysplasia and 0.15 in tumor; CK-4, 0.20 in dysplasia and 0.16 in tumor; and CK-14, 0.54 in dysplasia and 0.40 in tumor. Expression of two proteins was increased in dysplasia and tumor versus normal: cyclooxygenase-2, 1.35 in dysplasia and 2.32 in tumor and p53, 1.29 in dysplasia and 2.37 in tumor. Secreted protein, acidic and rich in cysteine, which is expressed in the adjacent stroma, was 1.56-fold higher in stroma underlying dysplasia and 6.20-fold increased in dysplastic stroma surrounding invasive tumor. These findings suggest that changes in protein expression can be detected during the transition to dysplasia and may be useful biomarkers.

Empirical evaluation of complex epidemiologic study designs: workplace exposure and cancer.

DC Deubner — 2008-03-01T09:41:20-00:00

J Occup Environ Med, Vol. 49, No. 9. (September 2007), pp. 953-959.

OBJECTIVE: To test whether a frequently used cohort-nested case-control study design exaggerated exposure-response relationships because of unrecognized study design bias. Our aim was to evaluate empirically the performance of this complex study design. METHODS: We applied the design from one such study to a closely related cohort using randomly selected probands as cases. Values for average exposures were assigned to probands equal to, greater than, and less than those assigned to controls (matches). RESULTS: Under certain lag scenarios, the nested study design produced higher average exposure in probands compared with their matches, even when this was clearly not the case. CONCLUSIONS: Empirical evaluation demonstrated that the study design produced a biased case-control lagged exposure difference under the null hypothesis and could not distinguish qualitatively between null and alternate hypotheses. Empirical evaluation provided a useful check on results generated from a complex study design. It gave useful insight into the behavior of the index study design that was not otherwise readily deducible.

Characterization of proteomic and metabolomic responses to dietary factors and supplements.

J Astle — 2008-02-22T01:56:38-00:00

J Nutr, Vol. 137, No. 12. (December 2007), pp. 2787-2793.

Over the past decade there has been a renewed interest in research and development of both dietary and nutritional supplements. Significant advancements have been made in the scientific assessment of the quality, safety, and efficacy of these products because of the strong interest in and financial support of these projects. As research in both fields continues to advance, opportunities to use new and innovative research technologies and methodologies, such as proteomics and metabolomics, are critical for the future progress of the science. The purpose of the symposium was to begin the process of communicating new innovative proteomic and metabolomic methodologies that may be applied by researchers in both the nutrition and the natural product communities. This symposium highlighted 2 proteomic approaches, protein fingerprinting in complex mixtures with peptoid microarrays and top-down mass spectrometry for annotation of gene products. Likewise, an overview of the methodologies used in metabolomic profiling of natural products was presented, and an illustration of an integrated metabolomics approach in nutrition research was highlighted.

Extracellular matrix deposition by primary human lung fibroblasts in response to TGF-beta 1 and TGF-beta 3

Oliver Eickelberg — 2008-01-27T16:06:22-00:00

Am J Physiol Lung Cell Mol Physiol, Vol. 276, No. 5. (1 May 1999), pp. L814-824.

Increased collagen and extracellular matrix (ECM) deposition within the lung is a characteristic feature of lung fibrosis. Transforming growth factor (TGF)-[beta] isoforms play a pivotal role in the production of collagen and ECM. In this study, we investigated the effects of TGF-[beta]1 and TGF-[beta]3 on the main processes controlling ECM deposition using primary human lung fibroblasts. We analyzed 1) collagen metabolism by [3H]proline incorporation, 2) matrix metalloproteinase (MMP) expression by substrate gel zymography, and 3) tissue inhibitor of metalloproteinases (TIMP) expression by Western blot analysis. TGF-[beta]1 and TGF-[beta]3 increased the percentage of secreted collagens in supernatants of primary fibroblasts from 8.0 +/- 1.2 (control) to 23.6 +/- 4.6 and 22.3 +/- 1.3%, respectively. The collagen percentage in deposited ECM was increased from 5.8 +/- 0.3 (control) to 9.0 +/- 0.5 and 8.8 +/- 0.5% by TGF-[beta]1 and TGF-[beta]3, respectively. Secretion of MMP-1 (interstitial collagenase) by fibroblasts was reduced by both TGF-[beta] isoforms, whereas secretion of MMP-2 (gelatinase A) was unaffected by either of the two isoforms. Both TGF-[beta] isoforms increased TIMP-1 protein expression, whereas TIMP-2 protein was decreased. We thus conclude that TGF-[beta]1 and TGF-[beta]3 are equally potent in increasing ECM deposition. Their fibrotic effect in lung fibroblasts results from 1) an increase in the secretion and deposition of total ECM and collagens, 2) a decrease in MMP-1 secretion, and 3) an increase of TIMP-1 expression.

Nutritional Interventions in Aging and Age-Associated Diseases

George Roth — 2007-11-06T08:30:32-00:00

Annals of the New York Academy of Sciences, Vol. 1114, No. 1. (October 2007), pp. 369-371.

Phagocyte-specific calcium-binding S100 proteins as clinical laboratory markers of inflammation.

D Foell — 2008-01-17T01:56:43-00:00

Clin Chim Acta, Vol. 344, No. 1-2. (June 2004), pp. 37-51.

The EF-hand homolog family of S100 proteins comprises the largest group of calcium-binding proteins. Within this S100 family, the phagocyte-specific calcium-binding proteins are pro-inflammatory molecules expressed and secreted by phagocytes, which play a pivotal role within the innate immune system. Although the exact biological functions of these proteins still remain to be defined in greater detail, there is evidence that they are involved in a pro-inflammatory axis associated with various inflammatory conditions. The three members of this group, S100A8, S100A9 and S100A12 are overexpressed at local sites of inflammation. High concentrations are found in synovial fluid, sputum, stool and blood plasma/serum during inflammation. Both the S100A8/S100A9 complex and S100A12 have been proven to be useful as diagnostic markers of inflammation especially in non-infectious inflammatory diseases such as arthritis, chronic inflammatory lung and bowel disease. They indicate phagocyte activation more sensitively than conventional parameters of inflammation. As a consequence, there is a strong correlation to the inflammation of various acute and chronic disorders, making these proteins sensitive parameters for the monitoring of disease activity and response to treatment in individual patients. The phagocyte-specific S100 proteins are able to indicate minimal residual inflammation, which is not detected by other diagnostic tests, and they may even be prospective markers for the outcome of patients. In this review, pro-inflammatory functions of S100 proteins and their usefulness as biomarkers of inflammation are presented.

Synthetic Microarray Data Generation with RANGE and NEMO.

James Long — 2007-11-10T10:29:00-00:00

Bioinformatics (3 November 2007)

MOTIVATION: For testing and sensitivity analysis purposes, it is beneficial to have known transcription networks of sufficient size and variability during development of microarray data and network deconvolution algorithms. Description of such networks in a simple language translatable to Systems Biology Markup Language would allow generation of model data for the networks. RESULTS: Described herein is software (RANGE: RAndom Network GEnerator) to generate large random transcription networks in the NEMO (NEtwork MOtif) language. NEMO is recognized by a grammar for transcription network motifs using lex and yacc to output Systems Biology Markup Language models for either specified or randomized gene input functions. These models of known networks may be input to a biochemical simulator, allowing the generation of synthetic microarray data. AVAILABILITY: http://range.sourceforge.net.

Vitamin D3 down-regulates monocyte TLR expression and triggers hyporesponsiveness to pathogen-associated molecular patterns.

K Sadeghi — 2007-12-21T03:36:08-00:00

Eur J Immunol, Vol. 36, No. 2. (February 2006), pp. 361-370.

Toll-like receptors (TLR) represent an ancient front-line defence system that enables the host organism to sense the presence of microbial components within minutes. As inducers of inflammation, TLR act as important triggers of distinct entities such as sepsis or autoimmune disease exacerbation. We report here that vitamin D3 [1alpha,25-dihydroxycholecalciferol, 1,25(OH)(2)D3] suppresses the expression of TLR2 and TLR4 protein and mRNA in human monocytes in a time- and dose-dependent fashion. Despite 1,25(OH)(2)D3-induced up-regulation of CD14, challenge of human monocytes with either LPS or lipoteichoic acid resulted in impaired TNF-alpha and procoagulatory tissue factor (CD142) production, emphasizing the critical role of TLR in the induction of inflammation. Moreover, reduced TLR levels in 1,25(OH)(2)D3-treated phagocytes were accompanied by impaired NF-kappaB/RelA translocation to the nucleus and by reduced p38 and p42/44 (extracellular signal-regulated kinase 1/2) phosphorylation upon TLR-ligand engagement. Both TLR down-regulation and CD14 up-regulation were substantially inhibited by the vitamin D receptor (VDR) antagonist ZK 159222, indicating that the immunomodulatory effect of 1,25(OH)(2)D3 on innate immunity receptors requires VDR transcription factor activation. Our data provide strong evidence that 1,25(OH)(2)D3 primes monocytes to respond less effectively to bacterial cell wall components in a VDR-dependent mechanism, most likely due to decreased levels of TLR2 and TLR4.

Phlorizin: a review.

JR Ehrenkranz — 2007-12-15T10:50:00-00:00

Diabetes Metab Res Rev, Vol. 21, No. 1. (b 2005), pp. 31-38.

The dihydrochalcone phlorizin is a natural product and dietary constituent found in a number of fruit trees. It has been used as a pharmaceutical and tool for physiology research for over 150 years. Phlorizin's principal pharmacological action is to produce renal glycosuria and block intestinal glucose absorption through inhibition of the sodium-glucose symporters located in the proximal renal tubule and mucosa of the small intestine.This review covers the role phlorizin has played in the history of diabetes mellitus and its use as an agent to understand fundamental concepts in renal physiology as well as summarizes the physiology of cellular glucose transport and the pathophysiology of renal glycosuria. It reviews the biology and pathobiology of glucose transporters and discusses the medical botany of phlorizin and the potential effects of plant flavonoids, such as phlorizin, on human metabolism. Lastly, it describes the clinical pharmacology and toxicology of phlorizin, including investigational uses of phlorizin and phlorizin analogs in the treatment of diabetes, obesity, and stress hyperglycemia.

CombiMatrix oligonucleotide arrays: genotyping and gene expression assays employing electrochemical detection.

AL Ghindilis — 2007-10-12T04:34:08-00:00

Biosens Bioelectron, Vol. 22, No. 9-10. (15 April 2007), pp. 1853-1860.

Electrochemical detection has been developed and assay performances studied for the CombiMatrix oligonucleotide microarray platform that contains 12,544 individually addressable microelectrodes (features) in a semiconductor matrix. The approach is based on the detection of redox active chemistries (such as horseradish peroxidase (HRP) and the associated substrate TMB) proximal to specific microarray electrodes. First, microarray probes are hybridized to biotin-labeled targets, second, the HRP-streptavidin conjugate binds to biotin, and enzymatic oxidation of the electron donor substrate then occurs. The detection current is generated due to electro-reduction of the HRP reaction product, and it is measured with the CombiMatrix ElectraSense Reader. Performance of the ElectraSense platform has been characterized using gene expression and genotyping assays to analyze: (i) signal to concentration dependence, (ii) assay resolution, (iii) coefficients of variation, (CV) and (iv) array-to-array reproducibility and data correlation. The ElectraSense platform was also compared to the standard fluorescent detection, and good consistency was observed between these two different detection techniques. A lower detection limit of 0.75 pM was obtained for ElectraSense as compared to the detection limit of 1.5 pM obtained for fluorescent detection. Thus, the ElectraSense platform has been used to develop nucleic acid assays for highly accurate genotyping of a variety of pathogens including bio-threat agents (such as Bacillus anthracis, Yersinia pestis, and other microorganisms including Escherichia coli, Bacillus subtilis, etc.) and common pathogens of the respiratory tract (e.g. influenza A virus).

Hypoxia modulates the effects of transforming growth factor-beta isoforms on matrix-formation by primary human lung fibroblasts.

E Papakonstantinou — 2007-10-09T02:14:29-00:00

Cytokine, Vol. 24, No. 1-2. (October 2003), pp. 25-35.

Chronic hypoxia is implicated in lung fibrosis, which is characterized by enhanced deposition of extracellular matrix (ECM) molecules. Transforming growth factor-beta (TGF-beta) plays a key role in fibroblast homeostasis and is involved in disease states characterized by excessive fibrosis, such as pulmonary fibrosis. In this study, we investigated if hypoxia modulates the effects of TGF-beta on the expression of gelatinases: matrix metalloproteinase (MMP)-2 and MMP-9, interstitial collagenases: MMP-1 and MMP-13, tissue inhibitors of MMP (TIMP), collagen type I and interleukin-6 (IL-6). Primary human lung fibroblasts, established from tissue biopsies, were cultivated under normoxia or hypoxia in the presence of TGF-beta1, TGF-beta2 or TGF-beta3. Gelatinases were assessed by gelatin zymography and collagenases, TIMP, collagen type I and IL-6 by ELISA. Under normoxia fibroblasts secreted MMP-2, collagenases, TIMP, collagen type I and IL-6. TGF-betas significantly decreased MMP-1 and increased TIMP-1, IL-6 and collagen type I. Hypoxia significantly enhanced MMP-2, and collagenases. Compared to normoxia, the combination of TGF-beta and hypoxia reduced MMP-1, and further amplified the level of TIMP, IL-6, and collagen type I. Thus, in human lung fibroblasts hypoxia significantly increases the TGF-betas-induced secretion of collagen type I and may be associated to the accumulation of ECM observed in lung fibrosis.

Systematic review: impact of health information technology on quality, efficiency, and costs of medical care.

B Chaudhry — 2006-06-07T03:45:18-00:00

Ann Intern Med, Vol. 144, No. 10. (16 May 2006), pp. 742-752.

BACKGROUND: Experts consider health information technology key to improving efficiency and quality of health care. PURPOSE: To systematically review evidence on the effect of health information technology on quality, efficiency, and costs of health care. DATA SOURCES: The authors systematically searched the English-language literature indexed in MEDLINE (1995 to January 2004), the Cochrane Central Register of Controlled Trials, the Cochrane Database of Abstracts of Reviews of Effects, and the Periodical Abstracts Database. We also added studies identified by experts up to April 2005. STUDY SELECTION: Descriptive and comparative studies and systematic reviews of health information technology. DATA EXTRACTION: Two reviewers independently extracted information on system capabilities, design, effects on quality, system acquisition, implementation context, and costs. DATA SYNTHESIS: 257 studies met the inclusion criteria. Most studies addressed decision support systems or electronic health records. Approximately 25% of the studies were from 4 academic institutions that implemented internally developed systems; only 9 studies evaluated multifunctional, commercially developed systems. Three major benefits on quality were demonstrated: increased adherence to guideline-based care, enhanced surveillance and monitoring, and decreased medication errors. The primary domain of improvement was preventive health. The major efficiency benefit shown was decreased utilization of care. Data on another efficiency measure, time utilization, were mixed. Empirical cost data were limited. LIMITATIONS: Available quantitative research was limited and was done by a small number of institutions. Systems were heterogeneous and sometimes incompletely described. Available financial and contextual data were limited. CONCLUSIONS: Four benchmark institutions have demonstrated the efficacy of health information technologies in improving quality and efficiency. Whether and how other institutions can achieve similar benefits, and at what costs, are unclear.

Intradialytic Parenteral Nutrition Does Not Improve Survival in Malnourished Hemodialysis Patients: A 2-Year Multicenter, Prospective, Randomized Study

Noel Cano — 2007-08-28T10:00:13-00:00

J Am Soc Nephrol, Vol. 18, No. 9. (1 September 2007), pp. 2583-2591.

Although intradialytic parenteral nutrition (IDPN) is a method used widely to combat protein-calorie malnutrition in hemodialysis patients, its effect on survival has not been thoroughly studied. We conducted a prospective, randomized trial in which 186 malnourished hemodialysis patients received oral nutritional supplements with or without 1 year of IDPN. IDPN did not improve 2-year mortality (primary end point), hospitalization rate, Karnofsky score, body mass index, or laboratory markers of nutritional status. Instead, both groups demonstrated improvement in body mass index and the nutritional parameters serum albumin and prealbumin (P < 0.05). Multivariate analysis showed that an increase in prealbumin of >30 mg/L within 3 months, a marker of nutritional improvement, independently predicted a 54% decrease in 2-year mortality, as well as reduced hospitalizations and improved general well-being as measured by the Karnofsky score. Therefore, although we found no definite advantage of adding IDPN to oral nutritional supplementation, this is the first prospective study demonstrating that an improvement in prealbumin during nutritional therapy is associated with a decrease in morbidity and mortality in malnourished hemodialysis patients. 10.1681/ASN.2007020184

An intervention to decrease catheter-related bloodstream infections in the ICU.

P Pronovost — 2007-08-15T09:04:59-00:00

N Engl J Med, Vol. 355, No. 26. (28 December 2006), pp. 2725-2732.

BACKGROUND: Catheter-related bloodstream infections occurring in the intensive care unit (ICU) are common, costly, and potentially lethal. METHODS: We conducted a collaborative cohort study predominantly in ICUs in Michigan. An evidence-based intervention was used to reduce the incidence of catheter-related bloodstream infections. Multilevel Poisson regression modeling was used to compare infection rates before, during, and up to 18 months after implementation of the study intervention. Rates of infection per 1000 catheter-days were measured at 3-month intervals, according to the guidelines of the National Nosocomial Infections Surveillance System. RESULTS: A total of 108 ICUs agreed to participate in the study, and 103 reported data. The analysis included 1981 ICU-months of data and 375,757 catheter-days. The median rate of catheter-related bloodstream infection per 1000 catheter-days decreased from 2.7 infections at baseline to 0 at 3 months after implementation of the study intervention (P< or =0.002), and the mean rate per 1000 catheter-days decreased from 7.7 at baseline to 1.4 at 16 to 18 months of follow-up (P<0.002). The regression model showed a significant decrease in infection rates from baseline, with incidence-rate ratios continuously decreasing from 0.62 (95% confidence interval [CI], 0.47 to 0.81) at 0 to 3 months after implementation of the intervention to 0.34 (95% CI, 0.23 to 0.50) at 16 to 18 months. CONCLUSIONS: An evidence-based intervention resulted in a large and sustained reduction (up to 66%) in rates of catheter-related bloodstream infection that was maintained throughout the 18-month study period.

Palmitoylated proteins: purification and identification.

J Wan — 2007-08-14T02:00:31-00:00

Nat Protoc, Vol. 2, No. 7. (2007), pp. 1573-1584.

This proteomic protocol purifies and identifies palmitoylated proteins (i.e., S-acylated proteins) from complex protein extracts. The method relies on an acyl-biotinyl exchange chemistry in which biotin moieties are substituted for the thioester-linked protein acyl-modifications through a sequence of three in vitro chemical steps: (i) blockade of free thiols with N-ethylmaleimide; (ii) cleavage of the Cys-palmitoyl thioester linkages with hydroxylamine; and (iii) labeling of thiols, newly exposed by the hydroxylamine, with biotin-HPDP (Biotin-HPDP-N-[6-(Biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide. The biotinylated proteins are then affinity-purified using streptavidin-agarose and identified by multi-dimensional protein identification technology (MuDPIT), a high-throughput, tandem mass spectrometry (MS/MS)-based proteomic technology. MuDPIT also affords a semi-quantitative analysis that may be used to assess the gross changes induced to the global palmitoylation profile by mutation or drugs. Typically, 2-3 weeks are required for this analysis.

Proteomic identification of palmitoylated proteins

Amy Roth — 2007-07-15T09:18:40-00:00

Methods, Vol. 40, No. 2. (October 2006), pp. 135-142.

A proteomic method that purifies and identifies palmitoylated proteins from complex protein extracts is described. Using the fatty acid exchange labeling chemistry (described in the preceding report), palmitoyl modifications are exchanged for biotinylated compounds, allowing the subset of palmitoyl-proteins to be affinity-purified and then identified by mass spectroscopic protein identification technologies. The advantages and pitfalls of this new technology are discussed within the context of the recent application of this method in the yeast Saccharomyces cerevisiae.