Offline Two-Dimensional Liquid Chromatography-Mass Spectrometry for Deep Annotation of the Fecal Metabolome Following Fecal Microbiota Transplantation.

Biological interpretation of untargeted LC-MS-based metabolomics data depends on accurate compound identification, but current techniques fall short of identifying most features that can be detected. The human fecal metabolome is complex, variable, incompletely annotated, and serves as an ideal matrix to evaluate novel compound identification methods. We devised an experimental strategy for compound annotation using multidimensional chromatography and semiautomated feature alignment and applied these methods to study the fecal metabolome in the context of fecal microbiota transplantation (FMT) for recurrent C. difficile infection. Pooled fecal samples were fractionated using semipreparative liquid chromatography and analyzed by an orthogonal LC-MS/MS method. The resulting spectra were searched against commercial, public, and local spectral libraries, and annotations were vetted using retention time alignment and prediction. Multidimensional chromatography yielded more than a 2-fold improvement in identified compounds compared to conventional LC-MS/MS and successfully identified several rare and previously unreported compounds, including novel fatty-acid conjugated bile acid species. Using an automated software-based feature alignment strategy, most metabolites identified by the new approach could be matched to features that were detected but not identified in single-dimensional LC-MS/MS data. Overall, our approach represents a powerful strategy to enhance compound identification and biological insight from untargeted metabolomics data.

Ras/ERK and PI3K/AKT signaling differentially regulate oncogenic ERG mediated transcription in prostate cells.

The TMPRSS2/ERG gene rearrangement occurs in 50% of prostate tumors and results in expression of the transcription factor ERG, which is normally silent in prostate cells. ERG expression promotes prostate tumor formation and luminal epithelial cell fates when combined with PI3K/AKT pathway activation, however the mechanism of synergy is not known. In contrast to luminal fates, expression of ERG alone in immortalized normal prostate epithelial cells promotes cell migration and epithelial to mesenchymal transition (EMT). Migration requires ERG serine 96 phosphorylation via endogenous Ras/ERK signaling. We found that a phosphomimetic mutant, S96E ERG, drove tumor formation and clonogenic survival without activated AKT. S96 was only phosphorylated on nuclear ERG, and differential recruitment of ERK to a subset of ERG-bound chromatin associated with ERG-activated, but not ERG-repressed genes. S96E did not alter ERG genomic binding, but caused a loss of ERG-mediated repression, EZH2 binding and H3K27 methylation. In contrast, AKT activation altered the ERG cistrome and promoted expression of luminal cell fate genes. These data suggest that, depending on AKT status, ERG can promote either luminal or EMT transcription programs, but ERG can promote tumorigenesis independent of these cell fates and tumorigenesis requires only the transcriptional activation function.

Modifying Chromatography Conditions for Improved Unknown Feature Identification in Untargeted Metabolomics.

Untargeted metabolomics is an essential component of systems biology research, but it is plagued by a high proportion of detectable features not identified with a chemical structure. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiments produce spectra that can be searched against databases to help identify or classify these unknowns, but many features do not generate spectra of sufficient quality to enable successful annotation. Here, we explore alterations to gradient length, mass loading, and rolling precursor ion exclusion parameters for reversed phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) that improve compound identification performance for human plasma samples. A manual review of spectral matches from the HILIC data set was used to determine reasonable thresholds for search score and other metrics to enable semi-automated MS/MS data analysis. Compared to typical LC-MS/MS conditions, methods adapted for compound identification increased the total number of unique metabolites that could be matched to a spectral database from 214 to 2052. Following data alignment, 68.0% of newly identified features from the modified conditions could be detected and quantitated using a routine 20-min LC-MS run. Finally, a localized machine learning model was developed to classify the remaining unknowns and select a subset that shared spectral characteristics with successfully identified features. A total of 576 and 749 unidentified features in the HILIC and RPLC data sets were classified by the model as high-priority unknowns or higher-importance targets for follow-up analysis. Overall, our study presents a simple strategy to more deeply annotate untargeted metabolomics data for a modest additional investment of time and sample.

Liquid chromatography above 20,000 PSI.

Continued improvements in HPLC have led to faster and more efficient separations than previously possible. One important aspect of these improvements has been the increase in instrument operating pressure and the advent of ultrahigh pressure LC (UHPLC). Commercial instrumentation is now capable of up to ~20 kpsi, allowing fast and efficient separations with 5-15 cm columns packed with sub-2 µm particles. Home-built instruments have demonstrated the benefits of even further increases in instrument pressure. The focus of this review is on recent advancements and applications in liquid chromatography above 20 kpsi. We outline the theory and advantages of higher pressure and discuss instrument hardware and design capable of withstanding 20 kpsi or greater. We also overview column packing procedures and stationary phase considerations for HPLC above 20 kpsi, and lastly highlight a few recent applicatioob pressure instruments for the analysis of complex mixtures.

Design and synthesis of fluorogenic substrate-based probes for detecting Cathepsin B activity.

Cathepsin B plays key roles in tumor progression with its overexpression being associated with invasive and metastatic phenotypes and is a primary target of protease activated antibody-directed prodrug therapy. It therefore represents a potential therapeutic and diagnostic target and effort has been made to develop fluorescent probes to report on Cathepsin B activity in cells and animal models of cancer. We have designed, synthesized, and thoroughly evaluated four novel "turn on" probes that employ a lysosomotropic dansylcadaverine dye to report on Cathepsin B activity. Enzyme activity assays using a recombinant human enzyme and cancer cell lysates coupled with confocal microscopy experiments demonstrated that one of the probes, derivatized with the self-immolative prodrug linker p-aminobenzyl alcohol, can selectively report on Cathepsin B in biological samples including live cells.

Oncogenic ETS Factors in Prostate Cancer.

Prostate cancer is unique among carcinomas in that a fusion gene created by a chromosomal rearrangement is a common driver of the disease. The TMPRSS2/ERG rearrangement drives aberrant expression of the ETS family transcription factor ERG in 50% of prostate tumors. Similar rearrangements promote aberrant expression of the ETS family transcription factors ETV1 and ETV4 in another 10% of cases. Together, these three ETS factors are thought to promote tumorigenesis in the majority of prostate cancers. A goal of precision medicine is to be able to apply targeted therapeutics that are specific to disease subtypes. ETS gene rearrangement positive tumors represent the largest molecular subtype of prostate cancer, but to date there is no treatment specific to this marker. In this chapter we will review the latest findings regarding the molecular mechanisms of ETS factor function in the prostate. These molecular details may provide a path towards new therapeutic targets for this subtype of prostate cancer. Further, we will describe efforts to target the oncogenic functions of ETS family transcription factors directly as well as indirectly.

Phosphorylation of the oncogenic transcription factor ERG in prostate cells dissociates polycomb repressive complex 2, allowing target gene activation.

In ∼50% of prostate cancers, chromosomal rearrangements cause the fusion of the promoter and 5'-UTR of the androgen-regulated TMPRSS2 (transmembrane protease, serine 2) gene to the open reading frame of ERG, encoding an ETS family transcription factor. This fusion results in expression of full-length or N-terminally truncated ERG protein in prostate epithelia. ERG is not expressed in normal prostate epithelia, but when expressed, it promotes tumorigenesis via altered gene expression, stimulating epithelial-mesenchymal transition, cellular migration/invasion, and transformation. However, limited knowledge about the molecular mechanisms of ERG function in prostate cells has hampered efforts to therapeutically target ERG. ERK-mediated phosphorylation of ERG is required for ERG functions in prostate cells, but the reason for this requirement is unknown. Here, we report a mechanism whereby ERK-mediated phosphorylation of ERG at one serine residue causes a conformational change that allows ERK phosphorylation at a second serine residue, Ser-96. We found that the Ser-96 phosphorylation resulted in dissociation of EZH2 and SUZ12, components of polycomb repressive complex 2 (PRC2), transcriptional activation of ERG target genes, and increased cell migration. Conversely, loss of ERG phosphorylation at Ser-96 resulted in recruitment of EZH2 across the ERG-cistrome and a genome-wide loss of ERG-mediated transcriptional activation and cell migration. In conclusion, our findings have identified critical molecular mechanisms involving ERK-mediated ERG activation that could be exploited for therapeutic intervention in ERG-positive prostate cancers.

A Cross-Sectional Study of Antibiotic Prescribing for Childhood Upper Respiratory Tract Infections in Irish General Practice

Introduction This study aimed to analyse antibiotic prescribing in cases of upper respiratory tract infection (URTI) in children under 6 years attending Irish daytime and out-of-hours General Practice (GP) services. There have been large scale changes in entitlements for free GP care for this group in recent years. Methods A cross-sectional study of children under 6 years with URTI presentations was performed, over a two-week period for three years from 2015 to 2017. Factors associated with antibiotic prescription and preferred antibiotic compliance were examined using multivariate logistic regression. Results 1,007 Under-6 patients presented with an URTI in our sample over the study period. Following introduction of free GP care, patients were 50% less likely to receive an antibiotic prescription. Overall antibiotic prescribing fell from 70% to 50% in daytime services and from 72% to 60% in the out-of-hours setting. Patients presenting to out-of-hours services were more likely to receive an antibiotic (OR: 1.42) and less likely to receive a deferred antibiotic (OR: 0.53). One quarter to one third of all prescriptions were for deferred antibiotics. Year-on-year trends showed a 13% decrease in prescriptions and 13% increase in preferred antibiotic use. Conclusion The introduction of free GP care led to significant reductions in antibiotic prescribing, which may be due to changes in health seeking behaviour by parents or other reasons. Antibiotic prescribing was more commonplace in the out-of-hours setting, and rates remains high by international standards. This study underlines the importance of ongoing work around GP antimicrobial stewardship, particularly in the out-of-hours setting.

Distinct subclonal tumour responses to therapy revealed by circulating cell-free DNA.

BACKGROUND: The application of precision medicine in oncology requires in-depth characterisation of a patient's tumours and the dynamics of their responses to treatment. PATIENTS AND METHODS: We used next-generation sequencing of circulating cell-free DNA (cfDNA) to monitor the response of a KIT p.L576P-mutant metastatic vaginal mucosal melanoma to sequential targeted, immuno- and chemotherapy. RESULTS: Despite a KIT mutation, the response to imatinib was mixed. Unfortunately, tumours were not accessible for molecular analysis. To study the mechanism underlying the mixed clinical response, we carried out whole-exome sequencing and targeted longitudinal analysis of cfDNA. This revealed two tumour subclones; one with a KIT mutation that responded to imatinib and a second KIT-wild-type subclone that did not respond to imatinib. Notably, the subclones also responded differently to immunotherapy. However, both subclones responded to carboplatin/paclitaxel, and although the KIT-wild-type subclone progressed after chemotherapy, it responded to subsequent re-administration of paclitaxel. CONCLUSION: We show that cfDNA can reveal tumour evolution and subclonal responses to therapy even when biopsies are not available.

Tumourigenic non-small-cell lung cancer mesenchymal circulating tumour cells: a clinical case study.

BACKGROUND: Over the past decade, numerous reports describe the generation and increasing utility of non-small-cell lung cancer (NSCLC) patient-derived xenografts (PDX) from tissue biopsies. While PDX have proven useful for genetic profiling and preclinical drug testing, the requirement of a tissue biopsy limits the available patient population, particularly those with advanced oligometastatic disease. Conversely, 'liquid biopsies' such as circulating tumour cells (CTCs) are minimally invasive and easier to obtain. Here, we present a clinical case study of a NSCLC patient with advanced metastatic disease, a never smoker whose primary tumour was EGFR and ALK wild-type. We demonstrate for the first time, tumorigenicity of their CTCs to generate a patient CTC-derived eXplant (CDX). PATIENTS AND METHODS: CTCs were enriched at diagnosis and again 2 months later during disease progression from 10 ml blood from a 48-year-old NSCLC patient and implanted into immunocompromised mice. Resultant tumours were morphologically, immunohistochemically, and genetically compared with the donor patient's diagnostic specimen. Mice were treated with cisplatin and pemetrexed to assess preclinical efficacy of the chemotherapy regimen given to the donor patient. RESULTS: The NSCLC CDX expressed lung lineage markers TTF1 and CK7 and was unresponsive to cisplatin and pemetrexed. Examination of blood samples matched to that used for CDX generation revealed absence of CTCs using the CellSearch EpCAM-dependent platform, whereas size-based CTC enrichment revealed abundant heterogeneous CTCs of which ∼80% were mesenchymal marker vimentin positive. Molecular analysis of the CDX, mesenchymal and epithelial CTCs revealed a common somatic mutation confirming tumour origin and showed CDX RNA and protein profiles consistent with the predominantly mesenchymal phenotype. CONCLUSIONS: This study shows that the absence of NSCLC CTCs detected by CellSearch (EpCAM(+)) does not preclude CDX generation, highlighting epithelial to mesenchymal transition and the functional importance of mesenchymal CTCs in dissemination of this disease.

Availability of Youth Services in U.S. Mental Health Treatment Facilities.

Despite concern about access to mental health (MH) services for youth, little is known about the specialty treatment infrastructure serving this population. We used national data to examine which types of MH treatment facilities (hospital- and community-based) were most likely to offer youth services and which types of communities were most likely to have this infrastructure. Larger (p < 0.001) and privately owned (p < 0.001) facilities were more likely to offer youth services. Rural counties, counties in which a majority of residents were nonwhite, and/or counties with a higher percentage of uninsured residents were less likely to have a community-based MH treatment facility that served youth (p < 0.001).

Preparation of high-efficiency HILIC capillary columns utilizing slurry packing at 2100 bar.

Complex mixture analysis requires high-efficiency chromatography columns. Although reversed phase liquid chromatography (RPLC) is the dominant approach for such mixtures, hydrophilic interaction liquid chromatography (HILIC) is an important complement to RPLC by enabling the separation of polar compounds. Chromatography theory predicts that small particles and long columns will yield high efficiency; however, little work has been done to prepare HILIC columns longer than 25 cm packed with sub-2 µm particles. In this work, we tested the slurry packing of 75 cm long HILIC columns with 1.7 µm bridged-ethyl-hybrid amide HILIC particles at 2,100 bar (30,000 PSI). Acetonitrile, methanol, acetone, and water were tested as slurry solvents, with acetonitrile providing the best columns. Slurry concentrations of 50-200 mg/mL were assessed, and while 50-150 mg/mL provided comparable results, the 150 mg/mL columns provided the shortest packing times (9 min). Columns prepared using 150 mg/mL slurries in acetonitrile yielded a reduced minimum plate height (hmin) of 3.3 and an efficiency of 120,000 theoretical plates for acenaphthene, an unretained solute. Para-toluenesulfonic acid produced the lowest hmin of 1.9 and the highest efficiency of 210,000 theoretical plates. These results identify conditions for producing high-efficiency HILIC columns with potential applications to complex mixture analysis.

Offline Two-dimensional Liquid Chromatography-Mass Spectrometry for Deep Annotation of the Fecal Metabolome.

Compound identification is an essential task in the workflow of untargeted metabolomics since the interpretation of the data in a biological context depends on the correct assignment of chemical identities to the features it contains. Current techniques fall short of identifying all or even most observable features in untargeted metabolomics data, even after rigorous data cleaning approaches to remove degenerate features are applied. Hence, new strategies are required to annotate the metabolome more deeply and accurately. The human fecal metabolome, which is the focus of substantial biomedical interest, is a more complex, more variable, yet lesser-investigated sample matrix compared to widely studied sample types like human plasma. This manuscript describes a novel experimental strategy using multidimensional chromatography to facilitate compound identification in untargeted metabolomics. Pooled fecal metabolite extract samples were fractionated using offline semi-preparative liquid chromatography. The resulting fractions were analyzed by an orthogonal LC-MS/MS method, and the data were searched against commercial, public, and local spectral libraries. Multidimensional chromatography yielded more than a 3-fold improvement in identified compounds compared to the typical single-dimensional LC-MS/MS approach and successfully identified several rare and novel compounds, including atypical conjugated bile acid species. Most features identified by the new approach could be matched to features that were detectable but not identifiable in the original single-dimension LC-MS data. Overall, our approach represents a powerful strategy for deeper annotation of the metabolome that can be implemented with commercially-available instrumentation, and should apply to any dataset requiring deeper annotation of the metabolome.

The Nathan Kline Institute cultural competency assessment scale: psychometrics and implications for disparity reduction.

The NKI Cultural Competency Assessment Scale measures organizational CC in mental health outpatient settings. We describe its development and results of tests of its psychometric properties. When tested in 27 public mental health settings, factor analysis discerned three factors explaining 65% of the variance; each factor related to a stage of implementation of CC. Construct validity and inter-rater reliability were satisfactory. In tests of predictive validity, higher scores on items related to linguistic and service accommodations predicted a reduction in service disparities for engagement and retention outcomes for Hispanics. Disparities for Blacks essentially persisted independent of CC scores.

Early weight gain trajectories in first episode anorexia: predictors of outcome for emerging adults in outpatient treatment.

BACKGROUND: Early response to treatment has been shown to be a predictor of later clinical outcomes in eating disorders (EDs). Specifically, early weight gain trajectories in anorexia nervosa (AN) have been shown to predict higher rates of later remission in inpatient treatment. However, no study has, as of yet, examined this phenomenon within outpatient treatment of first episode cases of AN or in emerging adults. METHODS: One hundred seven patients with AN, all between the ages of 16 and 25 and with an illness duration of < 3 years, received treatment via the first episode rapid early intervention in eating disorders (FREED) service pathway. Weight was recorded routinely across early treatment sessions and recovery outcomes (BMI > 18.5 kg/m2 and eating psychopathology) were assessed up to 1 year later. Early weight gain across the first 12 treatment sessions was investigated using latent growth mixture modelling to determine distinct classes of change. Follow-up clinical outcomes and remission rates were compared between classes, and individual and clinical characteristics at baseline (treatment start) were tested as potential predictors. RESULTS: Four classes of early treatment trajectory were identified. Three of these classes (n = 95), though differing in their early change trajectories, showed substantial improvement in clinical outcomes at final follow-up. One smaller class (n = 12), characterised by a 'higher' start BMI (> 17) and no early weight gain, showed negligible improvement 1 year later. Of the three treatment responding groups, levels of purging, depression, and patient reported carer expressed emotion (in the form of high expectations and low tolerance of the patient) determined class membership, although these findings were not significant after correcting for multiple testing. A higher BMI at treatment start was not sufficient to predict optimal clinical outcomes. CONCLUSION: First episode cases of AN treated via FREED fit into four distinct early response trajectory classes. These may represent subtypes of first episode AN patients. Three of these four trajectories included patients with substantial improvements 1 year later. For those in the non-response trajectory class, treatment adjustments or augmentations could be considered earlier, i.e., at treatment session 12.

A key feature of anorexia nervosa (AN) is an unhealthily low body weight. Previous studies show that more weight gained early in inpatient treatment leads to better outcomes. This study tried to see if this was also true for outpatients receiving treatment for the first time. All participants were emerging adults between the ages of 16 and 25 who had been ill for less than 3 years. Weight was recorded across the first 12 weekly treatment sessions. Statistics showed that the patients fit roughly into four different groups in early treatment, each with different starting weights and rates of weight gain in the first 12 treatment sessions. The group a patient belonged to could sometimes be predicted by vomiting behaviours, level of depression, and patients' perception of parental tolerance and expectations at the start of treatment. Out of the four groups, three did relatively well 1 year later, but one small group of patients did not. This small group had a higher starting weight than many of the other groups but did not gain any weight across the first 12 sessions. These patients could benefit from a change or increase in the amount or intensity of treatment after the first 12 treatment sessions.

Trends in the inpatient mental health treatment of children and adolescents in US community hospitals between 1990 and 2000.

CONTEXT: Previous work has demonstrated marked changes in inpatient mental health service use by children and adolescents in the 1980s and early 1990s, but more recent, comprehensive, nationally representative data have not been reported. OBJECTIVE: To describe trends in inpatient treatment of children and adolescents with mental disorders between 1990 and 2000. DESIGN AND SETTING: Analysis of the Healthcare Cost and Utilization Project Nationwide Inpatient Sample, a nationally representative sample of discharges from US community hospitals sponsored by the Agency for Healthcare Research and Quality. PATIENTS: Patients aged 17 years and younger discharged from US community hospitals with a principal diagnosis of a mental disorder. MAIN OUTCOME MEASURES: Changes in the number and population-based rate of discharges, total inpatient days and average length of stay, charges, diagnoses, dispositions, and patient demographic and hospital characteristics. RESULTS: Although the total number of discharges, population-based discharge rate, and daily charges did not significantly change between 1990 and 2000, the total number of inpatient days and mean charges per visit each fell by approximately one half. Median length of stay declined 63% over the decade from 12.2 days to 4.5 days. Declines in median and mean lengths of stay were observed for most diagnostic categories and remained significant after controlling for changes in background patient and hospital characteristics. Discharge rates for psychotic and mood disorders as well as intentional self-injuries increased while rates for adjustment disorders fell. Discharges to short-term, nursing, and other inpatient facilities declined. CONCLUSIONS: The period between 1990 and 2000 was characterized by a transformation in the length of inpatient mental health treatment for young people. Community hospitals evaluated, treated, and discharged mentally ill children and adolescents far more quickly than 10 years earlier despite higher apparent rates of serious illness and self-harm and fewer transfers to intermediate and inpatient care.

Epstein-Barr virus and Burkitt lymphoma.

Burkitt lymphoma (BL) is an aggressive B-cell malignancy with endemic, sporadic and immunodeficiency-associated variants. It has been known for many years that the fundamental transforming event in BL is the translocation of the MYC gene, and the events that bring about this translocation and those that allow cells to survive with the constitutive expression of MYC have been the subject of intense investigation. Epstein-Barr virus (EBV) infection, malaria, immunodeficiency and spontaneous, somatic mutation can all contribute to the origin and maintenance of this cancer and their mechanisms are the subject of this review.

Analysis of gene expression in a complex differentiation hierarchy by global amplification of cDNA from single cells.

BACKGROUND: Many differentiating tissues contain progenitor cells that differ in their commitment states but cannot be readily distinguished or segregated. Molecular analysis is therefore restricted to mixed populations or cell lines which may also be heterogeneous, and the critical differences in gene expression that might determine divergent development are obscured. In this study, we combined global amplification of mRNA transcripts in single cells with identification of the developmental potential of processed cells on the basis of the fates of their sibling cells from clonal starts. RESULTS: We analyzed clones of from four to eight hemopoietic precursor cells which had a variety of differentiative potentials; sibling cells generally each formed clones of identical composition in secondary culture. Globally amplified cDNA was prepared from individual precursors whose developmental potential was identified by tracking sibling fates. Further cDNA samples were prepared from terminally maturing, homogeneous hemopoietic cell populations. Together, the samples represented 16 positions in the hemopoietic developmental hierarchy. Expression patterns in the sample set were determined for 29 genes known to be involved in hemopoietic cell growth, differentiation or function. The cDNAs from a bipotent erythroid/megakaryocyte precursor and a bipotent neutrophil/macrophage precursor were subtractively hybridized, yielding numerous differentially expressed cDNA clones. Hybridization of such clones to the entire precursor sample set identified transcripts with consistent patterns of differential expression in the precursor hierarchy. CONCLUSIONS: Tracking of sibling fates reliably identifies the differentiative potential of a single cell taken for PCR analysis, and demonstrates the existence of a variety of distinct and stable states of differentiative commitment. Global amplification of cDNA from single precursor cells, identified by sibling fates, yields a true representation of lineage- and stage-specific gene expression, as confirmed by hybridization to a broad panel of probes. The results provide the first expression mapping of these genes that distinguishes between progenitors in different commitment states, generate new insights and predictions relevant to mechanism, and introduce a powerful set of tools for unravelling the genetic basis of lineage divergence.

Transactivation by the p65 subunit of NF-kappaB in response to interleukin-1 (IL-1) involves MyD88, IL-1 receptor-associated kinase 1, TRAF-6, and Rac1.

We have examined the involvement of components of the interleukin-1 (IL-1) signaling pathway in the transactivation of gene expression by the p65 subunit of NF-kappaB. Transient transfection of cells with plasmids encoding wild-type MyD88, IL-1 receptor-associated kinase 1 (IRAK-1), and TRAF-6 drove p65-mediated transactivation. In addition, dominant negative forms of MyD88, IRAK-1, and TRAF-6 inhibited the IL-1-induced response. In cells lacking MyD88 or IRAK-1, no effect of IL-1 was observed. Together, these results indicate that MyD88, IRAK-1, and TRAF-6 are important downstream regulators of IL-1-mediated p65 transactivation. We have previously shown that the low-molecular-weight G protein Rac1 is involved in this response. Constitutively active RacV12-mediated transactivation was not inhibited by dominant negative MyD88, while dominant negative RacN17 inhibited the MyD88-driven response, placing Rac1 downstream of MyD88 on this pathway. Dominant negative RacN17 inhibited wild-type IRAK-1- and TRAF-6-induced transactivation, and in turn, dominant negative IRAK-1 and TRAF-6 inhibited the RacV12-driven response, suggesting a mutual codependence of Rac1, IRAK-1, and TRAF-6 in regulating this pathway. Finally, Rac1 was found to associate with the receptor complex via interactions with both MyD88 and the IL-1 receptor accessory protein. A pathway emanating from MyD88 and involving IRAK-1, TRAF-6, and Rac1 is therefore involved in transactivation of gene expression by the p65 subunit of NF-kappaB in response to IL-1.

Entropy in protein folding and in protein-protein interactions.

The reduction of conformational entropy is a major barrier that has to be overcome in protein folding and binding. Changes in solvent entropy are also a major factor. Recent advances include clarification of the fundamental issues concerning the separation of entropy into components, the treatment of association entropy in binding, and the role of size and shape effects in solvation entropy. Advances in the application of entropy calculations include an emerging consensus for estimates of backbone and sidechain entropy loss in protein folding via use of numerically intensive methods for sampling, and use of the expanding protein-structure database.