Systematic screen for human disease genes in yeast.

High similarity between yeast and human mitochondria allows functional genomic study of Saccharomyces cerevisiae to be used to identify human genes involved in disease. So far, 102 heritable disorders have been attributed to defects in a quarter of the known nuclear-encoded mitochondrial proteins in humans. Many mitochondrial diseases remain unexplained, however, in part because only 40-60% of the presumed 700-1,000 proteins involved in mitochondrial function and biogenesis have been identified. Here we apply a systematic functional screen using the pre-existing whole-genome pool of yeast deletion mutants to identify mitochondrial proteins. Three million measurements of strain fitness identified 466 genes whose deletions impaired mitochondrial respiration, of which 265 were new. Our approach gave higher selection than other systematic approaches, including fivefold greater selection than gene expression analysis. To apply these advantages to human disorders involving mitochondria, human orthologs were identified and linked to heritable diseases using genomic map positions.

Preliminary validation of the Diversion Risk Scale (DRS): Using environmental and individual factors to predict opioid-related diversion events.

The standard of care calls for the assessment of patients with chronic pain prior to the initiation of opioids, with one part of this assessment including assessment of the risk of misuse of medications. However, traditional opioid risk assessment tools focus almost entirely on individual factors and on the risk of misuse and addiction to opioids. Diversion of opioid medications has been found to be not uncommon, but to date, there have been no assessment tools specifically designed to assess the risk of diversion. In this study, we developed a measure designed specifically to assess the risk of an opioid medication ending up in the hands of someone other than the chronic pain patient to whom they were prescribed. A 15-item measure, the Diversion Risk Scale, was created and administered to 85 patients at a chronic pain practice. Results found that the measure had acceptable predictive validity. It was moderately correlated with traditional opioid risk assessment tools and showed improved ability to predict specific indicators of diversion. Diversion has been an understudied phenomenon, and the clinical value of an assessment tool that can help predict diversion in the chronic pain population is discussed.

A comparison of common screening methods for predicting aberrant drug-related behavior among patients receiving opioids for chronic pain management.

OBJECTIVE: The ability to predict risk for violating opioid medication policies, known as aberrant drug-related behavior, is critical for providing optimal treatment. Many pain management centers measure risk using one of several partially validated measures: the Screener and Opioid Assessment for Patients with Pain (SOAPP), the Diagnosis, Intractability, Risk, and Efficacy inventory (DIRE), and/or the Opioid Risk Tool (ORT). However, little is known about how these measures compare with each other in predicting aberrant drug-related behavior and discontinuance of opioid pain medications. The current study aimed to address this research question. PATIENTS: Participants were 48 patients who attended a pain management center in Tennessee but were later discontinued from opioids for aberrant drug-related behavior. Patients referred for opioid medication for pain management participated in a semi-structured clinical interview with the staff psychologist and completed the aforementioned measures. Patients generally returned to the pain clinic on a monthly basis for medication management. Results. Analyses compared the sensitivity of each self-report measure and the clinical interview in predicting discontinuance for aberrant drug-related behavior. RESULTS: showed the highest sensitivity for the clinical interview (0.77) and the SOAPP (0.72), followed by the ORT (0.45) and the DIRE (0.17). Combining the clinical interview with the SOAPP increased sensitivity to 0.90. CONCLUSIONS: Among patients who were discontinued from opioids for aberrant drug-related behaviors, the clinical interview and the SOAPP were most effective at predicting risk at baseline. Implications for future research and clinical practice are discussed.

The Impact of Virtual Reality on Chronic Pain.

The treatment of chronic pain could benefit from additional non-opioid interventions. Virtual reality (VR) has been shown to be effective in decreasing pain for procedural or acute pain but to date there have been few studies on its use in chronic pain. The present study was an investigation of the impact of a virtual reality application for chronic pain. Thirty (30) participants with various chronic pain conditions were offered a five-minute session using a virtual reality application called Cool! Participants were asked about their pain using a 0-10 visual analog scale rating before the VR session, during the session and immediately after the session. They were also asked about immersion into the VR world and about possible side effects. Pain was reduced from pre-session to post-session by 33%. Pain was reduced from pre-session during the VR session by 60%. These changes were both statistically significant at the p < .001 level. Three participants (10%) reported no change between pre and post pain ratings. Ten participants (33%) reported complete pain relief while doing the virtual reality session. All participants (100%) reported a decrease in pain to some degree between pre-session pain and during-session pain. The virtual reality experience was found here to provide a significant amount of pain relief. A head mounted display (HMD) was used with all subjects and no discomfort was experienced. Only one participant noted any side effects. VR seems to have promise as a non-opioid treatment for chronic pain and further investigation is warranted.

Validation of a new risk assessment tool: the Brief Risk Questionnaire.

Opioids remain a common method of treating chronic pain conditions despite some controversy. In an effort to address some of the risks of opioid medications, opioid risk assessment has become a standard of care when opioids are used to treat a chronic pain condition. Research to date has found that many currently available patient-completed written questionnaires are relatively poor at identifying which patients will engage in medication aberrant behavior in the future. Clinical interview techniques have been found to provide better prediction, but practitioners often prefer the convenience of patient-completed tools. In this study, a new brief patient-completed risk tool, the Brief Risk Questionnaire (BRQ), was created and compared with a structured clinical interview and two commonly used patient-completed risk assessment tools: the Opioid Risk Tool (ORT) and Screener and Opioid Assessment for Patients with Pain-Revised (SOAPP-R). The different risk assessment measures were administered to 454 patients at a pain clinic and their prediction of medication aberrant behavior at 6-month follow-up was compared. Results found that the BRQ was able to predict future medication aberrant behavior better than the other two patient-completed risk measures and almost as well overall as a structured clinical interview rating system. This study indicates that the BRQ could be a useful new tool for clinicians in conducting opioid risk assessment.

Genotyping African haplotypes in ATM using a co-spotted single-base extension assay.

Human genetic analysis, including population genetic studies, increasingly calls for cost-effective, high-throughput methods for the rapid screening of single nucleotide polymorphisms (SNPs) across many individuals. The modified single-base extension assay described here (arrayed SBE) is a highly accurate and robust method for SNP genotyping that can deliver genotypes at 3.5 cents each, following PCR. Specifically, amino-modified probe/target pairs were prehybridized, then co-spotted in a microarray format prior to enzymatic addition of allele-specific nucleotides. Probe/target identity was determined solely by its physical location on the array rather than by hybridization to a complementary target, resulting in a call rate of 99-100%. These innovations result in an inexpensive, accurate assay with exceptional signal-to-noise ratios, depending on the glass surface employed. Comparison of glass slides from three different manufacturers indicated that aldehyde-based Zyomyx slides provided superior performance for this assay. Arrayed SBE was applied to study the geographic distribution of three African-specific haplotypes in the human ATM gene. Four selectively neutral markers, which define the haplotypes H5, H6, and H7, were screened in a total of 415 individuals. Region-specific haplotype frequencies were consistent with patterns of human migration across and outside of Africa, suggesting a possible haplotype origin in East Africa. Arrayed SBE was a robust tool for this analysis that could be applied to any situation requiring the genotyping of a few SNPs in many individuals.

Further validation of an opioid risk assessment tool: the Brief Risk Interview.

Opioids remain a common method of treating chronic pain conditions despite some controversy. In an effort to address some of the risks of opioid medications, opioid risk assessment has become a standard of care when opioids are used to treat a chronic pain condition. Research to date has found that clinical interviews may be superior to currently available patient-completed written questionnaires in identifying patients likely to engage in medication aberrant behavior. The Brief Risk Interview (BRI) has been developed as a risk assessment tool that has the sensitivity of a clinical interview while eliminating the need for the lengthy process of an interview. The current study compared the predictive ability of the BRI with two commonly used patient-completed risk assessment tools: the Opioid Risk Tool (ORT) and the Screener and Opioid Assessment for Patients with Pain-Revised (SOAPP-R). After clinical staff at a pain practice underwent a 1-hour training program, 124 consecutive new patients were evaluated using the BRI, ORT, and SOAPP-R. Follow-up data found that the BRI was a good predictor of medication aberrant behavior and offered better sensitivity and better overall predictive accuracy than the ORT or the SOAPP-R. Overall, it appears that the BRI is a valid risk assessment tool that, after a brief training session, can be used effectively by pain clinicians. Further study is needed in other practice settings and with larger sample sizes.

Preliminary data on a new opioid risk assessment measure: the Brief Risk Interview.

Risk assessment and stratification have become a central issue in prescribing opioids to patients with chronic pain. Research to date on various risk screening measures has shown that a clinical interview by an experienced clinician offers superior predictive ability in identifying patients who are more likely to engage in future medication aberrant behavior. The current study represents a pilot study of an interview rating scale that is designed to replicate this clinical assessment. This study compares the predictions of medication aberrant behavior made by the Opioid Risk Tool, the Screener and Opioid Assessment for Patients with Pain-Revised, and the new interview rating scale, the Brief Risk Interview (BRI). A sample of 196 patients was assessed by each of the three risk measures and then follow-up data were gathered at 6 months post interview to determine which patients had engaged in medication aberrant behavior and had been discharged from the practice. The BRI shows superior predictive ability in identifying patients who later engage in medication aberrant behavior. Although more study in other settings is needed, these preliminary data suggest that the Brief Risk Interview could be a useful tool for any pain clinician in assessing risk through the use of information gathered in a brief interview.

Effects of a single session group intervention for pain management in chronic pain patients: a pilot study.

INTRODUCTION: Behavioral and psychological interventions are key components of treating chronic pain. However, there are logistical barriers to providing such treatments, including a lack of psychological staff to provide such interventions and limited ability of patients with chronic pain to attend multiple sessions. As other areas of mental health have shown promise in providing single session interventions for various conditions, this pilot study hypothesized that a single group session for chronic pain patients could be helpful in decreasing patient pain catastrophizing. The five content areas addressed in the group were termed understanding, accepting, calming, balancing, and coping. METHODS: A pilot study was undertaken. Chronic pain patients were given a pre-group assessment, including the Pain Catastrophizing Scale with a follow-up assessment administered 3 months later. RESULTS: Fifty-three patients were studied. Results showed a significant decrease in overall pain catastrophizing scores at follow-up. A clear majority of patients also reported that the group was helpful and should be offered to other pain patients. CONCLUSION: This study suggests that a single session group can be a helpful intervention for patients with chronic pain.

A comparison of various risk screening methods in predicting discharge from opioid treatment.

OBJECTIVES: Risk assessment and stratification has become an important aspect of the prescribing of opioids to patients with chronic pain. There is little empirical data available on the sensitivity and specificity of commonly used risk assessment tools. This paper describes 2 studies that compare the prediction capabilities of various risk assessment tools. METHODS: The first study presents data on patients at a pain practice whose treatment with opioids was stopped due to their engaging in aberrant drug-related behavior. Patients were assessed with the Screener and Opioid Assessment for Patients with Pain-Revised (SOAPP-R), the Pain Medication Questionnaire, the Opioid Risk Tool, and a clinical interview. A second study compared the risk assessment measures, SOAPP-R, Pain Medication Questionnaire, Opioid Risk Tool, and a clinical interview. Data were gathered on whether patients had engaged in aberrant drug-related behavior at 6-month follow-up. RESULTS: Significant differences in the measures were found. Accuracy did not appear to be a function of the type of aberrant drug-related behavior that the patient engaged in for any of the measures. The clinical interview showed the best sensitivity of the 4 risk measures in predicting risk. The SOAPP-R showed the best sensitivity of the self-report measures. However, the SOAPP-R appears to overrate risk. DISCUSSION: Overall, these studies indicate that not all risk assessment tools are equal in their ability to accurately predict future aberrant drug-related behavior. It may be that written risk assessment tools that use more subtle items are better suited to certain patient populations.

Proteomic changes in cerebrospinal fluid of presymptomatic and affected persons carrying familial Alzheimer disease mutations.

OBJECTIVE: To identify cerebrospinal fluid (CSF) protein changes in persons who will develop familial Alzheimer disease (FAD) due to PSEN1 and APP mutations, using unbiased proteomics. DESIGN: We compared proteomic profiles of CSF from individuals with FAD who were mutation carriers (MCs) and related noncarriers (NCs). Abundant proteins were depleted and samples were analyzed using liquid chromatography-electrospray ionization-mass spectrometry on a high-resolution time-of-flight instrument. Tryptic peptides were identified by tandem mass spectrometry. Proteins differing in concentration between the MCs and NCs were identified. SETTING: A tertiary dementia referral center and a proteomic biomarker discovery laboratory. PARTICIPANTS: Fourteen FAD MCs (mean age, 34.2 years; 10 are asymptomatic, 12 have presenilin-1 [PSEN1 ] gene mutations, and 2 have amyloid precursor protein [APP ] gene mutations) and 5 related NCs (mean age, 37.6 years). RESULTS: Fifty-six proteins were identified, represented by multiple tryptic peptides showing significant differences between MCs and NCs (46 upregulated and 10 downregulated); 40 of these proteins differed when the analysis was restricted to asymptomatic individuals. Fourteen proteins have been reported in prior proteomic studies in late-onset AD, including amyloid precursor protein, transferrin, α(1)ß-glycoprotein, complement components, afamin precursor, spondin 1, plasminogen, hemopexin, and neuronal pentraxin receptor. Many other proteins were unique to our study, including calsyntenin 3, AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) 4 glutamate receptor, CD99 antigen, di- N-acetyl-chitobiase, and secreted phosphoprotein 1. CONCLUSIONS: We found much overlap in CSF protein changes between individuals with presymptomatic and symptomatic FAD and those with late-onset AD. Our results are consistent with inflammation and synaptic loss early in FAD and suggest new presymptomatic biomarkers of potential usefulness in drug development.

A comparison of methods of administering the opioid risk tool.

Risk assessment and stratification have become important aspects for the prescription of opioids to patients with chronic pain. Recent research has shown that the Opioid Risk Tool (ORT), a widely used risk assessment tool, has poor predictive abilities when compared with other risk assessment methods. This study compares two different methods of administration of the ORT to further study this issue. Patients at a pain practice were given an ORT to complete per the usual method of administration. In addition, a separate blinded ORT was completed by a psychologist after conducting a clinical interview with the patient. The results of the patient-completed ORT (PC-ORT) and the clinician-completed ORT (CC-ORT) were compared. There were significant differences found between the two, with the psychologist usually rating the patient higher in risk. The CC-ORT demonstrated better prediction of aberrant drug-taking behavior than the PC-ORT. The items that were discrepant between the two suggest that the differences were primarily due to comprehension issues. A strategy to help maximize the usefulness of the ORT derived from this finding and clinical experience is discussed.

Consistency of a two clinical site sample collection: a proteomics study.

PURPOSE: We investigated the ability to perform a clinical proteomic study using samples collected at different times from two independent clinical sites. EXPERIMENTAL DESIGN: Label-free 2-D-LC-MS proteomic analysis was used to differentially quantify tens of thousands of peptides from human plasma. We have asked whether samples collected from two sites, when analyzed by this type of peptide profiling, reproducibly contain detectable peptide markers that are differentially expressed in the plasma of disease (advanced renal cancer) patients relative to healthy normals. RESULTS: We have demonstrated that plasma proteins enriched in disease patients are indeed detected reproducibly in both clinical collections. Regression analysis, unsupervised hierarchical clustering and PCA detected no systematic bias in the data related to site of sample collection and processing. Using a genetic algorithm, support vector machine classification method, we were able to correctly classify disease samples at 88% sensitivity and 94% specificity using the second site as an independent validation set. CONCLUSIONS AND CLINICAL RELEVANCE: We conclude that multiple site collection, when analyzed by label-free 2-D-LC-MS, generates data that are sufficiently reproducible to guide reliable biomarker discovery.

Protein biomarker identification in the CSF of patients with CNS lymphoma.

PURPOSE: Elucidation of the CSF proteome may yield insights into the pathogenesis of CNS disease. We tested the hypothesis that individual CSF proteins distinguish CNS lymphoma from benign focal brain lesions. METHODS: We used a liquid chromatography/mass spectrometry-based method to differentially quantify and identify several hundred CSF proteins in CNS lymphoma and control patients. We used enzyme-linked immunosorbent assay (ELISA) to confirm one of these markers in an additional validation set of 101 cases. RESULTS: Approximately 80 CSF proteins were identified and found to be present at significantly different concentrations, both higher and lower, in training and test studies, which were highly concordant. To further validate these observations, we defined in detail the expression of one of these candidate biomarkers, antithrombin III (ATIII). ATIII RNA transcripts were identified within CNS lymphomas, and ATIII protein was localized selectively to tumor neovasculature. Determination of ATIII concentration by ELISA was significantly more accurate (> 75% sensitivity; > 98% specificity) than cytology in the identification of cancer. Measurement of CSF ATIII levels was found to potentially enhance the ability to diagnose and predict outcome. CONCLUSION: Our findings demonstrate for the first time that proteomic analysis of CSF yields individual biomarkers with greater sensitivity in the identification of cancer than does CSF cytology. We propose that the discovery of CSF protein biomarkers will facilitate early and noninvasive diagnosis in patients with lesions not amenable to brain biopsy, as well as provide improved surrogates of prognosis and treatment response in CNS lymphoma and brain metastasis.

Nonparametric mass calibration using hundreds of internal calibrants.

In situations where many molecular ions (>100) can be identified to the level of their elemental composition, such as in proteomics, metabolomics, and glycomics, a final mass calibration is possible for every sample without reliance on any analytical description of instrument behavior. This is achieved by applying a nonparametric calibration curve determined from the difference in observed, centroided m/z values of the known, internal calibrant molecular ions versus that calculated from their elemental compositions, over the m/z range. In examples here, proteomic data are examined for two sets of samples of complex mixtures composed of tryptic peptides from human and mouse blood proteins using high-resolution time-of-flight mass spectra from on-line liquid chromatography-mass spectrometry experiments. Resultant, postcalibration median absolute value mass errors and root-mean-square errors for peptides between 300 and 1100 m/z for many samples ranged from 3.1 to 4.4 and 5.2 to 6.9 ppm, respectively. The method may be applied to other types of mass spectrometers.

Genome sequencing and comparative analysis of Saccharomyces cerevisiae strain YJM789.

We sequenced the genome of Saccharomyces cerevisiae strain YJM789, which was derived from a yeast isolated from the lung of an AIDS patient with pneumonia. The strain is used for studies of fungal infections and quantitative genetics because of its extensive phenotypic differences to the laboratory reference strain, including growth at high temperature and deadly virulence in mouse models. Here we show that the approximately 12-Mb genome of YJM789 contains approximately 60,000 SNPs and approximately 6,000 indels with respect to the reference S288c genome, leading to protein polymorphisms with a few known cases of phenotypic changes. Several ORFs are found to be unique to YJM789, some of which might have been acquired through horizontal transfer. Localized regions of high polymorphism density are scattered over the genome, in some cases spanning multiple ORFs and in others concentrated within single genes. The sequence of YJM789 contains clues to pathogenicity and spurs the development of more powerful approaches to dissecting the genetic basis of complex hereditary traits.

A high-resolution map of transcription in the yeast genome.

There is abundant transcription from eukaryotic genomes unaccounted for by protein coding genes. A high-resolution genome-wide survey of transcription in a well annotated genome will help relate transcriptional complexity to function. By quantifying RNA expression on both strands of the complete genome of Saccharomyces cerevisiae using a high-density oligonucleotide tiling array, this study identifies the boundary, structure, and level of coding and noncoding transcripts. A total of 85% of the genome is expressed in rich media. Apart from expected transcripts, we found operon-like transcripts, transcripts from neighboring genes not separated by intergenic regions, and genes with complex transcriptional architecture where different parts of the same gene are expressed at different levels. We mapped the positions of 3' and 5' UTRs of coding genes and identified hundreds of RNA transcripts distinct from annotated genes. These nonannotated transcripts, on average, have lower sequence conservation and lower rates of deletion phenotype than protein coding genes. Many other transcripts overlap known genes in antisense orientation, and for these pairs global correlations were discovered: UTR lengths correlated with gene function, localization, and requirements for regulation; antisense transcripts overlapped 3' UTRs more than 5' UTRs; UTRs with overlapping antisense tended to be longer; and the presence of antisense associated with gene function. These findings may suggest a regulatory role of antisense transcription in S. cerevisiae. Moreover, the data show that even this well studied genome has transcriptional complexity far beyond current annotation.

Seasonal temperature fluctuations induces rapid inactivation of Cryptosporidium parvum.

This study measured the inactivation rate of bovine genotype A Cryptosporidium parvum oocysts attributable to diurnal oscillations of ambient temperature and solar radiation typical of California rangelands and dairies from spring through autumn. We first measured the relationship between air temperature and the internal temperature of bovine feces exposed to sunlight on commercial operations throughout California. Once maximum air temperature exceeded the mid 20 degrees C, diurnal thermal regimes of bovine fecal material exhibited peaks of over 40, 50, 60, and 70 degrees C. These diurnal thermal regimes were emulated using a thermocycler, with oocysts suspended in distilled water or fecal-water mix. Using oral inoculations of 10(5) C. parvum oocysts per neonatal Balb/c mouse (>1000-fold the ID50), no infections were observed using 1 to 5-day cycles of these thermal regimes. Loss of infectivity induced bythese thermal regimes was primarily due to partial or complete in vitro excystation during the first 24-h diurnal cycle and secondarily to thermal inactivation of the remaining intact or partial oocysts. These results suggest that as ambient conditions generate internal fecal temperatures > or = 40 degrees C via conduction, radiation, and convection, rapid environmental inactivation occurs at a rate of > or = 3.27 log reduction d(-1) for C. parvum oocysts deposited in the feces of cattle.

Elevated evolutionary rates in the laboratory strain of Saccharomyces cerevisiae.

By using the maximum likelihood method, we made a genome-wide comparison of the evolutionary rates in the lineages leading to the laboratory strain (S288c) and a wild strain (YJM789) of Saccharomyces cerevisiae and found that genes in the laboratory strain tend to evolve faster than in the wild strain. The pattern of elevated evolution suggests that relaxation of selection intensity is the dominant underlying reason, which is consistent with recurrent bottlenecks in the S. cerevisiae laboratory strain population. Supporting this conclusion are the following observations: (i) the increases in nonsynonymous evolutionary rate occur for genes in all functional categories; (ii) most of the synonymous evolutionary rate increases in S288c occur in genes with strong codon usage bias; (iii) genes under stronger negative selection have a larger increase in nonsynonymous evolutionary rate; and (iv) more genes with adaptive evolution were detected in the laboratory strain, but they do not account for the majority of the increased evolution. The present discoveries suggest that experimental and possible industrial manipulations of the laboratory strain of yeast could have had a strong effect on the genetic makeup of this model organism. Furthermore, they imply an evolution of laboratory model organisms away from their wild counterparts, questioning the relevancy of the models especially when extensive laboratory cultivation has occurred. In addition, these results shed light on the evolution of livestock and crop species that have been under human domestication for years.