Target-decoy false discovery rate estimation using Crema.

Assigning statistical confidence estimates to discoveries produced by a tandem mass spectrometry proteomics experiment is critical to enabling principled interpretation of the results and assessing the cost/benefit ratio of experimental follow-up. The most common technique for computing such estimates is to use target-decoy competition (TDC), in which observed spectra are searched against a database of real (target) peptides and a database of shuffled or reversed (decoy) peptides. TDC procedures for estimating the false discovery rate (FDR) at a given score threshold have been developed for application at the level of spectra, peptides, or proteins. Although these techniques are relatively straightforward to implement, it is common in the literature to skip over the implementation details or even to make mistakes in how the TDC procedures are applied in practice. Here we present Crema, an open-source Python tool that implements several TDC methods of spectrum-, peptide- and protein-level FDR estimation. Crema is compatible with a variety of existing database search tools and provides a straightforward way to obtain robust FDR estimates.

Introducing Molecular Hypernetworks for Discovery in Multidimensional Metabolomics Data.

Orthogonal separations of data from high-resolution mass spectrometry can provide insight into sample composition and address challenges of complete annotation of molecules in untargeted metabolomics. "Molecular networks" (MNs), as used in the Global Natural Products Social Molecular Networking platform, are a prominent strategy for exploring and visualizing molecular relationships and improving annotation. MNs are mathematical graphs showing the relationships between measured multidimensional data features. MNs also show promise for using network science algorithms to automatically identify targets for annotation candidates and to dereplicate features associated with a single molecular identity. This paper introduces "molecular hypernetworks" (MHNs) as more complex MN models able to natively represent multiway relationships among observations. Compared to MNs, MHNs can more parsimoniously represent the inherent complexity present among groups of observations, initially supporting improved exploratory data analysis and visualization. MHNs also promise to increase confidence in annotation propagation, for both human and analytical processing. We first illustrate MHNs with simple examples, and build them from liquid chromatography- and ion mobility spectrometry-separated MS data. We then describe a method to construct MHNs directly from existing MNs as their "clique reconstructions", demonstrating their utility by comparing examples of previously published graph-based MNs to their respective MHNs.

MS1Connect: a mass spectrometry run similarity measure.

MOTIVATION: Interpretation of newly acquired mass spectrometry data can be improved by identifying, from an online repository, previous mass spectrometry runs that resemble the new data. However, this retrieval task requires computing the similarity between an arbitrary pair of mass spectrometry runs. This is particularly challenging for runs acquired using different experimental protocols. RESULTS: We propose a method, MS1Connect, that calculates the similarity between a pair of runs by examining only the intact peptide (MS1) scans, and we show evidence that the MS1Connect score is accurate. Specifically, we show that MS1Connect outperforms several baseline methods on the task of predicting the species from which a given proteomics sample originated. In addition, we show that MS1Connect scores are highly correlated with similarities computed from fragment (MS2) scans, even though these data are not used by MS1Connect. AVAILABILITY AND IMPLEMENTATION: The MS1Connect software is available at https://github.com/bmx8177/MS1Connect. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The Crux Toolkit for Analysis of Bottom-Up Tandem Mass Spectrometry Proteomics Data.

The Crux tandem mass spectrometry data analysis toolkit provides a collection of algorithms for analyzing bottom-up proteomics tandem mass spectrometry data. Many publications have described various individual components of Crux, but a comprehensive summary has not been published since 2014. The goal of this work is to summarize the functionality of Crux, focusing on developments since 2014. We begin with empirical results demonstrating our recently implemented speedups to the Tide search engine. Other new features include a new score function in Tide, two new confidence estimation procedures, as well as three new tools: Param-medic for estimating search parameters directly from mass spectrometry data, Kojak for searching cross-linked mass spectra, and DIAmeter for searching data independent acquisition data against a sequence database.

Pooled analysis of radiation hybrids identifies loci for growth and drug action in mammalian cells.

Genetic screens in mammalian cells commonly focus on loss-of-function approaches. To evaluate the phenotypic consequences of extra gene copies, we used bulk segregant analysis (BSA) of radiation hybrid (RH) cells. We constructed six pools of RH cells, each consisting of ∼2500 independent clones, and placed the pools under selection in media with or without paclitaxel. Low pass sequencing identified 859 growth loci, 38 paclitaxel loci, 62 interaction loci, and three loci for mitochondrial abundance at genome-wide significance. Resolution was measured as ∼30 kb, close to single-gene. Divergent properties were displayed by the RH-BSA growth genes compared to those from loss-of-function screens, refuting the balance hypothesis. In addition, enhanced retention of human centromeres in the RH pools suggests a new approach to functional dissection of these chromosomal elements. Pooled analysis of RH cells showed high power and resolution and should be a useful addition to the mammalian genetic toolkit.

The evolution of mechanism of accommodation and a novel hypothesis.

Myopia and presbyopia are two major optometry problems facing the whole society. The mechanism of accommodation is strongly related to the treatments of myopia and presbyopia. However, the key mechanism of accommodation has puzzled us for over 400 years and is still not clear at present, leading to the stagnation of prevention and treatment of myopia and presbyopia. With the continued development of experimental technologies and equipment, the approaches to elucidate accommodation's intricacies have become more methodological and sophisticated. Fortunately, some significant progress has been made. This article is to review the evolution of the mechanism of accommodation. Helmholtz proposed a classical theory of "zonules relax during accommodation." In contrast, Schachar put forward a theory of "zonules taut during accommodation." Those hypotheses are relatively complete, but either do not fully explain everything about the accommodation mechanism or lack sufficient experimental and clinical evidence to support them. Then, some contentious issues are discussed in detail to find the truth. Finally, we proposed our hypothesis about accommodation based on the anatomy of the accommodative apparatus.

A Continuously Worn Dual Temperature Sensor System for Accurate Monitoring of Core Body Temperature from the Ear Canal.

The objective of this work was to develop a temperature sensor system that accurately measures core body temperature from an ear-worn device. Two digital temperature sensors were embedded in a hearing aid shell along the thermal gradient of the ear canal to form a linear heat balance relationship. This relationship was used to determine best fit parameters for estimating body temperature. The predicted body temperatures resulted in intersubject limits of agreement (LOA) of ±0.49 °C over a range of physiologic and ambient temperatures without calibration. The newly developed hearing aid-based temperature sensor system can estimate core body temperature at an accuracy level equal to or better than many devices currently on the market. An accurate, continuously worn, temperature monitoring and tracking device may help provide early detection of illnesses, which could prove especially beneficial during pandemics and in the elderly demographic of hearing aid wearers.

Improving Peptide-Level Mass Spectrometry Analysis via Double Competition.

The analysis of shotgun proteomics data often involves generating lists of inferred peptide-spectrum matches (PSMs) and/or of peptides. The canonical approach for generating these discovery lists is by controlling the false discovery rate (FDR), most commonly through target-decoy competition (TDC). At the PSM level, TDC is implemented by competing each spectrum's best-scoring target (real) peptide match with its best match against a decoy database. This PSM-level procedure can be adapted to the peptide level by selecting the top-scoring PSM per peptide prior to FDR estimation. Here, we first highlight and empirically augment a little known previous work by He et al., which showed that TDC-based PSM-level FDR estimates can be liberally biased. We thus propose that researchers instead focus on peptide-level analysis. We then investigate three ways to carry out peptide-level TDC and show that the most common method ("PSM-only") offers the lowest statistical power in practice. An alternative approach that carries out a double competition, first at the PSM and then at the peptide level ("PSM-and-peptide"), is the most powerful method, yielding an average increase of 17% more discovered peptides at 1% FDR threshold relative to the PSM-only method.

Sexual Identity and Birth Cohort Differences in Social Support and Its Link with Well-Being among Sexual Minority Individuals.

This study examined sexual identity and birth cohort differences in social support and its association with well-being, using a longitudinal national probability sample of 706 cisgender and non-binary sexual minority individuals from the USA. The data allowed for extensive descriptions of perceived social support and support networks across subgroups. Findings demonstrated that sexual identity and birth cohort differences in overall sizes of support networks and levels of perceived social support were small. Furthermore, fixed effects analyses indicated that changes in the size of respondents' social support networks were not related to well-being, with a one-person change being associated with a .04 SD change in well-being or less, depending on the indicator of well-being being tested. Moreover, changes in perceived social support were only limitedly related to changes in respondents' well-being, a 1-point change in the scale of perceived social support being associated with a .11 SD change in life-satisfaction. Associations were smaller for overall well-being or psychological distress, the other two indicators of well-being used. Together, these findings could imply that cross-sectional research has overestimated the relevance of social support for the well-being of sexual minority individuals, but also that general social support is insufficiently tailored to the support needs of the sexual minority population.

Social Change and the Health of Sexual Minority Individuals: Do the Effects of Minority Stress and Community Connectedness Vary by Age Cohort?

This study examined the extent to which social stress stemming from a stigmatized social status (i.e., minority stress) was associated with three domains of health in younger as compared with older age cohorts of sexual minority individuals. Data were analyzed from the Generations Study, a longitudinal study using a probability sample (N = 1518) of age cohorts of sexual minority individuals in the USA. Exposure to a variety of minority stressors was associated with poorer health for all age cohorts. We hypothesized that because of improved social and legal environments in recent years, the associations between minority stress and health would be diminished in the younger cohort. As expected, we found that the associations between some minority stressors and health outcomes were diminished in the younger cohort compared to older cohorts. Positive associations between community connectedness and mental health and social well-being were observed for all participants but were attenuated in the younger cohort. Findings demonstrate the continuing negative association between minority stress and health among sexual minorities, which, despite some attenuation, persists even for young cohorts of sexual minority individuals in a more equal and accepting social climate.

Accurately Assigning Peptides to Spectra When Only a Subset of Peptides Are Relevant.

The standard proteomics database search strategy involves searching spectra against a peptide database and estimating the false discovery rate (FDR) of the resulting set of peptide-spectrum matches. One assumption of this protocol is that all the peptides in the database are relevant to the hypothesis being investigated. However, in settings where researchers are interested in a subset of peptides, alternative search and FDR control strategies are needed. Recently, two methods were proposed to address this problem: subset-search and all-sub. We show that both methods fail to control the FDR. For subset-search, this failure is due to the presence of "neighbor" peptides, which are defined as irrelevant peptides with a similar precursor mass and fragmentation spectrum as a relevant peptide. Not considering neighbors compromises the FDR estimate because a spectrum generated by an irrelevant peptide can incorrectly match well to a relevant peptide. Therefore, we have developed a new method, "subset-neighbor search" (SNS), that accounts for neighbor peptides. We show evidence that SNS controls the FDR when neighbors are present and that SNS outperforms group-FDR, the only other method that appears to control the FDR relative to a subset of relevant peptides.

Full Sequencing of CRISPR/Cas9 Single Guide RNA (sgRNA) via Parallel Ribonuclease Digestions and Hydrophilic Interaction Liquid Chromatography-High-Resolution Mass Spectrometry Analysis.

CRISPR/Cas9 is a powerful genome editing approach in which a Cas9 enzyme and a single guide RNA (sgRNA) form a ribonucleoprotein complex effectively targeting site-specific cleavages of DNA. Accurate sequencing of sgRNA is critical to patient safety and is the expectation by regulatory agencies. In this paper, we present the full sequencing of sgRNA via parallel ribonuclease (RNase) T1, A, and U2 digestions and the simultaneous separation and identification of the digestion products by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution mass spectrometry (HRMS). When using RNase T1 digestion alone, a maximal sequence coverage of 81% was obtained excluding the nonunique fragments. Full sgRNA sequencing was achieved using unique fragments generated by RNase T1, A, and U2 parallel digestions. Thorough optimization of sgRNA digestions was performed by varying the nuclease-to-sgRNA ratio, buffer conditions, and reaction times. A biocompatible ethylene-bridged hybrid amide column was evaluated for the separation of RNase digestion products. To our knowledge, it is the first time that (i) RNA digests are separated and identified by HILIC-HRMS and (ii) chemically modified sgRNAs are directly sequenced via a bottom-up approach.

2018 YPIC Challenge: A Case Study in Characterizing an Unknown Protein Sample.

For the 2018 YPIC Challenge, contestants were invited to try to decipher two unknown English questions encoded by a synthetic protein expressed in Escherichia coli. In addition to deciphering the sentence, contestants were asked to determine the three-dimensional structure and detect any post-translation modifications left by the host organism. We present our experimental and computational strategy to characterize this sample by identifying the unknown protein sequence and detecting the presence of post-translational modifications. The sample was acquired with dynamic exclusion disabled to increase the signal-to-noise ratio of the measured molecules, after which spectral clustering was used to generate high-quality consensus spectra. De novo spectrum identification was used to determine the synthetic protein sequence, and any post-translational modifications introduced by E. coli on the synthetic protein were analyzed via spectral networking. This workflow resulted in a de novo sequence coverage of 70%, on par with sequence database searching performance. Additionally, the spectral networking analysis indicated that no systematic modifications were introduced on the synthetic protein by E. coli. The strategy presented here can be directly used to analyze samples for which no protein sequence information is available or when the identity of the sample is unknown. All software and code to perform the bioinformatics analysis is available as open source, and self-contained Jupyter notebooks are provided to fully recreate the analysis.

Survey of Nonalcoholic Fatty Liver Disease Knowledge, Nutrition, and Physical Activity Patterns Among the General Public in Beijing, China.

BACKGROUND: Despite high prevalence of nonalcoholic fatty liver disease in China, understanding of the disease appears to be low. AIMS: We assessed the knowledge of NAFLD among the public in Beijing, China, as well as diet and physical activity patterns, which may provide information useful for NAFLD prevention and management. METHODS: We surveyed adult patients and family members in the Peking University Health Science Center (PUHSC) ultrasound clinic and office staff in Beijing, China. Participants provided demographic and medical history data. NAFLD-related knowledge and diet and physical activity were assessed. RESULTS: A total of 1296 participants at the PUHSC clinic (51% female, median age 35, 61% college-educated) and 494 participants in offices (61% female, median age 43, 74% college-educated) completed the survey. Response rate was 68.4% and 96.7%, respectively. In clinic and offices, 44% versus 48% were overweight/obese, 5% had a history of diabetes in both groups, and 14% versus 23% had a personal history of NAFLD. Median knowledge score was 15 out of 25 in clinic versus 16 in offices. 44.9% reported minimal physical activity. Factors associated with higher NAFLD knowledge scores (> 16) on multivariate analysis included college education or higher (OR 1.7, p = 0.01), family history of hyperlipidemia (OR 1.96, p < 0.001), and number of sugary drinks per week (OR 0.74, p = 0.006). No factors were significantly associated with physical activity levels. CONCLUSIONS: Adults in Beijing had low knowledge about NAFLD, and most were not physically active. Programs to increase public awareness of NAFLD and promote physical activity are critical to curb this growing epidemic.

Combining High-Resolution and Exact Calibration To Boost Statistical Power: A Well-Calibrated Score Function for High-Resolution MS2 Data.

To achieve accurate assignment of peptide sequences to observed fragmentation spectra, a shotgun proteomics database search tool must make good use of the very high-resolution information produced by state-of-the-art mass spectrometers. However, making use of this information while also ensuring that the search engine's scores are well calibrated, that is, that the score assigned to one spectrum can be meaningfully compared to the score assigned to a different spectrum, has proven to be challenging. Here we describe a database search score function, the "residue evidence" (res-ev) score, that achieves both of these goals simultaneously. We also demonstrate how to combine calibrated res-ev scores with calibrated XCorr scores to produce a "combined p value" score function. We provide a benchmark consisting of four mass spectrometry data sets, which we use to compare the combined p value to the score functions used by several existing search engines. Our results suggest that the combined p value achieves state-of-the-art performance, generally outperforming MS Amanda and Morpheus and performing comparably to MS-GF+. The res-ev and combined p-value score functions are freely available as part of the Tide search engine in the Crux mass spectrometry toolkit ( http://crux.ms ).

Temporal Changes in the Modes of Hepatitis C Virus Transmission in the USA and Northern China.

BACKGROUND: Predominant modes of hepatitis C virus (HCV) transmission vary between countries and over time. AIMS: To compare HCV transmission modes in the USA and northern China. METHODS: We conducted a prospective study enrolling two cohorts of chronic HCV patients in the USA, and in China at Beijing, and at Gu'an and Kuancheng counties in Hebei. Patients self-reported the most likely source and year of infection. RESULTS: A total of 1957 patients were studied (1000 USA; 957 China-428 Beijing, 387 Gu'an, 142 Kuancheng). The predominant infection sources were transfusion (23.0%) and injection drug use (IDU) (32.1%) in the USA and transfusion (64.5%) in northern China. Within China, transfusion was the most common source in Beijing (62.1%) and Gu'an (88.1%), and medical procedures (35.9%) and IDU (12.0%) in Kuancheng. Infection via transfusion decreased significantly in the USA (35.1-4.6%) and Beijing (84.2-14.3%) but remained frequent in Gu'an (90.5-72.5%) over time. Infection via IDU decreased from 32.4% in those ≥61 years to 25.0% in those 41-50 years but increased to 46.5% in those ≤40 years in US patients and decreased over time from 38.7 to 1.9% in Kuancheng. Infection via medical procedures increased over time in Beijing (7.0-33.3%) and remained frequent in Kuancheng (45.2-31.1%). CONCLUSIONS: There are major differences in presumed HCV infection source between the USA and northern China. Favorable as well as worrisome changes in the modes of HCV transmission in both countries were observed.

Risk Assessment of Hepatocellular Carcinoma in Patients with Hepatitis C in China and the USA.

BACKGROUND: Hepatitis C (HCV) infection is an increasingly common cause of hepatocellular carcinoma (HCC) in China. AIMS: We aimed to determine differences in demographic and behavioral profiles associated with HCC in HCV+ patients in China and the USA. METHODS: Consecutive HCV+ patients were recruited from centers in China and the USA. Clinical data and lifestyle profiles were obtained through standardized questionnaires. Multivariable analysis was conducted to determine factors associated with HCC diagnosis within groups. RESULTS: We included 41 HCC patients from China and 71 from the USA, and 931 non-HCC patients in China and 859 in China. Chinese patients with HCC were significantly younger, less likely to be male and to be obese than US patients with HCC (all p < 0.001). Chinese patients with HCC had a significantly lower rate of cirrhosis diagnosis (36.6 vs. 78.9%, p < 0.001); however, they also had a higher rate of hepatitis B core antibody positivity (63.4 vs. 36.8%, p = 0.007). In a multivariable analysis of the entire Chinese cohort, age > 55, male sex, the presence of diabetes, and time from maximum weight were associated with HCC, while tea consumption was associated with a decreased HCC risk (OR 0.37, 95% CI 0.16-0.88). In the US cohort, age > 55, male sex, and cirrhosis were associated with HCC on multivariable analysis. CONCLUSIONS: With the aging Chinese population and increasing rates of diabetes, there will likely be continued increase in the incidence of HCV-related HCC in China. The protective effect of tea consumption on HCC development deserves further validation.

Survey of hepatitis B knowledge and stigma among chronically infected patients and uninfected persons in Beijing, China.

BACKGROUND & AIMS: Hepatitis B virus (HBV) infection carries substantial stigma in China. We surveyed HBV knowledge and stigma among chronic hepatitis B (CHB) patients and persons without HBV infection in Beijing, China. METHODS: Four hundred and thirty five CHB patients and 801 controls at Peking University People's Hospital were surveyed. RESULTS: Chronic hepatitis B patients were older (mean 46 vs. 39 years) and more often men (71 vs. 48%) than controls. Mean knowledge score was 11.9/15 for CHB and 9.3/15 for control patients (P < 0.001). Average stigma score was 22.1/39 for CHB and 19.2/30 for control patients. Controls expressed discomfort with close contact (45%) or sharing meals with CHB patients (39%) and believed CHB patients should not be allowed to work in restaurants (58%) or childcare (44%). Chronic hepatitis B patients felt that they were undesirable as spouses (33 vs. 17%) and brought trouble to their families (58 vs. 34%) more often than controls. Despite legal prohibitions, 40% of CHB patients were required to undergo pre-employment HBV testing, and 29% of these individuals thought that they lost job opportunities because of their disease status. 16% of CHB patients regretted disclosing their HBV status and disclosure was inversely associated with stigma. Higher stigma was associated with older age, lower education and lower knowledge score among controls; and with lower education, younger age, having undergone pre-employment HBV testing and regret disclosing their HBV status among CHB patients. CONCLUSION: Despite high prevalence of CHB in China, our study shows knowledge is limited and there is significant societal and internalized stigma associated with HBV infection.

A Comparative Study of Liver Disease Care in the USA and Urban and Rural China.

BACKGROUND: Chronic liver disease is a major cause of morbidity and mortality in the USA and China, but the etiology of liver disease, medical practice, and patient expectations in these two countries are different. AIMS: To compare patient knowledge about their liver disease, patient satisfaction with liver disease care, and patient medical decision-making preference in the USA and China. METHODS: Three cohorts of established adult patients with liver disease seen in liver clinics in Ann Arbor, USA, and Beijing (urban) and Hebei (rural), China, completed a survey between May and September 2014. RESULTS: A total of 990 patients (395 USA, 398 Beijing, and 197 Hebei) were analyzed. Mean liver disease knowledge score (maximum 5) in the USA, Beijing, and Hebei patients was 4.1, 3.6, and 3.0, respectively (p < 0.001). US patients had a greater preference for collaborative decision-making: 71.8 % preferred to make treatment decisions together with the doctor, while most Chinese patients (74.6 % Beijing and 84.8 % Hebei) preferred passive decision-making. Mean satisfaction score (maximum 85) in the USA was higher than in Beijing, which in turn was higher than in Hebei (78.2 vs. 66.5 vs. 60.3, p < 0.001). There was a positive correlation between liver disease knowledge score and satisfaction score (r = 0.27, p < 0.001) and with collaborative medical decision-making (r = 0.22, p < 0.001) when responses from all sites were combined. CONCLUSIONS: Liver disease knowledge and patient satisfaction were greatest in the USA, followed by Beijing and then Hebei patients. Understanding these differences and associated factors may help to improve patient outcomes.

Structure-based druggability assessment of the mammalian structural proteome with inclusion of light protein flexibility.

Advances reported over the last few years and the increasing availability of protein crystal structure data have greatly improved structure-based druggability approaches. However, in practice, nearly all druggability estimation methods are applied to protein crystal structures as rigid proteins, with protein flexibility often not directly addressed. The inclusion of protein flexibility is important in correctly identifying the druggability of pockets that would be missed by methods based solely on the rigid crystal structure. These include cryptic pockets and flexible pockets often found at protein-protein interaction interfaces. Here, we apply an approach that uses protein modeling in concert with druggability estimation to account for light protein backbone movement and protein side-chain flexibility in protein binding sites. We assess the advantages and limitations of this approach on widely-used protein druggability sets. Applying the approach to all mammalian protein crystal structures in the PDB results in identification of 69 proteins with potential druggable cryptic pockets.