Data Harmonization for a Molecularly Driven Health System.

Data commons have emerged as the best current method for enabling data aggregation across multiple projects and multiple data sources. Good data harmonization techniques are critical to maintain quality of data within a data commons, as well as to allow future meta-analysis across different data commons. We present some of the current best practices for data harmonization.

Cell-type-specific 3D epigenomes in the developing human cortex.

Lineage-specific epigenomic changes during human corticogenesis have been difficult to study owing to challenges with sample availability and tissue heterogeneity. For example, previous studies using single-cell RNA sequencing identified at least 9 major cell types and up to 26 distinct subtypes in the dorsal cortex alone1,2. Here we characterize cell-type-specific cis-regulatory chromatin interactions, open chromatin peaks, and transcriptomes for radial glia, intermediate progenitor cells, excitatory neurons, and interneurons isolated from mid-gestational samples of the human cortex. We show that chromatin interactions underlie several aspects of gene regulation, with transposable elements and disease-associated variants enriched at distal interacting regions in a cell-type-specific manner. In addition, promoters with increased levels of chromatin interactivity-termed super-interactive promoters-are enriched for lineage-specific genes, suggesting that interactions at these loci contribute to the fine-tuning of transcription. Finally, we develop CRISPRview, a technique that integrates immunostaining, CRISPR interference, RNAscope, and image analysis to validate cell-type-specific cis-regulatory elements in heterogeneous populations of primary cells. Our findings provide insights into cell-type-specific gene expression patterns in the developing human cortex and advance our understanding of gene regulation and lineage specification during this crucial developmental window.

An Inflection Point in Cancer Protein Biomarkers: What was and What's Next.

Biomarkers remain the highest value proposition in cancer medicine today-especially protein biomarkers. Despite decades of evolving regulatory frameworks to facilitate the review of emerging technologies, biomarkers have been mostly about promise with very little to show for improvements in human health. Cancer is an emergent property of a complex system, and deconvoluting the integrative and dynamic nature of the overall system through biomarkers is a daunting proposition. The last 2 decades have seen an explosion of multiomics profiling and a range of advanced technologies for precision medicine, including the emergence of liquid biopsy, exciting advances in single-cell analysis, artificial intelligence (machine and deep learning) for data analysis, and many other advanced technologies that promise to transform biomarker discovery. Combining multiple omics modalities to acquire a more comprehensive landscape of the disease state, we are increasingly developing biomarkers to support therapy selection and patient monitoring. Furthering precision medicine, especially in oncology, necessitates moving away from the lens of reductionist thinking toward viewing and understanding that complex diseases are, in fact, complex adaptive systems. As such, we believe it is necessary to redefine biomarkers as representations of biological system states at different hierarchical levels of biological order. This definition could include traditional molecular, histologic, radiographic, or physiological characteristics, as well as emerging classes of digital markers and complex algorithms. To succeed in the future, we must move past purely observational individual studies and instead start building a mechanistic framework to enable integrative analysis of new studies within the context of prior studies. Identifying information in complex systems and applying theoretical constructs, such as information theory, to study cancer as a disease of dysregulated communication could prove to be "game changing" for the clinical outcome of cancer patients.

A field guide to cultivating computational biology.

Evolving in sync with the computation revolution over the past 30 years, computational biology has emerged as a mature scientific field. While the field has made major contributions toward improving scientific knowledge and human health, individual computational biology practitioners at various institutions often languish in career development. As optimistic biologists passionate about the future of our field, we propose solutions for both eager and reluctant individual scientists, institutions, publishers, funding agencies, and educators to fully embrace computational biology. We believe that in order to pave the way for the next generation of discoveries, we need to improve recognition for computational biologists and better align pathways of career success with pathways of scientific progress. With 10 outlined steps, we call on all adjacent fields to move away from the traditional individual, single-discipline investigator research model and embrace multidisciplinary, data-driven, team science.

Gender Differences in Authorship and Quality of Anesthesia Clinical Practice Guidelines From 2016 to 2020 Using the Appraisal of Guidelines for Research and Evaluation II Instrument.

INTRODUCTION: Women continue to be underrepresented in academic anesthesiology. This study assessed guidelines in anesthesia journals over the past 5 years, evaluating differences in woman-led versus man-led guidelines in terms of author gender, quality, and changes over time. We hypothesized that anesthesia guidelines would be predominately man-led, and that there would be differences in quality between woman-led versus man-led guidelines. METHODS: All clinical practice guidelines published in the top 10 anesthesia journals were identified as per Clarivate Analytics Impact Factor between 2016 and 2020. Fifty-one guidelines were included for author, gender, and quality analysis using the Appraisal of Guidelines for Research & Evaluation (AGREE) II instrument. Each guideline was assessed across 6 domains and 23 items and given an overall score, overall quality score, and overall rating/recommendation. Stratified and trend analyses were performed for woman-led versus man-led guidelines. RESULTS: Fifty out of 51 guidelines were included: 1 was excluded due to unidentifiable first-author gender. In total, 255 of 1052 (24%) authors were women, and woman-led guidelines (woman-first author) represented 12 of 50 (24%) overall guidelines. Eighteen percent (9 of 50) of guidelines had all-male authors, and a majority (26 of 50, 52%) had less than one-third of female authors. The overall number and percentage of woman-led guidelines did not change over time. There was a significantly higher percentage of female authors in woman-led versus man-led guidelines, median 39% vs 20% (P = .012), as well as a significantly higher number of female coauthors in guidelines that were woman-led median 3.5 vs 1.0, P = .049. For quality, there was no significant difference in the overall rating or objective quality of woman- versus man-led guidelines. However, there was a significant increase in the overall rating of all the guidelines over time (P = .010), driven by the increase in overall rating among man-led guidelines, P = .002. The overall score of guidelines did not increase over time; however, they increased in man-led but not woman-led guidelines. There was no significant correlation between the percentage of female authors per guideline and either overall score or overall rating. CONCLUSIONS: There is a substantial disparity in the number of women leading and contributing to guidelines which has not improved over time. Woman-led guidelines included more women and a higher percentage of women. There was no difference in quality of guidelines by first-author gender or percentage of female authors. Further systematic and quota-driven sponsorship is needed to promote gender equity, diversity, and inclusion in anesthesia guidelines.

Paradoxical androgen receptor regulation by small molecule enantiomers.

Small molecules that target the androgen receptor (AR) are the mainstay of therapy for lethal castration-resistant prostate cancer (CRPC), yet existing drugs lose their efficacy during continued treatment. This evolution of resistance is due to heterogenous mechanisms which include AR mutations causing the identical drug to activate instead of inhibit the receptor. Understanding in molecular detail the paradoxical phenomenon wherein an AR antagonist is transformed into an agonist by structural mutations in the target receptor is thus of paramount importance. Herein, we describe a reciprocal paradox: opposing antagonist and agonist AR regulation determined uniquely by enantiomeric forms of the same drug structure. The antiandrogen BMS-641988, which has (R)-chirality at C-5 encompasses a previously uncharacterized (S)-stereoisomer that is, surprisingly, a potent agonist of AR, as demonstrated by transcriptional assays supported by cell imaging studies. This duality was reproduced in a series of novel compounds derived from the BMS-641988 scaffold. Coupled with in silico modeling studies, the results inform an AR model that explains the switch from potent antagonist to high-affinity agonist in terms of C-5 substituent steric interactions with helix 12 of the ligand binding site. They imply strategies to overcome AR drug resistance and demonstrate that insufficient enantiopurity in this class of AR antagonist can confound efforts to correlate structure with function.

Zebrafish model of RERE syndrome recapitulates key ophthalmic defects that are rescued by small molecule inhibitor of shh signaling.

BACKGROUND: RERE is a highly conserved transcriptional co-regulator that is associated with a human neurodevelopmental disorder with or without anomalies of the brain, eye, or heart (NEDBEH, OMIM: 616975). RESULTS: We show that the zebrafish rerea mutant (babyface) robustly recapitulates optic fissure closure defects resulting from loss of RERE function, as observed in humans. These defects result from expansion of proximal retinal optic stalk (OS) and reduced expression of some of the ventral retinal fate genes due to deregulated protein signaling. Using zebrafish and cell-based assays, we determined that NEDBEH-associated human RERE variants function as hypomorphs in their ability to repress shh signaling and some exhibit abnormal nuclear localization. Inhibiting shh signaling by the protein inhibitor HPI-1 rescues coloboma, confirming our observation that coloboma in rerea mutants is indeed due to deregulation of shh signaling. CONCLUSIONS: Zebrafish rerea mutants exhibit OS and optic fissure closure defects. The optic fissure closure defect was rescued by an shh signaling inhibitor, suggesting that this defect could arise due to deregulated shh signaling.

The theory of planned behavior and dietary behaviors in competitive women bodybuilders.

BACKGROUND: Women bodybuilders build their ideal physique by manipulating their diet, supplement, and exercise regimens to extreme levels. Excess protein intake and dietary supplement use is ubiquitous in women bodybuilders preparing for a competition, i.e., in-season competitors, however the impetus for these two dietary behaviors are relatively unknown. The Theory of Planned Behavior (TPB) has been used to explain dietary behaviors. The purpose of the study was to examine how the TPB can explain protein intake and dietary supplement use in in-season competitors. METHODS: Using a cross-sectional design, an online questionnaire was developed, validated, and administered to collect dietary supplement use, TPB variables, and other measures from 112 in-season competitors. Protein intake was assessed using multiple 24-h dietary recalls. Associations between TPB and protein intake and dietary supplement use were determined with multiple regression analysis while adjusting for confounders. RESULTS: For protein intake: attitude, subjective norm, and perceived behavioral control explained 8% of the variance in intention; subjective norm independently predicted intention. Behavioral beliefs predicted attitude; subjective norm was predicted by trainer/coach, workout partners, and social media influencers. For dietary supplement use: intention explained 5% of the variance in dietary supplement use; attitude, subjective norm, and perceived behavioral control together explained 38% of the variance in intention. Attitudes towards dietary supplements use were predicted by five factors (not a waste of money, help improve physique, sustain energy levels, provide enough calories, help with recovery). Primary determinants of subjective norm were fellow competitors, social media influencers, and trainer/coach. Perceived behavioral control was predicted by three factors (ease of purchase, affordability to purchase, availability to purchase). CONCLUSIONS: TPB predicted dietary supplement use in women bodybuilders during in-season but there was little evidence for the prediction of protein intake using the TPB. Health professionals should develop effective interventions using strategies that align health education messages with in-season competitors' outcome beliefs and collaborate with their referent others to influence safer and effective dietary supplement use.

Racial/Ethnic Disparities Impact the Real-World Effectiveness of a Multicomponent Maternal Smoking Cessation Program: Findings from the CTTP Cohort.

INTRODUCTION: Smoking during pregnancy adversely affects perinatal outcomes for both women and infants. We conducted a retrospective cohort study of the state-funded Comprehensive Tobacco Treatment Program (CTTP) - the largest maternal tobacco cessation program in San Bernardino County, California - to determine the real-world program effectiveness and to identify variables that can potentially improve effectiveness. METHODS: During 2012-2019, women who smoked during pregnancy were enrolled in CTTP's multicomponent behavioral smoking cessation program that implemented components of known efficacy (i.e., incentives, biomarker testing, feedback, and motivational interviewing). RESULTS: We found that 40.1% achieved prolonged abstinence by achieving weekly, cotinine-verified, 7-day abstinence during 6 to 8 weeks of enrollment. Using intention-to-treat analyses, we computed that the self-reported point prevalence abstinence rate (PPA) at the six-month telephone follow-up was 36.7%. Cohort members achieving prolonged abstinence during the CTTP were five times more likely to achieve PPA six months after CTTP. Several non-Hispanic ethnicities (Black, Native American, White, or More than one ethnicity) in the cohort were two-fold less likely (relative to Hispanics) to achieve prolonged abstinence during CTTP or PPA at six months after CTTP. This disparity was further investigated in mediation analysis. Variables such as quitting during the first trimester and smoking fewer cigarettes at enrollment were also associated with achieving PPA at six months. DISCUSSION: Racial/ethnic health disparities that have long been linked to a higher rate of maternal smoking persist even when the pregnant smoker enrolls in a smoking cessation program.

Social-Ecological Correlates of Loneliness Among Young Adult U.S. Males.

PURPOSE: Social disconnection, such as loneliness, is recognized as a significant public health concern in the United States, and young adult males may carry the greater burden of this issue when compared with their female peers. Little is known about the correlates of loneliness for this population. This study examines the social-ecological correlates of loneliness in young adult males. METHODS: Males, aged 18 to 25 years, in the United States were recruited to take part in a cross-sectional electronic survey. Loneliness was assessed as a composite measure. The social-ecological correlates consisted of intrapersonal-level (e.g., social-demographic characteristics), interpersonal-level (e.g., adverse childhood experiences), community-level (e.g., life expectancy at the county level), and societal-level (e.g., idealized masculine gender) variables. A four-block hierarchical regression was performed with each block representing the respective social-ecological level. RESULTS: Among the study sample (n = 495), the intra- and interpersonal variables significantly shared 10% and an incremental 3%, respectively, of the explained variance in loneliness. Mental health diagnosis (ß = 1.06, 95% confidence interval [CI]: [0.54, 1.59]), childhood physical and emotional abuse (ß = 0.21, 95% CI: [0.02, 0.39]), and childhood sexual abuse (ß = 0.30, 95% CI: [0.01, 0.60]) were significantly associated with greater loneliness. CONCLUSION: The findings highlight that the micro-level (intra- and interpersonal) correlates may be most important in predicting loneliness in young adult males. Specifically, young males with a mental health diagnosis and those with greater experiences of childhood adversity are at potentially greater risk for loneliness. Implications for research, programming, and policy are highlighted.

Quality of recent clinical practice guidelines in anaesthesia publications using the Appraisal of Guidelines for Research and Evaluation II instrument.

Clinical practice guidelines are a valuable resource aiding medical decision-making based on scientific evidence. In anaesthesia, guidelines are increasing in both number and scope, influencing individual practice and shaping local departmental policy. The aim of this review is to assess the quality of clinical practice guidelines published in high impact anaesthesia journals over the past 5 yr using the internationally validated Appraisal of Guidelines for Research and Evaluation (AGREE) II instrument. A literature search was conducted in Scopus to identify all guidelines published in the top 10 anaesthesia journals as per Clarivate Analytics Impact Factor from 2016 and 2020. Fifty-one guidelines were included for analysis by five independent appraisers using AGREE II. Each guideline was assessed across six domains and 23 items. Individual domain scores were calculated with a threshold agreed via consensus to represent high-quality guidelines. There was a significant increase in overall score over time (P=0.041), driven by Domain 3 (Rigour of Development, P=0.046). The raw overall score for Domain 3, however, was low. The other domains performed as expected based on previous studies, with Domains 1, 4, and 6 achieving high scores and Domains 2 and 5 incurring poor ratings. Most guidelines studied involved international collaboration but emerged from a single professional society. Use of an appraisal tool was stated as high but poorly detailed. The improvement in the overall score of guidelines and rigour of development is promising; however, only seven guidelines met high-quality criteria, suggesting room for improvement for the overall integrity of guidelines in anaesthesia.

A comparison of methods to assess cell mechanical properties.

The mechanical properties of cells influence their cellular and subcellular functions, including cell adhesion, migration, polarization, and differentiation, as well as organelle organization and trafficking inside the cytoplasm. Yet reported values of cell stiffness and viscosity vary substantially, which suggests differences in how the results of different methods are obtained or analyzed by different groups. To address this issue and illustrate the complementarity of certain approaches, here we present, analyze, and critically compare measurements obtained by means of some of the most widely used methods for cell mechanics: atomic force microscopy, magnetic twisting cytometry, particle-tracking microrheology, parallel-plate rheometry, cell monolayer rheology, and optical stretching. These measurements highlight how elastic and viscous moduli of MCF-7 breast cancer cells can vary 1,000-fold and 100-fold, respectively. We discuss the sources of these variations, including the level of applied mechanical stress, the rate of deformation, the geometry of the probe, the location probed in the cell, and the extracellular microenvironment.

Potential association between public medical insurance, waitlist mortality, and utilization of living donor liver transplantation: An analysis of the Scientific Registry of Transplant Recipients.

BACKGROUND: The Affordable Care Act (ACA) and subsequent Medicaid expansion has increased utilization of public health insurance. Living donor liver transplantation (LDLT) increases access to transplant and is associated with improved survival but consistently represents < 5% of LT in the United States. STUDY DESIGN: National registry data were analyzed to evaluate the impact of insurance payor on waitlist mortality and LDLT rates at LDLT centers since implementation of the ACA. RESULTS: Public insurance [Medicare RR 1.18 (1.13-1.22) P < .001, Medicaid RR 1.22 (1.18-1.27) P < .001], Latino ethnicity (P < .001), and lower education level (P = .02) were associated with increased waitlist mortality at LDLT centers. LDLT recipients were more likely to have private insurance (70.4% vs. 59.4% DDLT, P < .001), be Caucasian (92.1% vs. 83% DDLT, P < .001), and have post-secondary education (66.8% vs. 54.1% DDLT, P < .001). Despite 78% of LDLT centers being located in states with Medicaid expansion, there was no change in LDLT utilization among recipients with Medicaid (P = .196) or Medicare (P = .273). CONCLUSION: Despite Medicaid expansion, registry data suggests that patients with public medical insurance may experience higher waitlist mortality and underutilize LDLT at centers offering LDLT. It is possible that Medicaid expansion has not increased access to LDLT.

Generic Protocols for the Analytical Validation of Next-Generation Sequencing-Based ctDNA Assays: A Joint Consensus Recommendation of the BloodPAC's Analytical Variables Working Group.

Liquid biopsy, particularly the analysis of circulating tumor DNA (ctDNA), has demonstrated considerable promise for numerous clinical intended uses. Successful validation and commercialization of novel ctDNA tests have the potential to improve the outcomes of patients with cancer. The goal of the Blood Profiling Atlas Consortium (BloodPAC) is to accelerate the development and validation of liquid biopsy assays that will be introduced into the clinic. To accomplish this goal, the BloodPAC conducts research in the following areas: Data Collection and Analysis within the BloodPAC Data Commons; Preanalytical Variables; Analytical Variables; Patient Context Variables; and Reimbursement. In this document, the BloodPAC's Analytical Variables Working Group (AV WG) attempts to define a set of generic analytical validation protocols tailored for ctDNA-based Next-Generation Sequencing (NGS) assays. Analytical validation of ctDNA assays poses several unique challenges that primarily arise from the fact that very few tumor-derived DNA molecules may be present in circulation relative to the amount of nontumor-derived cell-free DNA (cfDNA). These challenges include the exquisite level of sensitivity and specificity needed to detect ctDNA, the potential for false negatives in detecting these rare molecules, and the increased reliance on contrived samples to attain sufficient ctDNA for analytical validation. By addressing these unique challenges, the BloodPAC hopes to expedite sponsors' presubmission discussions with the Food and Drug Administration (FDA) with the protocols presented herein. By sharing best practices with the broader community, this work may also save the time and capacity of FDA reviewers through increased efficiency.

Working Together with God: Religious Coping, Perceived Discrimination, and Hypertension.

This study examined the relationships of perceived discrimination and religious coping with hypertension in a sample of Black and White Seventh-day Adventists. Data come from a community-based sample of 6128 White American, 2253 African American and 927 Caribbean American adults (67% women; mean age = 62.9 years). Results indicate lifetime unfair treatment was significantly associated with hypertension regardless of race/ethnicity. Positive religious coping was associated with lower odds of hypertension and did not interact with unfair treatment. Both positive and negative religious coping were indirectly associated with increased hypertension risk through an increase in perceived discrimination.

Identification and Characterization of Circulating Tumor Cells in Men Who have Undergone Prostatectomy for Clinically Localized, High Risk Prostate Cancer.

PURPOSE: Approximately 15% of men with newly diagnosed prostate cancer have high risk features which increase the risk of recurrence and metastasis. Better predictive biomarkers could allow for earlier detection of biochemical recurrence and change surveillance and adjuvant treatment paradigms. Circulating tumor cells are thought to represent the earliest form of metastases. However, their role as biomarkers in men with high risk, localized prostate cancer is not well defined. MATERIALS AND METHODS: Two to 5 months after prostatectomy we obtained blood samples from 37 patients with high risk, localized prostate cancer, defined as stage T3a or higher, Gleason score 8 or greater, or prostate specific antigen 20 ng/ml or greater. Circulating tumor cells were enumerated using a commercial platform. Matched tumor and single circulating tumor cell sequencing was performed. RESULTS: Circulating tumor cells were detected in 30 of 37 samples (81.1%) with a median of 2.4 circulating tumor cells per ml (range 0 to 22.9). Patients with detectable circulating tumor cells showed a trend toward shorter recurrence time (p=0.12). All patients with biochemical recurrence had detectable circulating tumor cells. Androgen receptor over expression was detected in 7 of 37 patients (18.9%). Patients with biochemical recurrence had more circulating tumor cell copy number aberrations (p=0.027). Matched tumor tissue and single circulating tumor cell sequencing revealed heterogeneity. CONCLUSIONS: We noted a high incidence of circulating tumor cell detection after radical prostatectomy and shorter time to biochemical recurrence in men with a higher circulating tumor cell burden and more circulating tumor cell copy number aberrations. Genomic alterations consistent with established copy number aberrations in prostate cancer were detectable in circulating tumor cells but often discordant with cells analyzed in bulk from primary lesions. With further testing in appropriately powered cohorts early circulating tumor cell detection could be an informative biomarker to assist with adjuvant treatment decisions.

Physical activity energy expenditure and cardiometabolic health in three rural Kenyan populations.

OBJECTIVES: Physical activity is beneficial for metabolic health but the extent to which this may differ by ethnicity is still unclear. Here, the objective was to characterize the association between physical activity energy expenditure (PAEE) and cardiometabolic risk among the Luo, Kamba, and Maasai ethnic groups of rural Kenya. METHODS: In a cross-sectional study of 1084 rural Kenyans, free-living PAEE was objectively measured using individually-calibrated heart rate and movement sensing. A clustered metabolic syndrome risk score (zMS) was developed by averaging the sex-specific z-scores of five risk components measuring central adiposity, blood pressure, lipid levels, glucose tolerance, and insulin resistance. RESULTS: zMS was 0.08 (-0.09; -0.06) SD lower for every 10 kJ/kg/day difference in PAEE after adjustment for age and sex; this association was modified by ethnicity (interaction with PAEE P < 0.05). When adjusted for adiposity, each 10 kJ/kg/day difference in PAEE was predicted to lower zMS by 0.04 (-0.05, -0.03) SD, without evidence of interaction by ethnicity. The Maasai were predicted to have higher cardiometabolic risk than the Kamba and Luo at every quintile of PAEE, with a strong dose-dependent decreasing trend among all ethnicities. CONCLUSION: Free-living PAEE is strongly inversely associated with cardiometabolic risk in rural Kenyans. Differences between ethnic groups in this association were observed but were explained by differences in central adiposity. Therefore, targeted interventions to increase PAEE are more likely to be effective in subgroups with high central adiposity, such as Maasai with low levels of PAEE.

The Association between Affect and Biomarkers of Stress and Inflammation in Healthy Seventh-Day Adventists.

OBJECTIVE: To evaluate the association between positive and negative affective states with stress biomarkers, biomarkers of inflammation and blood pressure in a population of healthy Seventh-day Adventists. DESIGN: In a cross-sectional study, biomarkers were regressed on positive and negative affect and control variables among reportedly healthy 133 females and 100 males (35% Black and 65% White) who provided blood and urine samples following completion of a questionnaire and measurement of anthropometrics and vital signs. SETTING/LOCATION: Data were extracted from the Biological Manifestations of Religion Study, an NIA-funded study conducted in members of the entity who lived within driving distance of two clinic sites. OUTCOME MEASURES: The stress biomarkers, epinephrine, and norepinephrine, were measured in 12-hour overnight urine samples analyzed by high-performance liquid chromatography. Urinary cortisol was evaluated by enzyme-linked immunosorbent assay (ELISA) and normalized for urinary output (reported in µg/g creatinine). Serum DHEA-S (reported in µg/ml) was measured by ELISA. Inflammatory markers included CRP (ng/ml), IL-6, IL-10, and TNF-α, all analyzed in serum by ELISA, and the data expressed in pg/ml. RESULTS: Multiple linear regression analyses showed after controlling for age, gender, ethnicity, body mass index (BMI), education, socioeconomic status, exercise, and use of blood pressure medication, that negative affect was associated with higher levels of epinephrine (ß = .143; P = .030). Positive affect was not associated with the biomarkers. CONCLUSIONS: While negative affect was associated with a biomarker of sympathetic stimulation, positive affect was not protective against such stimulation.

Residual tissue repositories as a resource for population-based cancer proteomic studies.

BACKGROUND: Mass spectrometry-based proteomics has become a powerful tool for the identification and quantification of proteins from a wide variety of biological specimens. To date, the majority of studies utilizing tissue samples have been carried out on prospectively collected fresh frozen or optimal cutting temperature (OCT) embedded specimens. However, such specimens are often difficult to obtain, in limited in supply, and clinical information and outcomes on patients are inherently delayed as compared to banked samples. Annotated formalin fixed, paraffin embedded (FFPE) tumor tissue specimens are available for research use from a variety of tissue banks, such as from the surveillance, epidemiology and end results (SEER) registries' residual tissue repositories. Given the wealth of outcomes information associated with such samples, the reuse of archived FFPE blocks for deep proteomic characterization with mass spectrometry technologies would provide a valuable resource for population-based cancer studies. Further, due to the widespread availability of FFPE specimens, validation of specimen integrity opens the possibility for thousands of studies that can be conducted worldwide. METHODS: To examine the suitability of the SEER repository tissues for proteomic and phosphoproteomic analysis, we analyzed 60 SEER patient samples, with time in storage ranging from 7 to 32 years; 60 samples with expression proteomics and 18 with phosphoproteomics, using isobaric labeling. Linear modeling and gene set enrichment analysis was used to evaluate the impacts of collection site and storage time. RESULTS: All samples, regardless of age, yielded suitable protein mass after extraction for expression analysis and 18 samples yielded sufficient mass for phosphopeptide analysis. Although peptide, protein, and phosphopeptide identifications were reduced by 50, 20 and 76% respectively, from comparable OCT specimens, we found no statistically significant differences in protein quantitation correlating with collection site or specimen age. GSEA analysis of GO-term level measurements of protein abundance differences between FFPE and OCT embedded specimens suggest that the formalin fixation process may alter representation of protein categories in the resulting dataset. CONCLUSIONS: These studies demonstrate that residual FFPE tissue specimens, of varying age and collection site, are a promising source of protein for proteomic investigations if paired with rigorously verified mass spectrometry workflows.

Pyrobaculum igneiluti sp. nov., a novel anaerobic hyperthermophilic archaeon that reduces thiosulfate and ferric iron.

A novel anaerobic, hyperthermophilic archaeon was isolated from a mud volcano in the Salton Sea geothermal system in southern California, USA. The isolate, named strain 521T, grew optimally at 90 °C, at pH 5.5-7.3 and with 0-2.0 % (w/v) NaCl, with a generation time of 10 h under optimal conditions. Cells were rod-shaped and non-motile, ranging from 2 to 7 µm in length. Strain 521T grew only in the presence of thiosulfate and/or Fe(III) (ferrihydrite) as terminal electron acceptors under strictly anaerobic conditions, and preferred protein-rich compounds as energy sources, although the isolate was capable of chemolithoautotrophic growth. 16S rRNA gene sequence analysis places this isolate within the crenarchaeal genus Pyrobaculum. To our knowledge, this is the first Pyrobaculum strain to be isolated from an anaerobic mud volcano and to reduce only either thiosulfate or ferric iron. An in silico genome-to-genome distance calculator reported <25 % DNA-DNA hybridization between strain 521T and eight other Pyrobaculum species. Due to its genotypic and phenotypic differences, we conclude that strain 521T represents a novel species, for which the name Pyrobaculum igneiluti sp. nov. is proposed. The type strain is 521T (=DSM 103086T=ATCC TSD-56T).