Understanding and tackling the reproducibility crisis - Why we need to study scientists' trust in data.

In the life sciences, there is an ongoing discussion about a perceived 'reproducibility crisis'. However, it remains unclear to which extent the perceived lack of reproducibility is the consequence of issues that can be tackled and to which extent it may be the consequence of unrealistic expectations of the technical level of reproducibility. Large-scale, multi-institutional experimental replication studies are very cost- and time-intensive. This Perspective suggests an alternative, complementary approach: meta-research using sociological and philosophical methodologies to examine researcher trust in data. An improved understanding of the criteria used by researchers to judge data reliability will provide crucial, initial evidence on the actual scale of the reproducibility crisis and on measures to tackle it.

Dating first cases of COVID-19.

Questions persist as to the origin of the COVID-19 pandemic. Evidence is building that its origin as a zoonotic spillover occurred prior to the officially accepted timing of early December, 2019. Here we provide novel methods to date the origin of COVID-19 cases. We show that six countries had exceptionally early cases, unlikely to represent part of their main case series. The model suggests a likely timing of the first case of COVID-19 in China as November 17 (95% CI October 4). Origination dates are discussed for the first five countries outside China and each continent. Results infer that SARS-CoV-2 emerged in China in early October to mid-November, and by January, had spread globally. This suggests an earlier and more rapid timeline of spread. Our study provides new approaches for estimating dates of the arrival of infectious diseases based on small samples that can be applied to many epidemiological situations.

Understanding the drivers of expert opinion when classifying species as extinct.

The criteria as laid out by the International Union for the Conservation of Nature (IUCN) Red List are the gold standard by which the extinction risk of a species is assessed and where appropriate biological extinctions are declared. However, unlike all other categories, the category of extinct lacks a quantitative framework for assigning this category. Given its subjective nature, we surveyed expert assessors working on a diversity of taxa to explore the attributes they used to declare a species extinct. Using a choice experiment approach, we surveyed 674 experts from the IUCN Species Survival Commission specialist groups and taskforces. Data availability, time from the last sighting, detectability, habitat availability, and population decline were all important attributes favored by assessors when inferring extinction. Respondents with red-listing experience assigned more importance to the attributes data availability, time from the last sighting, and detectability when considering a species extinction, whereas those respondents working with well-known taxa gave more importance to the time from the last sighting. Respondents with no red-listing experience and those working with more well-known taxa (i.e., mammals and birds) were overall less likely to consider species extinct. Our findings on the importance assessors place on attributes used to declare a species extinct provide a basis for informing the development of specific criteria for more accurately assessing species extinctions.

Los criterios establecidos por la Unión Internacional para la Conservación de la Naturaleza (UICN) son la regla de oro con la cual se evalúa el riesgo de extinción de una especie y en donde se declaran las extinciones biológicas. Sin embargo, como con todas las demás categorías, la categoría "extinto" carece de un marco de trabajo cuantitativo para asignar esta categoría. Dada su naturaleza subjetiva, pedimos a los asesores expertos que trabajan con la diversidad de taxones que exploraran los atributos que usan para declarar extinta a una especie. Mediante un experimento de elección, sondeamos a 674 expertos de los grupos especialistas y de trabajo de la Comisión de Supervivencia de Especies de la UICN. La disponibilidad de datos, el tiempo desde la última detección, la detectabilidad, la disponibilidad del hábitat y la declinación poblacional fueron atributos importantes que los asesores favorecieron al inferir las extinciones. Los respondientes con experiencia con la lista roja les asignaron mayor importancia a los atributos de disponibilidad de datos, tiempo desde la última detección y detectabilidad cuando consideraron la extinción de una especie, mientras que los respondientes que trabajan con taxones conocidos le dieron más importancia al tiempo desde la última detección. En general fue menos probable que los respondientes sin experiencia con la lista roja y aquellos que trabajan con los taxones más conocidos (es decir, mamíferos y aves) consideraran extinta a una especie. Nuestros descubrimientos sobre la importancia que los asesores colocan sobre los atributos utilizados para declarar extinta a una especie proporcionan una base para orientar el desarrollo de criterios específicos para evaluar de manera más acertada las extinciones de las especies.

Hormetic association between perceived stress and human epigenetic aging based on resilience capacity.

Chronic stress is associated with deleterious health outcomes and mortality risk. A potential mechanism by which stress affects healthspan and lifespan is acceleration of cellular aging. Biologic age prediction models, termed epigenetic clocks, have been developed to estimate biologic age differences among people with the same chronologic age. This study evaluates the simultaneous impact of perceived chronic stress and resilience on Grim Age acceleration. The perceived stress score (PSS) and Connor-Davidson Resilience Scale (CD-RISC) were used to measure chronic stress and resilience, respectively. DNA was extracted from whole blood and analyzed using the MethylationEPIC BeadChip. GrimAge estimates were calculated using the methylation age calculator. Forty-seven business executives were categorized by levels of high or low stress and resilience scores. Compared to participants with low stress and high resilience, those with low stress and low resilience demonstrated the strongest association with Grim Age acceleration (p = 0.044), after controlling for age and estimated cellular proportions. Interestingly, among participants with low resilience, those with high perceived stress had a weaker association with Grim Age acceleration than participants with low perceived stress. However, among participants with high resilience, low perceived stress had a weaker association with Grim Age acceleration than high perceived stress. Our findings suggest that the impact of perceived stress on epigenetic age acceleration may differ based on resilience capacity, with a potential paradoxical beneficial effect among those with low resilience.

Preliminary Evaluation of a System with On-Body and Aerial Sensors for Monitoring Working Dogs.

This paper presents a system for behavioral, environmental, and physiological monitoring of working dogs using on-body and aerial sensors. The proof of concept study presented here includes two trained dogs performing nine scent detection tasks in an uncontrolled environment encompassing approximately two acres. The dogs were outfitted with a custom designed wearable harness to monitor their heart rate, activity levels and skin temperature. We utilized a commercially available micro-air vehicle to perform aerial sensing by tracking the terrain and movement of the dog in the outdoor space. The dogs were free to explore the space working at maximal speeds to complete a scent-based search-and-retrieval task. Throughout the experiment, the harness data was transferred to a base station via Wi-Fi in real-time. In this work, we also focused on testing the performance of a custom 3D electrode with application specific ergonomic improvements and adaptive filter processing techniques to recover as much electrocardiography data as possible during high intensity motion activity. We were able to recover and use 84% of the collected data where we observed a trend of heart rate generally increasing immediately after successful target localization. For tracking the dogs in the aerial video footage, we applied a state-of-the-art deep learning algorithm designed for online object tracking. Both qualitative and quantitative tracking results are very promising. This study presents an initial effort towards deployment of on-body and aerial sensors to monitor the working dogs and their environments during scent detection and search and rescue tasks in order to ensure their welfare, enable novel dog-machine interfaces, and allow for higher success rate of remote and automated task performance.

Suicidal Ideation Across Three Timepoints in Patients Discharged from Psychiatric Hospitalization.

This article describes the rate of suicidal ideation (SI) across three timepoints among treatment-seeking patients recently discharged from psychiatric hospitalization-a group that is at ultrahigh-risk for suicide. Retrospective chart review was used to quantify the rate of SI in 252 consecutive patients discharged to a post-hospital mental health clinic. Data include patients' lifetime history of SI, SI at the time of hospital intake, and SI at post-hospital outpatient clinic intake, as well as demographics and diagnosis. Overall, 67% of the sample reported a lifetime history of SI, 49% reported SI during hospital intake, and 6% reported SI at post-hospital clinic intake. Age was the only variable associated with history of SI (p = .04), with younger patients more likely (OR = 1.85) to report a history of SI. These results may help inform the development of interventions for the population of ultrahigh-risk patients being discharged from hospital after psychiatric care.

Machine Learning-Enabled Renal Cell Carcinoma Status Prediction Using Multiplatform Urine-Based Metabolomics.

Renal cell carcinoma (RCC) is diagnosed through expensive cross-sectional imaging, frequently followed by renal mass biopsy, which is not only invasive but also prone to sampling errors. Hence, there is a critical need for a noninvasive diagnostic assay. RCC exhibits altered cellular metabolism combined with the close proximity of the tumor(s) to the urine in the kidney, suggesting that urine metabolomic profiling is an excellent choice for assay development. Here, we acquired liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) data followed by the use of machine learning (ML) to discover candidate metabolomic panels for RCC. The study cohort consisted of 105 RCC patients and 179 controls separated into two subcohorts: the model cohort and the test cohort. Univariate, wrapper, and embedded methods were used to select discriminatory features using the model cohort. Three ML techniques, each with different induction biases, were used for training and hyperparameter tuning. Assessment of RCC status prediction was evaluated using the test cohort with the selected biomarkers and the optimally tuned ML algorithms. A seven-metabolite panel predicted RCC in the test cohort with 88% accuracy, 94% sensitivity, 85% specificity, and 0.98 AUC. Metabolomics Workbench Study IDs are ST001705 and ST001706.

Statistical inference of earlier origins for the first flaked stone technologies.

Identifying when hominins first produced Lomekwian, Oldowan, and Acheulean technologies is vital to multiple avenues of human origins research. Yet, like most archaeological endeavors, our understanding is currently only as accurate as the artifacts recovered and the sites identified. Here we use optimal linear estimation (OLE) modelling to identify the portion of the archaeological record not yet discovered, and statistically infer the date of origin of the earliest flaked stone technologies. These models provide the most accurate framework yet for understanding when hominins first produced these tool types. Our results estimate the Oldowan to have originated 2.617 to 2.644 Ma, 36,000 to 63,000 years earlier than current evidence. The Acheulean's origin is pushed back further through OLE, by at least 55,000 years to 1.815 to 1.823 Ma. We were unable to infer the Lomekwian's date of origin using OLE, but an upper bound of 5.1 million years can be inferred using alternative nonparametric techniques. These dates provide a new chronological foundation from which to understand the emergence of the first flaked stone technologies, alongside their behavioral and evolutionary implications. Moreover, they suggest there to be substantial portions of the artifact record yet to be discovered.

Ethics and governance for internet-based conservation science research.

Internet-based research is increasingly important for conservation science and has wide-ranging applications and contexts, including culturomics, illegal wildlife trade, and citizen science. However, online research methods pose a range of ethical and legal challenges. Online data may be protected by copyright, database rights, or contract law. Privacy rights may also restrict the use and access of data, as well as ethical requirements from institutions. Online data have real-world meaning, and the ethical treatment of individuals and communities must not be marginalized when conducting internet-based research. As ethics frameworks originally developed for biomedical applications are inadequate for these methods, we propose that research activities involving the analysis of preexisting online data be treated analogous to offline social science methods, in particular, nondeceptive covert observation. By treating internet users and their data with respect and due consideration, conservationists can uphold the public trust needed to effectively address real-world issues.

Ética y Gestión para la Investigación Científica de la Conservación Basada en Internet Resumen La investigación basada en internet es cada vez más importante para las ciencias de la conservación, además de tener contextos y aplicaciones de gran alcance como el análisis de textos, el mercado ilegal de fauna y la ciencia ciudadana. Sin embargo, los métodos de investigación en línea representan una gama de retos éticos y legales pues los datos virtuales pueden estar protegidos por derechos de autor, derechos de base de datos o leyes contractuales. Además, los derechos de privacidad pueden restringir el uso y el acceso a los datos, así como también los requerimientos éticos impuestos por las instituciones. Los datos virtuales tienen valor en el mundo real y el tratamiento ético de los individuos y de las comunidades no se debe marginalizar cuando se realiza una investigación por internet. Ya que los marcos éticos desarrollados originalmente para aplicarse en temas biomédicos son inadecuados para estos métodos, proponemos que las actividades de investigación que involucran el análisis de los datos virtuales preexistentes sean tratadas como análogas a los métodos no virtuales de las ciencias sociales, especialmente la observación encubierta no engañosa. Si se trata a los usuarios del internet y a sus datos con respeto y la consideración debida, los conservacionistas pueden mantener la confianza pública necesaria para tratar efectivamente los asuntos del mundo real.

Network analysis of a stakeholder community combatting illegal wildlife trade.

The illegal wildlife trade has emerged as a growing and urgent environmental issue. Stakeholders involved in the efforts to curb wildlife trafficking include nongovernmental organizations (NGOs), academia, and state government and enforcement bodies. The extent to which these stakeholders work and communicate among each other is fundamental to effectively combatting illicit trade. Using the United Kingdom as a case study, we used a social network analysis and semistructured interviews of stakeholders to assess communication relationships in the counter wildlife trafficking community. The NGOs consistently occupied 4 of the 5 most central positions in the generated networks, whereas academic institutions routinely occupied 4 of the 5 most peripheral positions. However, NGOs were the least diverse in their communication practices compared with the other stakeholder groups. Stakeholders identified personal relationships as the most important aspect of functioning communication. Participant insights also showed that stakeholder-specific variables (e.g., ethical and confidentiality concerns), competition, and fundraising can have a confounding effect on intercommunication. Evaluating communication networks and intrastakeholder communication trends is essential to creating cohesive, productive, and efficient responses to the challenges of combatting illegal wildlife trade. Article impact statement: Communication among those combatting illegal wildlife trade is confounded by stakeholder variables (ethics, confidentiality), competition, and fundraising.

Análisis de las Redes de una Comunidad de Activistas que Combate al Mercado Ilegal de Fauna Resumen El mercado ilegal de fauna ha emergido como un tema ambiental creciente y urgente. Los actores involucrados en los esfuerzos por detener el tráfico de fauna incluyen a las organizaciones no gubernamentales (ONGs), los académicos y a los cuerpos estatales de gobierno y aplicación de la ley. El grado hasta el que estos accionistas trabajen y se comuniquen entre sí es fundamental para combatir efectivamente el mercado ilícito. Con el Reino Unido como estudio de caso, usamos un análisis de redes sociales y entrevistas semiestructuradas con los actores para evaluar las relaciones de comunicación dentro de la comunidad que combate el tráfico de fauna. Las ONGs ocuparon sistemáticamente cuatro de las cinco posiciones más centrales en las redes generadas, mientras que las instituciones académicas ocuparon rutinariamente cuatro de las cinco posiciones más periféricas. Sin embargo, las ONGs fueron las menos diversas con sus prácticas de comunicación en comparación con los otros grupos de actores. Los actores identificaron a las relaciones personales como el aspecto más importante de la comunicación funcional. La percepción de los participantes también mostró que las variables específicas por accionista (p. ej.: preocupaciones éticas y de confidencialidad), la competencia y la captación de fondos pueden tener un efecto confuso sobre la intercomunicación. La evaluación de las tendencias en las redes de comunicación y la comunicación entre accionistas es esencial para la creación de respuestas cohesivas, productivas y eficientes ante los obstáculos que presenta el combate al comercio ilegal de fauna.

Social and non-social measures of cognition for predicting self-reported and informant-reported functional outcomes in early psychosis.

The main aim of this study was to investigate the individual contributions of neurocognitive and social-cognitive domains to self-reported and informant-reported functional outcome in early psychosis. We also sought to further characterize the nature of cognitive impairments in this sample and explore the interrelationships between the social-cognitive measures and how they correlate with measures of neurocognition and clinical symptoms. In this study, 70 patients (mean age: 24.1; 87.1% males) with primary psychotic disorder diagnosed in the previous 5 years were assessed on multiple neurocognitive (processing speed, attention, working memory, immediate verbal memory, delayed recall, visual reasoning, inhibition, planning, cognitive flexibility), and social-cognitive domains (theory of mind (ToM), emotion recognition, attributional style, metacognitive overconfidence) as well as measures of clinical symptoms. Functional outcome was assessed with three self-reports and two informant-reports. On average, patients performed one or more SD below healthy controls on measures of delayed recall, ToM and metacognitive overconfidence. Emotion recognition and ToM were intercorrelated and correlated with multiple neurocognitive domains and negative symptoms. Attributional style correlated with positive symptoms. In the context of multiple variables, self-reported functional outcomes were predicted by attributional style, whereas emotion recognition and immediate verbal memory predicted variance in informant-reported community functioning. These results support the suggestion of a likely distinction between the predictive factors for self-reported and informant-reported functional outcome in early psychosis and suggest that consideration of self-assessment of functional outcome is critical when attempting to evaluate the effects attributional style has on functional disability.

The Use of Access Groups for Engagement in Community Mental Health Post Hospitalization.

This paper describes a novel approach to engaging psychiatric patients in care after discharge from inpatient or ER treatment. The Access Group model provides rapid, flexible, high-volume intake for up to 20 patient referrals per day. Patients are scheduled for intake by referring hospital clinicians using an online scheduling software and can be seen within 1-3 days of hospital discharge. Access allows flexibility, easy rescheduling, and limited wait time. At intake, patients and family members participate in a structured group intake procedure from which they are scheduled for follow-up psychiatry, therapy, and social work appointments. Same-day appointments are available for urgent needs. Initial evidence suggests that the Access Group model is effective at providing rapid access to and engagement in care, and is acceptable to patients. Generalizability and funding approaches for the model are discussed.

On the functional extinction of the Passenger Pigeon.

The Passenger Pigeon (Ectopistes migratorius) was a social breeder, and it has been suggested that the species experienced functional extinction, defined as a total reproductive failure, prior to its actual extinction in the early years of the 20th century. We applied a novel randomization test based on the relative times of the most recent egg- and skin-specimen sightings (i.e., recorded date of specimen collection) to test for functional extinction. For a total of 6 eggs and 27 skins, the observed significance level was 0.38, which indicated that the species did not become functionally extinct. Thus, proposals to reverse its rapid decline in the late 19th century could have been successful.

Assessing the extent and nature of wildlife trade on the dark web.

Use of the internet as a trade platform has resulted in a shift in the illegal wildlife trade. Increased scrutiny of illegal wildlife trade has led to concerns that online trade of wildlife will move onto the dark web. To provide a baseline of illegal wildlife trade on the dark web, we downloaded and archived 9852 items (individual posts) from the dark web, then searched these based on a list of 121 keywords associated with illegal online wildlife trade, including 30 keywords associated with illegally traded elephant ivory on the surface web. Results were compared with items known to be illegally traded on the dark web, specifically cannabis, cocaine, and heroin, to compare the extent of the trade. Of these 121 keywords, 4 resulted in hits, of which only one was potentially linked to illegal wildlife trade. This sole case was the sale and discussion of Echinopsis pachanoi (San Pedro cactus), which has hallucinogenic properties. This negligible level of activity related to the illegal trade of wildlife on the dark web relative to the open and increasing trade on the surface web may indicate a lack of successful enforcement against illegal wildlife trade on the surface web.

Estimating the extent and structure of trade in horticultural orchids via social media.

The wildlife trade is a lucrative industry involving thousands of animal and plant species. The increasing use of the internet for both legal and illegal wildlife trade is well documented, but there is evidence that trade may be emerging on new online technologies such as social media. Using the orchid trade as a case study, we conducted the first systematic survey of wildlife trade on an international social-media website. We focused on themed forums (groups), where people with similar interests can interact by uploading images or text (posts) that are visible to other group members. We used social-network analysis to examine the ties between 150 of these orchid-themed groups to determine the structure of the network. We found 4 communities of closely linked groups based around shared language. Most trade occurred in a community that consisted of English-speaking and Southeast Asian groups. In addition to the network analysis, we randomly sampled 30 groups from the whole network to assess the prevalence of trade in cultivated and wild plants. Of 55,805 posts recorded over 12 weeks, 8.9% contained plants for sale, and 22-46% of these posts pertained to wild-collected orchids. Although total numbers of posts about trade were relatively small, the large proportion of posts advertising wild orchids for sale supports calls for better monitoring of social media for trade in wild-collected plants.

Social Cognition and Interaction Training (SCIT) for Adults with Psychotic Disorders: A Feasibility Study in Finland.

BACKGROUND: Social Cognition and Interaction Training (SCIT) is a psychosocial treatment designed to improve social functioning in schizophrenia by improving social cognition. Positive results have been reported from several studies, mainly from the USA, but more studies are needed to determine the feasibility of SCIT in different cultural contexts. AIMS: The objective of this study was to evaluate the feasibility and acceptability of the Finnish translation of SCIT in Finland. METHOD: This was an uncontrolled, within-group study. Thirty-three patients with psychotic disorders participated in SCIT groups and also received the standard services provided at their respective care facilities. We measured participant attendance, attrition and responses on feedback surveys. Participants also completed measures of emotion perception, Theory of Mind (ToM), attributional bias and metacognitive overconfidence both before and after SCIT. RESULTS: The attendance rate was high, attrition was low, and the patients expressed satisfaction with SCIT. Preliminary efficacy analyses showed a statistically significant pre to posttest improvement in emotion perception and ToM, but not attributional bias or overconfidence. CONCLUSIONS: SCIT is feasible and well accepted and may remediate social cognitive dysfunction in people with psychotic disorders in Finland.

What Patients With Severe Mental Illness Transitioning From Hospital to Community Have to Say About Care and Shared Decision-Making.

Shared decision-making (SDM) has been slow to disseminate in mental health. We conducted focus groups with ten individuals with serious mental illness (SMI) treated in a 90 day, outpatient transitional care clinic. Parallel groups were held with family caregivers (n = 8). Individuals with SMI wanted longer visits, to have their stories heard, more information about options presented simply, to hear from peers about similar experiences, and a bigger say in treatment choices. Caregivers wanted to be invited to participate to a larger extent.  Results suggest that after a decade, SDM may not have the expected penetration in community mental health.

Social cognition in schizophrenia: factor structure, clinical and functional correlates.

BACKGROUND: Social cognition is consistently impaired in people with schizophrenia, separable from general neurocognition, predictive of real-world functioning and amenable to psychosocial treatment. Few studies have empirically examined its underlying factor structure. AIMS: This study (1) examines the factor structure of social cognition in both a sample of individuals with schizophrenia-spectrum disorders and non-clinical controls and (2) explores relationships of factors to neurocognition, symptoms and functioning. METHOD: A factor analysis was conducted on social cognition measures in a sample of 65 individuals with schizophrenia or schizoaffective disorder, and 50 control participants. The resulting factors were examined for their relationships to symptoms and functioning. RESULTS: Results suggested a two-factor structure in the schizophrenia sample (social cognition skill and hostile attributional style) and a three-factor structure in the non-clinical sample (hostile attributional style, higher-level inferential processing and lower-level cue detection). In the schizophrenia sample, the social cognition skill factor was significantly related to negative symptoms and social functioning, whereas hostile attributional style predicted positive and general psychopathology symptoms. CONCLUSIONS: The factor structure of social cognition in schizophrenia separates hostile attributional style and social cognition skill, and each show differential relationships to relevant clinical variables in schizophrenia.

Observable Social Cognition--A Rating Scale: an interview-based assessment for schizophrenia.

INTRODUCTION: Individuals with schizophrenia consistently show impairments in social cognition (SC). SC has become a potential treatment target due to its association with functional outcomes. An alternative method of assessment is to administer an observer-based scale incorporating an informant's "first hand" impressions in ratings. METHODS: The present study used the Observable Social Cognition: A Rating Scale (OSCARS) in 62 outpatients and 50 non-psychiatric controls (NPCs) to assess performance in domains of SC (e.g. emotion perception, theory of mind). RESULTS: The OSCARS demonstrated sufficient internal consistency and test-retest reliability. Construct validity was assessed through an exploratory factor analysis. Patient OSCARS indices were not significantly correlated with measures of SC with the exception of aggressive attributional style. Individuals with less impairment in SC reacted more aggressively to ambiguous situations. NPC OSCARS were significantly correlated with measures of theory of mind and attributional style. In a combined sample of patients and controls, six of eight items were significantly correlated with the SC task assessing the same domain, providing modest evidence of convergent validity. In patients, the OSCARS was significantly correlated with measures of functional outcome and neurocognition. Last, the OSCARS was found to be significantly associated with functional outcome after the influence of objective measures of SC was statistically removed. CONCLUSIONS: The present study provides preliminary evidence that the OSCARS may be useful for clinicians in collecting data about patients' potential real-world SC deficits, in turn increasing the degree to which these impairments may be targeted in treatment.

von Hippel-Lindau exonic methylation analysis using MALDI-TOF mass spectrometry.

PURPOSE: Aberrant promoter methylation turns off gene expression and is involved in human malignancy. Studies show that first exon methylation has a tighter association with gene silencing compared to promoter methylation or gene mutation. However, to our knowledge the clinical importance of exonic methylation in renal cell carcinoma is unknown. We analyzed renal cell carcinoma for VHL gene exonic methylation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. MATERIALS AND METHODS: In 48 institutionally banked renal cell carcinoma patient tissue samples VHL exon sequencing was done as well as methylation analysis of promoter and exon 1 by mass spectrometry or conventional bisulfite analysis. Methylated human lymphocytic DNA (0% and 100%), nontemplate distilled H2O, and the UOK121 and UOK171 human renal cell carcinoma cell lines served as assay controls. Samples were considered hypermethylated if a CpG site showed greater than 50% methylation. RESULTS: Nine of the 43 patient samples read by our exon 1 assay had methylated VHL exon 1 sites, including 3 showing hypermethylation. The exon 1 methylation assay was robust and reproducible. Samples with exon 1 hypermethylation showed no exonic mutations. All samples assayed at VHL exon 2 were hypermethylated. CONCLUSIONS: To assay renal cell carcinoma tumors for VHL methylation matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is robust and reproducible, and capable of quantifying the methylation status of individual DNA bases. Exon 1 methylation may be an alternate mechanism of VHL gene silencing in renal cell carcinoma in addition to mutation and promoter methylation. Applying this assay in patient populations may allow enhanced diagnosis or tumor typing in the future.