Proteomic analysis of chromophobe renal cell carcinoma and benign renal oncocytoma biopsies reveals shared metabolic dysregulation.

BACKGROUND: This study investigates the proteomic landscapes of chromophobe renal cell carcinoma (chRCC) and renal oncocytomas (RO), two subtypes of renal cell carcinoma that together account for approximately 10% of all renal tumors. Despite their histological similarities and shared origins, chRCC is a malignant tumor necessitating aggressive intervention, while RO, a benign growth, is often subject to overtreatment due to difficulties in accurate differentiation. METHODS: We conducted a label-free quantitative proteomic analysis on solid biopsies of chRCC (n = 5), RO (n = 5), and normal adjacent tissue (NAT, n = 5). The quantitative analysis was carried out by comparing protein abundances between tumor and NAT specimens. Our analysis identified a total of 1610 proteins across all samples, with 1379 (85.7%) of these proteins quantified in at least seven out of ten LC‒MS/MS runs for one renal tissue type (chRCC, RO, or NAT). RESULTS: Our findings revealed significant similarities in the dysregulation of key metabolic pathways, including carbohydrate, lipid, and amino acid metabolism, in both chRCC and RO. Compared to NAT, both chRCC and RO showed a marked downregulation in gluconeogenesis proteins, but a significant upregulation of proteins integral to the citrate cycle. Interestingly, we observed a distinct divergence in the oxidative phosphorylation pathway, with RO showing a significant increase in the number and degree of alterations in proteins, surpassing that observed in chRCC. CONCLUSIONS: This study underscores the value of integrating high-resolution mass spectrometry protein quantification to effectively characterize and differentiate the proteomic landscapes of solid tumor biopsies diagnosed as chRCC and RO. The insights gained from this research offer valuable information for enhancing our understanding of these conditions and may aid in the development of improved diagnostic and therapeutic strategies.

Absolute quantitative proteomics using the total protein approach to identify novel clinical immunohistochemical markers in renal neoplasms.

BACKGROUND: Renal neoplasms encompass a variety of malignant and benign tumors, including many with shared characteristics. The diagnosis of these renal neoplasms remains challenging with currently available tools. In this work, we demonstrate the total protein approach (TPA) based on high-resolution mass spectrometry (MS) as a tool to improve the accuracy of renal neoplasm diagnosis. METHODS: Frozen tissue biopsies of human renal tissues [clear cell renal cell carcinoma (n = 7), papillary renal cell carcinoma (n = 5), chromophobe renal cell carcinoma (n = 5), and renal oncocytoma (n = 5)] were collected for proteome analysis. Normal adjacent renal tissue (NAT, n = 5) was used as a control. Proteins were extracted and digested using trypsin, and the digested proteomes were analyzed by label-free high-resolution MS (nanoLC-ESI-HR-MS/MS). Quantitative analysis was performed by comparison between protein abundances of tumors and NAT specimens, and the label-free and standard-free TPA was used to obtain absolute protein concentrations. RESULTS: A total of 205 differentially expressed proteins with the potential to distinguish the renal neoplasms were found. Of these proteins, a TPA-based panel of 24, including known and new biomarkers, was selected as the best candidates to differentiate the neoplasms. As proof of concept, the diagnostic potential of PLIN2, TUBB3, LAMP1, and HK1 was validated using semi-quantitative immunohistochemistry with a total of 128 samples assessed on tissue micro-arrays. CONCLUSIONS: We demonstrate the utility of combining high-resolution MS and the TPA as potential new diagnostic tool in the pathology of renal neoplasms. A similar TPA approach may be implemented in any cancer study with solid biopsies.

The role of internal auditing in promoting accountability in Higher Education Institutions / El papel de la auditoría interna en la promoción de la accountability en las Instituciones de Educación Superior / O papel da auditoria interna na promoção da accountability nas Instituições de Ensino Superior

Abstract This paper discusses the link between accountability and internal auditing, particularly analyzing the extent to which the latter contributes to improve the former, in Higher Education Institutions (HEIs). This study applied a questionnaire to the management boards of a sample of HEIs, to empirical analyze the relationship between internal auditing and accountability. The main focus was on internal auditing carried out by the offices or departments in those institutions. The paper contributes to understand how management boards perceive internal auditing to foster transparency and accountability in HEIs, allowing to corroborate that auditing, and particularly internal auditing, favors the institutions' accountability. In effect, it promotes the principles underlying accountability practices. The information provided in the scope of internal audits is acknowledged as contributing to improve management effectiveness and helping in decision-making. HEIs wanting to create an internal auditing office or to enhance the role of an existing one, should develop this office's activities so that it becomes an instrument to support accountability and good governance of the organization. The sample consisted of Portuguese public HEIs, universities and polytechnics. Despite a certain international convergence regarding this type of public sector organizations, and regarding their purposes and governance, certain contextual specificities might limit the generalization of the findings for other jurisdictions.

Resumen Este artículo discute la asociación entre la accountability y la auditoría interna, en particular analizando hasta qué punto esta última contribuye a mejorar la primera, en las Instituciones de Educación Superior (IES). Para analizar empíricamente la relación entre la auditoría interna y la accountability, el estudio se basa en los resultados de una encuesta enviada a los consejos de administración de una muestra de IES, con el enfoque principal en la auditoría interna realizada por las oficinas o departamentos en esas instituciones. El artículo contribuye a comprender cómo los consejos de administración perciben la auditoría interna para fomentar la transparencia y accountability en las IES, lo que permite corroborar que la auditoría, y particularmente la auditoría interna, sirve a la accountability. De hecho, ella promueve los principios subyacentes a las prácticas de accountability. Se reconoce que la información proporcionada en el ámbito de las auditorías internas contribuye a mejorar la efectividad de la gestión y ayuda en la toma de decisiones. Las IES que desean crear una oficina de auditoría interna o mejorar el papel de una existente, deben desarrollar las actividades de esta oficina para que se convierta en un instrumento de apoyo a la accountability y al buen gobierno de la organización. La muestra consistió en IES públicas portuguesas, universidades y politécnicos. A pesar de una cierta convergencia internacional con respecto a este tipo de organizaciones, así como con respecto a sus propósitos y gobernanza, ciertas especificidades contextuales pueden limitar la generalización de los resultados a otras jurisdicciones.

Resumo Este artigo discute a associação entre accountability e auditoria interna, analisando particularmente até que ponto a última contribui para melhorias na primeira, nas Instituições de Ensino Superior (IES). Para analisar empiricamente a relação entre auditoria interna e accountability, o estudo baseia-se nos resultados de um questionário enviado aos órgãos de gestão de uma amostra de IES, com o foco principal na auditoria interna desenvolvida pelos gabinetes ou departamentos nessas instituições. O artigo contribui para entender como os órgãos de gestão percebem a auditoria interna para promover a transparência e a accountability nas IES, permitindo corroborar que a auditoria e, principalmente, a auditoria interna, serve a accountability. Com efeito, ela promove os princípios subjacentes às práticas de accountability. A informação proporcionada no âmbito das auditorias internas é reconhecida como contribuindo para melhorar a eficácia da gestão e ajudar na tomada de decisões. As IES que desejem criar um gabinete de auditoria interna ou aprimorar o papel de um gabinete já existente, devem desenvolver as atividades desse serviço de modo a que se torne um instrumento para apoiar a accountability e a boa governança da organização. A amostra foi constituída por IES públicas portuguesas, universidades e institutos politécnicos. Apesar de existir uma certa convergência internacional em relação a este tipo de organizações do setor público, bem como em relação aos seus propósitos e governança, certas especificidades contextuais podem limitar a generalização dos resultados desta pesquisa, para outras jurisdições.

Ultrasonic-assisted extraction and digestion of proteins from solid biopsies followed by peptide sequential extraction hyphenated to MALDI-based profiling holds the promise of distinguishing renal oncocytoma from chromophobe renal cell carcinoma.

A novel analytical approach is proposed to discriminate between solid biopsies of chromophobe renal cell carcinoma (chRCC) and renal oncocytoma (RO). The method comprises the following steps: (i) ultrasonic extraction of proteins from solid biopsies, (ii) protein depletion with acetonitrile, (iii) ultrasonic assisted in-solution digestion using magnetic nanoparticle with immobilized trypsin, (iv) C18 tip-based preconcentration of peptides, (v) sequential extraction of the peptides with ACN, (vi) MALDI-snapshot of the extracts and (vii) investigation of the extract containing the most discriminating features using high resolution mass spectrometry. With this approach we have been able to differentially cluster renal oncocytoma and chromophobe renal cell carcinoma and identified 18 proteins specific to chromophobe and seven unique to renal oncocytoma. Chromophobes express proteins associated with ATP function (ATP5I & 5E; VATE1 & G2; ADT2), glycolysis (PGK1) and neuromedin whilst oncocytomas express ATP5H, ATPA, DEPD7 and TRIPB thyroid receptor interacting protein.

Proteomic changes driven by urban pollution suggest particulate matter as a deregulator of energy metabolism, mitochondrial activity, and oxidative pathways in the rat brain.

The adverse effects of air pollution have been long studied in the lung and respiratory systems, but the molecular changes that this causes at the central nervous system level have yet to be fully investigated and understood. To explore the evolution with time of protein expression levels in the brain of rats exposed to particulate matter of different sizes, we carried out two-dimensional gel electrophoresis followed by determination of dysregulated proteins through Coomassie blue staining-based densities (SameSpots software) and subsequent protein identification using MALDI-based mass spectrometry. Expression differences in dysregulated proteins were found to be statistically significant with p-value <0.05. A systems biology-based approach was utilized to determine critical biochemical pathways involved in the rats' brain response. Our results suggest that rats' brains have a particulate matter size dependent-response, being the mitochondrial activity and the astrocyte function severely affected. Our proteomic study confirms the dysregulation of different biochemical pathways involving energy metabolism, mitochondrial activity, and oxidative pathways as some of the main effects of PM exposure on the rat brain. SIGNIFICANCE: Rat brains exposed to particulate matter with origin in car engines are affected in two main areas: mitochondrial activity, by the dysregulation of many pathways linked to the respiratory chain, and neuronal and astrocytic function, which stimulates brain changes triggering tumorigenesis and neurodegeneration.

Development of a Robust Ultrasonic-Based Sample Treatment To Unravel the Proteome of OCT-Embedded Solid Tumor Biopsies.

An effective three-step proteomics workflow is proposed to overcome the pitfalls caused by polymers present in optimum cutting temperature (OCT)-embedded tissue during its preparation for mass spectrometry analysis. First, the OCT-embedded tissue biopsies are cleaned using ethanol and water in a sequential series of ultrasonic washes in an ultrasound bath (35 kHz ultrasonic frequency, 100% ultrasonic amplitude, 2 min of ultrasonic duty time). Second, a fast ultrasonic-assisted extraction of proteins is done using an ultrasonic probe (30 kHz ultrasonic frequency, 50% ultrasonic amplitude, 2 min of ultrasonic duty time, 1 mm diameter tip). Third, a rapid ultrasonic digestion of complex proteomes is performed using a microplate horn assembly device (20 kHz ultrasonic frequency, 25% ultrasonic amplitude, 4 min of ultrasonic duty time). As a proof of concept, the new workflow was applied to human normal and tumor kidney biopsies including chromophobe renal cell carcinomas (chRCCs) and renal oncocytomas (ROs). A successful cluster of proteomics profiles was obtained comprising 511 and 172 unique proteins found in chRCC and RO samples, respectively. The new method provides high sample throughput and comprehensive protein recovery from OCT samples.

S2P: A software tool to quickly carry out reproducible biomedical research projects involving 2D-gel and MALDI-TOF MS protein data.

BACKGROUND AND OBJECTIVE: 2D-gel electrophoresis is widely used in combination with MALDI-TOF mass spectrometry in order to analyze the proteome of biological samples. For instance, it can be used to discover proteins that are differentially expressed between two groups (e.g. two disease conditions, case vs. control, etc.) thus obtaining a set of potential biomarkers. This procedure requires a great deal of data processing in order to prepare data for analysis or to merge and integrate data from different sources. This kind of work is usually done manually (e.g. copying and pasting data into spreadsheet files), which is highly time consuming and distracts the researcher from other important, core tasks. Moreover, engaging in a repetitive process in a non-automated, handling-based manner is prone to error, thus threatening reliability and reproducibility. The objective of this paper is to present S2P, an open source software to overcome these drawbacks. METHODS: S2P is implemented in Java on top of the AIBench framework, and relies on well-established open source libraries to accomplish different tasks. RESULTS: S2P is an AIBench based desktop multiplatform application, specifically aimed to process 2D-gel and MALDI-mass spectrometry protein identification-based data in a computer-aided, reproducible manner. Different case studies are presented in order to show the usefulness of S2P. CONCLUSIONS: S2P is open source and free to all users at http://www.sing-group.org/s2p. Through its user-friendly GUI interface, S2P dramatically reduces the time that researchers need to invest in order to prepare data for analysis.

Unparalleled sample treatment throughput for proteomics workflows relying on ultrasonic energy.

We report on the new microplate horn ultrasonic device as a powerful tool to speed proteomics workflows with unparalleled throughput. 96 complex proteomes were digested at the same time in 4min. Variables such as ultrasonication time, ultrasonication amplitude, and protein to enzyme ratio were optimized. The "classic" method relying on overnight protein digestion (12h) and the sonoreactor-based method were also employed for comparative purposes. We found the protein digestion efficiency homogeneously distributed in the entire microplate horn surface using the following conditions: 4min sonication time and 25% amplitude. Using this approach, patients with lymphoma and myeloma were classified using principal component analysis and a 2D gel-mass spectrometry based approach. Furthermore, we demonstrate the excellent performance by using MALDI-mass spectrometry based profiling as a fast way to classify patients with rheumatoid arthritis, systemic lupus erythematosus, and ankylosing spondylitis. Finally, the speed and simplicity of this method were demonstrated by clustering 90 patients with knee osteoarthritis disease (30), with a prosthesis (30, control group) and healthy individuals (30) with no history of joint disease. Overall, the new approach allows profiling a disease in just one week while allows to match the minimalism rules as outlined by Halls.