Comprehensive Metabolomics and Lipidomics Profiling of Prostate Cancer Tissue Reveals Metabolic Dysregulations Associated with Disease Development.

Prostate cancer (PCa) is a global health problem that affects millions of men every year. In the past decade, metabolomics and related subareas, such as lipidomics, have demonstrated an enormous potential to identify novel mechanisms underlying PCa development and progression, providing a good basis for the development of new and more effective therapies and diagnostics. In this study, a multiplatform metabolomics and lipidomics approach, combining untargeted mass spectrometry (MS) and nuclear magnetic resonance (NMR)-based techniques, was applied to PCa tissues to investigate dysregulations associated with PCa development, in a cohort of 40 patients submitted to radical prostatectomy for PCa. Results revealed significant alterations in the levels of 26 metabolites and 21 phospholipid species in PCa tissue compared with adjacent nonmalignant tissue, suggesting dysregulation in 13 metabolic pathways associated with PCa development. The most affected metabolic pathways were amino acid metabolism, nicotinate and nicotinamide metabolism, purine metabolism, and glycerophospholipid metabolism. A clear interconnection between metabolites and phospholipid species participating in these pathways was observed through correlation analysis. Overall, these dysregulations may reflect the reprogramming of metabolic responses to produce high levels of cellular building blocks required for rapid PCa cell proliferation.

Urinary Volatilomics Unveils a Candidate Biomarker Panel for Noninvasive Detection of Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer usually associated with asymptomatic development and risk of systemic progression. Hence, reliable molecular biomarkers of ccRCC are needed to provide early and minimally invasive detection. In this study, urinary volatilome profiling of patients diagnosed with ccRCC (n = 75), and cancer-free controls (n = 75), was performed to investigate the presence of a volatile signature characteristic of ccRCC. Volatile organic compounds (VOCs) in general, and more specifically volatile carbonyl compounds (VCCs), present in urine were extracted by headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Supervised multivariate models showed a good discriminatory power of ccRCC patients from controls in urine. Overall, 22 volatile metabolites were found significantly altered between the two groups, including aldehydes, ketones, aromatic hydrocarbons, and terpenoids. A candidate six-biomarker panel, comprising octanal, 3-methylbutanal, benzaldehyde, 2-furaldehyde, 4-heptanone, and p-cresol, depicted the best performance for ccRCC detection with 83% sensitivity, 79% specificity, and 81% accuracy. Moreover, the ccRCC urinary volatilome signature suggested dysregulation of energy metabolism and overexpression of enzymes associated with carcinogenesis. These findings provide the molecular basis for the fine-tuning of gas-sensing materials for application in the development of a bioelectronic sensor.

New findings on urinary prostate cancer metabolome through combined GC-MS and 1H NMR analytical platforms.

INTRODUCTION: The inherent sensitivity of metabolomics allows the detection of subtle alterations in biological pathways, making it a powerful tool to study biomarkers and the mechanisms that underlie cancer. OBJECTIVES: The purpose of this work was to characterize the urinary metabolic profile of prostate cancer (PCa) patients and cancer-free controls to obtain a holistic coverage of PCa metabolome. METHODS: Two groups of samples, a training set (n = 41 PCa and n = 42 controls) and an external validation set (n = 18 PCa and n = 18 controls) were analyzed using a dual analytical platform, namely gas chromatography-mass spectrometry (GC-MS) and proton nuclear magnetic resonance spectroscopy (1H NMR). RESULTS: The multivariate analysis models revealed a good discrimination between cases and controls with an AUC higher than 0.8, a sensitivity ranging from 67 to 89%, a specificity ranging from 74 to 89% and an accuracy from 73 to 86%, considering the training and external validation sets. A total of 28 metabolites (15 from GC-MS and 13 from 1H NMR) accounted for the separation. These discriminant metabolites are involved in 14 biochemical pathways, indicating that PCa is highly linked to dysregulation of metabolic pathways associated with amino acids and energetic metabolism. CONCLUSION: These findings confirmed the complementary information provided by GC-MS and 1H NMR, enabling a more comprehensive picture of the altered metabolites, underlying pathways and deepening the understanding of PCa development and progression.

Identification of a biomarker panel for improvement of prostate cancer diagnosis by volatile metabolic profiling of urine.

BACKGROUND: The lack of sensitive and specific biomarkers for the early detection of prostate cancer (PCa) is a major hurdle to improve patient management. METHODS: A metabolomics approach based on GC-MS was used to investigate the performance of volatile organic compounds (VOCs) in general and, more specifically, volatile carbonyl compounds (VCCs) present in urine as potential markers for PCa detection. RESULTS: Results showed that PCa patients (n = 40) can be differentiated from cancer-free subjects (n = 42) based on their urinary volatile profile in both VOCs and VCCs models, unveiling significant differences in the levels of several metabolites. The models constructed were further validated using an external validation set (n = 18 PCa and n = 18 controls) to evaluate sensitivity, specificity and accuracy of the urinary volatile profile to discriminate PCa from controls. The VOCs model disclosed 78% sensitivity, 94% specificity and 86% accuracy, whereas the VCCs model achieved the same sensitivity, a specificity of 100% and an accuracy of 89%. Our findings unveil a panel of 6 volatile compounds significantly altered in PCa patients' urine samples that was able to identify PCa, with a sensitivity of 89%, specificity of 83%, and accuracy of 86%. CONCLUSIONS: It is disclosed a biomarker panel with potential to be used as a non-invasive diagnostic tool for PCa.

NMR-based metabolomics studies of human prostate cancer tissue.

INTRODUCTION: Prostate cancer (PCa) is one of the most prevalent cancers in men worldwide. Serum prostate-specific antigen (PSA) remains the most used biomarker in the detection and management of patients with PCa, in spite of the problems related with its low specificity, false positive rate and overdiagnosis. Furthermore, PSA is unable to discriminate indolent from aggressive PCa, which can lead to overtreatment. Early diagnosed and treated PCa can have a good prognosis and is potentially curable. Therefore, the discovery of new biomarkers able to detect clinically significant aggressive PCa is urgently needed. METHODS: This revision was based on an electronic literature search, using Pubmed, with Nuclear Magnetic Resonance (NMR), tissue and prostate cancer as keywords. All metabolomic studies performed in PCa tissues by NMR spectroscopy, from 2007 until March 2018, were included in this review. RESULTS: In the context of cancer, metabolomics allows the analysis of the entire metabolic profile of cancer cells. Several metabolic alterations occur in cancer cells to sustain their abnormal rates of proliferation. NMR proved to be a suitable methodology for the evaluation of these metabolic alterations in PCa tissues, allowing to unveil alterations in citrate, spermine, choline, choline-related compounds, lactate, alanine and glutamate. CONCLUSION: The study of the metabolic alterations associated with PCa progression, accomplished by the analysis of PCa tissue by NMR, offers a promising approach for elucidating biochemical pathways affected by PCa and also for discovering new clinical biomarkers. The main metabolomic alterations associated with PCa development and promising biomarker metabolites for diagnosis of PCa were outlined.

Neuroscience: unveiling the brain of Saimiri collinsi (Squirrel Monkey).

Behavioral intelligence among non-human primates is a somewhat puzzling area to study, since it is closely linked to the morphology of the central nervous system. However, this morphology is still unknown to science in the case of Saimiri collinsi. As a means of assisting future studies on the social behavior of this species, we analyzed the brains of six adult females from the National Primate Center / Pará, which were sent to the Animal Morphological Research Laboratory at the Federal Rural University of Amazônia after death by natural causes. The animals were fixed in formaldehyde solution (10%) and dissected. The brains were covered externally by the dura mater, with long cerebral hemispheres, although there was a complete absence of gyri. The internal structures which form a part of the telencephalon, diencephalon, midbrain, hindbrain and myelencephalon were identified, with the exception of the mammillary bodies. This condition, when linked to the lissencephaly found in these individuals raises questions about the real learning abilities of this species.

Morphological Characterization of Diaphragm in Common Squirrel Monkey (Saimiri sciureus).

The wall of the diaphragm can be affected by congenital or acquired alterations which allow the passage of viscera between the abdominal and chest cavities, allowing the formation of a diaphragmatic hernia. We characterized morphology and performed biometrics of the diaphragm in the common squirrel monkey Saimiri sciureus. After fixation, muscle fragments were collected and processed for optical microscopy. In this species the diaphragm muscle is attached to the lung by phrenopericardial ligament. It is also connected to the liver via the coronary and falciform ligaments. The muscle is composed of three segments in total: 1) sternal; 2) costal, and 3) a segment consisting of right and left diaphragmatic pillars. The anatomical structures analyzed were similar to those reported for other mammals. Histological analysis revealed stable, organized muscle fibers with alternation of light and dark streaks, indicating transverse striation.

Morphology of the Diaphragm Muscle in Southern Tamandua (Tamandua tetradactyla) and its Importance in Cases of Traumatic Hernia.

The wall of the diaphragm can be affected by changes caused by physical trauma, allowing the passage of viscera between the abdominal cavity and thoracic cavity, thus reducing the space for pulmonary expansion, leading to the formation of hernia and possible death. Thus, we aimed to characterize, size and determine the topography of the diaphragmatic muscle in the Southern Tamandua, since clinical and surgical activities in wild animals have become a reality more and more present in veterinary medicine. We used six adult animals, x-rayed and dissected, followed by collection of fragments of muscular portions for histological analysis. Initially we observed that the animals presented 17 thoracic vertebrae, 3 lumbar vertebrae and 5 sacral vertebrae. The diaphragm was conformed by three segments: sternal, costal and right and left diaphragm pillar, with presence of tendinous centre that housed the passage of the caudal vena cava, called foramen of the caudal vena cava. Dorsally to the tendinous centre, already in the muscle portion, we located the esophageal and aortic hiatus. These findings, as well as the microscopic, were equivalent to that found in the general literature also, corroborating with descriptions already carried out in other mammals' diaphragms.

Topography and morphometry of the infraorbital and mandibular foramen of the common sloth (Bradypus variegatus) for use in the application of perineural anaesthesia.

A local anaesthetic block performed in cranial and mandibular foramina contributes towards analgesia and the reduction of systemic anaesthesia in procedures in the oral cavity. However, anatomical differences between in wild animals may require adaptations to the anaesthetic and surgical manoeuvres of the head used in domestic animals. With that in mind, we aimed to describe the topography and morphometry of the infraorbital and mandibular foramina in Bradypus variegatus, to support perineural anaesthetic approaches in the cranio-mandibular region of this species. We demonstrated that in B. variegatus there is no significant variability in the morphometric data, allowing access to the studied foramina to occur in a similar way in adult individuals. Finally, we understand that the techniques for the infraorbital and mandibular foramen needed to be adapted due to the anatomical singularities of the species, which culminated in new reference points for more assertive needle positioning.

Fetal development of the urinary tract of Cuniculus paca.

The species Cuniculus paca is highly subject to predation, whether natural or anthropogenic, and the ability of species to withstand different levels of depredation depends directly on their reproductive dynamics. However, there is little literature on the developmental biology of this species, and so the aim of this paper was to describe the fetal development of the urinary tract of C. paca through morphological analysis. Twelve specimens with estimated gestational ages of between 75 and 157 days were used, divided into 3 groups. We found the urinary tract in pelvic-abdominal topography with macroscopic differentiation between the organs already present in the first ages studied; in addition, the microscopic structural pattern changed little between the groups. This evidence reinforces the precocial development of these individuals. RESEARCH HIGHLIGHTS: With the results obtained for development of urinary tract in Cuniculus paca reinforces the precocial development of these individuals. The urinary tract had pelvic-abdominal topography with macroscopic differentiation between the organs already present in the first ages studied. The microscopic structural pattern changed little between the groups. At all analyzed fetal ages, the cortical region of kidney was characterized by the presence of glomeruli arranged throughout the region, formed by capillary plexuses surrounded by a glomerular capsule. In addition, the cortical region also presented convoluted tubules with cubic epithelial tissue cells and a brush border. The presence of a developed macula densa was observed next to the glomeruli, suggesting the initial formation of the fetal juxtaglomerular apparatus.

Topography landmarks of the infraorbital, mandibular and mentual foramens of Saimiri collinsi (Osgood, 1916) for anesthetic access and blocks.

Due to events related to the urbanization process, specimens of Saimiri collinsi are often referred to veterinarians specializing in the treatment of wild animals. With these professionals and the oral health of this species in mind, we evaluated the skull and the exact location of the infraorbital, mentual and mandibular foramens, with the aim of supporting the anesthetic block for dental procedures in Saimiri collinsi. The infraorbital foramen was located in the maxillary bone and was arranged with one on each side, except in one individual, with a pair in each antimer. The mentual foramen was located in the diastema between the canine tooth and the lateral incisor. The mandibular foramen was located medially on the ramus of the mandible, close to the mandibular incisure. The distances between the foramina and the main reference points, were greater in females than in males (p < 0.05). For access purposes, in the foramens investigated we suggest using a Gingival Needle 30G 21 mm Short, positioned externally at 15º to the maxillary bone to access the infraorbital foramen. Externally, perpendicular to the chin, in the diastema between the lower lateral incisor tooth and the canine tooth, to approach the mentual foramen, and ventral to the edge of the mandibular body, at a 90º angle, to access the mandibular foramen.

Aortic branches and rete mirabile of the limbs of two-toed sloth (Choloepus didactylus).

Choloepus didactylus has reduced metabolism and difficulty in thermoregulation owing to its low body mass, and there are few studies related to the vascularization of abdominal and thoracic organs in this species. Therefore, we macroscopically described the arteries that comprise the aortic arch, thoracic aorta, and abdominal aorta. Six specimens were used, and their arterial systems filled with red latex before fixation in 10% formaldehyde, and fragments of the rete mirabile were processed for histological analysis using light and scanning electron microscopy. In these species, the aortic arch had two branches: the brachiocephalic trunk and left subclavian artery. The initial portion of the abdominal aorta presented four different ramifications, besides to the peculiarities of the adrenal, renal, and iliac arteries. Microscopy of the rete mirabile revealed a muscular artery surrounded by smaller muscular arteries, veins, nerves, and lymphatic tissue joined by connective tissue. Thus, the data obtained have clinical and surgical importance, with applicability in procedures involving vascularization of the thoracic and abdominal organs. We suggest that the rete mirabile is an efficient thermoregulatory structure because it allows the accumulation of blood and the countercurrent heat exchange, as there is no blood mixing.

Horse spleen segmentation technique as large animal model of preclinical trials.

The parenchymal distribution of the splenic artery was studied in order to obtain anatomical basis for partial splenectomy. Thirty two spleens were studied, 26 spleens of healthy horses weighing 320 to 450 kg, aged 3 to 12 years and 6 spleens of fetus removed from slaughterhouse. The spleens were submitted to arteriography and scintigraphy in order to have their vascular pattern examined and compared to the external aspect of the organ aiming establish anatomo-surgical segments. All radiographs were photographed with a digital camera and the digital images were submitted to a measuring system for comparative analysis of areas of dorsal and ventral anatomo-surgical segments. Anatomical investigations into the angioarchitecture of the equine spleen showed a paucivascular area, which coincides with a thinner external area, allowing the organ to be divided in two anatomo-surgical segments of approximately 50% of the organ each.

Urogenital development and sexual differentiation in males of the species Dasyprocta fuliginosa.

Thirteen black agouti (Dasyprocta fuliginosa) male fetuses, acquired in two areas of the Amazon Forest, were used for the purpose of morphologically describing the fetal male urogenital organs with a focus on addressing histological and macroscopic aspects. The organs of interest were dissected and photographed in situ and ex situ. Fragments were collected and subjected to routine histological processing for inclusion in paraffin, which was cut and stained by haematoxylin and eosin and Gomori's Trichrome methods and subsequently analysed in a light microscope. The results showed that the urinary tract is like that of domestic animals and is composed of smooth unilobed kidneys covered by a dense capsule of connective tissue and divided into two well-defined regions, cortical and medullary. Ureters, urethra and urinary bladder also showed macroscopic and microscopic characteristics similar to those of domestic animals. The penis of these animals has fibroelastic characteristics, with numerous keratinized structures at its apex. In the middle third of the penis, a "U" shaped penile flexure was seen; the glans penis is covered by a keratinized epidermis containing horny spicules. The presence of a penile bone in an endochondral ossification process was observed, being more developed in gestational ages greater than 76 days. The annex glands were not observed, probably because they were fetuses; only the ampulla of the ductus deferens was identified.

Comparative morphology of the paw pad of the main arboreal Xenarthras from Amazon.

Paw pads are specializations of the integument and important shock absorbers of the locomotor system, as well as pressure, pain, temperature, storage and excretion sensors. Aiming to describe the paw pad morphology of the main arboreal xenarthras species in the Amazon, 16 animals were studied, Bradypus variegatus (6), Choloepus didactylus (5), Tamandua tetradactyla (3) and Cyclopes didactylus (2) that after death were donated to the Animal Morphological Research Laboratory (LaPMA / Ufra). The corpses were thawed and fixed with 10% aqueous formalin solution. The paw pads were measured, photographed and removed by skin incision dorsally to them. Fragments were used for routine histological processing, using two staining techniques: Hematoxylin-Eosin (HE) and Gomori's Trichrome, in sections of 6 to 8 µm. Choloepus didactylus, Tamandua tetradactyla and Cyclopes didactylus have digital paw pads, one in each digit, and one palm, as well as a plantar, whose shapes and colours are distinct from each other. Bradypus variegatus, however, has only one palmar and one plantar pad. Histologically, they have keratinized stratified squamous epithelium, supported by a large amount of collagen fibres and fibroblast cords in the dermis and hypodermis. Groups of eccrine sweat glands were observed in the reticular dermis of C. didactylus, B. variegatus, T. tetradactyla and only in the hypodermis of Cyclopes didactylus.

Enantioselectivity of Pentedrone and Methylone on Metabolic Profiling in 2D and 3D Human Hepatocyte-like Cells.

Pentedrone and methylone can express stereoselectivity in toxicokinetic and toxicodynamic processes. Similarly, their chiral discrimination in metabolism, which was not yet evaluated, can result in different metabolic profiles and subsequent hepatotoxic effects. Therefore, the aim of this work was to assess, for the first time, both the hepatic cytotoxic and metabolic profile of pentedrone and methylone enantiomers using physiologically relevant in vitro models. The hepatotoxicity of these compounds was observed in a concentration-dependent manner in human stem-cell-derived hepatocyte-like cells (HLCs) cultured under 3D (3D-HLCs) and 2D (2D-HLCs) conditions. Enantioselectivity, on the other hand, was only shown for pentedrone (1 mM) in 3D-HLCs, being R-(-)-pentedrone the most cytotoxic. Furthermore, the metabolic profile was initially evaluated in human liver microsomes (HLM) and further demonstrated in 3D-HLCs and 2D-HLCs applying a gas chromatography coupled to a mass spectrometer (GC-MS) technique. Methylone and pentedrone showed distinct and preferential metabolic routes for their enantiomers, resulting in the production of differentiated metabolites; R-(+)-methylone and R-(-)-pentedrone are the most metabolized enantiomers. In conclusion, the results demonstrated enantioselectivity for pentedrone and methylone in the metabolic processes, with enantioselectivity in cytotoxicity for pentedrone.

Morphology of the male reproductive tract of Eira barbara.

Eira barbara, popularly known as irara, is a medium-sized carnivore member of the Mustelidae family. Despite its important role in the ecosystems in which its lives, data on the internal morphology of E. barbara remains scarce. Therefore, this study aimed to provide knowledge regarding the anatomy of the male reproductive system of this species to improve understanding of its reproduction to inform its conservation. We studied seven specimens who had died after being run over. The specimens were dissected for the evaluation of the reproductive system, which comprised a globular scrotum; a small pendulum covered with light-coloured hair; a pair of testicles of firm consistency and ellipsoid shape and suspended in the scrotum by the spermatic cord; a paired duct system; an ampoule of the deferens duct and prostate constituting the set of attached glands; a urethra divided into pelvic and penile portions; a penis with a baculum having a novel "C" shaped apex; and a prepuce. Microscopically, the testicular parenchyma consisted of seminiferous tubules separated by intertubular spaces formed by loose connective tissue, fibrocytes, Leydig cells, and blood and lymph vessels. The epididymis was surrounded by a capsule of dense connective tissue and extended to form septa. The baculum was microscopically classified as a compact bone containing several bony lamellae with osteocytes and osteoblasts. The macro and microscopic findings were generally similar to those of domestic carnivores, with some notable differences.

Ocular microscopy of Bradypus variegatus.

Bradypus variegatus, popularly known as common sloth or brown-throated sloth, is a species with wide geographical distribution in Brazil. Expand on the literature about the species, we aimed to describe the morphology of its eye and its main annexes. For this study, six frozen specimens donated to the Animal Morphological Research Laboratory of the Federal Rural University of Amazonia were used. The specimens were fixed in a 10% aqueous solution of formaldehyde. The structures that constitute the ocular apparatus were identified and described after dissection and histological analysis using light microscopy. The bulbar conjunctiva presented a stratified cuboidal epithelium, with a considerable number of serous glands. The stroma layer and the posterior limiting lamina were found well defined in the species. The corneoscleral meshwork possessed a large opening for drainage of aqueous humour, followed by a wide retinal region, in which a layer of photoreceptors was visualized. Cones and rods could not be discriminated from each other. Microscopy of the medial angle of the eye revealed the cartilage and glands of the third eyelid, and a well-developed lacrimal gland structure. Bradypus variegatus distinguishing features and similarities in comparison with other domestic and wild mammal and bird species already described were investigated, which expounded the limited knowledge that is presently available on the morphology of wild fauna. The data extend the limited knowledge that are presently available on the ocular histology of B. variegatus.

Primary results of STRONG: An open-label, multicenter, phase 3b study of fixed-dose durvalumab monotherapy in previously treated patients with urinary tract carcinoma.

BACKGROUND: Prior durvalumab (anti-PD-L1 agent) studies in platinum-refractory metastatic urothelial carcinoma evaluated a dose of 10 mg/kg administered every two weeks. The nonrandomised phase 3b STRONG study (NCT03084471) evaluated the safety and efficacy of fixed-dose durvalumab at a more convenient dosing schedule in a previously treated patient population, more similar to a real-world clinical setting. PATIENTS AND METHODS: 867 patients with urothelial or nonurothelial urinary tract carcinoma (UTC) who progressed on or after platinum or nonplatinum chemotherapy were treated with durvalumab 1500 mg every four weeks; 87% had an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0-1, and 13% had an ECOG PS of 2. The primary end-point was the incidence of adverse events of special interest (AESIs), including immune-mediated AEs (imAEs). Secondary and exploratory end-points included overall survival (OS), objective response rate (ORR) and disease control rate (at six and 12 months) (DCR). RESULTS: AESIs of any grade were reported in 51% of patients (8% grade ≥ 3). The incidence of imAEs was 11% (2% grade ≥ 3). The median OS was 7.0 months (95% confidence interval [CI]: 6.4-8.2) and ORR was 18% (95% CI: 14.8-20.6), with complete responses in 5% of patients and a DCR at six months of 19% (95% CI: 16.1-22.1). CONCLUSION: Fixed-dose durvalumab monotherapy every four weeks has an acceptable safety profile and yields durable clinical activity in previously chemotherapy-treated patients with UTC. Safety and efficacy are consistent with previous durvalumab studies and other anti-PD-1/PD-L1 agents in this setting. CLINICALTRIALS. GOV IDENTIFIER: NCT03084471https://clinicaltrials.gov/ct2/show/NCT03084471.

Epigenomics in Hurthle Cell Neoplasms: Filling in the Gaps Towards Clinical Application.

It has been widely described that cancer genomes have frequent alterations to the epigenome, including epigenetic silencing of various tumor suppressor genes with functions in almost all cancer-relevant signalling pathways, such as apoptosis, cell proliferation, cell migration and DNA repair. Epigenetic alterations comprise DNA methylation, histone modification, and microRNAs dysregulated expression and they play a significant role in the differentiation and proliferation properties of TC. In this review, our group assessed the published evidence on the tumorigenic role of epigenomics in Hurthle cell neoplasms (HCN), highlighting the yet limited, heteregeneous and non-validated data preventing its current use in clinical practice, despite the well developed assessment techniques available. The identified evidence gaps call for a joint endeavour by the medical community towards a deeper and more systematic study of HCN, aiming at defining epigenetic markers in early diagnose, allowing for accurate stratification of maligancy and disease risk and for effective systemic treatment.