Coupled Mass-Spectrometry-Based Lipidomics Machine Learning Approach for Early Detection of Clear Cell Renal Cell Carcinoma.

A discovery-based lipid profiling study of serum samples from a cohort that included patients with clear cell renal cell carcinoma (ccRCC) stages I, II, III, and IV (n = 112) and controls (n = 52) was performed using ultraperformance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry and machine learning techniques. Multivariate models based on support vector machines and the LASSO variable selection method yielded two discriminant lipid panels for ccRCC detection and early diagnosis. A 16-lipid panel allowed discriminating ccRCC patients from controls with 95.7% accuracy in a training set under cross-validation and 77.1% accuracy in an independent test set. A second model trained to discriminate early (I and II) from late (III and IV) stage ccRCC yielded a panel of 26 compounds that classified stage I patients from an independent test set with 82.1% accuracy. Thirteen species, including cholic acid, undecylenic acid, lauric acid, LPC(16:0/0:0), and PC(18:2/18:2), identified with level 1 exhibited significantly lower levels in samples from ccRCC patients compared to controls. Moreover, 3α-hydroxy-5α-androstan-17-one 3-sulfate, cis-5-dodecenoic acid, arachidonic acid, cis-13-docosenoic acid, PI(16:0/18:1), PC(16:0/18:2), and PC(O-16:0/20:4) contributed to discriminate early from late ccRCC stage patients. The results are auspicious for early ccRCC diagnosis after validation of the panels in larger and different cohorts.

Mass Spectrometry-Based Metabolic Fingerprinting Contributes to Unveil the Role of RSUME in Renal Cell Carcinoma Cell Metabolism.

Clear cell renal cell carcinoma (ccRCC) is a heterogeneous disease with 50-80% patients exhibiting mutations in the von Hippel-Lindau (VHL) gene. RSUME (RWD domain (termed after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases)-containing protein small ubiquitin-related modifier (SUMO) enhancer) acts as a negative regulator of VHL function in normoxia. A discovery-based metabolomics approach was developed by means of ultraperformance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (MS) for fingerprinting the endometabolome of a human ccRCC cell line 786-O and three other transformed cell systems (n = 102) with different expressions of RSUME and VHL. Cross-validated orthogonal projection to latent structures discriminant analysis models were built on positive, negative, and a combination of positive- and negative-ion mode MS data sets. Discriminant feature panels selected by an iterative multivariate classification allowed differentiating cells with different expressions of RSUME and VHL. Fifteen identified discriminant metabolites with level 1, including glutathione, butyrylcarnitine, and acetylcarnitine, contributed to understand the role of RSUME in ccRCC. Altered pathways associated with the RSUME expression were validated by biological and bioinformatics analyses. Combined results showed that in the absence of VHL, RSUME is involved in the downregulation of the antioxidant defense system, whereas in the presence of VHL, it acts in rerouting energy-related pathways, negatively modulating the lipid utilization, and positively modulating the fatty acid synthesis, which may promote deposition in droplets.

Metabolic Footprinting of a Clear Cell Renal Cell Carcinoma in Vitro Model for Human Kidney Cancer Detection.

A protocol for harvesting and extracting extracellular metabolites from an in vitro model of human renal cell lines was developed to profile the exometabolome by means of a discovery-based metabolomics approach using ultraperformance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry. Metabolic footprints provided by conditioned media (CM) samples ( n = 66) of two clear cell Renal Cell Carcinoma (ccRCC) cell lines with different genetic backgrounds and a nontumor renal cell line, were compared with the human serum metabolic profile of a pilot cohort ( n = 10) comprised of stage IV ccRCC patients and healthy individuals. Using a cross-validated orthogonal projection to latent structures-discriminant analysis model, a panel of 21 discriminant features selected by iterative multivariate classification, allowed differentiating control from tumor cell lines with 100% specificity, sensitivity, and accuracy. Isoleucine/leucine, phenylalanine, N-lactoyl-leucine, and N-acetyl-phenylalanine, and cysteinegluthatione disulfide (CYSSG) were identified by chemical standards, and hydroxyprolyl-valine was identified with MS and MS/MS experiments. A subset of 9 discriminant features, including the identified metabolites except for CYSSG, produced a fingerprint of classification value that enabled discerning ccRCC patients from healthy individuals. To our knowledge, this is the first time that N-lactoyl-leucine is associated with ccRCC. Results from this study provide a proof of concept that CM can be used as a serum proxy to obtain disease-related metabolic signatures.

Bromopyrrole alkaloids isolated from the Patagonian bryozoan Aspidostoma giganteum.

Nine new bromopyrrole alkaloids, aspidostomides A-H and aspidazide A (1-9), were isolated from the Patagonian bryozoan Aspidostoma giganteum. Aspidostomides A-H have dibromotyrosine- or bromotryptophan-derived moieties forming either linear amides or pyrroloketopiperazine-type lactams with a bromopyrrole carboxylic acid as a common structural motif. On the other hand, aspidazide A is a rare asymmetric acyl azide formed by an N-N link of two different pyrroloketopiperazine lactams and is the first isolated compound of this class from marine invertebrates. This work is the first report of secondary metabolites isolated from a bryozoan from the Patagonian region. The structures of compounds 1-9 were elucidated by spectroscopic methods and chemical transformations. One of these compounds, aspidostomide E (5), was moderately active against the 786-O renal carcinoma cell line.

Effect of Angeli's salt on the glutamate/glutamine cycle activity and on glutamate excitotoxicity in the hamster retina.

Glutamate is the main excitatory neurotransmitter in the retina, but it is toxic when present in excessive amounts. It is well known that NO is involved in glutamate excitotoxicity, but information regarding the possibility that NO-related species could reciprocally affect glutamate synaptic levels was not previously provided. The dependence of glutamatergic neurons upon glia via the glutamate/glutamine cycle to provide the precursor for neurotransmitter glutamate is well established. The aim of the present work was to comparatively analyze the effect of nitroxyl and NO on the retinal glutamate/glutamine cycle in vitro activity. For this purpose, Angeli's salt (AS) and diethylamine NONOate (DEA/NO) were used as nitroxyl and NO donor, respectively. AS and DEA/NO significantly decreased retinal l-glutamate uptake and glutamine synthetase activity, but only AS decreased l-glutamine influx. Dithiothreitol prevented all the effects of AS and DEA/NO. The intravitreal injection of DEA/NO (but not AS) or a supraphysiological concentration of glutamate induced retinal histological alterations. Although AS could increase glutamate synaptic concentration in vitro, the histological alterations induced by glutamate were abrogated by AS. These results suggest that nitroxyl could regulate the hamster retinal glutamatergic pathway by acting through differential mechanisms at pre- and postsynaptic level.

Synthesis of steroidal quinones and hydroquinones from bile acids by Barton radical decarboxylation and benzoquinone addition. Studies on their cytotoxic and antifungal activities.

Twelve new hydroquinones and quinones (4a-c to 7a-c) derived from free or peracetylated bile acids were prepared by a Barton decarboxylation reaction, with subsequent trapping of the resulting free radical by benzoquinone. All new compounds were completely characterized by 2D NMR techniques and screened for antifungal and cytotoxic activity. One of the new hydroquinones (7b) showed promising results against the human pancreatic ductal carcinoma cell line PANC1, with similar cytotoxic activity as the commercial chemotherapy drug doxorubicin.

Wall structures of myocardial precapillary arterioles and postcapillary venules reexamined and reconstructed in vitro for studies on barrier functions.

The barrier functions of myocardial precapillary arteriolar and postcapillary venular walls (PCA or PCV, respectively) are of considerable scientific and clinical interest (regulation of blood flow and recruitment of immune defense). Using enzyme histochemistry combined with confocal microscopy, we reexamined the cell architecture of human PCA and PVC and reconstructed appropriate in vitro models for studies of their barrier functions. Contrary to current opinion, the PCA endothelial tube is encompassed not by smooth muscle cells but rather by a concentric layer of pericytes cocooned in a thick, microparticle-containing extracellular matrix (ECM) that contributes substantially to the tightness of the arteriolar wall. This core tube extends upstream into the larger arterioles, there additionally enwrapped by smooth muscle. PCV consist of an inner layer of large, contractile endothelial cells encompassed by a fragile, wide-meshed pericyte network with a weakly developed ECM. Pure pericyte and endothelial cell preparations were isolated from PCA and PCV and grown in sandwich cultures. These in vitro models of the PCA and PCV walls exhibited typical histological and functional features. In both plasma-like (PLM) and serum-containing (SCM) media, the PCA model (including ECM) maintained its low hydraulic conductivity (L(P) = 3.24 ± 0.52·10(-8)cm·s(-1)·cmH(2)O(-1)) and a high selectivity index for transmural passage of albumin (SI(Alb) = 0.95 ± 0.02). In contrast, L(P) and SI(Alb) in the PCV model (almost no ECM) were 2.55 ± 0.32·10(-7)cm·s(-1)·cmH(2)O(-1) and 0.88 ± 0.03, respectively, in PLM, and 1.39 ± 0.10·10(-6)cm·s(-1)·cmH(2)O(-1) and 0.49 ± 0.04 in SCM. With the use of these models, systematic, detailed studies on the regulation of microvascular barrier properties now appear to be feasible.

Regulation of coronary venular barrier function by blood borne inflammatory mediators and pharmacological tools: insights from novel microvascular wall models.

We hypothesized that postcapillary venules play a central role in the control of the tightness of the coronary system as a whole, particularly under inflammatory conditions. Sandwich cultures of endothelial cells and pericytes of precapillary arteriolar or postcapillary venular origin from human myocardium as models of the respective vascular walls (sandwich cultures of precapillary arteriolar or postcapillary venular origin) were exposed to thrombin and components of the acutely activatable inflammatory system, and their hydraulic conductivity (L(P)) was registered. L(P) of SC-PAO remained low under all conditions (3.24 ± 0.52·10(-8)cm·s(-1)·cmH(2)O(-1)). In contrast, in the venular wall model, PGE(2), platelet-activating factor (PAF), leukotriene B(4) (LTB(4)), IL-6, and IL-8 induced a prompt, concentration-dependent, up to 10-fold increase in L(P) with synergistic support when combined. PAF and LTB(4) released by metabolically cooperating platelets, and polymorphonuclear leucocytes (PMNs) caused selectively venular endothelial cells to contract and to open their clefts widely. This breakdown of the barrier function was preventable and even reversible within 6-8 h by the presence of 50 µM quercetin glucuronide (QG). LTB(4) synthesis was facilitated by biochemical involvement of erythrocytes. Platelets segregated in the arterioles and PMNs in the venules of blood-perfused human myocardium (histological studies on donor hearts refused for heart transplantation). Extrapolating these findings to the coronary microcirculation in vivo would imply that the latter's complex functionality after accumulation of blood borne inflammatory mediators can change rapidly due to selective breakdown of the postcapillary venular barrier. The resulting inflammatory edema and venulo-thrombosis will severely impair myocardial performance. The protection afforded by QG could be of particular relevance in the context of cardiosurgical intervention.

Isolation, bulk cultivation, and characterization of coronary microvascular pericytes: the second most frequent myocardial cell type in vitro.

Densely arranged pericytes engird the endothelial tube of all coronary microvessels. Since the experimental access to these abundant cells in situ is difficult, a prerequisite for broader investigation is the availability of sufficient numbers of fully differentiated pericytes in homogenous culture. To reach this goal, we applied strictly standardized cell isolation techniques, optimized culture methods and specific histological staining. Approximately 1,000-fold enriched pericytes were proteolytically detached from highly purified coronary microvascular networks (density gradient centrifugation) of eight mammalian species including human. Addition of species-autologous fetal or neonatal serum (10-20% vol/vol) was a precondition for longer term survival of homogenous pericyte cultures. This ensured optimal growth (doubling time <14 h) and full expression of pericyte-specific markers. In 3-mo, 10(10) pericytes (15 g) could be cultivated from 1 bovine heart. Pericytes could be stored in liquid N(2), recultured, and passaged repeatedly without loss of typical features. In cocultures with EC or vascular smooth muscle cells, pericytes transferred fluorescent calcein to each other and to EC via their antler-like extensions, organized angiogenetic sprouting of vessels, and rapidly activated coagulation factors X and II via tissue factor and prothrombinase. The interconnected pericytes of the coronary system are functionally closely correlated with the vascular endothelium and may play key roles in the adjustment of local blood flow, the regulation of angiogenic processes, and the induction of procoagulatory processes. Their successful bulk cultivation enables direct experimental access under defined in vitro conditions and the isolation of pericyte specific antigens for the production of specific antibodies.

Differential effect of acute and persistent Junin virus infections on the nucleo-cytoplasmic trafficking and expression of heterogeneous nuclear ribonucleoproteins type A and B.

Heterogeneous nuclear ribonucleoproteins A and B (hnRNPs A/B), cellular RNA-binding proteins that participate in splicing, trafficking, translation and turnover of mRNAs, have been implicated in the life cycles of several cytoplasmic RNA viruses. Here, we demonstrate that silencing of hnRNPs A1 and A2 significantly reduces the replication of the arenavirus Junín virus (JUNV), the aetiological agent of Argentine haemorrhagic fever. While acute JUNV infection did not modify total levels of expression of hnRNPs A/B in comparison with uninfected cells, non-cytopathic persistent infection exhibited low levels of these cell proteins. Furthermore, acutely infected cells showed a cytoplasmic relocalization of overexpressed hnRNP A1, probably related to the involvement of this protein in virus replicative cycle. This cytoplasmic accumulation was also observed in cells expressing viral nucleoprotein (N), and co-immunoprecipitation studies revealed the interaction between hnRNP A1 and N protein. By contrast, a predominantly nuclear distribution of overexpressed hnRNP A1 was found during persistent infection, even in the presence of endogenous or overexpressed N protein, indicating a differential modulation of nucleo-cytoplasmic trafficking in acute and persistent JUNV infections.