Practicability of clinical application of bladder cancer molecular classification and additional value of epithelial-to-mesenchymal transition: prognostic value of vimentin expression.

BACKGROUND: Bladder cancer (BlCa) taxonomy has proved its impact in patient outcome and selection for targeted therapies, but such transcriptomic-based classification has not yet translated to routine practice. Moreover, epithelial-to-mesenchymal transition (EMT) has shown relevance in acquisition of more aggressive BlCa phenotype. We aimed to test the usefulness of the molecular classification, as defined by immunohistochemistry (a routinely performed and easy-to-implement technique), in a well-defined BlCa cohort of both non-muscle invasive (NMIBC) and muscle invasive (MIBC) disease. Also, we aimed to assess the additional prognostic value of the mesenchymal marker vimentin to the stratification strategy. METHODS: A total of 186 samples were available. Immunohistochemistry/RT-qPCR for luminal markers GATA3/FOXA1, basal markers KRT5/KRT6A and vimentin were performed. RESULTS: mRNA expression levels of the markers positively correlated with immunoexpression scores. We found substantial overlapping in immunoexpression of luminal and basal markers, evidencing tumor heterogeneity. In MIBC, basal tumors developed recurrence more frequently. NMIBC patients with higher vimentin immunoexpression endured poorer disease-free survival, and increased expression was observed from normal bladder-NMIBC-MIBC-metastases. CONCLUSIONS: The classification has the potential to be implemented in routine, but further adjustments in practical scoring should be defined; focusing on additional markers, including those related to EMT, may further refine BlCa molecular taxonomy.

A Secreted Phospholipase A2 Induces Formation of Smooth Muscle Foam Cells Which Transdifferentiate to Macrophage-Like State.

Vascular smooth muscle cells (VSMCs) loaded with lipid droplets (LDs) are markers of atherosclerosis. In this disease, inflammatory Group IIA-secreted phospholipase A2s (GIIA sPLA2s) are highly expressed in VSMCs, but their actions in these cells are unknown. Here, we investigated the ability of myotoxin III (MT-III), an ophidian GIIA sPLA2 sharing structural and functional features with mammalian GIIA sPLA2s, to induce LD formation and lipid metabolism factors involved in this effect. Modulation of VSMC phenotypes by this sPLA2 was also evaluated. Incubation of VSMCs with MT-III significantly increased the number of LDs. MT-III upregulated scavenger receptor type 1 (SR-A1) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) protein expression and enhanced acetylated-low density lipoprotein (acLDL) uptake by VSMCs, revealing the ability of a GIIA PLA2 to modulate scavenger receptor activities. MT-III induced translocation and protein expression of PPAR-γ and -ß/δ. Inhibition of peroxisome proliferator-activated receptors (PPARs) and diacylglycerol O-acyltransferase (DGAT) and acyl-CoA:cholesterolacyltransferase (ACAT) enzymes abrogated MT-III-induced LD formation. Moreover, in response to MT-III, VSMCs acquired phagocytic activity and expressed macrophage markers CD68 and MAC-2. In conclusion, MT-III is able to stimulate VSMCs and recruit factors involved in lipid uptake and metabolism, leading to the formation of VSMC-derived foam cells with acquisition of macrophage-like markers and functions.

A Snake Venom-Secreted Phospholipase A2 Induces Foam Cell Formation Depending on the Activation of Factors Involved in Lipid Homeostasis.

MT-III, a snake venom GIIA sPLA2, which shares structural and functional features with mammalian GIIA sPLA2s, activates macrophage defense functions including lipid droplet (LDs) formation, organelle involved in both lipid metabolism and inflammatory processes. Macrophages (MΦs) loaded with LDs, termed foam cells, characterize early blood vessel fatty-streak lesions during atherosclerosis. However, the factors involved in foam cell formation induced by a GIIA sPLA2 are still unknown. Here, we investigated the participation of lipid homeostasis-related factors in LD formation induced by MT-III in macrophages. We found that MT-III activated PPAR-γ and PPAR-ß/δ and increased the protein levels of both transcription factors and CD36 in macrophages. Pharmacological interventions evidenced that PPAR-γ, PPAR-ß/δ, and CD36 as well as the endoplasmic reticulum enzymes ACAT and DGAT are essential for LD formation. Moreover, PPAR-ß/δ, but not PPAR-γ, is involved in MT-III-induced PLIN2 protein expression, and both PPAR-ß/δ and PPAR-γ upregulated CD36 protein expression, which contributes to MT-III-induced COX-2 expression. Furthermore, production of 15-d-PGJ2, an activator of PPARs, induced by MT-III, was dependent on COX-1 being LDs an important platform for generation of this mediator.

Effect of photobiomodulation on endothelial cell exposed to Bothrops jararaca venom.

Bleeding is a common feature in envenoming caused by Bothrops snake venom due to extensive damage to capillaries and venules, producing alterations in capillary endothelial cell morphology. It has been demonstrated, in vivo, that photobiomodulation (PBM) decreases hemorrhage after venom inoculation; however, the mechanism is unknown. Thus, the objective was to investigate the effects of PBM on a murine endothelial cell line (tEnd) exposed to Bothrops jararaca venom (BjV). Cells were exposed to BjV and irradiated once with either 660- or 780-nm wavelength laser light at energy densities of 4 and 5 J/cm(2), respectively, and irradiation time of 10 s. Cell integrity was analyzed by crystal violet and cell viability/mitochondrial metabolism by MTT assay. The release of lactic dehydrogenase (LDH) was quantified as a measure of cell damage. In addition, cytokine IL1-ß levels were measured in the supernatant. PBM at 660 and 780 nm wavelength was able to increase cellular viability and decrease the release of LDH and the loss of cellular integrity. In addition, the concentration of pro-inflammatory cytokine IL1-ß was reduced after PBM by both wavelengths. The data reported herein indicates that irradiation with red or near-infrared laser resulted in protection on endothelial cells after exposure to Bothrops venom and could be, at least in part, a reasonable explanation by the beneficial effects of PBM inhibiting the local effects induced by Bothrops venoms, in vivo.

Nutrient variability and its influence on nitrogen processes in a highly turbid tropical estuary (Bangpakong, Gulf of Thailand).

Estuarine ecosystems in SE Asia have been poorly studied when compared to other tropical environments. Important gaps exist particularly in the understanding of their biogeochemical function and contribution to global change. In this work we looked into N-turnover in the water column and sediments of the Bangpakong estuary (13°N). A seasonal sampling program was performed along the salinity gradient covering different stretches of the estuary (68km). Key physical and chemical characteristics were also monitored in order to unravel possible environmental controls. Results showed the occurrence of active denitrification in sediments (5.7-50.9nmol N-N2/(cm(3)·hr)), and water column (3.5-1044pmol N-N2/(cm(3)·hr)). No seasonal or spatial variability was detected for denitrification potential in sediment samples. However, in the water column, the denitrification activity peaked during the transition season in the downstream sites coinciding with high turbidity levels. Therefore, in that period of the year, the water column compartment may be an important contributor to nitrate reduction within the estuary. The rather low nitrification rates detected were not always measurable, probably due to the reduced oxygen content and high siltation. This study is one of the few dealing simultaneously with sediments and water column processes in a highly turbid tropical estuary. Therefore, it emerges as a valuable contribution for the understanding of the dynamics of the nitrogen cycle in tropical environments by exploring the role of estuarine N microbial activity in reducing the effects of increased nitrogen loads.

Endurance training prevents TWEAK but not myostatin-mediated cardiac remodelling in cancer cachexia.

Strategies to prevent tumour burden-induced cardiac remodelling that might progress to heart failure are necessary to improve patients' health outcomes and tolerability to cancer therapies. Exercise has been suggested as a measure to prevent cardiac damage; however, its effectiveness on regulating cardiac remodelling secondary to cancer was never addressed. Using an animal model of mammary tumorigenesis, we studied the impact of 35weeks of endurance training on heart, focusing on the signalling pathways modulated by pro-inflammatory and wasting cytokines. The cardiac fibrosis and myofiber disorganization induced by tumour burden was paralleled by the increase of myostatin and TWEAK with the activation of signalling pathways involving Smad-3, NF-κB, TRAF-6 and atrogin-1. The activation of Akt/mTOR was observed in heart from rats with tumours, for which contributed the extracellular matrix. Endurance training prevented the increase of serum and cardiac TWEAK promoted by cancer, as well as the activation of NF-κB, TRAF6, atrogin-1 and p70S6K in heart. Data highlight the impact of exercise in the modulation of signalling pathways activated by wasting cytokines and the resulting outcomes on heart adaptation. Future studies focused on the cellular pathways underlying cardiac remodelling will assist in the development of exercise programs targeting cancer-related cardiac alterations.

Is brain natriuretic peptide a reliable biomarker of hydration status in all peritoneal dialysis patients?

BACKGROUND: Achievement of euvolemia is a fundamental challenge in the peritoneal dialysis (PD) population. Bioimpedance spectroscopy (BIS) is one of the best techniques for routine assessment of hydration status (HS) in PD, but in recent years, the role of brain natriuretic peptides (BNP) in the assessment of volume status has gained interest. The aim of this study was to investigate the relation between BNP and volume status as measured by BIS in PD patients and to assess how these variables correlate according to the time that a patient has been on PD. METHODS: We prospectively studied 68 PD patients from whom measurements of BNP and assessments of HS by BIS were performed every 3 months. Three groups were defined based on HS: group A, measurements of HS <-1.1 liters (underhydrated); group B, measurements of HS between -1.1 and +1.1 liters (normohydrated), and group C, measurements of HS >+1.1 liters (overhydrated). Measurements were also separated according to the time on PD (<6 vs. ≥6 months). Correlation between HS and BNP was performed using Spearman's correlation. RESULTS: We performed a total of 478 measurements of HS and BNP. There was a statistically significant difference in BNP (p < 0.001) among three HS groups, with higher levels of BNP detected in overhydrated patients. We found a positive correlation between HS and BNP (rs = 0.28; p <0.001) that seemed stronger in the first 6 months on PD (rs = 0.42; p = 0.006). CONCLUSIONS: BNP correlated positively with fluid overload measured by HS, and this correlation was stronger in the first 6 months on PD.

An Asp49 phospholipase A2 from snake venom induces cyclooxygenase-2 expression and prostaglandin E2 production via activation of NF-κB, p38MAPK, and PKC in macrophages.

Phospholipases A2 (PLA2) are key enzymes for production of lipid mediators. We previously demonstrated that a snake venom sPLA2 named MT-III leads to prostaglandin (PG)E2 biosynthesis in macrophages by inducing the expression of cyclooxygenase-2 (COX-2). Herein, we explored the molecular mechanisms and signaling pathways leading to these MT-III-induced effects. Results demonstrated that MT-III induced activation of the transcription factor NF-κB in isolated macrophages. By using NF-κB selective inhibitors, the involvement of this factor in MT-III-induced COX-2 expression and PGE2 production was demonstrated. Moreover, MT-III-induced COX-2 protein expression and PGE2 release were attenuated by pretreatment of macrophages with SB202190, and Ly294002, and H-7-dihydro compounds, indicating the involvement of p38MAPK, PI3K, and PKC pathways, respectively. Consistent with this, MT-III triggered early phosphorylation of p38MAPK, PI3K, and PKC. Furthermore, SB202190, H-7-dihydro, but not Ly294002 treatment, abrogated activation of NF-κB induced by MT-III. Altogether, these results show for the first time that the induction of COX-2 protein expression and PGE2 release, which occur via NF-κB activation induced by the sPLA2-MT-III in macrophages, are modulated by p38MAPK and PKC, but not by PI3K signaling proteins.

A strategy to potentiate Cd phytoremediation by saltmarsh plants - autochthonous bioaugmentation.

The recovery of estuarine environments is in need. Phytoremediation could be a valid option to reduce pollution while preserving natural biodiversity. In this work, estuarine sediments colonized by Juncus maritimus or Phragmites australis were spiked with cadmium in the absence and in the presence of an autochthonous microbial consortium resistant to the metal. The aim of this study was to increase the potential for cadmium phytoremediation that these two halophyte plants have shown. Experiments were carried out in greenhouses with an automatic irrigation system that simulated estuarine tidal cycles. After 2 months, Cd concentration in P. australis stems increased up to 7 times when the rhizosphere was inoculated with the microbial consortium. So, P. australis phytoextraction potential was increased through autochthonous bioaugmentation. As for J. maritimus, up to 48% more Cd (total amount) was observed in its belowground tissues after being subjected to autochthonous bioaugmentation. Therefore, the phytostabilization potential of this plant was promoted. For both plants this increase in cadmium uptake did not cause significant signs of toxicity. Therefore, the addition of autochthonous microorganisms resistant to cadmium seems to be a valuable strategy to potentiate phytoremediation of this metal in saltmarshes, being useful for the recovery of moderately impacted estuaries. This will contribute for an effective management of these areas. Research on this topic regarding estuarine ecosystems, especially saltmarshes, is, to our knowledge, inexistent.

Role of nitric oxide and signaling pathways modulating the stimulatory effect of snake venom secretory PLA2S on non-opsonized zymosan phagocytosis by macrophages.

The phagocytic activity of macrophages activated with MT-II, a Lys-49 PLA2 homolog, and MT-III, an Asp-49 PLA2, from Bothrops asper snake venom, was investigated in this study using a pharmacological approach. Stimulating thioglycollate-elicited macrophages with both venom components enhanced their ability to phagocytose non-opsonized zymosan particles. MT-II and MT-III-induced phagocytosis was drastically inhibited by pretreating cells with L-NAME, aminoguanidine or L-NIL, cNOS or iNOS inhibitors, or with ODQ (sGC inhibitor) or Rp-cGMPS (PKG inhibitor). These results indicate that the NO/sGC/GMP/PKG pathway plays an essential role in the ß-glucan-mediated phagocytosis induced in macrophages by these venom-secretory PLA2s.

PKC-α is involved in the signaling of phagocytosis induced by two snake venom secretory PLA2S in macrophages.

Phagocytosis, an essential process for host defense, requires the coordination of a variety of signaling reactions. MT-II, an enzymatically inactive Lys49 phospholipase A2 (PLA2) homolog, and MT-III, a catalytically-active Asp49 PLA2, are known to activate phagocytosis in macrophages. In this study, the signaling pathways mediating phagocytosis, focusing on protein kinases, were investigated. Macrophages from male Swiss mice peritoneum were obtained 96 h after intraperitoneal thioglycolate injection. Phagocytosis was evaluated using non-opsonized zymosan particles in the presence or absence of specific inhibitors, as well as PKC and PKC-α localization by confocal microscopy. Moreover, protein kinase C (PKC) activity was assessed by γP32 ATP in macrophages stimulated by both PLA2s. Data showed that both sPLA2s increased phagocytosis. Cytochalasin D, staurosporine/H7, wortmannin, and herbimycin, inhibitors of actin polymerization, PKC, phosphoinositide 3-kinase (PI3K), and protein tyrosine kinase (PTK), respectively, significantly reduced phagocytosis induced by both PLA2s. PKC activity was increased in macrophages stimulated by both PLA2s. Actin polymerization and talin were evidenced by immunofluorescence and talin was recruited 5 min after both PLA2s stimulation. PKC and PKC-α localization within the cell were increased after 60 min of MT-II and MT-III stimulation. These data suggest that the effect of both PLA2s depends on actin cytoskeleton rearrangements and the activation of PKC, PI3K, and PTK signaling events required for phagocytosis.

Prognostic Markers in Pediatric Acute Liver Failure.

Introduction: Acute liver failure (ALF), although rare in children, is a complex progressive pathology, with multisystem involvement and high mortality. Isolated variables or those included in prognostic scores have been studied, to optimize organ allocation. However, its validation is challenging. This study aimed to assess the accuracy of several biomarkers and scores as predictors of prognosis in pediatric ALF (PALF). Methods: An observational study with retrospective data collection, including all cases of ALF, was defined according to the criteria of the Pediatric Acute Liver Failure Study Group, admitted to a pediatric intensive care unit (PICU) for 28 years. Two groups were defined: spontaneous recovery (SR) and non-SR (NSR) - submitted to liver transplantation (LT) or death at PICU discharge. Results: Fifty-nine patients were included, with a median age of 24 months, and 54% were female. The most frequent etiologies were metabolic (25.4%) and infectious (18.6%); 32.2% were undetermined. SR occurred in 21 patients (35.6%). In NSR group (N = 38, 64.4%), 25 required LT (42.4%) and 19 died (32.2%), 6 (15.7%) of whom after LT. The accuracy to predict NSR was acceptable for lactate at admission (AUC 0.72; 95% CI: 0.57-0.86; p = 0.006), ammonia peak (AUC 0.72; 95% CI: 0.58-0.86; p = 0.006), and INR peak (AUC 0.70; 95% CI: 0.56-0.85; p = 0.01). The cut-off value for lactate at admission was 1.95 mmol/L (sensitivity 78.4% and specificity 61.9%), ammonia peak was 64 µmol/L (sensitivity 100% and specificity 38.1%), and INR peak was 4.8 (sensitivity 61.1% and specificity 76.2%). Lactate on admission was shown to be an independent predictor of NSR on logistic regression model. Two prognostic scores had acceptable discrimination for NSR, LIU (AUC 0.73; 95% CI: 0.59-0.87; p = 0.004) and PRISM (AUC 0.71; 95% CI: 0.56-0.86; p = 0.03). In our study, the PALF delta score (PALF-ds) had lower discrimination capacity (AUC 0.63; 95% CI: 0.47-0.78; p = 0.11). Conclusions: The lactate at admission, an easily obtained parameter, had a similar capacity than the more complex scores, LIU and PRISM, to predict NSR. The prognostic value in our population of the promising dynamic score, PALF-ds, was lower than expected.

Introdução: A falência hepática aguda (FHA), apesar de rara em pediatria, é uma patologia complexa, com envolvimento multissistémico e elevada mortalidade. Têm sido estudadas variáveis isoladas ou incluídas em scores de prognóstico, com o objetivo de otimizar a alocação de órgãos. No entanto, a sua validação apresenta alguns desafios. O presente estudo tem como objetivo avaliar a precisão de vários biomarcadores e scores, como preditores de prognóstico na FHA. Métodos: Estudo observacional com método de colheita de dados retrospetivo, tendo como critérios de inclusão os casos de FHA, definida de acordo com os critérios da Pediatric Acute Liver Failure Study Group, admitidos numa Unidade de Cuidados Intensivos Pediátricos (UCIP) num período de 28 anos. Definiram-se 2 grupos: recuperação espontânea (RE) e sem recuperação espontânea (SRE) ­ doentes submetidos a transplante hepático (TRH) ou morte na alta da UCIP. Resultados: Incluíram-se 59 doentes, com mediana de idade de 24 meses, 54% do sexo feminino. As etiologias mais frequentes foram a metabólica (25.4%) e a infeciosa (18.6%); em 32.2% foi indeterminada. Apresentaram RE 21 doentes (35.6%). No grupo SRE (N = 38, 64.4%), 25 necessitaram de TRH (42.4%) e 19 faleceram (32.2%), dos quais 6 (15.7%) tinham sido submetidos a TRH. A precisão prognóstica para a ausência de recuperação espontânea foi aceitável para o lactato na admissão (AUC 0.72; IC 95%: 0.57­0.86; p = 0.006), amónia máxima (AUC 0.72; IC 95%: 0.58­0.86; p = 0.006) e INR máximo (AUC 0.70; IC 95%: 0.56­0.85; p = 0.01). O valor de cut-off do lactato na admissão foi de 1.95 mmol/L (sensibilidade 78.4% e especificidade 61.9%) e da amónia máxima foi de 64 umol/L (sensibilidade 100% e especificidade 38.1%). O lactato à admissão mostrou ser um fator independente para NSR, no modelo de regressão logística. Os scores LIU e PRISM apresentaram curvas ROC com aceitável capacidade de discriminação para a ausência de recuperação espontânea, com AUC de 0.73 (IC 95%: 0.59­0.87; p = 0.004) e 0.71 (IC 95%: 0.56­0.86; p = 0.03), respetivamente. No nosso estudo, o score PALF-Delta (PALF-ds) teve uma menor capacidade de discriminação (AUC 0.63; IC 95%: 0.47­0.78; p = 0.11). Conclusões: O lactato na admissão, um parâmetro de fácil obtenção, teve uma capacidade semelhante aos scores mais complexos, LIU e PRISM, para predizer a ausência de recuperação espontânea. O valor prognóstico nesta série, do promissor score dinâmico PALF-ds, foi inferior ao esperado.

From the Gut to the Brain: Is Microbiota a New Paradigm in Parkinson's Disease Treatment?

Parkinson's disease (PD) is recognized as the second most prevalent primary chronic neurodegenerative disorder of the central nervous system. Clinically, PD is characterized as a movement disorder, exhibiting an incidence and mortality rate that is increasing faster than any other neurological condition. In recent years, there has been a growing interest concerning the role of the gut microbiota in the etiology and pathophysiology of PD. The establishment of a brain-gut microbiota axis is now real, with evidence denoting a bidirectional communication between the brain and the gut microbiota through metabolic, immune, neuronal, and endocrine mechanisms and pathways. Among these, the vagus nerve represents the most direct form of communication between the brain and the gut. Given the potential interactions between bacteria and drugs, it has been observed that the therapies for PD can have an impact on the composition of the microbiota. Therefore, in the scope of the present review, we will discuss the current understanding of gut microbiota on PD and whether this may be a new paradigm for treating this devastating disease.

Distinct disease-modifying therapies are associated with different blood immune cell profiles in people with relapsing-remitting multiple sclerosis.

Disease modifying therapies (DMTs) used for treating people with relapsing-remitting multiple sclerosis (pwRRMS) target the immune system by different mechanisms of action. However, there is a lack of a comprehensive assessment of their effects on the immune system in comparison to treatment-naïve pwRRMS. Herein, we evaluated the numbers of circulating B cells, CD4+ and CD8+ T cells, regulatory T cells (Tregs), natural killer (NK) cells and NKT cells, and their subsets, in pwRRMS who were treatment-naïve or treated with different DMTs. Compared to treatment-naïve pwRRMS, common and divergent effects on immune system cells were observed on pwRRMS treated with different DMTs, with no consistent pattern across all therapies in any of the cell populations analysed. PwRRMS treated with fingolimod, dimethyl fumarate (DMF), or alemtuzumab have reduced numbers of CD4+ and CD8+ T cells, as well as Treg subsets, with fingolimod causing the most pronounced decrease in T cell subsets. In contrast, teriflunomide and interferon (IFN) ß have minimal impact on T cells, and natalizumab marginally increases the number of memory T cells in the blood. The effect of DMTs on the B cell, NKT and NK cell subsets is highly variable with alemtuzumab inducing a strong increase in the number of the most immature NK cells and its subsets. This study comprehensively evaluates the magnitude of the effect of different DMTs on blood immune cells providing a better understanding of therapy outcome. Furthermore, the lack of a discernible pattern in the effects of DMTs on blood immune cells suggests that multiple immune cells can independently modulate the disease.

Tumor Necrosis Factor-Alpha Modulates Expression of Genes Involved in Cytokines and Chemokine Pathways in Proliferative Myoblast Cells.

Skeletal muscle regeneration after injury is a complex process involving inflammatory signaling and myoblast activation. Pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) are key mediators, but their effects on gene expression in proliferating myoblasts are unclear. We performed the RNA sequencing of TNF-α treated C2C12 myoblasts to elucidate the signaling pathways and gene networks regulated by TNF-α during myoblast proliferation. The TNF-α (10 ng/mL) treatment of C2C12 cells led to 958 differentially expressed genes compared to the controls. Pathway analysis revealed significant regulation of TNF-α signaling, along with the chemokine and IL-17 pathways. Key upregulated genes included cytokines (e.g., IL-6), chemokines (e.g., CCL7), and matrix metalloproteinases (MMPs). TNF-α increased myogenic factor 5 (Myf5) but decreased MyoD protein levels and stimulated the release of MMP-9, MMP-10, and MMP-13. TNF-α also upregulates versican and myostatin mRNA. Overall, our study demonstrates the TNF-α modulation of distinct gene expression patterns and signaling pathways that likely contribute to enhanced myoblast proliferation while suppressing premature differentiation after muscle injury. Elucidating the mechanisms involved in skeletal muscle regeneration can aid in the development of regeneration-enhancing therapeutics.

Snake and arthropod venoms: Search for inflammatory activity in human cells involved in joint diseases.

Most anti-inflammatory drugs currently adopted to treat chronic inflammatory joint diseases can alleviate symptoms but they do not lead to remission. Therefore, new and more efficient drugs are needed to block the course of joint inflammatory diseases. Animal venoms, rich in bioactive compounds, can contribute as valuable tools in this field of research. In this study, we first demonstrate the direct action of venoms on cells that constitute the articular joints. We established a platform consisting of cell-based assays to evaluate the release of cytokines (IL-6, IL-8, TNFα, IL-1ß, and IL-10) by human chondrocytes, synoviocytes and THP1 macrophages, as well as the release of neuropeptides (substance-P and ß-endorphin) by differentiated sensory neuron-like cells, 24 h after stimulation of cells with 21 animal venoms from snake and arthropod species, sourced from different taxonomic families and geographic origins. Results demonstrated that at non-cytotoxic concentrations, the venoms activate at varying degrees the secretion of inflammatory mediators involved in the pathology of articular diseases, such as IL-6, IL-8, and TNF-α by chondrocytes, synoviocytes, and macrophages and of substance P by neuron-like cells. Venoms of the Viperidae snake family were more inflammatory than those of the Elapidae family, while venoms of Arthropods were less inflammatory than snake venoms. Notably, some venoms also induced the release of the anti-inflammatory IL-10 by macrophages. However, the scorpion Buthus occitanus venom induced the release of IL-10 without increasing the release of inflammatory cytokines by macrophages. Since the cell types used in the experiments are crucial elements in joint inflammatory processes, the results of this work may guide future research on the activation of receptors and inflammatory signaling pathways by selected venoms in these particular cells, aiming at discovering new targets for therapeutic intervention.

EHMT2/G9a and EZH2: Epimarkers in testicular germ cell tumors.

BACKGROUND: Testicular germ cell tumors remain the most frequent solid malignancies in young males. Despite excellent prognosis, the fact that only 60% of patients at diagnosis have elevated serum tumor markers (dependent on stage and histology) and the poor quality of life of patients who develop resistance to chemotherapy cannot be neglected. Consequently, it is mandatory to bring out novel biomarkers. OBJECTIVES: The main goal was to evaluate EZH2 and EHMT2/G9a immunoexpression in a well-characterized patients' cohort of primary and metastatic testicular germ cell tumors, seeking associations with clinicopathological features and discovering differential immunoexpression patterns among specific subtypes. MATERIALS AND METHODS: First, an in silico analysis of the Cancer Genome Atlas database was performed regarding EZH2 and EHMT2/G9a. Then, immunohistochemistry for EZH2 and EHMT2/G9a was carried out in a cohort of testicular germ cell tumor patients, comprising 155 chemo-naïve primary tumors and 11 chemo-treated metastases. Immunoexpression was evaluated using a digital pathology analysis software. RESULTS: Higher EZH2 and EHMT2/G9a expression levels were found in non-seminoma in the in silico analysis, particularly in embryonal carcinoma. Through digital pathology analysis, non-seminomas showed significantly higher EZH2 and EHMT2/G9a immunoexpression, with embryonal carcinoma showing higher expression. Moreover, mixed tumors with 50% or more of embryonal carcinoma component revealed the highest nuclei positivity for both biomarkers. Cisplatin-exposed metastases demonstrated a higher EZH2-positive nuclei and H-score, as well as higher EHMT2/G9a-positive nuclei. DISCUSSION AND CONCLUSION: Overall, our data suggest that EZH2 and EHMT2/G9a might be associated with greater aggressiveness and, eventually, involved in the metastatic setting, paving the way for testing targeted therapies.

Fluid balance and urine volume are independent predictors of mortality in acute kidney injury.

INTRODUCTION: In ICUs, both fluid overload and oliguria are common complications associated with increased mortality among critically ill patients, particularly in acute kidney injury (AKI). Although fluid overload is an expected complication of oliguria, it remains unclear whether their effects on mortality are independent of each other. The aim of this study is to evaluate the impact of both fluid balance and urine volume on outcomes and determine whether they behave as independent predictors of mortality in adult ICU patients with AKI. METHODS: We performed a secondary analysis of data from a multicenter, prospective cohort study in 10 Italian ICUs. AKI was defined by renal sequential organ failure assessment (SOFA) score (creatinine >3.5 mg/dL or urine output (UO) <500 mL/d). Oliguria was defined as a UO <500 mL/d. Mean fluid balance (MFB) and mean urine volume (MUV) were calculated as the arithmetic mean of all daily values. Use of diuretics was noted daily. To assess the impact of MFB and MUV on mortality of AKI patients, multivariate analysis was performed by Cox regression. RESULTS: Of the 601 included patients, 132 had AKI during their ICU stay and the mortality in this group was 50%. Non-surviving AKI patients had higher MFB (1.31 ± 1.24 versus 0.17 ± 0.72 L/day; P <0.001) and lower MUV (1.28 ± 0.90 versus 2.35 ± 0.98 L/day; P <0.001) as compared to survivors. In the multivariate analysis, MFB (adjusted hazard ratio (HR) 1.67 per L/day, 95%CI 1.33 to 2.09; <0.001) and MUV (adjusted HR 0.47 per L/day, 95%CI 0.33 to 0.67; <0.001) remained independent risk factors for 28-day mortality after adjustment for age, gender, diabetes, hypertension, diuretic use, non-renal SOFA and sepsis. Diuretic use was associated with better survival in this population (adjusted HR 0.25, 95%CI 0.12 to 0.52; <0.001). CONCLUSIONS: In this multicenter ICU study, a higher fluid balance and a lower urine volume were both important factors associated with 28-day mortality of AKI patients.

Biodegradation of petroleum hydrocarbons in estuarine sediments: metal influence.

In this work, the potential effect of metals, such as Cd, Cu and Pb, on the biodegradation of petroleum hydrocarbons in estuarine sediments was investigated under laboratory conditions. Sandy and muddy non-vegetated sediments were collected in the Lima River estuary (NW Portugal) and spiked with crude oil and each of the metals. Spiked sediments were left in the dark under constant shaking for 15 days, after which crude oil biodegradation was evaluated. To estimate microbial abundance, total cell counts were obtained by DAPI staining and microbial community structure was characterized by ARISA. Culturable hydrocarbon degraders were determined using a modified most probable number protocol. Total petroleum hydrocarbons concentrations were analysed by Fourier Transform Infrared Spectroscopy after their extraction by sonication, and metal contents were determined by atomic absorption spectrometry. The results obtained showed that microbial communities had the potential to degrade petroleum hydrocarbons, with a maximum of 32 % degradation obtained for sandy sediments. Both crude oil and metals changed the microbial community structure, being the higher effect observed for Cu. Also, among the studied metals, only Cu displayed measurable deleterious effect on the hydrocarbons degradation process, as shown by a decrease in the hydrocarbon degrading microorganisms abundance and in the hydrocarbon degradation rates. Both degradation potential and metal influence varied with sediment characteristics probably due to differences in contaminant bioavailability, a feature that should be taken into account in developing bioremediation strategies for co-contaminated estuarine sites.