Human proximal tubular cells can form calcium phosphate deposits in osteogenic culture: role of cell death and osteoblast-like transdifferentiation

Abstract Nephrocalcinosis is a clinicopathological entity characterized by microscopic calcium crystals in the renal parenchyma, within the tubular lumen or in the interstitium. Crystal binding to tubular cells may be the cause underlying nephrocalcinosis and nephrolithiasis. Pathological circumstances, such as acute cortical necrosis, may induce healthy cells to acquire a crystal-binding phenotype. The present study aimed to investigate whether human renal proximal tubular cells (HK-2 cells) can form calcium phosphate deposits under osteogenic conditions, and whether apoptosis and/or osteogenic-like processes are involved in cell calcification. HK-2 cells were cultured in standard or osteogenic medium for 1, 5, and 15 days. Von Kossa staining and ESEM were used to analyze crystal deposition. Apoptosis was investigated, analyzing caspase activation by in-cell Western assay, membrane translocation of phosphotidylserine by annexin V-FITC/propidium iodide staining, and DNA fragmentation by TUNEL assay. qRT/PCR, immunolabeling and cytochemistry were performed to assess osteogenic activation (Runx2, Osteonectin, Osteopontin and ALP), and early genes of apoptosis (BAX, Bcl-2). HK-2 cell mineralization was successfully induced on adding osteogenic medium. Calcium phosphate deposition increased in a time-dependent manner, and calcified cell aggregates exhibited characteristic signs of apoptosis. At 15 days, calcifying HK-2 cells revealed osteogenic markers, such as Runx2, ALP, osteonectin and osteopontin. Monitoring the processes at 1, 5, and 15 days showed apoptosis starting already after 5 days of osteogenic induction, when the first small calcium phosphate crystals began to appear on areas where cell aggregates were in apoptotic conditions. The cell death process proved caspase-dependent. The importance of apoptosis was reinforced by the time-dependent increase in BAX expression, starting from day 1. These findings strongly support the hypothesis that apoptosis triggered HK-2 calcification even before any calcium phosphate crystal deposition or acquisition of an osteogenic phenotype

Elevated urinary levels of urokinase-type plasminogen activator receptor (uPAR) in pancreatic ductal adenocarcinoma identify a clinically high-risk group

BACKGROUND: The urokinase plasminogen activator receptor is highly expressed and its gene is amplified in about 50% of pancreatic ductal adenocarcinomas; this last feature is associated with worse prognosis. It is unknown whether the level of its soluble form (suPAR) in urine may be a diagnostic-prognostic marker in these patients. METHODS: The urinary level of suPAR was measured in 146 patients, 94 pancreatic ductal adenocarcinoma and 52 chronic pancreatitis. Urine from 104 healthy subjects with similar age and gender distribution served as controls. suPAR levels were normalized with creatinine levels (suPAR/creatinine, ng/mg) to remove urine dilution effect. RESULTS: Urinary suPAR/creatinine values of pancreatic ductal adenocarcinoma patients were significantly higher (median 9.8; 25(th)-75(th )percentiles 5.3-20.7) than those of either healthy donors (median 0; 0-0.5) or chronic pancreatitis patients (median 2.7; 0.9-4.7). The distribution of values among cancer patients was widespread and asymmetric, 53% subjects having values beyond the 95(th )percentile of healthy donors. The values of suPAR/creatinine did not correlate with tumour stage, Ca19-9 or CEA levels. Higher values correlated with poor prognosis among non-resected patients at univariate analysis; multivariate Cox regression identified high urinary suPAR/creatinine as an independent predictor of poor survival among all cancer patients (odds ratio 2.10, p = 0.0023), together with tumour stage (stage III odds ratio 2.65, p = 0.0017; stage IV odds ratio 4.61, p < 0.0001) and female gender (odds ratio 1.85, p = 0.01). CONCLUSIONS: A high urinary suPAR/creatinine ratio represents a useful marker for the identification of a subset of patients with poorer outcome

Lithium niobate micromachining for the fabrication of microfluidic droplet generators

In this paper, we present the first microfluidic junctions for droplet generation directly engraved on lithium niobate crystals by micromachining techniques, preparatory to a fully integrated opto-microfluidics lab-on-chip system. In particular, laser ablation technique and the mechanical micromachining technique are exploited to realise microfluidic channels in T-and cross junction configurations. The quality of both lateral and bottom surfaces of the channels are therefore compared together with a detailed study of their roughness measured by means of atomic force microscopy in order to evaluate the final performance achievable in an optofluidic device. Finally, the microfluidics performances of these water-in-oil droplets generators are investigated depending on these micromachining techniques, with particular focus on a wide range of droplet generation rates

Changes in inflammatory biomarkers in HCV-infected patients undergoing direct acting antiviral-containing regimens with or without interferon

Background and aims Increased levels of chemokine interferon-gamma (IFN-γ)-inducible protein-10 (CXCL10), soluble CD163 (sCD163) and soluble CD14 (sCD14) have been reported in HCV infection. The aim of this study was to compare, sCD163 and sCD14 levels in HCV-infected patients undergoing direct acting antiviral (DAA)-containing regimens with or without interferon (IFN). Methods sCD163, sCD14 and CXCL10 were longitudinally measured by ELISA in 159 plasma samples from 25 HCV-infected patients undergoing IFN-based treatment plus telaprevir or boceprevir and 28 HCV infected subjects treated with DAA IFN-free regimens. Twenty-five healthy donors (HD) were included as controls. Results At baseline CXCL10, sCD163 and sCD14 levels were higher in HCV-infected patients than in HD. CXCL10 and sCD163 levels were significantly decreased in responder (R) patients who achieved sustained virological response (SVR), with both IFN-based and IFN-free regimens, while they were persistently elevated in non-responders (NR) patients who stopped IFN-based treatments because of failure or adverse events. Conversely, sCD14 levels were apparently unchanged during therapy, but at the end of treatment the levels reached normal ranges. Comparing the two regimens, the extent of CXCL10 reduction was more pronounced in patients undergoing DAA IFN-free therapies, whereas sCD163 and sCD14 reduction was similar in the two groups. Interestingly, only in IFN-based regimens baseline sCD163 levels were significantly higher in NR than in R patients, while in the IFN-free treatment group also patients with highsCD163 plasma levels obtained SVR. At the end of therapy, even if the biomarkers were largely decreased, their levels remained significantly higher compared to HD. Only in the early fibrosis stages, sCD163 values tended to normalize. Conclusions These results indicate that IFN-free regimens including newer DAA induce an early and marked decrease in circulating inflammatory biomarkers. However, the full normalization of biomarkers was not obtained, especially in patients with advanced fibrosis, thus underlying the need for a treatment in the early stages of HCV infection

Novel Nanogels Loaded with Mn(II) Chelates as Effective and Biologically Stable MRI Probes

Here it is described nanogels (NG) based on a chitosan matrix, which are covalently stabilized by a bisamide derivative of Mn-t-CDTA (t-CDTA = trans-1,2-diaminocyclohexane-N,N,N & PRIME;,N & PRIME;-tetraacetic acid). the Mn(II) complex acts both as a contrast medium and as a cross-linking agent. These nanogels are proposed as an alternative to the less stable paramagnetic nanogels obtained by electrostatic interactions between the polymeric matrix and paramagnetic Gd(III) chelates. The present novel nanogels show: i) relaxivity values seven times higher than that of typical monohydrated Mn(II) chelates at the clinical fields, thanks to the combination of a restricted mobility of the complex with a fast exchange of the metal-bound water molecule; ii) high stability of the formulation over time at pH 5 and under physiological conditions, thus excluding metal leaking or particles aggregation; iii) good extravasation and accumulation, with a maximum contrast achieved at 24 h post-injection in mice bearing subcutaneous breast cancer tumor; iv) high T-1 contrast (1 T) in the tumor 24 h post-injection. These improved properties pave the way for the use of these paramagnetic nanogels as promising magnetic resonance imaging (MRI) probes for in vitro and in vivo preclinical applications

ANALYSIS OF THE TARGET DECOMPOSITION TECHNIQUE ATTRIBUTES AND POLARIMETRIC RATIOS TO DISCRIMINATE LAND USE AND LAND COVER CLASSES OF THE TAPAJÓS REGION

This study aims to analyze the capability of the target decomposition techniques and the polarimetric ratios applied to the ALOS/PALSAR-2 satellite polarimetric images to discriminate the land use and land cover classes in the Tapajós National Forest region, Pará State. Three full polarimetric ALOS/PALSAR-2, level 1 single look complex scenes were selected to generate the coherence and the covariance matrices to derive the Cloude-Pottier and the Freeman-Durden target decomposition attributes. From the radiometrically calibrated PALSAR-2 images, we generated the backscatter coefficients, the cross polarized ratio (RC; HV/HH), the parallel polarized ratio (RP; VV/HH) and the Radar Forest Degradation Index (RFDI). The images resulting from these polarimetric attributes were processed by the Maximum Likelihood (MAXVER) classifier coupled with the Iterated Conditional Modes (ICM) contextual algorithm. We found that the classifications derived from the target decomposition attributes, mainly from the CloudePottier technique, with a Kappa index of 0.75, presented a significant higher performance than those derived from the RC ratio, RP ratio, and RFDI

Do estádio para o estúdio: representação do corpo masculino e virilidade na capa da revista Placar

Essa pesquisa analisa como ocorreram as mudanças de representação do corpo masculino veiculado na capa da revista Placar. Há 30 anos, as imagens desses corpos dialogavam com representações de homens em atividade, atualmente, essa representação se torna estética, planejada e organizada no sentido de serem imagens de estúdios. Percebe-se a relação do corpo masculino sem máculas, afastado das relações de sofrimento e dor e a reconfiguração da masculinidade e virilidade

Desempenho de leitões alimentados com semente de canola extrusada ou não

Foi realizado um experimento para avaliar o desempenho de leit&otilde;es na fase de creche alimentados com diferentes n&iacute;veis de inclus&atilde;o da semente de canola extrusada ou n&atilde;o e sua viabilidade econ&ocirc;mica. Foram utilizados 56 leit&otilde;es, com peso vivo m&eacute;dio inicial de 15,03 &plusmn; 1,8kg, distribu&iacute;dos em um delineamento inteiramente casualizado, com sete tratamentos, quatro repeti&ccedil;&otilde;es e dois animais por unidade experimental. Os tratamentos consistiram em uma ra&ccedil;&atilde;o &agrave; base de milho e farelo de soja, tr&ecirc;s ra&ccedil;&otilde;es com inclus&atilde;o de 6; 12 e 18% de semente de canola integral mo&iacute;da (SCI) e tr&ecirc;s ra&ccedil;&otilde;es com inclus&atilde;o de 6; 12 e 18% de semente de canola extrusada (SCE). N&atilde;o foram observadas diferen&ccedil;as (P&gt;0,05) no desempenho dos leit&otilde;es, entretanto, ocorreu redu&ccedil;&atilde;o linear (P&lt;0,05) no consumo de ra&ccedil;&atilde;o e no custo da ra&ccedil;&atilde;o por quilograma de peso vivo ganho para as ra&ccedil;&otilde;es contendo SCI e SCE, &agrave; medida que o n&iacute;vel de inclus&atilde;o destes alimentos aumentou. A semente de canola, na forma extrusada ou n&atilde;o, pode ser inclu&iacute;da na ra&ccedil;&atilde;o de leit&otilde;es em fase de creche em at&eacute; 18%, sem prejudicar o desempenho. A inclus&atilde;o de 18% da SCE proporcionou menores custos por quilograma de leit&atilde;o produzido