Long non-coding RNA INXS is a critical mediator of BCL-XS induced apoptosis.

BCL-X mRNA alternative splicing generates pro-apoptotic BCL-XS or anti-apoptotic BCL-XL gene products and the mechanism that regulates splice shifting is incompletely understood. We identified and characterized a long non-coding RNA (lncRNA) named INXS, transcribed from the opposite genomic strand of BCL-X, that was 5- to 9-fold less abundant in tumor cell lines from kidney, liver, breast and prostate and in kidney tumor tissues compared with non-tumors. INXS is an unspliced 1903 nt-long RNA, is transcribed by RNA polymerase II, 5'-capped, nuclear enriched and binds Sam68 splicing-modulator. Three apoptosis-inducing agents increased INXS lncRNA endogenous expression in the 786-O kidney tumor cell line, increased BCL-XS/BCL-XL mRNA ratio and activated caspases 3, 7 and 9. These effects were abrogated in the presence of INXS knockdown. Similarly, ectopic INXS overexpression caused a shift in splicing toward BCL-XS and activation of caspases, thus leading to apoptosis. BCL-XS protein accumulation was detected upon INXS overexpression. In a mouse xenograft model, intra-tumor injections of an INXS-expressing plasmid caused a marked reduction in tumor weight, and an increase in BCL-XS isoform, as determined in the excised tumors. We revealed an endogenous lncRNA that induces apoptosis, suggesting that INXS is a possible target to be explored in cancer therapies.

Iron Regulates Cellular Proliferation by Enhancing the Expression of Glucose Transporter GLUT3 in the Liver.

Iron is often accumulated in the liver during pathological conditions such as cirrhosis and cancer. Elevated expression of glucose transporters GLUT1 and GLUT3 is associated with reduced overall survival in patients with hepatocellular carcinoma. However, it is not known whether iron can regulate glucose transporters and contribute to tumor proliferation. In the present study, we found that treatment of human liver cell line HepG2 with ferric ammonium citrate (FAC) resulted in a significant upregulation of GLUT3 mRNA and protein in a dose-dependent manner. Similarly, iron accumulation in mice fed with high dietary iron as well as in mice injected intraperitoneally with iron dextran enhanced the GLUT3 expression drastically in the liver. We demonstrated that iron-induced hepatic GLUT3 upregulation is mediated by the LKB1/AMPK/CREB1 pathway, and this activation was reversed when treated with iron chelator deferiprone. In addition, inhibition of GLUT3 using siRNA prevented iron-mediated increase in the expression of cell cycle markers and cellular hyperproliferation. Furthermore, exogenous sodium beta-hydroxybutyrate treatment prevented iron-mediated hepatic GLUT3 activation both in vitro and in vivo. Together, these results underscore the importance of iron, AMPK, CREB1 and GLUT3 pathways in cell proliferation and highlight the therapeutic potential of sodium beta-hydroxybutyrate in hepatocellular carcinoma with high GLUT3 expression.

Differential Proteins Expression Distinguished Between Patients With Infectious and Noninfectious Uveitis.

PURPOSE: We investigated the aqueous humor proteome and associated plasma proteome in patients with infectious or noninfectious uveitis. METHODS: AH and plasma were obtained from 28 patients with infectious uveitis (IU), 29 patients with noninfectious uveitis (NIU) and 35 healthy controls undergoing cataract surgery. The proteins profile was analyzed by SomaScan technology. RESULTS: We found 1844 and 2484 proteins up-regulated and 124 and 161 proteins down-regulated in the AH from IU and NIU groups, respectively. In the plasma, three proteins were up-regulated in NIU patients, and one and five proteins were down-regulated in the IU and NIU patients, respectively. The results of pathway enrichment analysis for both IU and NIU groups were related mostly to inflammatory and regulatory processes. CONCLUSION: SomaScan was able to detect novel AH and plasma protein biomarkers in IU and NIU patients. Also, the unique proteins found in both AH and plasma suggest a protein signature that could distinguish between infectious and noninfectious uveitis.

Ecology of anopheline mosquitoes (Diptera: Culicidae) in the Central Atlantic Forest Biodiversity Corridor, southeastern Brazil.

Knowledge of the fauna composition of anopheline mosquitoes, their ecological aspects and behavior, and influence of climatic variables on their population dynamics can help in understanding the transmission of Plasmodium parasites and thus develop more efficient strategies for the control of malaria. In the Central Atlantic Forest Biodiversity Corridor, southeastern Brazil, foci of introduced malaria have been reported among people returning from the Amazon region, north Brazil. Our objective was to evaluate and compare the anopheline fauna from a preserved environment and an adjacent peridomiciliary modified environment at the Central Atlantic Forest Biodiversity Corridor. We collected anopheline mosquitoes on a monthly basis from June 2004 to May 2006 from both these environments to understand the ecological aspects and their association with the occurrence of malaria. We captured 5,491 anopheline mosquitoes belonging to two subgenera and 11 species and studied the correlations between anopheline mosquito species and climatic variables. We considered Anopheles darlingi (Root) as the principal malaria vector and Anopheles albitarsis s. l. (Arribalzaga) as the secondary vector.

ZONAB promotes proliferation and represses differentiation of proximal tubule epithelial cells.

Epithelial polarization modulates gene expression. The transcription factor zonula occludens 1 (ZO-1)-associated nucleic acid binding protein (ZONAB) can shuttle between tight junctions and nuclei, promoting cell proliferation and expression of cyclin D1 and proliferating cell nuclear antigen (PCNA), but whether it also represses epithelial differentiation is unknown. Here, during mouse kidney ontogeny and polarization of proximal tubular cells (OK cells), ZONAB and PCNA levels decreased in parallel and inversely correlated with increasing apical differentiation, reflected by expression of megalin/cubilin, maturation of the brush border, and extension of the primary cilium. Conversely, ZONAB reexpression and loss of apical differentiation markers provided a signature for renal clear cell carcinoma. In confluent OK cells, ZONAB overexpression increased proliferation and PCNA while repressing megalin/cubilin expression and impairing differentiation of the brush border and primary cilium. Reporter and chromatin immunoprecipitation assays demonstrated that megalin and cubilin are ZONAB target genes. Sparsely plated OK cells formed small islands composed of distinct populations: Cells on the periphery, which lacked external tight junctions, strongly expressed nuclear ZONAB, proliferated, and failed to differentiate; central cells, surrounded by continuous junctions, lost nuclear ZONAB, stopped proliferating, and engaged in apical differentiation. Taken together, these data suggest that ZONAB is an important component of the mechanisms that sense epithelial density and participates in the complex transcriptional networks that regulate the switch between proliferation and differentiation.

Single photon emission-computed tomography (SPECT) for functional investigation of the proximal tubule in conscious mice.

Noninvasive analysis of renal function in conscious mice is necessary to optimize the use of mouse models. In this study, we evaluated whether single photon emission-computed tomography (SPECT) using specific radionuclear tracers can be used to analyze changes in renal proximal tubule functions. The tracers included (99m)TC- dimercaptosuccinic acid ((99m)Tc-DMSA), which is used for cortex imaging; (99m)Tc-mercaptoacetyltriglycine ((99m)Tc-MAG3), used for dynamic renography; and (123)I-beta(2)-microglobulin, which monitors receptor-mediated endocytosis. (99m)Tc-DMSA SPECT imaging was shown to delineate the functional renal cortex with a approximately 1-mm spatial resolution and accumulated in the cortex reaching a plateau 5 h after injection. The cortical uptake of (99m)Tc-DMSA was abolished in Clcn5 knockout mice, a model of proximal tubule dysfunction. Dynamic renography with (99m)Tc-MAG3 in conscious mice demonstrated rapid extraction from blood, renal accumulation, and subsequent tubular secretion. Anesthesia induced a significant delay in the (99m)Tc-MAG3 clearance. The tubular reabsorption of (123)I-beta(2)-microglobulin was strongly impaired in the Clcn5 knockout mice, with defective tubular processing and loss of the native tracer in urine, reflecting proximal tubule dysfunction. Longitudinal studies in a model of cisplatin-induced acute tubular injury revealed a correlation between tubular recovery and (123)I-beta(2)-microglobulin uptake. These data show that SPECT imaging with well-validated radiotracers allows in vivo investigations of specific proximal tubule functions in conscious mice.

Setting New Routes for Antifungal Drug Discovery Against Pathogenic Fungi.

Fungal diseases are life-threatening to human health and responsible for millions of deaths around the world. Fungal pathogens lead to a high number of morbidity and mortality. Current antifungal treatment comprises drugs, such as azoles, echinocandins, and polyenes and the cure is not guaranteed. In addition, such drugs are related to severe side effects and the treatment lasts for an extended period. Thus, setting new routes for the discovery of effective and safe antifungal drugs should be a priority within the health care system. The discovery of alternative and efficient antifungal drugs showing fewer side effects is time-consuming and remains a challenge. Natural products can be a source of antifungals and used in combinatorial therapy. The most important natural products are antifungal peptides, antifungal lectins, antifungal plants, and fungi secondary metabolites. Several proteins, enzymes, and metabolic pathways could be targets for the discovery of efficient inhibitor compounds and recently, heat shock proteins, calcineurin, salinomycin, the trehalose biosynthetic pathway, and the glyoxylate cycle have been investigated in several fungal species. HSP protein inhibitors and echinocandins have been shown to have a fungicidal effect against azole-resistant fungi strains. Transcriptomic and proteomic approaches have advanced antifungal drug discovery and pointed to new important specific-pathogen targets. Certain enzymes, such as those from the glyoxylate cycle, have been a target of antifungal compounds in several fungi species. Natural and synthetic compounds inhibited the activity of such enzymes and reduced the ability of fungal cells to transit from mycelium to yeast, proving to be promisor antifungal agents. Finally, computational biology has developed effective approaches, setting new routes for early antifungal drug discovery since normal approaches take several years from discovery to clinical use. Thus, the development of new antifungal strategies might reduce the therapeutic time and increase the quality of life of patients.

Expression patterns of the aquaporin gene family during renal development: influence of genetic variability.

High-throughput analyses have shown that aquaporins (AQPs) belong to a cluster of genes that are differentially expressed during kidney organogenesis. However, the spatiotemporal expression patterns of the AQP gene family during tubular maturation and the potential influence of genetic variation on these patterns and on water handling remain unknown. We investigated the expression patterns of all AQP isoforms in fetal (E13.5 to E18.5), postnatal (P1 to P28), and adult (9 weeks) kidneys of inbred (C57BL/6J) and outbred (CD-1) mice. Using quantitative polymerase chain reaction (PCR), we evidenced two mRNA patterns during tubular maturation in C57 mice. The AQPs 1-7-11 showed an early (from E14.5) and progressive increase to adult levels, similar to the mRNA pattern observed for proximal tubule markers (Megalin, NaPi-IIa, OAT1) and reflecting the continuous increase in renal cortical structures during development. By contrast, AQPs 2-3-4 showed a later (E15.5) and more abrupt increase, with transient postnatal overexpression. Most AQP genes were expressed earlier and/or stronger in maturing CD-1 kidneys. Furthermore, adult CD-1 kidneys expressed more AQP2 in the collecting ducts, which was reflected by a significant delay in excreting a water load. The expression patterns of proximal vs. distal AQPs and the earlier expression in the CD-1 strain were confirmed by immunoblotting and immunostaining. These data (1) substantiate the clustering of important genes during tubular maturation and (2) demonstrate that genetic variability influences the regulation of the AQP gene family during tubular maturation and water handling by the mature kidney.

Control of neuronal branching by the death receptor CD95 (Fas/Apo-1).

The CD95 (Apo-1/Fas)/CD95 ligand (CD95L) system is best characterized as a trigger of apoptosis. Nevertheless, despite broad expression of CD95L and CD95 in the developing brain, absence of functional CD95 (lpr mice) or CD95L (gld mice) does not alter neuronal numbers. Here, we report that in embryonic hippocampal and cortical neurons in vivo and in vitro CD95L does not induce apoptosis. Triggering of CD95 in cultured immature neurons substantially increases neurite branches by promoting their formation. The branching increase occurs in a caspase-independent and death domain-dependent manner and is paralleled by an increase in the nonphosphorylated form of Tau. Most importantly, lpr and gld mutants exhibit a reduced number of dendritic branches in vivo at the time when synapse formation takes place. These data reveal a novel function for the CD95 system and add to the picture of guidance molecules in the developing brain.

Solid state characterization and theoretical study of non-linear optical properties of a Fluoro-N-Acylhydrazide derivative.

In this work we determine the linear and non-linear optical properties of a Fluoro-N-Acylhydrazide derivative (FBHZ), using a combined supermolecule approach and an iterative scheme of electrostatic polarization, where the atoms of neighbouring molecules are represented by point charges. Our results for non-linear optics (NLO) are comparable to those found experimentally, suggesting that FBHZ constitutes an attractive object for future studies and for use as an interesting material for third-order NLO applications. The dynamic electrical properties of FBHZ in different solvent media are reported. Its molecular properties are closely related to supramolecular features; accordingly, we analysed all its crystal structure properties via intermolecular interactions in the solid state, using X-ray crystallography data and Hirshfeld surface (HS), including thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and hot-stage microscopy (HSM), where the results reveal crystal stability in respect to temperature variation.

CD95 maintains stem cell-like and non-classical EMT programs in primary human glioblastoma cells.

Glioblastoma (GBM) is one of the most aggressive types of cancer with limited therapeutic options and unfavorable prognosis. Stemness and non-classical epithelial-to-mesenchymal transition (ncEMT) features underlie the switch from normal to neoplastic states as well as resistance of tumor clones to current therapies. Therefore, identification of ligand/receptor systems maintaining this privileged state is needed to devise efficient cancer therapies. In this study, we show that the expression of CD95 associates with stemness and EMT features in GBM tumors and cells and serves as a prognostic biomarker. CD95 expression increases in tumors and with tumor relapse as compared with non-tumor tissue. Recruitment of the activating PI3K subunit, p85, to CD95 death domain is required for maintenance of EMT-related transcripts. A combination of the current GBM therapy, temozolomide, with a CD95 inhibitor dramatically abrogates tumor sphere formation. This study molecularly dissects the role of CD95 in GBM cells and contributes the rational for CD95 inhibition as a GBM therapy.

Signaling transcript profile of the asexual intraerythrocytic development cycle of Plasmodium falciparum induced by melatonin and cAMP.

According to the World Health Organization (WHO), Plasmodium falciparum is the deadliest parasite among all species. This parasite possesses the ability to sense molecules, including melatonin (MEL) and cAMP, and modulate its cell cycle accordingly. MEL synchronizes the development of this malaria parasite by activating several cascades, including the generation of the second messenger cAMP. Therefore, we performed RNA sequencing (RNA-Seq) analysis in P. falciparum erythrocytic stages (ring, trophozoite and schizont) treated with MEL and cAMP. To investigate the expression profile of P. falciparum genes regulated by MEL and cAMP, we performed RNA-Seq analysis in three P. falciparum strains (control, 3D7; protein kinase 7 knockout, PfPK7-; and PfPK7 complement, PfPK7C). In the 3D7 strain, 38 genes were differentially expressed upon MEL treatment; however, none of the genes in the trophozoite (T) stage PfPK7- knockout parasites were differentially expressed upon MEL treatment for 5 hours compared to untreated controls, suggesting that PfPK7 may be involved in the signaling leading to differential gene expression. Moreover, we found that MEL modified the mRNA expression of genes encoding membrane proteins, zinc ion-binding proteins and nucleic acid-binding proteins, which might influence numerous functions in the parasite. The RNA-Seq data following treatment with cAMP show that this molecule modulates different genes throughout the intraerythrocytic cycle, namely, 75, 101 and 141 genes, respectively, in the ring (R), T and schizont (S) stages. Our results highlight P. falciparum's perception of the external milieu through the signaling molecules MEL and cAMP, which are able to drive to changes in gene expression in the parasite.

Therapeutic neutralization of CD95-ligand and TNF attenuates brain damage in stroke.

Stroke is the third most common cause of death in the Western world. The mechanisms of brain damage in the affected areas are largely unknown. Hence, rational treatment strategies are limited. Previous experimental evidence suggested that cerebral lesions were less prominent in CD95 (APO-1/Fas)-deficient (lpr) than in wild-type mice. Additional results strongly suggested that the CD95-ligand (CD95L) was a major cause of neuronal autocrine suicide in the penumbra. These data and the assumption that death-receptor systems might determine stroke-related damage in the brain prompted us to examine these systems in in vitro and in vivo models of ischemia. We showed that hybrids of TNF-deficient and gld mice were strongly resistant towards stroke-induced damage. To determine the mechanism of action of TNF and CD95L, we separately investigated their influence on primary ischemic death and secondary inflammatory injury. Inhibition of both TNF and CD95L in vitro prevented death of primary neurons induced by oxygen-glucose deprivation and reperfusion. The recruitment of inflammatory cells to the ischemic hemisphere was abrogated in the absence of both TNF and CD95L. Significantly, mice injected with a mixture of neutralizing anti-TNF and anti-CD95L antibodies 30 min after induction of stroke showed a marked decrease in both infarct volumes and mortality. Accordingly, the locomotor performance of these animals was not significantly impaired in comparison to sham-operated animals. These data reveal that inhibition of TNF and CD95L blocks stroke-related damage at two levels, the primary ischemic and the secondary inflammatory injury. These results offer new approaches in stroke treatment.

CD95 promotes metastatic spread via Sck in pancreatic ductal adenocarcinoma.

Cancer stem cells (CSCs) have been implicated in the initiation and maintenance of tumour growth as well as metastasis. Recent reports link stemness to epithelial-mesenchymal transition (EMT) in cancer. However, there is still little knowledge about the molecular markers of those events. In silico analysis of RNA profiles of 36 pancreatic ductal adenocarcinomas (PDAC) reveals an association of the expression of CD95 with EMT and stemness that was validated in CSCs isolated from PDAC surgical specimens. CD95 expression was also higher in metastatic pancreatic cells than in primary PDAC. Pharmacological inhibition of CD95 activity reduced PDAC growth and metastasis in CSC-derived xenografts and in a murine syngeneic model. On the mechanistic level, Sck was identified as a novel molecule indispensable for CD95's induction of cell cycle progression. This study uncovers CD95 as a marker of EMT and stemness in PDAC. It also addresses the molecular mechanism by which CD95 drives tumour growth and opens tantalizing therapeutic possibilities in PDAC.

DNA typing of short tandem repeat loci on Y-chromosome of Greek population.

Haplotype, allele frequencies and population data of nine Y-chromosome STR loci, DYS385I, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, were determined from a sample of 69 unrelated Greek male individuals.

An examination of morphometric variations in a neotropical toad population (Proceratophrys cristiceps, Amphibia, Anura, Cycloramphidae).

The species Proceratophrys cristiceps belongs to the genus Proceratophrys within the family Cycloramphidae. These amphibians are found exclusively in South America in the morphoclimatic domain of the semi-arid depression zones in northeastern Brazil known as the Caatinga. We examined intrapopulational variation using univariate and multivariate statistics with traditional and geometric morphometrics, which supported the existence of two morphotypes of this species. Our results indicated significant degrees of variation in skeletal characteristics between some natural populations of this species. Careful analyses of variability levels are fundamental to avoid taxonomic errors, principally in populations that demonstrate characteristics intimately associated with their area of occurrence, as is the case of Proceratophrys cristiceps.

Homozygosity for uromodulin disorders: FJHN and MCKD-type 2.

BACKGROUND: Autosomal-dominant medullary cystic kidney disease type 2 (MCKD2) and familial juvenile hyperuricemic nephropathy (FJHN) are heritable renal diseases with autosomal-dominant transmission and shared features, including polyuria, progressive renal failure, and abnormal urate handling, which leads to hyperuricemia and gout. Mutations of the UMOD gene, disrupting the tertiary structure of uromodulin, cause MCKD2 and FJHN. METHODS: Haplotype analysis of a large Spanish family with MCKD was carried out to determinate genetic linkage to MCKD2 locus. Mutation detection was performed by direct sequencing of the UMOD gene. The level of Tamm-Horsfall protein in the urine was measured by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. RESULTS: Linkage to MCKD2 locus was demonstrated (LOD score: 4.13), and a known pathogenic uromodulin mutation was found in exon 4, corresponding to Cys255Tyr, disrupting the light chain binding domain of the protein. In this consanguineous family there were three patients homozygous for the C255Y mutation, and multiple heterozygous cases, allowing the MCKD phenotypes associated with one or two mutant alleles to be compared. The homozygous individuals survived to adulthood, although presenting an earlier onset of hyperuricemia and faster progression to end-stage renal disease than heterozygous individuals. Western analysis revealed lower levels of urine THP in one heterozygous patient compared with a normal control patient, both with normal renal function. CONCLUSION: The study shows that individuals with two UMOD mutations are viable, but they do have more severe disease on average than heterozygotes. This family sheds light on the possible disease mechanism in this disorder.

Ototoxicidade / Ototoxicity

Faz-se um estudo clínico apoiados em revisäo bibliográfica da Ototoxicidade, procurando uma análise das alteraçöes histopatológicas e características clínicas; ressalta-se a importância do tratamento preventivo como forma ideal para o controle da patologia