TFE3-Splicing Factor Fusions Represent Functional Drivers and Druggable Targets in Translocation Renal Cell Carcinoma.

TFE3 is a member of the basic helix-loop-helix leucine zipper MiT transcription factor family, and its chimeric proteins are associated with translocation renal cell carcinoma (tRCC). Despite the variety of gene fusions, most TFE3 fusion partner genes are related to spliceosome machinery. Dissecting the function of TFE3 fused to spliceosome machinery factors (TFE3-SF) could direct the development of effective therapies for this lethal disease, which is refractory to standard treatments for kidney cancer. Here, by using a combination of in silico structure prediction, transcriptome profiling, molecular characterization, and high-throughput high-content screening (HTHCS), we interrogated a number of oncogenic mechanisms of TFE3-SF fusions. TFE3-SF fusions drove the transformation of kidney cells and promoted distinct oncogenic phenotypes in a fusion partner-dependent manner, differentially altering the transcriptome and RNA splicing landscape and activating different oncogenic pathways. Inhibiting TFE3-SF dimerization reversed its oncogenic activity and represented a potential target for therapeutic intervention. Screening the FDA-approved drugs library LOPAC and a small-molecule library (Microsource) using HTHCS combined with FRET technology identified compounds that inhibit TFE3-SF dimerization. Hit compounds were validated in 2D and 3D patient-derived xenograft models expressing TFE3-SF. The antihistamine terfenadine decreased cell proliferation and reduced in vivo tumor growth of tRCC. Overall, these results unmask therapeutic strategies to target TFE3-SF dimerization for treating patients with tRCC. SIGNIFICANCE: TFE3-splicing factor fusions possess both transcription and splicing factor functions that remodel the transcriptome and spliceosome and can be targeted with dimerization inhibitors to suppress the growth of translocation renal cell carcinoma.

Left atrial veno-arterial extracorporeal membrane oxygenation in post-myocardial infarction ventricular septal defect with cardiogenic shock: a case report.

Background: Ventricular septal defect (VSD) complicating acute myocardial infarction (MI) represents a life-threatening condition and has a mortality of >90% if left untreated. Case summary: A 53-year-old man with a prior medical history of diabetes and hypertension presented with cardiogenic shock secondary to VSD as a mechanical complication of non-reperfused inferior MI. Discussion: The choice of mechanical support can be difficult in this type of patient. Given the risk of an increased shunt because of veno-arterial extracorporeal membrane oxygenation (VA-ECMO) and the increase in left ventricle (LV) afterload, several measures were taken to plan the best ECMO configuration. Given the absence of any real improvement in the LV and an elevated residual ratio between pulmonary and systemic flow (Qp/Qs), the final decision was to switch to left atrial VA-ECMO (LAVA-ECMO). The use of LAVA-ECMO improved the patient's haemodynamics and allowed his condition to stabilize; LAVA-ECMO is feasible and may be effective as a mechanical circulatory support (MCS) strategy for patients in cardiogenic shock due to VSD as a mechanical complication of acute MI.

Activation of the integrated stress response (ISR) pathways in response to Ref-1 inhibition in human pancreatic cancer and its tumor microenvironment.

Pancreatic cancer or pancreatic ductal adenocarcinoma (PDAC) is characterized by a profound inflammatory tumor microenvironment (TME) with high heterogeneity, metastatic propensity, and extreme hypoxia. The integrated stress response (ISR) pathway features a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) and regulate translation in response to diverse stress conditions, including hypoxia. We previously demonstrated that eIF2 signaling pathways were profoundly affected in response to Redox factor-1 (Ref-1) knockdown in human PDAC cells. Ref-1 is a dual function enzyme with activities of DNA repair and redox signaling, responds to cellular stress, and regulates survival pathways. The redox function of Ref-1 directly regulates multiple transcription factors including HIF-1α, STAT3, and NF-κB, which are highly active in the PDAC TME. However, the mechanistic details of the crosstalk between Ref-1 redox signaling and activation of ISR pathways are unclear. Following Ref-1 knockdown, induction of ISR was observed under normoxic conditions, while hypoxic conditions were sufficient to activate ISR irrespective of Ref-1 levels. Inhibition of Ref-1 redox activity increased expression of p-eIF2 and ATF4 transcriptional activity in a concentration-dependent manner in multiple human PDAC cell lines, and the effect on eIF2 phosphorylation was PERK-dependent. Treatment with PERK inhibitor, AMG-44 at high concentrations resulted in activation of the alternative ISR kinase, GCN2 and induced levels of p-eIF2 and ATF4 in both tumor cells and cancer-associated fibroblasts (CAFs). Combination treatment with inhibitors of Ref-1 and PERK enhanced cell killing effects in both human pancreatic cancer lines and CAFs in 3D co-culture, but only at high doses of PERK inhibitors. This effect was completely abrogated when Ref-1 inhibitors were used in combination with GCN2 inhibitor, GCN2iB. We demonstrate that targeting of Ref-1 redox signaling activates the ISR in multiple PDAC lines and that this activation of ISR is critical for inhibition of the growth of co-culture spheroids. Combination effects were only observed in physiologically relevant 3D co-cultures, suggesting that the model system utilized can greatly affect the outcome of these targeted agents. Inhibition of Ref-1 signaling induces cell death through ISR signaling pathways, and combination of Ref-1 redox signaling blockade with ISR activation could be a novel therapeutic strategy for PDAC treatment.

Aminoacylation-defective bi-allelic mutations in human EPRS1 associated with psychomotor developmental delay, epilepsy, and deafness.

Aminoacyl-tRNA synthetases are enzymes that ensure accurate protein synthesis. Variants of the dual-functional cytoplasmic human glutamyl-prolyl-tRNA synthetase, EPRS1, have been associated with leukodystrophy, diabetes and bone disease. Here, we report compound heterozygous variants in EPRS1 in a 4-year-old female patient presenting with psychomotor developmental delay, seizures and deafness. Functional studies of these two missense mutations support major defects in enzymatic function in vitro and contributed to confirmation of the diagnosis.

Polyphenon E Effects on Gene Expression in PC-3 Prostate Cancer Cells.

Polyphenon E (Poly E) is a standardized, caffeine-free green tea extract with defined polyphenol content. Poly E is reported to confer chemoprotective activity against prostate cancer (PCa) progression in the TRAMP model of human PCa, and has shown limited activity against human PCa in human trials. The molecular mechanisms of the observed Poly E chemopreventive activity against PCa are not fully understood. We hypothesized that Poly E treatment of PCa cells induces gene expression changes, which could underpin the molecular mechanisms of the limited Poly E chemoprevention activity against PCa. PC-3 cells were cultured in complete growth media supplemented with varied Poly E concentrations for 24 h, then RNA was isolated for comparative DNA microarray (0 vs. 200 mg/L Poly E) and subsequent TaqMan qRT-PCR analyses. Microarray data for 54,613 genes were filtered for >2-fold expression level changes, with 8319 genes increased and 6176 genes decreased. Eight genes involved in key signaling or regulatory pathways were selected for qRT-PCR. Two genes increased expression significantly, MXD1 (13.98-fold; p = 0.0003) and RGS4 (21.98-fold; p = 0.0011), by qRT-PCR. MXD1 and RGS4 significantly increased gene expression in Poly E-treated PC-3 cells, and the MXD1 gene expression increases were Poly E dose-dependent.

GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis.

A stress adaptation pathway termed the integrated stress response has been suggested to be active in many cancers including prostate cancer (PCa). Here, we demonstrate that the eIF2 kinase GCN2 is required for sustained growth in androgen-sensitive and castration-resistant models of PCa both in vitro and in vivo, and is active in PCa patient samples. Using RNA-seq transcriptome analysis and a CRISPR-based phenotypic screen, GCN2 was shown to regulate expression of over 60 solute-carrier (SLC) genes, including those involved in amino acid transport and loss of GCN2 function reduces amino acid import and levels. Addition of essential amino acids or expression of 4F2 (SLC3A2) partially restored growth following loss of GCN2, suggesting that GCN2 targeting of SLC transporters is required for amino acid homeostasis needed to sustain tumor growth. A small molecule inhibitor of GCN2 showed robust in vivo efficacy in androgen-sensitive and castration-resistant mouse models of PCa, supporting its therapeutic potential for the treatment of PCa.

Prostate cancer is the fourth most common cancer worldwide, affecting over a million people each year. Existing drug treatments work by blocking the effects or reducing the levels of the hormone testosterone. However, these drug regimens are not always effective, so finding alternative treatments is an important area of research. One option is to target the 'integrated stress response', a pathway that acts as a genetic switch, turning on a group of genes that counteract cellular stress and are essential for the survival of cancer cells. The reason cancer cells are under stress is because they are hungry. They need to make a lot of proteins and other metabolic intermediates to grow and divide, which means they need plenty of amino acids, the building blocks that make up proteins and fuel metabolism. Amino acids enter cells through molecular gates called amino acid transporters, and scientists think the integrated stress response might play a role in this process. One of the integrated stress response components is a protein called General Control Nonderepressible 2, or GCN2 for short. In healthy cells, this protein helps to boost amino acid levels when supplies start to run low. Cordova et al. examined human prostate cancer cells to find out what role GCN2 plays in this cancer. In both lab-grown cells and tissue from patients, GCN2 was active and played a critical role in prostate tumor growth by turning on the genes for amino acid transporters to increase the levels of amino acids entering the cancer cells. Deleting the gene for GCN2, or blocking its effects with an experimental drug, slowed the growth of cultured prostate cancer cells and reduced tumor growth in mice. In these early experiments, Cordova et al. did not notice any toxic side effects to healthy tissues. If GCN2 works in the same way in humans as it does in mice, blocking it might help to control prostate cancer growth. The integrated stress response is also active in other cancer types, so the same logic might apply to different tumors. However, before GCN2 blockers can become treatments, researchers need a more complete understanding of their molecular effects.

Osteoprotegerin Is a Better Predictor for Cardiovascular and All-Cause Mortality than Vascular Calcifications in a Multicenter Cohort of Patients on Peritoneal Dialysis.

The purpose of this study was to compare vascular calcification (VC), serum osteoprotegerin (OPG) levels, and other biochemical markers to determine their value as available predictors of all-cause and cardiovascular (CV) mortality in patients on peritoneal dialysis (PD). A total of 197 patients were recruited from seven dialysis centers in Mexico City. VC was assessed with multi-slice computed tomography, measured using the calcification score (CaSc). OPG, albumin, calcium, hsC-reactive protein, phosphorous, osteocalcin, total alkaline phosphatase, and intact parathormone were also analyzed. Follow-up and mortality analyses were assessed using the Cox regression model. The mean age was 43.9 ± 12.9 years, 64% were males, and 53% were diabetics. The median OPG was 11.28 (IQR: 7.6−17.4 pmol/L), and 42% of cases had cardiovascular calcifications. The median VC was 424 (IQR:101−886). During follow-up (23 ± 7 months), there were 34 deaths, and 44% were cardiovascular in origin. In multivariable analysis, OPG was a significant predictor for all-cause (HR 1.08; p < 0.002) and CV mortality (HR 1.09; p < 0.013), and performed better than VC (HR 1.00; p < 0.62 for all-cause mortality and HR 1.00; p < 0.16 for CV mortality). For each mg/dL of albumin-corrected calcium, there was an increased risk for CV mortality, and each g/dL of albumin decreased the risk factor for all-cause mortality. OPG levels above 14.37 and 13.57 pmol/L showed the highest predictive value for all-cause and CV mortality in incident PD patients and performed better than VC.

Vascular amyloid accumulation alters the gabaergic synapse and induces hyperactivity in a model of cerebral amyloid angiopathy.

Cerebral amyloid angiopathy (CAA) is typified by the cerebrovascular deposition of amyloid. The mechanisms underlying the contribution of CAA to neurodegeneration are not currently understood. Although CAA is highly associated with the accumulation of ß-amyloid (Aß), other amyloids are known to associate with the vasculature. Alzheimer's disease (AD) is characterized by parenchymal Aß deposition and intracellular accumulation of tau as neurofibrillary tangles (NFTs), affecting synapses directly, leading to behavioral and physical impairment. CAA increases with age and is present in 70%-97% of individuals with AD. Studies have overwhelmingly focused on the connection between parenchymal amyloid accumulation and synaptotoxicity; thus, the contribution of vascular amyloid is mostly understudied. Here, synaptic alterations induced by vascular amyloid accumulation and their behavioral consequences were characterized using a mouse model of Familial Danish dementia (FDD), a neurodegenerative disease characterized by the accumulation of Danish amyloid (ADan) in the vasculature. The mouse model (Tg-FDD) displays a hyperactive phenotype that potentially arises from impairment in the GABAergic synapses, as determined by electrophysiological analysis. We demonstrated that the disruption of GABAergic synapse organization causes this impairment and provided evidence that GABAergic synapses are impaired in patients with CAA pathology. Understanding the mechanism that CAA contributes to synaptic dysfunction in AD-related dementias is of critical importance for developing future therapeutic interventions.

Prevalencia de alteraciones posturales de la columna vertebral, asociada al carente habito deportivo en jóvenes de 17 a 22 años de edad de abril-junio del 2017 / PREVALENCE OF POSTURAL ALTERATIONS OF THE SPINAL COLUMN, ASSOCIATED WITH THE LACK OF SPORT HABIT, IN YOUNG PEOPLE FROM 17 TO 22 YEARS OLD, 2017 / PREVALENCE OF POSTURAL ALTERATIONS OF THE SPINAL COLUMN, ASSOCIATED WITH THE LACK OF SPORT HABIT, IN YOUNG PEOPLE FROM 17 TO 22 YEARS OLD, 2017

Las modificaciones patológicas que afectan la postura son cada vez más frecuentes. Estudios recientes en América Latina, demuestran que las alterciones de la columna vertebral ha experimentado un incremento en la población infantil. Pese a que en nuestro país los estudios al respecto son escasos, existe una publicación en la Revista Ciencia, Tecnologia e Innovación la cual indica que 34 % de los joves sobrellevan una alteración en la columna vertebral. Las alteraciones de postura se van presentando a lo largo de la vida, afectando tanto a personas adultas y ancianas como a jóvenes en edad de desarrollo, que experimentan significativas adaptaciones para lacancear las nuevas proporciones de la musculatura profunda del rquis, favorecerán en un futuro a la aparición de problemas de espalda

Targeting the FKBP51/GR/Hsp90 Complex to Identify Functionally Relevant Treatments for Depression and PTSD.

Genetic and epigenetic alterations in FK506-binding protein 5 ( FKBP5) have been associated with increased risk for psychiatric disorders, including post-traumatic stress disorder (PTSD). Some of these common variants can increase the expression of FKBP5, the gene that encodes FKBP51. Excess FKBP51 promotes hypothalamic-pituitary-adrenal (HPA) axis dysregulation through altered glucocorticoid receptor (GR) signaling. Thus, we hypothesized that GR activity could be restored by perturbing FKBP51. Here, we screened 1280 pharmacologically active compounds and identified three compounds that rescued FKBP51-mediated suppression of GR activity without directly activating GR. One of the three compounds, benztropine mesylate, disrupted the association of FKBP51 with the GR/Hsp90 complex in vitro. Moreover, we show that removal of FKBP51 from this complex by benztropine restored GR localization in ex vivo brain slices and primary neurons from mice. In conclusion, we have identified a novel disruptor of the FKBP51/GR/Hsp90 complex. Targeting this complex may be a viable approach to developing treatments for disorders related to aberrant FKBP51 expression.

Trifunctional High-Throughput Screen Identifies Promising Scaffold To Inhibit Grp94 and Treat Myocilin-Associated Glaucoma.

Gain-of-function mutations within the olfactomedin (OLF) domain of myocilin result in its toxic intracellular accumulation and hasten the onset of open-angle glaucoma. The absence of myocilin does not cause disease; therefore, strategies aimed at eliminating myocilin could lead to a successful glaucoma treatment. The endoplasmic reticulum Hsp90 paralog Grp94 accelerates OLF aggregation. Knockdown or pharmacological inhibition of Grp94 in cells facilitates clearance of mutant myocilin via a non-proteasomal pathway. Here, we expanded our support for targeting Grp94 over cytosolic paralogs Hsp90α and Hsp90ß. We then developed a high-throughput screening assay to identify new chemical matter capable of disrupting the Grp94/OLF interaction. When applied to a blind, focused library of 17 Hsp90 inhibitors, our miniaturized single-read in vitro thioflavin T -based kinetics aggregation assay exclusively identified compounds that target the chaperone N-terminal nucleotide binding site. In follow up studies, one compound (2) decreased the extent of co-aggregation of Grp94 with OLF in a dose-dependent manner in vitro, and enabled clearance of the aggregation-prone full-length myocilin variant I477N in cells without inducing the heat shock response or causing cytotoxicity. Comparison of the co-crystal structure of compound 2 and another non-selective hit in complex with the N-terminal domain of Grp94 reveals a docking mode tailored to Grp94 and explains its selectivity. A new lead compound has been identified, supporting a targeted chemical biology assay approach to develop a protein degradation-based therapy for myocilin-associated glaucoma by selectively inhibiting Grp94.

The influence of poly(ethylene oxide) and illumination on the copper electrodeposition process onto n-Si(100).

In this study, we examined the influence of illumination and the presence of poly(ethylene oxide) (PEO) as an additive for the copper electrodeposition process onto n-Si(100). The study was carried out by means of cyclic voltammetry (CV) and the potential steps method, from which the corresponding nucleation and growth mechanism (NGM) were determined. Likewise, a morphologic analysis of the deposits obtained at different potential values by means of atomic force microscopy (AFM) was carried out. In a first stage, Mott-Schottky measurements so as to characterize the energetics of the semiconductor/electrolyte interface were made. Also, parallel capacity measurements were carried out in order to determine the surface state density of the substrate. It was found that when PEO concentration is increased, the number of these surface states decreases. The CV results indicated that the presence of PEO inhibits the photoelectrochemical reaction of oxide formation on the surface of the semiconductor. This allows a decrease in the overpotential associated with the electrodeposition process. The analysis of the j/t transients shows that the NGM corresponds to progressive three-dimensional (3D) diffusional controlled (PN3D(Diff)), which was confirmed by the AFM technique. Neither illumination nor the presence of PEO changes the mechanisms. Their influence is in that they diminish the size of the nuclei and the speed with which these are formed, which produces a more homogeneous electrodeposit.

Electrosynthesis of Cu-Se films on copper electrodes in alkaline media: a voltammetric, electrochemical quartz crystal microbalance and I/t transient study.

The electroformation of Cu-Se phases, obtained by selenizing a thin film of copper deposited on the quartz/gold electrode system, was studied with an electrochemical quartz crystal microbalance (EQCM) and by cyclic voltammetry (CV) in an alkaline solution (0.05 M Na(2)B(4)O(7)) containing selenide ion. Potentiodynamic parameters showed that the formation of the initial Cu-Se phases (Cu(2-x)Se/Cu(3)Se(2)) is ruled by an irreversible diffusion controlled mechanism, where a first electron transfer is the rate-determining step. A CV study was also performed with a bulk copper electrode in 1 M NaOH solution containing selenide ion. The deconvolution of the anodic and cathodic I/E profiles corresponding to the electroformation and electroreduction of the Cu-Se film formed allowed us to establish that, depending on the anodic potential limit of the potentiodynamic scan, the Cu-Se phases formed were either a mixture of Cu(2)(-x)Se/Cu(3)Se(2) or Cu(2-x)Se/Cu(3)Se(2)/CuSe. An EQCM study showed that, during the initial stage of Cu-Se phase electroformation, water molecules were released from the electrode. In advanced stages of the process, when the electrode was completely covered by Cu-Se compounds, selenide anions were adsorbed on the formed phase. When the anodic potential limit was extended to -0.2 V, copper oxide compounds were formed. The analysis of the cathodic charge related to Cu-Se phase electroreduction and Energy Dispersive X-ray Spectroscopy (EDXS) analysis confirmed that when the anodic limit was -0.8 V, a mixture of different Cu-Se phases was formed. A I/t transient study performed with a bulk copper electrode in alkaline solution containing selenide established that the nucleation and growth mechanism (NGM) of the Cu-Se phases takes place through an initial bidimensional-instantaneous nucleation (IN2D), followed by four bidimensional-progressive nucleations (PN2D). These results and atomic force microscopy (AFM) experiences supported that the growth of the Cu-Se films occurs through a layer-by-layer mechanism.

Chronocoulometric study of the electrochemistry of Prussian blue.

During the electrochemical oxidation of Prussian blue (PB) to Prussian yellow (PY), an electrocatalytic oxygen production proceeds at the electrode when aqueous electrolyte solutions are used. The formed oxygen is scavenged by the PY, probably by absorption, and it is consumed during the electrochemical reduction of PY to PB by a heterogeneous chemical reaction of PB with oxygen to PY and hydrogen peroxide. Because of this catalytic regeneration of PY, it is impossible to determine the amount of low-spin iron by chronocoulometry using a potential program in which PB is first oxidized to PY and then the charge is measured to reduce PY to PB. The latter charge is biased by the electrocatalytic PY regeneration.

New aspects of the electroadsorption of ethyl xanthate on copper electrodes.

The interaction of the ethyl xanthate (EX) anion with a copper electrode in a borate buffer solution, pH 9.2, has been investigated by cyclic voltammetry (CV), electrochemical quartz crystal microbalance (EQCM), electrochemical impedance spectroscopy (EIS), and measurements of contact angle (CA) under controlled potential. The results obtained allow establishing that, in the potential range from -0.80 and -0.60 V, two parallel reactions were characterized. These reactions were the ethyl xanthate electroadsorption and the hydrogen evolution reaction (HER). This last reaction has not been described by previous authors. Besides, the EIS measurements show that the mechanism of the HER on copper electrodes is not affected by the presence of a ethyl xanthate species. The EQCM study shows that in the electrodesorption process the departure of each ethyl xanthate species from the copper electrode is accompanied with the simultaneous entry of four to five water molecules. This fact is in accordance with the number of copper atoms involved in the adsorption of one ethyl xanthate species.

A chemical, morphological, and electrochemical (XPS, SEM/EDX, CV, and EIS) analysis of electrochemically modified electrode surfaces of natural chalcopyrite (CuFeS2) and pyrite (FeS2) in alkaline solutions.

Electrodic surfaces of natural chalcopyrite and natural pyrite minerals (El Teniente mine, Chile) have been studied by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy including microanalysis (SEM/EDX). For comparison, fractured and polished mineral surfaces were also studied by XPS. In both electrodes, the formation of Fe(III) species containing oxygen were detected and Cu(II) species containing oxygen were additionally detected for chalcopyrite at advanced oxidation states. The presence of Cu(II) species containing oxygen was not detected by XPS for the initial oxidation states of the chalcopyrite. For pyrite, the present results do not allow confirmation of the presence of polysulfurs such as have been previously proposed. In both minerals, the measurements of SEM and EDX show relevant alterations in the respective surfaces when different potential values were applied. The chalcopyrite surface shows the formation of protrusions with a high concentration of oxygen. The pyrite surface shows a layer of modified material with high oxygen content. The modifications detected by XPS, SEM, and EDX allowed the explanation of the complexity of the equivalent circuit used to simulate the experimental EIS data. At high oxidation states, both minerals showed a pseudoinductive loop in the equivalent circuit, which was due to the active electrodissolution of the minerals which takes place through a surface film previously formed.