Correction: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3'-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis.

[This corrects the article DOI: 10.1371/journal.pgen.1004749.].

AP-1γ2 is an adaptor protein 1 variant required for endosome-to-Golgi trafficking of the mannose-6-P receptor (CI-MPR) and ATP7B copper transporter.

Selective retrograde transport from endosomes back to the trans-Golgi network (TGN) is important for maintaining protein homeostasis, recycling receptors, and returning molecules that were transported to the wrong compartments. Two important transmembrane proteins directed to this pathway are the Cation-Independent Mannose-6-phosphate receptor (CI-MPR) and the ATP7B copper transporter. Among CI-MPR functions is the delivery of acid hydrolases to lysosomes, while ATP7B facilitates the transport of cytosolic copper ions into organelles or the extracellular space. Precise subcellular localization of CI-MPR and ATP7B is essential for the proper functioning of these proteins. This study shows that both CI-MPR and ATP7B interact with a variant of the clathrin adaptor 1 (AP-1) complex that contains a specific isoform of the γ-adaptin subunit called γ2. Through synchronized anterograde trafficking and cell-surface uptake assays, we demonstrated that AP-1γ2 is dispensable for ATP7B and CI-MPR exit from the TGN while being critically required for ATP7B and CI-MPR retrieval from endosomes to the TGN. Moreover, AP-1γ2 depletion leads to the retention of endocytosed CI-MPR in endosomes enriched in retromer complex subunits. These data underscore the importance of AP-1γ2 as a key component in the sorting and trafficking machinery of CI-MPR and ATP7B, highlighting its essential role in the transport of proteins from endosomes.

HIV-1 Nef Changes the Proteome of T Cells Extracellular Vesicles Depleting IFITMs and Other Antiviral Factors.

Extracellular vesicles (EVs) are biomolecule carriers for intercellular communication in health and disease. Nef is a HIV virulence factor that is released from cells within EVs and is present in plasma EVs of HIV-1 infected individuals. We performed a quantitative proteomic analysis to fully characterize the Nef-induced changes in protein composition of T cell-derived EVs and identify novel host targets of HIV. Several proteins with well-described roles in infection or not previously associated with HIV pathogenesis were specifically modulated by Nef in EVs. Among the downregulated proteins are the interferon-induced transmembrane 1, 2, and 3 (IFITM1-3) proteins, broad-spectrum antiviral factors known to be cell-to-cell transferable by EVs. We demonstrate that Nef depletes IFITM1-3 from EVs by excluding these proteins from the plasma membrane and lipid rafts, which are sites of EVs biogenesis in T cells. Our data establish Nef as a modulator of EVs' global protein content and as an HIV factor that antagonizes IFITMs.

Prevalence of levator ani muscle injuries in primiparous women after delivery and their influence on pelvic floor disorders-systematic review.

BACKGROUND: Studies show a significant association between the first vaginal delivery and injuries of the levator ani muscle (LAM), which can cause pelvic floor disorders (PFDs). OBJECTIVES: This study aims to identify the prevalence of short and long-term LAM injuries after vaginal delivery in primiparous women and its influence on PFDs. METHOD: A systematic review was conducted according to the PRISMA methodology. The databases used were Pubmed, Cochrane, and PEDro. The quality assessment of the evidence was carried out using the Critical Appraisal Skills Programme (CASP). Both the selection of studies and their evaluation were done by two researchers and a third reviewer in cases of disagreement. RESULTS: From the search, 57 articles were gathered, and 19 were included to match the eligibility criteria. The prevalence of avulsion of the LAM was found in association with vaginal delivery between 13% and 28% ≤ 1 year after delivery and between 16% and 29% > 1 year after delivery. Ballooning was detected between 20% and 37% ≤ 1 year, and 33% of women > 1 year after delivery, appearing to be more common when compared to avulsion. Pelvic organ prolapse (POP) was considered the most common disorder associated with injuries of the LAM, and there seems to be some connection with sexual dysfunction. CONCLUSION: Avulsion of the LAM and ballooning of the hiatal area have a high prevalence in primiparous women after vaginal delivery and have a strong direct relation to the development of POP.

Genetic Ablation of Inositol 1,4,5-Trisphosphate Receptor Type 2 (IP3R2) Fails to Modify Disease Progression in a Mouse Model of Spinocerebellar Ataxia Type 3.

Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disease caused by an abnormal polyglutamine expansion within the ataxin-3 protein (ATXN3). This leads to neurodegeneration of specific brain and spinal cord regions, resulting in a progressive loss of motor function. Despite neuronal death, non-neuronal cells, including astrocytes, are also involved in SCA3 pathogenesis. Astrogliosis is a common pathological feature in SCA3 patients and animal models of the disease. However, the contribution of astrocytes to SCA3 is not clearly defined. Inositol 1,4,5-trisphosphate receptor type 2 (IP3R2) is the predominant IP3R in mediating astrocyte somatic calcium signals, and genetically ablation of IP3R2 has been widely used to study astrocyte function. Here, we aimed to investigate the relevance of IP3R2 in the onset and progression of SCA3. For this, we tested whether IP3R2 depletion and the consecutive suppression of global astrocytic calcium signalling would lead to marked changes in the behavioral phenotype of a SCA3 mouse model, the CMVMJD135 transgenic line. This was achieved by crossing IP3R2 null mice with the CMVMJD135 mouse model and performing a longitudinal behavioral characterization of these mice using well-established motor-related function tests. Our results demonstrate that IP3R2 deletion in astrocytes does not modify SCA3 progression.

Revisiting Proteasome Inhibitors: Molecular Underpinnings of Their Development, Mechanisms of Resistance and Strategies to Overcome Anti-Cancer Drug Resistance.

Proteasome inhibitors have shown relevant clinical activity in several hematological malignancies, namely in multiple myeloma and mantle cell lymphoma, improving patient outcomes such as survival and quality of life, when compared with other therapies. However, initial response to the therapy is a challenge as most patients show an innate resistance to proteasome inhibitors, and those that respond to the therapy usually develop late relapses suggesting the development of acquired resistance. The mechanisms of resistance to proteasome inhibition are still controversial and scarce in the literature. In this review, we discuss the development of proteasome inhibitors and the mechanisms of innate and acquired resistance to their activity-a major challenge in preclinical and clinical therapeutics. An improved understanding of these mechanisms is crucial to guiding the design of new and more effective drugs to tackle these devastating diseases. In addition, we provide a comprehensive overview of proteasome inhibitors used in combination with other chemotherapeutic agents, as this is a key strategy to combat resistance.

Attitudes of dentists toward maintaining, repairing, or replacing direct composite resin restorations.

This study aimed to report the reasons for replacement of direct composite resin restorations in dental practices in Brazil. The study used a convenience sample of 213 dentists. A questionnaire was developed from previously validated instruments and sent electronically to prospective participants. The questions pertained to professional characteristics; criteria most frequently used in determining whether a direct composite resin restoration warranted replacement; and respondents' choice of treatment options (maintain, repair, or replace the restoration) in various clinical scenarios based on FDI World Dental Federation evaluation criteria, which are categorized into 3 groups: esthetic, functional, and biological properties. The descriptive statistical analysis was conducted using percentage frequencies, and associations between variables were tested using chi-square tests (α = 0.05). According to 47.9% of respondents, staining was the esthetic property that most often warranted replacement. For the functional property, 53.8% of respondents reported that fracture of material and retention was the most important factor indicating the need for replacement. For the clinical scenarios, 41.7% of respondents chose restoration replacement when evaluating esthetic properties, 59.8% when evaluating functional properties, and 64.4% when evaluating biological properties. Replacement, rather than maintenance or repair, was the most frequently reported clinical decision, and the dentists' professional profiles influenced treatment recommendations, with specialists exhibiting a slightly lower frequency of recommendations for replacement of direct composite resin restorations.

Two Functional Variants of AP-1 Complexes Composed of either γ2 or γ1 Subunits Are Independently Required for Major Histocompatibility Complex Class I Downregulation by HIV-1 Nef.

The HIV-1 accessory protein Nef downregulates the cell surface expression of major histocompatibility complex class I (MHC-I) molecules to facilitate virus spreading. The Nef-induced downregulation of MHC-I molecules such as HLA-A requires the clathrin adaptor protein 1 (AP-1) complex. The cooperative interaction of Nef, AP-1, and the cytosolic tail (CT) of HLA-A leads to a redirection of HLA-A targeting from the trans-Golgi network (TGN) to lysosomes for degradation. Although the γ-adaptin subunit of AP-1 has two distinct isoforms (γ1 and γ2), which may form two AP-1 complex variants, so far, only the importance of AP-1γ1 in MHC-I downregulation by Nef has been investigated. Here, we report that the AP-1γ2 isoform also participates in this process. We found that AP-1γ2 forms a complex with Nef and HLA-A2_CT and that this interaction depends on the Y320 residue in HLA-A2_CT and Nef expression. Moreover, Nef targets AP-1γ1 and AP-1γ2 to different compartments in T cells, and the depletion of either AP-1 variant impairs the Nef-mediated reduction of total endogenous HLA-A levels and rescues HLA-A levels on the cell surface. Finally, immunofluorescence and immunoelectron microscopy analyses reveal that the depletion of γ2 in T cells compromises both the Nef-mediated retention of HLA-A molecules in the TGN and targeting to multivesicular bodies/late endosomes. Altogether, these results show that in addition to AP-1γ1, Nef also requires the AP-1γ2 variant for efficient MHC-I downregulation.IMPORTANCE HIV-1 Nef mediates evasion of the host immune system by inhibiting MHC-I surface presentation of viral antigens. To achieve this goal, Nef modifies the intracellular trafficking of MHC-I molecules in several ways. Despite being the subject of intense study, the molecular details underlying these modifications are not yet fully understood. Adaptor protein 1 (AP-1) plays an essential role in the Nef-mediated downregulation of MHC-I molecules such as HLA-A in different cell types. However, AP-1 has two functionally distinct variants composed of either γ1 or γ2 subunit isoforms. Because previous studies on the role of AP-1 in MHC-I downregulation by Nef focused on AP-1γ1, an important open question is the participation of AP-1γ2 in this process. Here, we show that AP-1γ2 is also essential for Nef-mediated depletion of surface HLA-A molecules in T cells. Our results indicate that Nef hijacks AP-1γ2 to modify HLA-A intracellular transport, redirecting these proteins to lysosomes for degradation.

Adult polyglucosan body disease-an atypical compound heterozygous with a novel GBE1 mutation.

INTRODUCTION: Adult polyglucosan body disease (APBD) is an autosomal recessive leukodystrophy characterized by neurogenic bladder starting after 40 years old, spastic paraparesis and peripheral neuropathy. It is mainly resultant from the GBE1 homozygous p.Tyr329Ser (c.986A>C) mutation, especially in Ashkenazi-Jewish patients, although some cases of compound heterozygous have been reported. A genotype-phenotype correlation is not established, but atypical phenotypes have been described mainly in non-p.Tyr329Ser pathogenic variants. CASE REPORT: We describe an atypical case in a 62-year-old Portuguese woman, presenting the typical clinical triad of APBD plus prominent autonomic dysfunction, suggested by orthostatic hypotension and thermoregulatory dysfunction; she has compound heterozygous GBE1 mutations, namely, p.Asn541Asp (c.1621A>G) and p.Arg515Gly (c.1543C>G), the last one not yet reported in literature and whose pathogenicity was suggested by bioinformatics analysis and confirmed by sural nerve biopsy that showed intra-axonal polyglucosan bodies. DISCUSSION: Besides the report of a novel GBE1 mutation, this case also expands the phenotypic spectrum of this disorder, reinforcing autonomic dysfunction as a possible and prominent manifestation of APBD, mimicking autosomal dominant leukodystrophy with autonomic disease in some way. Therefore, we questioned a possible relationship between this genotype and the phenotype marked by dysautonomia. Additionally, we review previously reported cases of APBD in non-homozygous p.Tyr329Ser patients with atypical phenotypes.

Evaluation of the In Vitro and In Vivo Efficacy of Ruthenium Polypyridyl Compounds against Breast Cancer.

The clinical success of cisplatin, carboplatin, and oxaliplatin has sparked the interest of medicinal inorganic chemistry to synthesize and study compounds with non-platinum metal centers. Despite Ru(II)-polypyridyl complexes being widely studied and well established for their antitumor properties, there are not enough in vivo studies to establish the potentiality of this type of compound. Therefore, we report to the best of our knowledge the first in vivo study of Ru(II)-polypyridyl complexes against breast cancer with promising results. In order to conduct our study, we used MCF7 zebrafish xenografts and ruthenium complexes [Ru(bipy)2(C12H8N6-N,N)][CF3SO3]2Ru1 and [{Ru(bipy)2}2(µ-C12H8N6-N,N)][CF3SO3]4Ru2, which were recently developed by our group. Ru1 and Ru2 reduced the tumor size by an average of 30% without causing significant signs of lethality when administered at low doses of 1.25 mg·L-1. Moreover, the in vitro selectivity results were confirmed in vivo against MCF7 breast cancer cells. Surprisingly, this work suggests that both the mono- and the dinuclear Ru(II)-polypyridyl compounds have in vivo potential against breast cancer, since there were no significant differences between both treatments, highlighting Ru1 and Ru2 as promising chemotherapy agents in breast cancer therapy.

Neurotoxic effects of MPTP on mouse cerebral cortex: Modulation of neuroinflammation as a neuroprotective strategy.

Parkinson's disease (PD) is a progressive neurological disorder, mainly characterized by the progressive loss of dopaminergic neurons in the Substantia nigra pars compacta (SNpc) and by the presence of intracellular inclusions, known as Lewy bodies. Despite SNpc being considered the primary affected region in PD, the neuropathological features are confined solely to the nigro-striatal axis. With disease progression other brain regions are also affected, namely the cerebral cortex, although the spreading of the neurologic damage to this region is still not completely unraveled. Tauroursodeoxycholic acid (TUDCA) is an endogenous bile acid that has been shown to have antioxidant properties and to exhibit a neuroprotective effect in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mice model of PD. Moreover, TUDCA anti-inflammatory properties have been reported in glial cells, making it a prominent therapeutic agent in PD. Here, we used C57BL/6 mice injected with MPTP in a sub-acute paradigm aiming to investigate if the neurotoxic effects of MPTP could be extended to the cerebral cortex. In parallel, we evaluated the anti-oxidant, neuroprotective and anti-inflammatory effects of TUDCA. The anti-inflammatory mechanisms elicited by TUDCA were further dissected in microglia cells. Our results show that MPTP leads to a decrease of ATP and activated AMP-activated protein kinase levels in mice cortex, and to a transient increase in the expression of antioxidant downstream targets of nuclear factor erythroid 2 related factor 2 (Nrf-2), and parkin. Notably, MPTP increases pro-inflammatory markers, while down-regulating the expression of the anti-inflammatory protein Annexin-A1 (ANXA1). Importantly, we show that TUDCA treatment prevents the deleterious effects of MPTP, sustains increased levels of antioxidant enzymes and parkin, and most of all negatively modulates neuroinflammation and up-regulates ANXA1 expression. Additionally, results from cellular models using microglia corroborate TUDCA modulation of ANXA1 synthesis, linking inhibition of neuroinflammation and neuroprotection by TUDCA.

Mean platelet volume and mechanical thrombectomy.

BACKGROUND AND PURPOSE: Mean Platelet Volume (MPV) is a marker of platelet activity and it is an independent predictor for long-term outcome in stroke patients. The aim of this study was to evaluate the association between baseline MPV value and clinical outcome at 90-days in anterior circulation stroke and large vessel occlusion (LVO) patients submitted to mechanical thrombectomy (MT). METHODS: We conducted a prospective observational cohort study in acute ischemic stroke (AIS) patients submitted to MT between January 2017 and May 2018. MPV was measured at admission. Patients were initially stratified into two groups according to the mean MPV level. We also compared groups that were stratified according to the MPV cut-off obtained by Peng F et al (10,4 fL) and performed analyses among MPV terciles. RESULTS: A total of 129 patients were included. Mean level of MPV was 10,9 fL. Patients with embolic stroke of undetermined source (ESUS) had significantly higher rates of good outcome at 3 months compared with large-artery atherosclerotic disease and cardioembolism [(82,9%) vs (78,3%) vs (55,2%); p=0,009]. There were no statistically significant differences in the mean MPV value (p=0,222), successful recanalization (p=0,464) and mortality (p=0,343) when evaluated for all TOAST etiologies. There were no statistically significant differences between the two groups according to the MPV level (10,4 and 10,9 fL) or between the terciles (lowest tertile <10,3 fL, median 10,3 - 11,3 fL, highest >11,3fL) concerning functional outcome at 3 months (p=0,357; p=0,24 and p=0,558, respectively), successful recanalization (p=0,108; p=0,582 and p=0,899, respectively) or mortality at 3 months (p=0,465; p=0,061 and p=0,484, respectively). CONCLUSION: Our study did not find an association between elevated MPV and worse outcome at 3 months in patients with acute anterior circulation stroke and LVO treated with MT. Since ischemic strokes have different pathophysiologic mechanisms, MPV may have distinct prognostic value according to each stroke etiology.

Primary Thrombectomy Versus Combined Mechanical Thrombectomy and Intravenous Thrombolysis in Large Vessel Occlusion Acute Ischemic Stroke.

Mechanical thrombectomy (MT) in combination with intravenous thrombolysis (IVT) is the standard of care for patients with acute ischemic stroke with anterior circulation large vessel occlusion. The particular benefit of IVT in these patients is unknown. We performed a retrospective analysis of patients submitted to MT at our center between January 2015 and June 2017. Functional outcome was prospectively assessed using modified Rankin scale (mRS) at 3 months. A total of 234 patients were enrolled, 152 (65%) in the combined treatment group and 82 (35%) in the direct MT group. Patients receiving combined treatment had a higher frequency of intracranial internal carotid artery occlusion (48 [31.6%] versus 16 [19.5%], P = .048) and significantly less strokes of cardioembolic etiology (72 [47.4%] versus 57 [69.5%], P = .01). Other baseline characteristics did not differ between the 2 groups. Good functional outcome at 3 months (mRS 0-2) was trending toward being higher in patients in the combined treatment group (98 [64.9%] versus 42 [52.5%], P = .066). Rates of symptomatic intracranial hemorrhage (5 [3.3%] versus 4 [4.9%], P = .723) and mortality (15 [9.9%] versus 14 [17.5%], P = .099) did not differ between groups. In multivariate logistic regression analysis, we did not find a statistically significant association between the use of IVT and any of the outcomes studied. Our results suggest that combined treatment carries similar effectiveness and safety than direct MT. Randomized controlled trials regarding this subject are warranted.

Influence of different application protocols of universal adhesive system on the clinical behavior of Class I and II restorations of composite resin - a randomized and double-blind controlled clinical trial.

BACKGROUND: Multimode adhesives incorporate the versatility of adapting to various clinical situations by its capacity to be used in different protocols. This study aimed to evaluate the clinical behavior of composite resin direct restorations (Class I and II) performed with different universal dentin adhesive application protocols comparing adapted FDI and adapted USPHS criteria. METHODS: The current study is a randomized, double-blind, split-mouth, and convenience sample controlled clinical trial. The participants (age ≥ 18 years) had restorative need of Class I and/or II due to the presence of carious lesions and/or unsatisfactory restorations in at least three dental elements. Each participant received three application protocols for Scotchbond Universal adhesive (3M ESPE), one in each tooth to be restored: ER = etch-and-rinse + adhesive (n = 50); SEE = selective enamel etch + adhesive (n = 50) and SE = self-etch adhesive (n = 50). All teeth were restored in a similar way using Filtek™ Supreme composite resin (3M ESPE). Restorations were evaluated using the adapted FDI and adapted USPHS criteria, at baseline after 7 to 21 (12.02 ± 5.68) days (T1; n = 50 per group) and after 12 to 20 (15.8 ± 2.7) months (T2; n = 46 per group) by two previously calibrated evaluators (Kappa > 0.80). The statistical tests were performed between groups (Friedman), intragroup (Wilcoxon), and between the criteria considering acceptable and not acceptable restorations (McNemar), α = 0.05. RESULTS: A statistically significant difference was observed only for the property "superficial staining", between groups at T2 (p = 0.01) for ER (n = 13 with score 2 or more) and SEE (n = 3 with score 2 or more) and intragroup for ER (T1, n = 1 with score 2 or more; T2, n = 13 with score 2 or more, p = 0.001) and SE (T1, n = 0 with score 2 or more; T2, n = 8 with score 2 or more p = 0.007). For the other comparisons between groups, intragroup, and between the adapted FDI and adapted USPHS criteria, there were no statistically significant differences (p ≥ 0.05). CONCLUSIONS: It can be concluded that the different application protocols of the universal adhesive resulted in clinically "acceptable" restorations after 15.8 ± 2.7 months of follow-up. Adapted FDI and adapted USPHS criteria provided similar results to each other. TRIAL REGISTRATION: Number in Brazilian Registry of Clinical Trials (ReBEC): RBR-9p3hdp. Registered 24 May 2015.

Neuroprotective Effects of Creatine in the CMVMJD135 Mouse Model of Spinocerebellar Ataxia Type 3.

BACKGROUND AND OBJECTIVE: Mitochondrial dysfunction has been implicated in several neurodegenerative diseases. Creatine administration increases concentration of the energy buffer phosphocreatine, exerting protective effects in the brain. We evaluate whether a creatine-enriched diet would be beneficial for a mouse model of spinocerebellar ataxia type 3, a genetically defined neurodegenerative disease for which no treatment is available. METHODS: We performed 2 independent preclinical trials using the CMVMJD135 mouse model (treating 2 groups of animals with different disease severity) and wild-type mice, to which 2% creatine was provided for 19 (preclinical trial 1) or 29 (preclinical trial 2) weeks, starting at a presymptomatic age. Motor behavior was evaluated at several time points from 5 to 34 weeks of age, and neuropathological studies were performed at the end of each trial. RESULTS: Creatine supplementation led to an overall improvement in the motor phenotype of CMVMJD135 mice in both trials, rescuing motor balance and coordination and also restored brain weight, mitigated astrogliosis, and preserved Calbindin-positive cells in the cerebellum. Moreover, a reduction of mutant ataxin-3 aggregates occurred despite maintained steady-state levels of the protein and the absence of autophagy activation. Creatine treatment also restored the expression of the mitochondrial mass marker Porin and reduced the expression of antioxidant enzymes Heme oxygenase 1 (HO1) and NAD(P)H Quinone Dehydrogenase 1 (NQO1), suggesting a beneficial effect at the level of mitochondria and oxidative stress. CONCLUSIONS: Creatine slows disease progression and improves motor dysfunction as well as ameliorates neuropathology of the CMVMJD135 animals, supporting this as a useful strategy to slow the progression of spinocerebellar ataxia type 3. © 2018 International Parkinson and Movement Disorder Society.

Time to Reset the Definition of Successful Revascularization in Endovascular Treatment of Acute Ischemic Stroke.

BACKGROUND: A 2013 consensus statement recommended the use of the modified Treatment In Cerebral Ischemia (mTICI) scale to evaluate angiographic revascularization after endovascular treatment (EVT) of acute ischemic stroke due to its higher inter-rater agreement and capacity of clinical outcome prediction. The current definition of successful revascularization includes the achievement of grades mTICI 2b or 3. However, mTICI 2b grade encompasses a large heterogeneity of revascularization states, and prior studies suggested that the magnitude of benefit derived from mTICI 2b and mTICI 3 does not seem to be equivalent. In a way to restrain the referred heterogeneity, Goyal et al. [J Neurointerv Surg 2014; 6: 83-86] proposed a revised mTICI scale that includes a 2c grade (rTICI). METHODS: Retrospective analysis of prospectively collected data from consecutive cases of EVT for anterior circulation large-vessel occlusion, performed between January 2015 and July 2017. Patients with mTICI 2b or 3 grades were reclassified according to the rTICI scale, and the outcomes between the 3 revascularization grades (rTICI 2b, 2c, 3) compared. RESULTS: Our study population of 226 patients (64 rTICI 2b, 30 rTICI 2c, 132 rTICI 3) has a mean age of 71 years, 48.2% males, median baseline NIHSS of 16 (13-19) and ASPECTS of 8 (7-9). The 3 revascularization grades are represented by homogeneous populations. Logistic regression analysis showed statistically significant higher rates of functional independence at 3 months (65.9 vs. 50.0%; adjusted OR 0.39, 95% CI 0.18-0.86), with lower rates of mortality (8.3 vs. 15.6%; adjusted OR 3.54, 95% CI 1.14-10.97) and intracranial hemorrhage (ICH) in rTICI 3 than 2b groups. When comparing rTICI 3 with 2c groups, there were only statistically significant differences in the total ICH rate (8.3 vs. 26.7%; adjusted OR 7.08, 95% CI 1.80-27.82) but not in symptomatic ICH. CONCLUSIONS: These results corroborate the scarce prior findings suggesting that patients with rTICI 2c grade should be reported separately, since they have similar outcomes to rTICI 3, and better than rTICI 2b patients. Therefore, we suggest resetting the angiographic revascularization endpoint to perfect revascularization (rTICI 2c or 3 grades), a target that neurointerventionalists should strive to achieve.

RuII( p-cymene) Compounds as Effective and Selective Anticancer Candidates with No Toxicity in Vivo.

Ruthenium(II) complexes are currently considered a viable alternative to the widely used platinum complexes as efficient anticancer agents. We herein present the synthesis and characterization of half-sandwich ruthenium compounds with the general formula [Ru( p-cymene)(L-N,N)Cl][CF3SO3] (L = 3,6-di-2-pyridyl-1,2,4,5-tetrazine (1) 6,7-dimethyl-2,3-bis(pyridin-2-yl)quinoxaline (2)), which have been synthesized by substitution reactions from the precursor dimer [Ru( p-cymene)(Cl)(µ-Cl)]2 and were characterized by elemental analysis, mass spectrometry, 1H NMR, UV-vis, and IR spectroscopy, conductivity measurements, and cyclic voltammetry. The molecular structure for complex 2 was determined by single-crystal X-ray diffraction. The cytotoxic activity of these compounds was evaluated against human tumor cells, namely ovarian carcinoma A2780 and breast MCF7 and MDAMB231 adenocarcinoma cells, and against normal primary fibroblasts. Whereas the cytotoxic activity of 1 is moderate, IC50 values found for 2 are among the lowest previously reported for Ru( p-cymene) complexes. Both compounds present no cytotoxic effect in normal human primary fibroblasts when they are used at the IC50 concentration in A2780 and MCF7 cancer cells. Their antiproliferative capacity is associated with a combined mechanism of apoptosis and autophagy. A strong interaction with DNA was observed for both with a binding constant value of the same magnitude as that of the classical intercalator [Ru(phen)2(dppz)]2+. Both complexes bind to human serum albumin with moderate to strong affinity, with conditional binding constants (log Kb) of 4.88 for complex 2 and 5.18 for complex 1 in 2% DMSO/10 mM Hepes pH7.0 medium. The acute toxicity was evaluated in zebrafish embryo model using the fish embryo acute toxicity test (FET). Remarkably, our results show that compounds 1 and 2 are not toxic/lethal even at extremely high concentrations. The novel compounds reported herein are highly relevant antitumor metallodrug candidates, given their in vitro cytotoxicity toward cancer cells and the lack of in vivo toxicity.

The role of the mammalian DNA end-processing enzyme polynucleotide kinase 3'-phosphatase in spinocerebellar ataxia type 3 pathogenesis.

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3'-P and 5'-OH, are processed by mammalian polynucleotide kinase 3'-phosphatase (PNKP), a bifunctional enzyme with 3'-phosphatase and 5'-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14-41 to 55-82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP's 3' phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3'-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients' brain. Finally, long amplicon quantitative PCR analysis of human MJD patients' brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.

Dominant negative effect of polyglutamine expansion perturbs normal function of ataxin-3 in neuronal cells.

The physiological function of Ataxin-3 (ATXN3), a deubiquitylase (DUB) involved in Machado-Joseph Disease (MJD), remains elusive. In this study, we demonstrate that ATXN3 is required for neuronal differentiation and for normal cell morphology, cytoskeletal organization, proliferation and survival of SH-SY5Y and PC12 cells. This cellular phenotype is associated with increased proteasomal degradation of α5 integrin subunit (ITGA5) and reduced activation of integrin signalling and is rescued by ITGA5 overexpression. Interestingly, silencing of ATXN3, overexpression of mutant versions of ATXN3 lacking catalytic activity or bearing an expanded polyglutamine (polyQ) tract led to partially overlapping phenotypes. In vivo analysis showed that both Atxn3 knockout and MJD transgenic mice had decreased levels of ITGA5 in the brain. Furthermore, abnormal morphology and reduced branching were observed both in cultured neurons expressing shRNA for ATXN3 and in those obtained from MJD mice. Our results show that ATXN3 rescues ITGA5 from proteasomal degradation in neurons and that polyQ expansion causes a partial loss of this cellular function, resulting in reduced integrin signalling and neuronal cytoskeleton modifications, which may be contributing to neurodegeneration.

Novel insights into the antioxidant role of tauroursodeoxycholic acid in experimental models of Parkinson's disease.

Impaired mitochondrial function and generation of reactive oxygen species are deeply implicated in Parkinson's disease progression. Indeed, mutations in genes that affect mitochondrial function account for most of the familial cases of the disease, and post mortem studies in sporadic PD patients brains revealed increased signs of oxidative stress. Moreover, exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a mitochondrial complex I inhibitor, leads to clinical symptoms similar to sporadic PD. The bile acid tauroursodeoxycholic acid (TUDCA) is an anti-apoptotic molecule shown to protect against MPTP-induced neurodegeneration in mice, but the mechanisms involved are still incompletely identified. Herein we used MPTP-treated mice, as well as primary cultures of mice cortical neurons and SH-SY5Y cells treated with MPP+ to investigate the modulation of mitochondrial dysfunction by TUDCA in PD models. We show that TUDCA exerts its neuroprotective role in a parkin-dependent manner. Overall, our results point to the pharmacological up-regulation of mitochondrial turnover by TUDCA as a novel neuroprotective mechanism of this molecule, and contribute to the validation of TUDCA clinical application in PD.