The selective cytotoxicity of new triazene compounds to human melanoma cells.

Metastatic melanoma still remains one the most difficult cancers to overcome. The aim of our research was the design of anti-tumour triazene compounds 3 for application to a melanoma-specific therapy. The strategy exploits the unique enzyme pathway of melanin biosynthesis for conversion of non-toxic prodrugs into toxic drugs in the melanoma cell. The compounds 3 were designed by coupling two active moieties, the alkylating triazenes and different tyrosinase substrates. All compounds 3 revealed to be chemically stable in isotonic phosphate buffer (PBS) at physiologic pH (t½≥48h), and most of them showed to be slowly hydrolysed in human plasma (1.5≤t½ (h)≤161). Compounds 3c-n revealed to be excellent tyrosinase substrates (0.74≤t½ (min)≤6) with the best tyrosinase substrate 3l releasing MMT 45s after tyrosinase activation. Structure-activity relationship studies allowed the identification of the better structural features for enzyme affinity. Furthermore, the derivatives 3l and 3m showed cell selectivity with significant cytotoxic effects (IC50 values of 46-65µM) against melanoma cell lines with tyrosinase overexpression MNT-1 and B16F10.

Ezh2, the histone methyltransferase of PRC2, regulates the balance between self-renewal and differentiation in the cerebral cortex.

Multipotent progenitor cells of the cerebral cortex balance self-renewal and differentiation to produce complex neural lineages in a fixed temporal order in a cell-autonomous manner. We studied the role of the polycomb epigenetic system, a chromatin-based repressive mechanism, in controlling cortical progenitor cell self-renewal and differentiation. We found that the histone methyltransferase of polycomb repressive complex 2 (PCR2), enhancer of Zeste homolog 2 (Ezh2), is essential for controlling the rate at which development progresses within cortical progenitor cell lineages. Loss of function of Ezh2 removes the repressive mark of trimethylated histone H3 at lysine 27 (H3K27me3) in cortical progenitor cells and also prevents its establishment in postmitotic neurons. Removal of this repressive chromatin modification results in marked up-regulation in gene expression, the consequence of which is a shift in the balance between self-renewal and differentiation toward differentiation, both directly to neurons and indirectly via basal progenitor cell genesis. Although the temporal order of neurogenesis and gliogenesis are broadly conserved under these conditions, the timing of neurogenesis, the relative numbers of different cell types, and the switch to gliogenesis are all altered, narrowing the neurogenic period for progenitor cells and reducing their neuronal output. As a consequence, the timing of cortical development is altered significantly after loss of PRC2 function.

Aberrant splicing exonizes C9ORF72 repeat expansion in ALS/FTD.

A nucleotide repeat expansion (NRE) in the first annotated intron of the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). While C9 NRE-containing RNAs can be translated into several toxic dipeptide repeat proteins, how an intronic NRE can assess the translation machinery in the cytoplasm remains unclear. By capturing and sequencing NRE-containing RNAs from patient-derived cells, we found that C9 NRE was exonized by the usage of downstream 5' splice sites and exported from the nucleus in a variety of spliced mRNA isoforms. C9ORF72 aberrant splicing was substantially elevated in both C9 NRE+ motor neurons and human brain tissues. Furthermore, NREs above the pathological threshold were sufficient to activate cryptic splice sites in reporter mRNAs. In summary, our results revealed a crucial and potentially widespread role of repeat-induced aberrant splicing in the biogenesis, localization, and translation of NRE-containing RNAs.

Human sensorimotor organoids derived from healthy and amyotrophic lateral sclerosis stem cells form neuromuscular junctions.

Human induced pluripotent stem cells (iPSC) hold promise for modeling diseases in individual human genetic backgrounds and thus for developing precision medicine. Here, we generate sensorimotor organoids containing physiologically functional neuromuscular junctions (NMJs) and apply the model to different subgroups of amyotrophic lateral sclerosis (ALS). Using a range of molecular, genomic, and physiological techniques, we identify and characterize motor neurons and skeletal muscle, along with sensory neurons, astrocytes, microglia, and vasculature. Organoid cultures derived from multiple human iPSC lines generated from individuals with ALS and isogenic lines edited to harbor familial ALS mutations show impairment at the level of the NMJ, as detected by both contraction and immunocytochemical measurements. The physiological resolution of the human NMJ synapse, combined with the generation of major cellular cohorts exerting autonomous and non-cell autonomous effects in motor and sensory diseases, may prove valuable to understand the pathophysiological mechanisms of ALS.

Effect of Ezogabine on Cortical and Spinal Motor Neuron Excitability in Amyotrophic Lateral Sclerosis: A Randomized Clinical Trial.

Importance: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of the motor nervous system. Clinical studies have demonstrated cortical and spinal motor neuron hyperexcitability using transcranial magnetic stimulation and threshold tracking nerve conduction studies, respectively, although metrics of excitability have not been used as pharmacodynamic biomarkers in multi-site clinical trials. Objective: To ascertain whether ezogabine decreases cortical and spinal motor neuron excitability in ALS. Design, Setting, and Participants: This double-blind, placebo-controlled phase 2 randomized clinical trial sought consent from eligible participants from November 3, 2015, to November 9, 2017, and was conducted at 12 US sites within the Northeast ALS Consortium. Participants were randomized in equal numbers to a higher or lower dose of ezogabine or to an identical matched placebo, and they completed in-person visits at screening, baseline, week 6, and week 8 for clinical assessment and neurophysiological measurements. Interventions: Participants were randomized to receive 600 mg/d or 900 mg/d of ezogabine or a matched placebo for 10 weeks. Main Outcomes and Measures: The primary outcome was change in short-interval intracortical inhibition (SICI; SICI-1 was used in analysis to reflect stronger inhibition from an increase in amplitude) from pretreatment mean at screening and baseline to the full-dose treatment mean at weeks 6 and 8. The secondary outcomes included levels of cortical motor neuron excitability (including resting motor threshold) measured by transcranial magnetic stimulation and spinal motor neuron excitability (including strength-duration time constant) measured by threshold tracking nerve conduction studies. Results: A total of 65 participants were randomized to placebo (23), 600 mg/d of ezogabine (23), and 900 mg/d of ezogabine (19 participants); 45 were men (69.2%) and the mean (SD) age was 58.3 (8.8) years. The SICI-1 increased by 53% (mean ratio, 1.53; 95% CI, 1.12-2.09; P = .009) in the 900-mg/d ezogabine group vs placebo group. The SICI-1 did not change in the 600-mg/d ezogabine group vs placebo group (mean ratio, 1.15; 95% CI, 0.87-1.52; P = .31). The resting motor threshold increased in the 600-mg/d ezogabine group vs placebo group (mean ratio, 4.61; 95% CI, 0.21-9.01; P = .04) but not in the 900-mg/d ezogabine group vs placebo group (mean ratio, 1.95; 95% CI, -2.64 to 6.54; P = .40). Ezogabine caused a dose-dependent decrease in excitability by several other metrics, including strength-duration time constant in the 900-mg/d ezogabine group vs placebo group (mean ratio, 0.73; 95% CI, 0.60 to 0.87; P < .001). Conclusions and Relevance: Ezogabine decreased cortical and spinal motor neuron excitability in participants with ALS, suggesting that such neurophysiological metrics may be used as pharmacodynamic biomarkers in multisite clinical trials. Trial Registration: ClinicalTrials.gov Identifier: NCT02450552.

Pharmacological Profiling of Purified Human Stem Cell-Derived and Primary Mouse Motor Neurons.

Directed differentiation of human pluripotent stem cells (hPSCs) has enabled the generation of specific neuronal subtypes that approximate the intended primary mammalian cells on both the RNA and protein levels. These cells offer unique opportunities, including insights into mechanistic understanding of the early driving events in neurodegenerative disease, replacement of degenerating cell populations, and compound identification and evaluation in the context of precision medicine. However, whether the derived neurons indeed recapitulate the physiological features of the desired bona fide neuronal subgroups remains an unanswered question and one important for validating stem cell models as accurate functional representations of the primary cell types. Here, we purified both hPSC-derived and primary mouse spinal motor neurons in parallel and used extracellular multi-electrode array (MEA) recording to compare the pharmacological sensitivity of neuronal excitability and network function. We observed similar effects for most receptor and channel agonists and antagonists, supporting the consistency between human PSC-derived and mouse primary spinal motor neuron models from a physiological perspective.

Evidence for a retinal progenitor cell in the postnatal and adult mouse.

Progress in cell therapy for retinal disorders has been challenging. Recognized retinal progenitors are a heterogeneous population of cells that lack surface markers for the isolation of live cells for clinical implementation. In the present application, our objective was to use the stem cell factor receptor c-Kit (CD117), a surface marker, to isolate and evaluate a distinct progenitor cell population from retinas of postnatal and adult mice. Here we report that, by combining traditional methods with fate mapping, we have identified a c-Kit-positive (c-Kit+) retinal progenitor cell (RPC) that is self-renewing and clonogenic in vitro, and capable of generating many cell types in vitro and in vivo. Based on cell lineage tracing, significant subpopulations of photoreceptors in the outer nuclear layer and bipolar, horizontal, amacrine and Müller cells in the inner nuclear layer are the progeny of c-Kit+ cells in vivo. The RPC progeny contributes to retinal neurons and glial cells, which are responsible for the conversion of light into visual signals. The ability to isolate and expand in vitro live c-Kit+ RPCs makes them a future therapeutic option for retinal diseases.

Single-cell analysis of the fate of c-kit-positive bone marrow cells.

The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart.

Biomaterials from blends of fluoropolymers and corn starch-implant and structural aspects.

The development of polymeric blends to be used as matrices for bone regeneration is a hot topic nowadays. In this article we report on the blends composed by corn starch and poly(vinylidene fluoride), PVDF, or poly(vinylidene fluoride-trifluoroethylene), P(VDF-TrFE), to obtain biocompatible materials. Blends were produced by compressing/annealing and chemically/structurally characterized by micro-Raman scattering and Fourier transform infrared (FTIR) absorption spectroscopies, dynamic mechanical analysis (DMA) and scanning electron microscopy (SEM), besides in vivo study to evaluate the tissue response. Vibrational spectroscopy reveals no chemical interaction between the polymers and starch, absence of material degradation due to compressing/annealing process or organism implantation, and maintenance of α and ferroelectric crystalline phases of PVDF and P(VDF-TrFE), respectively. As a consequence of absence of interaction between polymers and starch, it was possible to identify by SEM each material, with starch acting as filler. Elastic modulus (E') obtained from DMA measurement, independent of the material proportion used in blends, reaches values close to those of cancellous bone. Finally, the in vivo study in animals shows that the blends, regardless of the composition, were tolerated by cancellous bone.

Synthesis and evaluation of N-acylamino acids derivatives of triazenes. Activation by tyrosinase in human melanoma cell lines.

In this research work we report the synthesis of a new series of triazene prodrugs designed for Melanocyte-Directed Enzyme Prodrug Therapy (MDEPT). These compounds are derived from the N-acyltyrosine amino acid - a good enzyme substrate for the tyrosinase enzyme, which is significantly overexpressed in melanoma cells. We analysed their chemical stability and plasma enzymatic hydrolysis, and we also evaluated the release of the antitumoral drug in the presence of the tyrosinase. Subsequently, we performed the evaluation of the prodrug cytotoxicity in melanoma cell lines with different levels of tyrosinase activity. Prodrug 5c showed the highest cytotoxicity against melanoma cell lines, and this effect correlated well with the tyrosinase activity suggesting that prodrug cytotoxicity is tyrosinase-dependent.

Subcutaneous tissue reaction and cytotoxicity of polyvinylidene fluoride and polyvinylidene fluoride-trifluoroethylene blends associated with natural polymers.

Cytotoxicity and subcutaneous tissue reaction of innovative blends composed by polyvinylidene fluoride and polyvinylidene fluoride-trifluoroethylene associated with natural polymers (natural rubber and native starch) forming membranes were evaluated, aiming its applications associated with bone regeneration. Cytotoxicity was evaluated in mouse fibroblasts culture cells (NIH3T3) using trypan blue staining. Tissue response was in vivo evaluated by subcutaneous implantation of materials in rats, taking into account the presence of necrosis and connective tissue capsule around implanted materials after 7, 14, 21, 28, 35, 60, and 100 days of surgery. The pattern of inflammation was evaluated by histomorphometry of the inflammatory cells. Chemical and morphological changes of implanted materials after 60 and 100 days were evaluated by Fourier transform infrared (FTIR) absorption spectroscopy and scanning electron microscopy (SEM) images. Cytotoxicity tests indicated a good tolerance of the cells to the biomaterial. The in vivo tissue response of all studied materials showed normal inflammatory pattern, characterized by a reduction of polymorphonuclear leukocytes and an increase in mononuclear leukocytes over the time (p < 0.05 Kruskal-Wallis). On day 60, microscopic analysis showed regression of the chronic inflammatory process around all materials. FTIR showed no changes in chemical composition of materials due to implantation, whereas SEM demonstrated the delivery of starch in the medium. Therefore, the results of the tests performed in vitro and in vivo show that the innovative blends can further be used as biomaterials.

Projeto Hemiplegia: um modelo de fisioterapia em grupo para hemiplégicos crônicos / Project Hemiplegia: a model of group physical therapy of chronic hemiplegic patients

Objetivo: Este trabalho teve por objetivo caracterizar os pacientes e as atividades realizadas no Projeto Hemiplegia, que consiste em encontros de portadores de hemiplegia para realização de fisioterapia em grupo. Método: A caracterização dos pacientes foi feita por meio da análise de seus prontuários e da aplicação da Escala de Equilíbrio de Berg (EEB), e a descrição do projeto pela análise da filmagem de 18 sessões do projeto. Resultados: Dos pacientes analisados 15 pertenciam ao sexo masculino e 4 ao feminino, com idade média de59,63 ± 11,1 anos. O tempo médio de seqüela pós-AVC foi de 5,17 ± 3,92 anos. As médias alcançadas nas avaliações da EEB foram 43,00 ± 10,48 pontos na 1ª avaliação e 44,95 ± 9,69 pontos na 2ª avaliação. A análise comparativa entre as duas avaliações determinou que houve diferença estatística significativa entre elas(p<0,05). A conduta fisioterapêutica caracteriza-se pela utilização de exercícios ativos, alongamentos, exercícios de equilíbrio e coordenação e atividades lúdicas. Conclusão: O Projeto Hemiplegia demonstra que a terapia em grupo pode ser uma boa opção terapêutica capaz de impedir complicações que possam determinar uma maior deterioração da capacidade funcional e da dependência, também garante a manutenção ou melhora do equilíbrio, melhorando assim a qualidade de vida desses pacientes.

Objective: The aim of this study was to characterize the patients and the physical activities performed in theHemiplegia Project. This project consisted of a group of hemiplegic patients that get together to performgroup physical therapy. Method: The profile of the patients was made through the analysis of their medicalrecords and the use of Berg balance scale. The outline of the project was made by analyzing the recordingimages of 18 sessions of the program. Results: Of the 19 patients enrolled, 15 were male and 4 female. Meanage was 59.63 ± 11.1 years. The median known duration of sequela after a stroke (CVA) was 5.17 ± 3.92 years.The average scores reached in the BBS evaluations were 43.00 ± 10.48 points in 1st evaluation and 44.95 ± 9.69points in 2nd evaluation. A comparative analysis was performed to test differences between the two evaluations.There were no significant statistical differences between the two evaluations (p £ 0.05). The average time ofsequel after AVC was 5.17 ± 3.92 years. The physical therapy session is characterized for the use of activeexercises, stretching, equilibrium and motor coordination exercises, and playful activities. Conclusion: TheHemiplegia Project demonstrates that the group therapy can be a good therapeutical option to preventcomplications that may determine a much large deterioration of the functional capacity and the dependence.Also the group therapy assures the maintenance of the equilibrium or improves it, thus improving the qualityof life of these patients.