From Neuronal Differentiation of iPSCs to 3D Neural Organoids: Modeling of Neurodegenerative Diseases

In the last decade, the finding that somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) leads to a great improvement of research involving the use of differentiated stem cells as model of diseases. In the field of neurodegeneration, iPSC technology allowed to culture in vitro all the types of patient-specific neurons, not only helping the discovery of diseases’ etiopathology but also testing new drugs with a personalized medicine approach. Moreover, iPSCs can be combined with the 3D bioprinting technology, allowing physiological cell-to-cell interactions, given by a combination of several biomaterials, scaffolds, and cells. This technology combines bioplotter and biomaterials which can encapsulate several types of cells, e.g., iPSCs or differentiated neurons, to develop an innovative cellular model. iPSCs and 3D cell cultures’ technologies represent the first step to obtain a more reliable model, like an organoid to facilitate neurodegenerative diseases’ investigation

RNA Metabolism and Therapeutics in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disorder characterized by the selective death of upper and lowers motor neurons in spinal cord, brain stem, and motor cortex, which leads to paralysis and death within 2–3 years of onset. Deeply sequencing technologies, to simultaneously analyze the transcriptional expression of thousands of genes, offered new possibilities to focus on ALS pathogenesis and, most notably, to find new potential targets for novel treatments. The present book chapter illustrates recent advances in transcriptomic studies in animal models and human samples and in new molecular targets related to ALS pathogenesis and disease progression. Additionally, new insights into the involvement of altered transcriptional profiles of noncoding RNAs (microRNA and lncRNA) and ALS-associated ribosomal binding proteins have been investigated, to understand the functional consequences of extensive RNA dysregulation in ALS. Attention has been also turned on how transcriptome alterations could highlight new molecular targets for drug development

SOD1 Transcriptional and Posttranscriptional Regulation and Its Potential Implications in ALS

Copper-zinc superoxide dismutase (SOD1) is a detoxifying enzyme localized in the cytosol, nucleus, peroxisomes, and mitochondria. The discovery that mutations in SOD1 gene cause a subset of familial amyotrophic lateral sclerosis (FALS) has attracted great attention, and studies to date have been mainly focused on discovering mutations in the coding region and investigation at protein level. Considering that changes in SOD1 mRNA levels have been associated with sporadic ALS (SALS), a molecular understanding of the processes involved in the regulation of SOD1 gene expression could not only unravel novel regulatory pathways that may govern cellular phenotypes and changes in diseases but also might reveal therapeutic targets and treatments. This review seeks to provide an overview of SOD1 gene structure and of the processes through which SOD1 transcription is controlled. Furthermore, we emphasize the importance to focus future researches on investigating posttranscriptional mechanisms and their relevance to ALS

Fermentation of sugar cane juice (Sacharum officinarum) cultivar RB 7515 by wild yeasts resistant to UVC

Commercial alcoholic fermentations coexist with microbial contamination come together withsugar cane or industrial water. The contaminants are bacteria or wild yeast that also results reduction in yield. The contaminants control use acid treatment and antibiotics. Disinfection by ultraviolet radiation is efficient in transparent liquid and has as advantage leaving noresidues. Reports on the UV effect over microorganisms in turbid and colored liquids are scarce. The research evaluated the use of commercial UVC lamp in continuous reactor for reducing microbial load and its effect on the fermentation of sugarcane cultivar RB 7515. The sugar cane juice and tap water used in dilution were both exposed to doses of 0, 500, 1000, 2000 and 4000 Joules/ljuice. The counts were made in specific culture media. The dose 500 Joules/liter was enough to eliminate the coliforms, but survived yeast and bacteria without difference to the others dose. The treated sugar cane juice was diluted to 12 Brix and fermented by the surviving wild yeasts untilBrix stabilization. The alcoholic graduation was 5° GL for all the treatment. It was proved the ability of wild yeasts resistant to UVC even with long fermentation times.Fermentações alcoólicas comerciais convivem com contaminações por microrganismos presentes nos colmos de cana ou na água industrial. Os contaminantes são bactérias ou leveduras “selvagens” que resultam em prejuízosde rendimento. O controle destas contaminações é feito com ácido e antibiótico. A desinfecção por radiação ultravioleta é eficiente em líquidos transparentes e apresenta vantagem por não deixar resíduos. Relatos do efeito sobre microrganismos em líquidos turvos e coloridos são escassos. A pesquisa avaliou o uso de lâmpada comercial de radiação UVC para redução de carga microbiana e seu efeito sobre a fermentação do caldo de cana cultivar RB 7515. O caldo brutoe água usados na diluição a Brix 12,0 foram submetidos àsdoses de 0, 500, 1000, 2000 e 4000 Joules/l. As contagens foram feitas em meios de cultivo específicos. A dose 500 Joules/Lfoi suficiente para eliminar os coliformes, mas sobreviveram leveduras e bactérias mesófilas. Doses superiores a 500 Joules/leliminaram todos os microrganismos. O caldo tratado diluído a 12º Brix foi fermentado com a leveduras sobrevivente até estabilização do Brix, proporcionando vinho a 5°GLpara todos os tratamentos. Considerou-se que a potencialidade de leveduras selvagens resistentes àradiação UVCfoi comprovada

Altered immune system in frailty: Genetics and diet may influence inflammation.

Frailty is a complex geriatric syndrome associated with biological vulnerability to stressors and decreased physiological reserve. Its etiology and pathogenesis are not completely understood, although various causes and complex pathways have been proposed. Immune system alterations (immunosenescence and "InflammAging") have been suggested to contribute to frailty, but a precise causative role of such alterations remains to be determined. Genetic studies support the suggestion of immune system involvement in frailty: genetic variants in genes involved in immune system function have been associated with the syndrome. Interestingly, nutritional status, through its effects on cellular metabolism, may also influence the immune system, i.e. hormone and cytokine (mainly adipocytokine) levels, and immune cell populations and function, increasing inflammation and contributing to frailty. This review aims to discuss the role of immune system alterations in frailty, analyzing the role of genetic factors in frailty onset and the impact of diet on inflammation and, in turn, on frailty

Antimicrobial activity of ceftobiprole against Gram-negative and Gram-positive pathogens: results from INVITA-A-CEFTO Brazilian study

Ceftobiprole is a broad-spectrum cephalosporin with potent activity against staphylococci, including those resistant to oxacillin, as well as against most Gram-negative bacilli including Pseudomonas aeruginosa. In this study, the in vitro activity of ceftobiprole and comparator agents was tested against bacterial isolates recently collected from Brazilian private hospitals. A total of 336 unique bacterial isolates were collected from hospitalized patients between February 2008 and August 2009. Each hospital was asked to submit 100 single bacterial isolates responsible for causing blood, lower respiratory tract or skin and soft tissue infections. Bacterial identification was confirmed and antimicrobial susceptibility testing was performed using CLSI microdilution method at a central laboratory. The CLSI M100-S21 (2011) was used for interpretation of the antimicrobial susceptibility results. Among the 336 pathogens collected, 255 (75.9%) were Gram-negative bacilli and 81 (24.1%) were Gram-positive cocci. Although ceftobiprole MIC50 values for oxacillin resistant strains were two-fold higher than for methicillin susceptible S. aureus, ceftobiprole inhibited 100% of tested S. aureus at MICs 6 µg/mL for both species. Our results showed that ceftobiprole has potent activity against staphylococci and E. faecalis, which was superior to that of vancomycin. Our data also indicates that ceftobiprole demonstrated potency comparable to that of cefepime and ceftazidime against key Gram-negative species.Janssen-CilagUniversidade Federal de São Paulo (UNIFESP) Post-graduation Course in SciencesUNIFESPUNIFESP, Post-graduation Course in SciencesSciEL

HuD regulates SOD1 expression during oxidative stress in differentiated neuroblastoma cells and sporadic ALS motor cortex.

The neuronal RNA-binding protein (RBP) HuD plays an important role in brain development, synaptic plasticity and neurodegenerative diseases such as Parkinson's (PD) and Alzheimer's (AD). Bioinformatics analysis of the human SOD1 mRNA 3' untranslated region (3'UTR) demonstrated the presence of HuD binding adenine-uridine (AU)-rich instability-conferring elements (AREs). Using differentiated SH-SY5Y cells along with brain tissues from sporadic amyotrophic lateral sclerosis (sALS) patients, we assessed HuD-dependent regulation of SOD1 mRNA. In vitro binding and mRNA decay assays demonstrate that HuD specifically binds to SOD1 ARE motifs promoting mRNA stabilization. In SH-SY5Y cells, overexpression of full-length HuD increased SOD1 mRNA and protein levels while a dominant negative form of the RBP downregulated its expression. HuD regulation of SOD1 mRNA was also found to be oxidative stress (OS)-dependent, as shown by the increased HuD binding and upregulation of this mRNA after H2O2 exposure. This treatment also induced a shift in alternative polyadenylation (APA) site usage in SOD1 3'UTR, increasing the levels of a long variant bearing HuD binding sites. The requirement of HuD for SOD1 upregulation during oxidative damage was validated using a specific siRNA that downregulated HuD protein levels to 36% and prevented upregulation of SOD1 and 91 additional genes. In the motor cortex from sALS patients, we found increases in SOD1 and HuD mRNAs and proteins, accompanied by greater HuD binding to this mRNA as confirmed by RNA-immunoprecipitation (RIP) assays. Altogether, our results suggest a role of HuD in the post-transcriptional regulation of SOD1 expression during ALS pathogenesis

SOD1 and DJ-1 Converge at Nrf2 Pathway: A Clue for Antioxidant Therapeutic Potential in Neurodegeneration

Neurodegenerative diseases share diverse pathological features and among these oxidative stress (OS) plays a leading role. Impaired activity and reduced expression of antioxidant proteins have been reported as common events in several aging-associated disorders. In this review paper, we first provide an overview of the involvement of reactive oxygen species- (ROS-) induced oxidative damage in Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). Subsequently, we focus on DJ-1 and SOD1 proteins, which are involved in PD and ALS and also exert a prominent role in the interaction between redox homeostasis and neurodegeneration. Interestingly, recent studies demonstrated that DJ-1 and SOD1 are both tightly connected with Nrf2 protein, a transcriptional factor and master regulator of the expression of many antioxidant/detoxification genes. Nrf2 is emerging as a key neuroprotective protein in neurodegenerative diseases, since it helps neuronal cells to cope with toxic insults and OS. We herein summarize the recent literature providing a detailed picture of the promising therapeutic efficacy of Nrf2 natural and synthetic inducers as disease-modifying molecules for the treatment of neurodegenerative diseases

TDP-43 deficiency links Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage

TDP-43 is a DNA and RNA binding protein involved in RNA processing and with structural resemblance to heterogeneous ribonucleoproteins (hnRNPs), whose depletion sensitizes neurons to double strand DNA breaks (DSBs). Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder, in which 97% of patients are familial and sporadic cases associated with TDP-43 proteinopathies and conditions clearing TDP-43 from the nucleus, but we know little about the molecular basis of the disease. Here, we prove that mislocalization of mutated TDP-43 (A382T) in transfected neuronal SH-SY5Y and lymphoblastoid cell lines (LCLs) from an ALS patient cause R-loop accumulation, and R loop-dependent increased DSBs and Fanconi Anemia repair centers. Similar results were observed in a non-neuronal model of HeLa cells depleted of TDP-43. These results uncover a new role of TDP-43 in the control of co-transcriptional R-loops and the maintenance of genome integrity by preventing harmful R-loop accumulation. Our findings thus link TDP-43 pathology to increased R-loops and R loop-mediated DNA damage opening the possibility that R-loop modulation in TDP-43-defective cells might help develop ALS therapies