Molecular and Cellular Mechanisms Affected in ALS

Amyotrophic lateral sclerosis (ALS) is a terminal late-onset condition characterized by the loss of upper and lower motor neurons. Mutations in more than 30 genes are associated to the disease, but these explain only ~20% of cases. The molecular functions of these genes implicate a wide range of cellular processes in ALS pathology, a cohesive understanding of which may provide clues to common molecular mechanisms across both familial (inherited) and sporadic cases and could be key to the development of effective therapeutic approaches. Here, the different pathways that have been investigated in ALS are summarized, discussing in detail: mitochondrial dysfunction, oxidative stress, axonal transport dysregulation, glutamate excitotoxicity, endosomal and vesicular transport impairment, impaired protein homeostasis, and aberrant RNA metabolism. This review considers the mechanistic roles of ALS-associated genes in pathology, viewed through the prism of shared molecular pathways

Molecular and Cellular Mechanisms Affected in ALS

Amyotrophic lateral sclerosis (ALS) is a terminal late-onset condition characterized by the loss of upper and lower motor neurons. Mutations in more than 30 genes are associated to the disease, but these explain only ~20% of cases. The molecular functions of these genes implicate a wide range of cellular processes in ALS pathology, a cohesive understanding of which may provide clues to common molecular mechanisms across both familial (inherited) and sporadic cases and could be key to the development of effective therapeutic approaches. Here, the different pathways that have been investigated in ALS are summarized, discussing in detail: mitochondrial dysfunction, oxidative stress, axonal transport dysregulation, glutamate excitotoxicity, endosomal and vesicular transport impairment, impaired protein homeostasis, and aberrant RNA metabolism. This review considers the mechanistic roles of ALS-associated genes in pathology, viewed through the prism of shared molecular pathways

Smart energy monitoring and power quality performance based evaluation of 100-kW grid tied PV system

Globally, the demand for energy from renewable sources is growing due to the increasing electricity consumption and the pollution of fossil fuels. The government has framed various policies to facilitate green energy generation, encouraging renewable energy source usage through PV installations in multiple sectors, including educational institutions. The primary objective of this paper is to propose a methodological approach for analysing the performance of the installed PV system on the rooftop of a university building in Tamil Nadu, India. The site selected is favourable for electricity generation from PV systems with an average global solar radiation of 5.82 kWh/m2day. Solar energy changes periodically with annual and daily variations and is not steady due to seasonal changes. The step-by-step performance assessment and the annual performance of the 100-kW solar PV system, which was instituted in 2019, with the forecasted parameters, are presented in this paper. Therefore, the assessment analysis is carried out in four phases: feasibility assessment, Energy yield assessment, Life cycle assessment, and Power quality assessment. To improve the solar PV output and efficiency, considering the solar irradiation, temperature, wind velocity, etc., PV yield is measured to evaluate the PV system\u27s energy metrics. This paper also considers the carbon credits earned, solar power generated in the location, and the payback period. The power quality assessment is carried out in this paper to test the PV plant\u27s compliance with effective grid integration

Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles

BACKGROUND: The cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards MNs, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Because MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle-secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology. METHODS: Sporadic ALS patients were confirmed to be ALS according to El Escorial criteria and were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS-R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n = 27) or deltoids of aged-matched healthy subjects (n = 30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RT-qPCR, and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human-derived iPSC MNs and on healthy human myotubes, with untreated cells used as controls. RESULTS: An accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing. Compared with vesicles from healthy control myotubes, when administered to healthy MNs the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. The RNA-processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient MNs was abolished by anti-CD63 immuno-blocking of vesicle uptake. CONCLUSIONS: ALS muscle vesicles are shown to be toxic to MNs, which establishes the skeletal muscle as a potential source of vesicle-mediated toxicity in ALS

Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles

Background: The cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards MNs, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Because MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle-secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology. Methods: Sporadic ALS patients were confirmed to be ALS according to El Escorial criteria and were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS-R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n = 27) or deltoids of aged-matched healthy subjects (n = 30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RT-qPCR, and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human-derived iPSC MNs and on healthy human myotubes, with untreated cells used as controls. Results: An accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing. Compared with vesicles from healthy control myotubes, when administered to healthy MNs the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. The RNA-processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient MNs was abolished by anti-CD63 immuno-blocking of vesicle uptake. Conclusions: ALS muscle vesicles are shown to be toxic to MNs, which establishes the skeletal muscle as a potential source of vesicle-mediated toxicity in ALS

Randomized Clinical Trial of High-Dose Rifampicin With or Without Levofloxacin Versus Standard of Care for Pediatric Tuberculous Meningitis: The TBM-KIDS Trial

Background. Pediatric tuberculous meningitis (TBM) commonly causes death or disability. In adults, high-dose rifampicin may reduce mortality. The role of fluoroquinolones remains unclear. There have been no antimicrobial treatment trials for pediatric TBM. Methods. TBM-KIDS was a phase 2 open-label randomized trial among children with TBM in India and Malawi. Participants received isoniazid and pyrazinamide plus: (i) high-dose rifampicin (30 mg/kg) and ethambutol (R30HZE, arm 1); (ii) high-dose rifampicin and levofloxacin (R30HZL, arm 2); or (iii) standard-dose rifampicin and ethambutol (R15HZE, arm 3) for 8 weeks, followed by 10 months of standard treatment. Functional and neurocognitive outcomes were measured longitudinally using Modified Rankin Scale (MRS) and Mullen Scales of Early Learning (MSEL). Results. Of 2487 children prescreened, 79 were screened and 37 enrolled. Median age was 72 months; 49%, 43%, and 8% had stage I, II, and III disease, respectively. Grade 3 or higher adverse events occurred in 58%, 55%, and 36% of children in arms 1, 2, and 3, with 1 death (arm 1) and 6 early treatment discontinuations (4 in arm 1, 1 each in arms 2 and 3). By week 8, all children recovered to MRS score of 0 or 1. Average MSEL scores were significantly better in arm 1 than arm 3 in fine motor, receptive language, and expressive language domains (P < .01). Conclusions. In a pediatric TBM trial, functional outcomes were excellent overall. The trend toward higher frequency of adverse events but better neurocognitive outcomes in children receiving high-dose rifampicin requires confirmation in a larger trial. Clinical Trials Registration. NCT02958709

Role of microRNA deregulation in the pathogenesis of Machado-Joseph disease

Tese de doutoramento em Biologia Experimental e Biomedicina, na especialidade de Neurociências e Doença, apresentada ao Instituto de Investigação Interdisciplinar da Universidade de CoimbraA doença de Machado-Joseph (DMJ), também conhecida como ataxia espinocerebelosa do tipo 3, é uma doença neurodegenerativa autossómica dominante causada por uma expansão no número de repetições do trinucleótido CAG na região codificante do gene MJD1, a qual é traduzida num trato poliglutamínico expandido na proteína ataxina-3. Esta proteína mutada resulta em disfunção neuronal e neurodegenerescência em diferentes regiões do cérebro. Até à data, não foi ainda desenvolvida nenhuma terapia capaz de bloquear ou atrasar a progressão da DMJ. Estudos recentes sugerem que a desregulação transcripcional desempenha um papel importante na DMJ. Apesar de diferentes transcritos terem sido implicados na patogénese da DMJ, nos últimos anos a desregulação de microRNAs observada em outras doenças neurodegenerativas sugeriu um potencial papel destas moléculas na DMJ. Assim sendo, o objectivo deste projecto foi estudar o papel da desregulação de microRNAs na DMJ e simultaneamente desenvolver e avaliar o potencial de uma nova estratégia terapêutica baseada em microRNAs recorrendo para isso a um modelo lentiviral da DMJ em murganho. Na primeira parte deste projecto, descrita no capítulo 2, recorrendo à tecnologia de microarray de miRNAs identificámos diversos miRNAs cuja expressão se encontra alterada num modelo lentiviral estriatal da DMJ em murganho. De entre o conjunto de miRNAs com expressão alterada, os níveis do miRNA miR-10b encontravam-se particularmente elevados. Para além disso, os níveis de expressão do miR-10b foram também avaliados em outros modelos de DMJ, verificando-se um aumento significativo nos níveis deste miRNA no cerebelo de murganhos transgénicos DMJ observando-se também uma tendência para aumento em tecido humano post-mortem de cerebelo de doentes DMJ. Assim sendo, focámo-nos neste miRNA (miR-10b) para avaliar os efeitos terapêuticos da sua modulação no contexto da DMJ. Antes de avançar para um modelo em murganho da DMJ, começámos por avaliar o efeito de um inibidor do miR-10b num organismo mais simples, a Drosophila. A inibição do miR-10b em Drosophilas DMJ resultou numa extensão do seu período de vida. Estes resultados sugerem assim que os miRNAs desregulados no modelo lentiviral de DMJ poderão contribuir para a patogénese da doença e que a reposição dos seus níveis, em Drosophila, leva a um aumento do seu tempo de vida implicando assim este miRNA no processo patológico da DMJ. No capítulo 3, recorrendo à utilização de lentivírus codificantes para um inibidor do miR-10b baseado na tecologia tough-decoy, conseguimos re-estabelecer os níveis normais de miR-10b no estriado de um modelo lentiviral DMJ em murganho. Para além disso, identificámos a transtirretina como um alvo do miR-10b. A inibição do miR-10b no modelo lentiviral DMJ levou a uma redução do número de inclusões de positivas para ataxina-3 e ubiquitina ao mesmo tempo que levou a uma diminuição da disfunção neuronal associada a este modelo. Em conclusão, esta tese reúne evidências que demonstram que a desregulação de miRNAs é uma componente da DMJ, identificando pela primeira vez o miRNA miR-10b como estando sobrexpresso no contexto da doença. Para além disso, foi validado um novo alvo deste miRNA, a transtirretina, um gene previamente associado ao desenvolvimento de outras doenças neurodegenerativas. Simultaneamente, a inibição do miR-10b, num modelo de murganho da DMJ levou a uma melhoria das manifestações da doença. Em suma, este estudo não só identificou uma nova via patógenica na DMJ como também gerou uma nova oportunidade para o desenvolvimento de uma estratégia terapêutica baseada em miRNAs para o tratamento da DMJ.Machado-Joseph disease (MJD) / Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disorder caused by the over-repetition of the CAG trinucleotide in the coding region of MJD1 gene which translates into an expanded polyglutamine tract within the affected protein ataxin-3. The toxic mutant ataxin-3 protein results in neuronal dysfunction and neurodegeneration in many brain regions. Until now, there is no therapy to prevent or delay the disease progression in MJD. Recent evidence suggests transcriptional dysregulation may play a crucial role in MJD. Even though different transcripts have been shown to contribute to the pathogenesis of MJD, in recent years the evidence regarding dysregulation of microRNAs in other neurodegenerative disorders was suggestive of a potential role in MJD. Therefore, the aim of this project to study the role of microRNA dysregulation in MJD and to develop a microRNA based therapeutic strategy towards MJD. In the first part of this project, described in chapter 2, using a miRNA microarray we identified numerous miRNAs that are differentially expressed in a lentiviral MJD mouse model. Notably, of all the differentially expressed miRs, miR-10b expression levels were robustly up-regulated. miR-10b expression levels were also evaluated in other MJD models and found to be significantly up-regulated in a transgenic mouse model of MJD and to show a trend towards upregulation in human post-mortem brain tissue. Our results suggest that microRNAs are dysregulated in MJD potentially contributing to the pathogenesis of the disease. We next investigated whether a miR-10 inhibitor would modify disease progression in a small organism, drosophila. MJD drosophila treated with a miR-10b repressor resulted in the elongation of the life-span of MJD flies. Our results suggest miR-10b dysregulation could contribute to the pathogenesis of MJD and that re-establishing the normal levels of miR-10b may be protective in MJD. In chapter 3, taking advantage of lentiviral based delivery of a miR-10b tough-decoy inhibitor, we re-established the normal levels of miR-10b in the striatal lentiviral MJD mouse model. Notably, our study identifies transthyretin as a target of miR-10b. Importantly, neuronal dysfunction, ubiquitin-positive inclusions and ataxin-3 positive inclusions were reduced upon miR-10b TuD inhibitor treatment in the striatal lentiviral MJD mouse model. In summary, the present thesis provides evidence for a dysregulation of microRNAs in MJD, showing, for the first time, that miR-10b is upregulated in MJD. Moreover, we have validated a novel target gene for miR-10b, transthyretin, a gene previously implicated in other neurodegenerative diseases. Importantly, inhibition of mir-10b in vivo in a MJD mouse model was effective in ameliorating the disease manifestations. Altogether, we believe our study identifies a novel disease pathway in MJD and, at the same time, opens a new door for the development of a miR-based therapeutic strategy towards MJD.Richard Chin and Lily Lock Machado-Joseph Research FundNational Ataxia FoundationMarie Curie ITN –Treat PolyQSétimo Programa Estrutural da União Europeia, Agência Executiva de Investigação - FP7-PEOPLE-2010-ITN com o acordo de subvenção número 264508 TreatPoly

Tfh1 Cells in Germinal Centers During Chronic HIV/SIV Infection

T follicular helper CD4 cells (Tfh) are essential for the development and maintenance of germinal center (GC) reactions, a critical process that promotes the generation of long-lived high affinity humoral immunity. It is becoming increasingly evident that GC-Tfh cells are heterogeneous in nature with some cellular characteristics associated with a Th1, Th2, and Th17 phenotype. Emerging studies suggest that GC-Tfh cells are directed to differentiate into distinct phenotypes during chronic HIV/SIV infection and these changes in GC-Tfh cells can greatly impact the B cell response and subclass of antibodies generated. Studies in HIV-infected humans have shown that certain Tfh phenotypes are associated with the generation of broadly neutralizing antibody responses. Moreover, the susceptibility of particular GC-Tfh subsets to HIV infection within the secondary lymphoid sites can also impact GC-Tfh/B cell interactions. In this review, we discuss the recent advances that show Tfh heterogeneity during chronic HIV/SIV infection. In particular, we will discuss the dynamics of GC-Tfh cells, their altered differentiation state and function, and their impact on B cell responses during HIV/SIV infection. In addition, we will also discuss the potential role of a recently described novel subset of follicular homing CXCR5+ CD8 T cells (Tfc) and their importance in contributing to control of chronic HIV/SIV infection. A better understanding of the mechanistic role of follicular homing CD4 and CD8 T cells during HIV/SIV infection will aid in the design of vaccines and therapeutic strategies to prevent and treat HIV/AIDS

Tfh1 Cells in Germinal Centers During Chronic HIV/SIV Infection

T follicular helper CD4 cells (Tfh) are essential for the development and maintenance of germinal center (GC) reactions, a critical process that promotes the generation of long-lived high affinity humoral immunity. It is becoming increasingly evident that GC-Tfh cells are heterogeneous in nature with some cellular characteristics associated with a Th1, Th2, and Th17 phenotype. Emerging studies suggest that GC-Tfh cells are directed to differentiate into distinct phenotypes during chronic HIV/SIV infection and these changes in GC-Tfh cells can greatly impact the B cell response and subclass of antibodies generated. Studies in HIV-infected humans have shown that certain Tfh phenotypes are associated with the generation of broadly neutralizing antibody responses. Moreover, the susceptibility of particular GC-Tfh subsets to HIV infection within the secondary lymphoid sites can also impact GC-Tfh/B cell interactions. In this review, we discuss the recent advances that show Tfh heterogeneity during chronic HIV/SIV infection. In particular, we will discuss the dynamics of GC-Tfh cells, their altered differentiation state and function, and their impact on B cell responses during HIV/SIV infection. In addition, we will also discuss the potential role of a recently described novel subset of follicular homing CXCR5+ CD8 T cells (Tfc) and their importance in contributing to control of chronic HIV/SIV infection. A better understanding of the mechanistic role of follicular homing CD4 and CD8 T cells during HIV/SIV infection will aid in the design of vaccines and therapeutic strategies to prevent and treat HIV/AIDS

Біоінженерні наночастинки металів та оксидів металів для фотокаталітичного і біологічного застосування (огляд)

In this modern era, (M/MO-NPs) Metal/metal oxide nanoparticles are utilized in various areas. The growth of nanoparticles is tremendous in our daily activities like beauty products, doses, delivery of drugs, and outfit. They can be set up by numerous strategies, for example, green amalgamation and the ordinary compound blend techniques. Green synthesis incorporates endless increase to deliver M/MO-NPs with requesting properties. In Bioengineering, the "green" combination has increased massive consideration as a trustworthy, dependable, and natural benevolent convention for orchestrating a wide scope of Nanomaterials including M/MO-NPs and bio propelled materials. Be that as it may, the utilization of plant extracts for this intention is profitable over organisms because of usability and less biohazard. Union of metal and MO-NPs by using the extract of plant fluid arrangement have increased consideration toward the green methodology and with no antagonistic impact on nature. The current article intends to survey the advancement made lately on nanoparticle biosynthesis by organisms. These microbial assets incorporate microorganisms, organisms, yeast, green growth, and viruses. This study predominantly centers on the biosynthesis of the most usually examined M/MO-NPs, for example, copper, cadmium, noble metals, platinum, titanium oxide, palladium, zinc oxide, and cadmium sulfide.У сучасну епоху (M / MO-NPs) наночастинки металу / оксиду металу використовуються в різних областях. Для повсякденного життя сьогодні використовують велику кількість наночастинок, наприклад, для косметичних засобів, дозування та доставки ліків, для легкої промисловості, тощо. Для їх створення використовують різноманітні стратегії: дослідження натуральних речовин (зелений синтез) та стандартні методи створення сумішей сполук. Зелений синтез включає постійний ріст доставки M / MO-NPs із потрібними властивостями. У галузі біоінженерії посилена увага спостерігається саме для "зеленої" комбінації, як до надійної та природно сприятливої конвенції для формування широкого спектру наноматеріалів, включаючи M/MO-NPs та біологічні матеріали. Інтригуючими є фотокаталітичні та біологічні застосування наночастинок оксиду металу, що охоплює широкий спектр передових досліджень та нових розробок у цій галузі. Використання рослинних екстрактів є вигідним для організму через зручність та меншу біологічну небезпеку. Поєднання металів та MO-NPs за допомогою екстракту рослинної рідини збільшило увагу до зеленої методики і не має антагоністичного впливу на природу. Ця стаття має на меті проаналізувати досягнення, зроблені останнім часом щодо біосинтезу наночастинок організмами, зокрема, мікроорганізмами, дріжджами, зеленими утвореннями та вірусами. Дане дослідження переважно зосереджено на біосинтезі найбільш часто досліджуваних M / MO-NPs, наприклад, міді, кадмію, благородних металів, платини, оксиду титану, паладію, оксиду цинку та сульфіду кадмію