Serotonergic signalling suppresses ataxin 3 aggregation and neurotoxicity in animal models of Machado-Joseph disease.

Polyglutamine diseases are a class of dominantly inherited neurodegenerative disorders for which there is no effective treatment. Here we provide evidence that activation of serotonergic signalling is beneficial in animal models of Machado-Joseph disease. We identified citalopram, a selective serotonin reuptake inhibitor, in a small molecule screen of FDA-approved drugs that rescued neuronal dysfunction and reduced aggregation using a Caenorhabditis elegans model of mutant ataxin 3-induced neurotoxicity. MOD-5, the C. elegans orthologue of the serotonin transporter and cellular target of citalopram, and the serotonin receptors SER-1 and SER-4 were strong genetic modifiers of ataxin 3 neurotoxicity and necessary for therapeutic efficacy. Moreover, chronic treatment of CMVMJD135 mice with citalopram significantly reduced ataxin 3 neuronal inclusions and astrogliosis, rescued diminished body weight and strikingly ameliorated motor symptoms. These results suggest that small molecule modulation of serotonergic signalling represents a promising therapeutic target for Machado-Joseph disease.

Development and comprehensive characterization of porcine hepatocellular carcinoma for translational liver cancer investigation.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. New animal models that faithfully recapitulate human HCC phenotypes are required to address unmet clinical needs and advance standard-of-care therapeutics. This study utilized the Oncopig Cancer Model to develop a translational porcine HCC model which can serve as a bridge between murine studies and human clinical practice. Reliable development of Oncopig HCC cell lines was demonstrated through hepatocyte isolation and Cre recombinase exposure across 15 Oncopigs. Oncopig and human HCC cell lines displayed similar cell cycle lengths, alpha-fetoprotein production, arginase-1 staining, chemosusceptibility, and drug metabolizing enzyme expression. The ability of Oncopig HCC cells to consistently produce tumors in vivo was confirmed via subcutaneous (SQ) injection into immunodeficient mice and Oncopigs. Reproducible development of intrahepatic tumors in an alcohol-induced fibrotic microenvironment was achieved via engraftment of SQ tumors into fibrotic Oncopig livers. Whole-genome sequencing demontrated intrahepatic tumor tissue resembled human HCC at the genomic level. Finally, Oncopig HCC cells are amenable to gene editing for development of personalized HCC tumors. This study provides a novel, clinically-relevant porcine HCC model which holds great promise for improving HCC outcomes through testing of novel therapeutic approaches to accelerate and enhance clinical trials.

Mitochondrial Stress Restores the Heat Shock Response and Prevents Proteostasis Collapse during Aging.

In Caenorhabditis elegans, the programmed repression of the heat shock response (HSR) accompanies the transition to reproductive maturity, leaving cells vulnerable to environmental stress and protein aggregation with age. To identify the factors driving this event, we performed an unbiased genetic screen for suppressors of stress resistance and identified the mitochondrial electron transport chain (ETC) as a central regulator of the age-related decline of the HSR and cytosolic proteostasis. Mild downregulation of ETC activity, either by genetic modulation or exposure to mitochondria-targeted xenobiotics, maintained the HSR in adulthood by increasing HSF-1 binding and RNA polymerase II recruitment at HSF-1 target genes. This resulted in a robust restoration of cytoplasmic proteostasis and increased vitality later in life, without detrimental effects on fecundity. We propose that low levels of mitochondrial stress regulate cytoplasmic proteostasis and healthspan during aging by coordinating the long-term activity of HSF-1 with conditions preclusive to optimal fitness.

Use of transcranial direct current stimulation for the treatment of auditory hallucinations of schizophrenia - a systematic review.

INTRODUCTION: Auditory hallucinations are defined as experiences of auditory perceptions in the absence of a provoking external stimulus. They are the most prevalent symptoms of schizophrenia with high capacity for chronicity and refractoriness during the course of disease. The transcranial direct current stimulation (tDCS) - a safe, portable, and inexpensive neuromodulation technique - has emerged as a promising treatment for the management of auditory hallucinations. OBJECTIVE: The aim of this study is to analyze the level of evidence in the literature available for the use of tDCS as a treatment for auditory hallucinations in schizophrenia. METHODS: A systematic review was performed, searching in the main electronic databases including the Cochrane Library and MEDLINE/PubMed. The searches were performed by combining descriptors, applying terms of the Medical Subject Headings (MeSH) of Descriptors of Health Sciences and descriptors contractions. PRISMA protocol was used as a guide and the terms used were the clinical outcomes ("Schizophrenia" OR "Auditory Hallucinations" OR "Auditory Verbal Hallucinations" OR "Psychosis") searched together ("AND") with interventions ("transcranial Direct Current Stimulation" OR "tDCS" OR "Brain Polarization"). RESULTS: Six randomized controlled trials that evaluated the effects of tDCS on the severity of auditory hallucinations in schizophrenic patients were selected. Analysis of the clinical results of these studies pointed toward incongruence in the information with regard to the therapeutic use of tDCS with a view to reducing the severity of auditory hallucinations in schizophrenia. Only three studies revealed a therapeutic benefit, manifested by reductions in severity and frequency of auditory verbal hallucinations in schizophrenic patients. CONCLUSION: Although tDCS has shown promising results in reducing the severity of auditory hallucinations in schizophrenic patients, this technique cannot yet be used as a therapeutic alternative due to lack of studies with large sample sizes that portray the positive effects that have been described.

Immiscible phase filter extraction and equivalent amplification of genotypes 1-6 of hepatitis C RNA: The building blocks for point-of-care diagnosis.

The lack of hepatitis C virus (HCV) diagnostic tests designed for use in decentralized settings is a major obstacle for providing access to treatment and prevention services particularly in low and middle income countries. Here we describe the development and validation of two building blocks of the HCV Quant Assay, a test in development for point-of-care use: 1) an RT-qPCR assay with noncompetitive internal control that equivalently detects the 6 major HCV genotypes and 2) an automated sample prep method using immiscible phase filter technology. This novel assay has wide dynamic range of HCV quantification and a limit of detection of 30IU/ml with 200µl specimen volume. In a preliminary study of 61 clinical specimens, the HCV Quant Assay demonstrated 100% sensitivity and specificity and gave comparable viral load results across 4 logs of IU/ml when compared to the Abbott RealTime HCV Assay.

Filtration Isolation of Nucleic Acids: A Simple and Rapid DNA Extraction Method.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 µl whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

The nematode Caenorhabditis elegans displays a chemotaxis behavior to tuberculosis-specific odorants.

A simple, affordable diagnostic test for pulmonary tuberculosis (TB) is urgently needed to improve detection of active Mycobacterium tuberculosis. Recently, it has been suggested that animal behavior can be used as a biosensor to signal the presence of human disease. For example, the giant African pouched rats can detect tuberculosis by sniffing sputum specimens while trained honeybees respond to three of the volatile organic compounds (VOCs) detected in the breath of TB positive patients by proboscis extension. However, both rats and honeybees require animal housing facilities and professional trainers, which are outside the scope of most disease testing facilities. Here, we report that the innate olfactory behavioral response of the roundworm nematode Caenorhabditis elegans can be used to detect the TB-specific VOCs methyl p-anisate, methyl nicotinate, methyl phenylacetate and o-phenylanisole, in chemotaxis assays. Dauer larvae, a long-lived stress resistant alternative development state of C. elegans in which the animals can survive for extended periods of time in dry conditions with no food, were also demonstrated to detect the VOCs. We propose that exposing naive dauer larvae to TB-related VOCs and recording their response in this behavioral assay could lead to the development of a new method for TB diagnostics using breath as the sample type.

Investigating the spreading and toxicity of prion-like proteins using the metazoan model organism C. elegans.

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.