SUcceSS, SUrgery for Spinal Stenosis: Protocol of a randomised, placebo-controlled trial

© Author(s) (or their employer(s)) 2019. Introduction: Central lumbar spinal stenosis (LSS) is a common cause of pain, reduced function and quality of life in older adults. Current management of LSS includes surgery to decompress the spinal canal and alleviate symptoms. However, evidence supporting surgical decompression derives from unblinded randomised trials with high cross-over rates or cohort studies showing modest benefits. This protocol describes the design of the SUrgery for Spinal Stenosis (SUcceSS) trial-the first randomised placebo-controlled trial of decompressive surgery for symptomatic LSS. Methods and analysis: SUcceSS will be a prospectively registered, randomised placebo-controlled trial of decompressive spinal surgery. 160 eligible participants (80 participants/group) with symptomatic LSS will be randomised to either surgical spinal decompression or placebo surgical intervention. The placebo surgical intervention is identical to surgical decompression in all other ways with the exception of the removal of any bone or ligament. All participants and assessors will be blinded to treatment allocation. Outcomes will be assessed at baseline and at 3, 6, 12 and 24 months. The coprimary outcomes will be function measured with the Oswestry Disability Index and the proportion of participants who have meaningfully improved their walking capacity at 3 months postrandomisation. Secondary outcomes include back pain intensity, lower limb pain intensity, disability, quality of life, anxiety and depression, neurogenic claudication score, perceived recovery, treatment satisfaction, adverse events, reoperation rate and rehospitalisation rate. Those who decline to be randomised will be invited to participate in a parallel observational cohort. Data analysis will be blinded and by intention to treat. A trial-based cost-effectiveness analysis will determine the potential incremental cost per quality-adjusted life year gained. Ethics and dissemination: Ethics approval has been granted by the NSW Health (reference:17/247/POWH/601) and the Monash University (reference: 12371) Human Research Ethics Committees. Dissemination of results will be via journal articles and presentations at national and international conferences

Methamphetamine Inhibits the Glucose Uptake by Human Neurons and Astrocytes: Stabilization by Acetyl-L-Carnitine

Methamphetamine (METH), an addictive psycho-stimulant drug exerts euphoric effects on users and abusers. It is also known to cause cognitive impairment and neurotoxicity. Here, we hypothesized that METH exposure impairs the glucose uptake and metabolism in human neurons and astrocytes. Deprivation of glucose is expected to cause neurotoxicity and neuronal degeneration due to depletion of energy. We found that METH exposure inhibited the glucose uptake by neurons and astrocytes, in which neurons were more sensitive to METH than astrocytes in primary culture. Adaptability of these cells to fatty acid oxidation as an alternative source of energy during glucose limitation appeared to regulate this differential sensitivity. Decrease in neuronal glucose uptake by METH was associated with reduction of glucose transporter protein-3 (GLUT3). Surprisingly, METH exposure showed biphasic effects on astrocytic glucose uptake, in which 20 µM increased the uptake while 200 µM inhibited glucose uptake. Dual effects of METH on glucose uptake were paralleled to changes in the expression of astrocytic glucose transporter protein-1 (GLUT1). The adaptive nature of astrocyte to mitochondrial β-oxidation of fatty acid appeared to contribute the survival of astrocytes during METH-induced glucose deprivation. This differential adaptive nature of neurons and astrocytes also governed the differential sensitivity to the toxicity of METH in these brain cells. The effect of acetyl-L-carnitine for enhanced production of ATP from fatty oxidation in glucose-free culture condition validated the adaptive nature of neurons and astrocytes. These findings suggest that deprivation of glucose-derived energy may contribute to neurotoxicity of METH abusers

Retrograde trafficking of β-dystroglycan from the plasma membrane to the nucleus

β-Dystroglycan (β-DG) is a transmembrane protein with critical roles in cell adhesion, cytoskeleton remodeling and nuclear architecture. This functional diversity is attributed to the ability of β-DG to target to, and conform specific protein assemblies at the plasma membrane (PM) and nuclear envelope (NE). Although a classical NLS and importin α/β mediated nuclear import pathway has already been described for β-DG, the intracellular trafficking route by which β-DG reaches the nucleus is unknown. In this study, we demonstrated that β-DG undergoes retrograde intracellular trafficking from the PM to the nucleus via the endosome-ER network. Furthermore, we provided evidence indicating that the translocon complex Sec61 mediates the release of β-DG from the ER membrane, making it accessible for importins and nuclear import. Finally, we show that phosphorylation of β-DG at Tyr890 is a key stimulus for β-DG nuclear translocation. Collectively our data describe the retrograde intracellular trafficking route that β-DG follows from PM to the nucleus. This dual role for a cell adhesion receptor permits the cell to functionally connect the PM with the nucleus and represents to our knowledge the first example of a cell adhesion receptor exhibiting retrograde nuclear trafficking and having dual roles in PM and NE

Prediction of susceptibility to major depression by a model of interactions of multiple functional genetic variants and environmental factors

Major depressive disorder (MDD) is the most common psychiatric disorder and the second overall cause of disability. Even though a significant amount of the variance in the MDD phenotype is explained by inheritance, specific genetic variants conferring susceptibility to MDD explain only a minimal proportion of MDD causality. Moreover, genome-wide association studies have only identified two small-sized effect loci that reach genome-wide significance. In this study, a group of Mexican-American patients with MDD and controls recruited for a pharmacogenetic study were genotyped for nonsynonymous single-nucleotide polymorphisms (nsSNPs) and used to explore the interactions of multiple functional genetic variants with risk-classification tree analysis. The risk-classification tree analysis model and linkage disequilibrium blocks were used to replicate exploratory findings in the database of genotypes and phenotypes (dbGaP) for major depression, and pathway analysis was performed to explore potential biological mechanisms using the branching events. In exploratory analyses, we found that risk-classification tree analysis, using 15 nsSNPs that had a nominal association with MDD diagnosis, identified multiple increased-MDD genotype clusters and significant additive interactions in combinations of genotype variants that were significantly associated with MDD. The results in the dbGaP for major depression disclosed a multidimensional dependent phenotype constituted of MDD plus significant modifiers (smoking, marriage status, age, alcohol abuse/dependence and gender), which then was used for the association tree analysis. The reconstructed tree analysis for the dbGaP data showed robust reliability and replicated most of the genes involved in the branching process found in our exploratory analyses. Pathway analysis using all six major events of branching (PSMD9, HSD3B1, BDNF, GHRHR, PDE6C and PDLIM5) was significant for positive regulation of cellular and biological processes that are relevant to growth and organ development. Our findings not only provide important insights into the biological pathways underlying innate susceptibility to MDD but also offer a predictive framework based on interactions of multiple functional genetic variants and environmental factors. These findings identify novel targets for therapeutics and for translation into preventive, clinical and personalized health care

State-of-the art data normalization methods improve NMR-based metabolomic analysis

Extracting biomedical information from large metabolomic datasets by multivariate data analysis is of considerable complexity. Common challenges include among others screening for differentially produced metabolites, estimation of fold changes, and sample classification. Prior to these analysis steps, it is important to minimize contributions from unwanted biases and experimental variance. This is the goal of data preprocessing. In this work, different data normalization methods were compared systematically employing two different datasets generated by means of nuclear magnetic resonance (NMR) spectroscopy. To this end, two different types of normalization methods were used, one aiming to remove unwanted sample-to-sample variation while the other adjusts the variance of the different metabolites by variable scaling and variance stabilization methods. The impact of all methods tested on sample classification was evaluated on urinary NMR fingerprints obtained from healthy volunteers and patients suffering from autosomal polycystic kidney disease (ADPKD). Performance in terms of screening for differentially produced metabolites was investigated on a dataset following a Latin-square design, where varied amounts of 8 different metabolites were spiked into a human urine matrix while keeping the total spike-in amount constant. In addition, specific tests were conducted to systematically investigate the influence of the different preprocessing methods on the structure of the analyzed data. In conclusion, preprocessing methods originally developed for DNA microarray analysis, in particular, Quantile and Cubic-Spline Normalization, performed best in reducing bias, accurately detecting fold changes, and classifying samples

The impact of changes to heroin supply on blood-borne virus notifications and injecting related harms in New South Wales, Australia

BACKGROUND: In early 2001 Australia experienced a sudden and unexpected disruption to heroin availability, know as the 'heroin shortage'. This 'shortage has been linked to a decrease in needle and syringe output and therefore possibly a reduction in injecting drug use. We aimed to examine changes, if any, in blood-borne viral infections and presentations for injecting related problems related to injecting drug use following the reduction heroin availability in Australia, in the context of widespread harm reduction measures. METHODS: Time series analysis of State level databases on HIV, hepatitis B, hepatitis C notifications and hospital and emergency department data. Examination of changes in HIV, hepatitis B, hepatitis C notifications and hospital and emergency department admissions for injection-related problems following the onset of the heroin shortage; non-parametric curve-fitting of number of hepatitis C notifications among those aged 15–19 years. RESULTS: There were no changes observed in hospital visits for injection-related problems. There was no change related to the onset heroin shortage in the number of hepatitis C notifications among persons aged 15–19 years, but HCV notifications have subsequently decreased in this group. No change occurred in HIV and hepatitis B notifications. CONCLUSION: A marked reduction in heroin supply resulted in no increase in injection-related harm at the community level. However, a delayed decrease in HCV notifications among young people may be related. These changes occurred in a setting with widespread, publicly funded harm reduction initiatives

β-Hydroxy-β-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells

β-Hydroxy-β-methylbutyrate (HMB) has been shown to enhance cell survival, differentiation and protein turnover in muscle, mainly activating phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases/ extracellular-signal-regulated kinases (MAPK/ERK) signaling pathways. Since these two pathways are related to neuronal survival and differentiation, in this study, we have investigated the neurotrophic effects of HMB in mouse neuroblastoma Neuro2a cells. In Neuro2a cells, HMB promotes differentiation to neurites independent from any effects on proliferation. These effects are mediated by activation of both the PI3K/Akt and the extracellular-signal-regulated kinases (ERK1/2) signaling as demonstrated by the use of specific inhibitors of these two pathways. As myocyte-enhancer factor 2 (MEF2) family of transcription factors are involved in neuronal survival and plasticity, the transcriptional activity and protein levels of MEF2 were also evaluated. HMB promoted MEF2-dependent transcriptional activity mediated by the activation of Akt and ERK1/2 pathways. Furthermore, HMB increases the expression of brain glucose transporters 1 (GLUT1) and 3 (GLUT3), and mTOR phosphorylation, which translates in a higher protein synthesis in Neuro2a cells. Furthermore, Torin1 and rapamycin effects on MEF2 transcriptional activity and HMB-dependent neurite outgrowth support that HMB acts through mTORC2. Together, these findings provide clear evidence to support an important role of HMB in neurite outgrowth.This project has been funded by Abbott Nutrition R&D

Predicting the impact of Lynch syndrome-causing missense mutations from structural calculations

Accurate methods to assess the pathogenicity of mutations are needed to fully leverage the possibilities of genome sequencing in diagnosis. Current data-driven and bioinformatics approaches are, however, limited by the large number of new variations found in each newly sequenced genome, and often do not provide direct mechanistic insight. Here we demonstrate, for the first time, that saturation mutagenesis, biophysical modeling and co-variation analysis, performed in silico, can predict the abundance, metabolic stability, and function of proteins inside living cells. As a model system, we selected the human mismatch repair protein, MSH2, where missense variants are known to cause the hereditary cancer predisposition disease, known as Lynch syndrome. We show that the majority of disease-causing MSH2 mutations give rise to folding defects and proteasome-dependent degradation rather than inherent loss of function, and accordingly our in silico modeling data accurately identifies disease-causing mutations and outperforms the traditionally used genetic disease predictors. Thus, in conclusion, in silico biophysical modeling should be considered for making genotype-phenotype predictions and for diagnosis of Lynch syndrome, and perhaps other hereditary diseases

Chemokine Transfer by Liver Sinusoidal Endothelial Cells Contributes to the Recruitment of CD4+ T Cells into the Murine Liver

Leukocyte adhesion and transmigration are central features governing immune surveillance and inflammatory reactions in body tissues. Within the liver sinusoids, chemokines initiate the first crucial step of T-cell migration into the hepatic tissue. We studied molecular mechanisms involved in endothelial chemokine supply during hepatic immune surveillance and liver inflammation and their impact on the recruitment of CD4+ T cells into the liver. In the murine model of Concanavalin A-induced T cell-mediated hepatitis, we showed that hepatic expression of the inflammatory CXC chemokine ligands (CXCL)9 and CXCL10 strongly increased whereas homeostatic CXCL12 significantly decreased. Consistently, CD4+ T cells expressing the CXC chemokine receptor (CXCR)3 accumulated within the inflamed liver tissue. In histology, CXCL9 was associated with liver sinusoidal endothelial cells (LSEC) which represent the first contact site for T-cell immigration into the liver. LSEC actively transferred basolaterally internalized CXCL12, CXCL9 and CXCL10 via clathrin- coated vesicles to CD4+ T cells leading to enhanced transmigration of CXCR4+ total CD4+ T cells and CXCR3+ effector/memory CD4+ T cells, respectively in vitro. LSEC-expressed CXCR4 mediated CXCL12 transport and blockage of endothelial CXCR4 inhibited CXCL12-dependent CD4+ T-cell transmigration. In contrast, CXCR3 was not involved in the endothelial transport of its ligands CXCL9 and CXCL10. The clathrin-specific inhibitor chlorpromazine blocked endothelial chemokine internalization and CD4+ T-cell transmigration in vitro as well as migration of CD4+ T cells into the inflamed liver in vivo. Moreover, hepatic accumulation of CXCR3+ CD4+ T cells during T cell-mediated hepatitis was strongly reduced after administration of chlorpromazine. These data demonstrate that LSEC actively provide perivascularly expressed homeostatic and inflammatory chemokines by CXCR4- and clathrin-dependent intracellular transport mechanisms thereby contributing to the hepatic recruitment of CD4+ T-cell populations during immune surveillance and liver inflammation

Evaluation and Characterization of Bacterial Metabolic Dynamics with a Novel Profiling Technique, Real-Time Metabolotyping

BACKGROUND: Environmental processes in ecosystems are dynamically altered by several metabolic responses in microorganisms, including intracellular sensing and pumping, battle for survival, and supply of or competition for nutrients. Notably, intestinal bacteria maintain homeostatic balance in mammals via multiple dynamic biochemical reactions to produce several metabolites from undigested food, and those metabolites exert various effects on mammalian cells in a time-dependent manner. We have established a method for the analysis of bacterial metabolic dynamics in real time and used it in combination with statistical NMR procedures. METHODOLOGY/PRINCIPAL FINDINGS: We developed a novel method called real-time metabolotyping (RT-MT), which performs sequential (1)H-NMR profiling and two-dimensional (2D) (1)H, (13)C-HSQC (heteronuclear single quantum coherence) profiling during bacterial growth in an NMR tube. The profiles were evaluated with such statistical methods as Z-score analysis, principal components analysis, and time series of statistical TOtal Correlation SpectroScopY (TOCSY). In addition, using 2D (1)H, (13)C-HSQC with the stable isotope labeling technique, we observed the metabolic kinetics of specific biochemical reactions based on time-dependent 2D kinetic profiles. Using these methods, we clarified the pathway for linolenic acid hydrogenation by a gastrointestinal bacterium, Butyrivibrio fibrisolvens. We identified trans11, cis13 conjugated linoleic acid as the intermediate of linolenic acid hydrogenation by B. fibrisolvens, based on the results of (13)C-labeling RT-MT experiments. In addition, we showed that the biohydrogenation of polyunsaturated fatty acids serves as a defense mechanism against their toxic effects. CONCLUSIONS: RT-MT is useful for the characterization of beneficial bacterium that shows potential for use as probiotic by producing bioactive compounds