Cerebellar Purkinje cells (PCs) are the sole output neurons of the cerebellar cortex, and damage to PCs results in motor deficits. Spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease), a hereditary neurodegenerative disease, is caused by an abnormal expansion of the polyglutamine tract in the causative ATXN3 protein. SCA3 affects a wide range of cells in the central nervous system, including those in the cerebellum. To unravel SCA3 pathology, we used adeno-associated virus serotype 9 (AAV9) vectors to express full-length ATXN3 with an abnormally expanded 89 polyglutamine stretch (ATXN3[Q89]) in cerebellar neurons of mature wild-type mice. Mice expressing ATXN3[Q89] exhibited motor impairment in a manner dependent on the viral titer. Immunohistochemistry of the cerebellum showed ubiquitinated nuclear aggregates in PCs; degeneration of PC dendrites; and a significant decrease in multiple proteins including retinoid-related orphan receptor α (RORα), a transcription factor, and type 1 metabotropic glutamate receptor (mGluR1) signaling molecules. Patch clamp analysis of ATXN3[Q89]-expressing PCs revealed marked defects in mGluR1 signaling. Notably, the emergence of behavioral, morphological, and functional defects was inhibited by a single injection of SR1078, an RORα/γ agonist. These results suggest that RORα plays a key role in mutant ATXN3-mediated aberrant phenotypes and that the pharmacological enhancement of RORα could function as a method for therapeutic intervention in SCA3.

The biochemical properties of muscle extracellular matrix are essential for stem cell adhesion, motility, proliferation and myogenic development. Recombinant elastin-like polypeptides are synthetic polypeptides that, besides maintaining some properties of the native protein, can be tailored by fusing bioactive sequences to their C-terminal. Our laboratory synthesized several Human Elastin-Like Polypeptides (HELP) derived from the sequence of human tropoelastin. Here, we developed a novel HELP family member by fusing the elastin-like backbone to the sequence of human Epidermal Growth Factor. We employed this synthetic protein, named HEGF, either alone or in combination with other proteins of the HELP family carrying RGD-integrin binding sites, as adhesion substrate for C2C12 myoblasts and satellite cells primary cultures. Adhesion of myoblasts to HEGF-based substrates induced scattering, decreased adhesion and cytoskeleton assembly; the concomitant presence of the RGD motifs potentiated all these effects. Recombinant substrates induced myoblasts proliferation, differentiation and the development of multinucleated myotubes, thus favoring myoblasts expansion and preserving their myogenic potential. The effects induced by adhesion substrates were inhibited by AG82 (Tyrphostin 25) and herbimycin A, indicating their dependence on the activation of both the EGF receptor and the tyrosine kinase c-src. Finally, HEGF increased the number of muscle stem cells (satellite cells) derived from isolated muscle fibers in culture, thus highlighting its potential as a novel substrate for skeletal muscle regeneration strategies.

Sodium deficiency elevates aldosterone, which in addition to epithelial tissues acts on the brain to promote dysphoric symptoms and salt intake. Aldosterone boosts the activity of neurons that express 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), a hallmark of aldosterone-sensitive cells. To better characterize these neurons, we combine immunolabeling and in situ hybridization with fate mapping and Cre-conditional axon tracing in mice. Many cells throughout the brain have a developmental history of Hsd11b2 expression, but in the adult brain one small brainstem region with a leaky blood-brain barrier contains HSD2 neurons. These neurons express Hsd11b2, Nr3c2 (mineralocorticoid receptor), Agtr1a (angiotensin receptor), Slc17a6 (vesicular glutamate transporter 2), Phox2b, and Nxph4; many also express Cartpt or Lmx1b. No HSD2 neurons express cholinergic, monoaminergic, or several other neuropeptidergic markers. Their axons project to the parabrachial complex (PB), where they intermingle with AgRP-immunoreactive axons to form dense terminal fields overlapping FoxP2 neurons in the central lateral subnucleus (PBcL) and pre-locus coeruleus (pLC). Their axons also extend to the forebrain, intermingling with AgRP- and CGRP-immunoreactive axons to form dense terminals surrounding GABAergic neurons in the ventrolateral bed nucleus of the stria terminalis (BSTvL). Sparse axons target the periaqueductal gray, ventral tegmental area, lateral hypothalamic area, paraventricular hypothalamic nucleus, and central nucleus of the amygdala. Dual retrograde tracing revealed that largely separate HSD2 neurons project to pLC/PB or BSTvL. This projection pattern raises the possibility that a subset of HSD2 neurons promotes the dysphoric, anorexic, and anhedonic symptoms of hyperaldosteronism via AgRP-inhibited relay neurons in PB.

Patient-derived cancer organoid culture is an important live material that reflects clinical heterogeneity. However, the limited amount of organoids available for each case as well as the considerable amount of time and cost to expand in vitro makes it impractical to perform high-throughput drug screening using organoid cultures from multiple patients. Here, we report an advanced system for the high-throughput screening of 2427 drugs using the cancer tissue-originated spheroid (CTOS) method. In this system, we apply the CTOS method in an ex vivo platform from xenograft tumors, using machines to handle CTOS and reagents, and testing a CTOS reference panel of multiple CTOS lines for the hit drugs. CTOS passages in xenograft tumors resulted in minimal changes of morphological and genomic status, and xenograft tumor generation efficiently expanded the number of CTOS to evaluate multiple drugs. Our panel of colorectal cancer CTOS lines exhibited diverse sensitivities to the hit compounds, demonstrating the usefulness of this system for investigating highly heterogeneous disease.

The circadian clock and associated feeding rhythms have a profound impact on metabolism and the gut microbiome. To what extent microbiota reciprocally affect daily rhythms of physiology in the host remains elusive. Here, we analyzed transcriptome and metabolome profiles of male and female germ-free mice. While mRNA expression of circadian clock genes revealed subtle changes in liver, intestine, and white adipose tissue, germ-free mice showed considerably altered expression of genes associated with rhythmic physiology. Strikingly, the absence of the microbiome attenuated liver sexual dimorphism and sex-specific rhythmicity. The resulting feminization of male and masculinization of female germ-free animals is likely caused by altered sexual development and growth hormone secretion, associated with differential activation of xenobiotic receptors. This defines a novel mechanism by which the microbiome regulates host metabolism.

Alt a 1 family proteins (AA1s) have only been observed in the Dothideomycetes and Sordariomycetes classes of fungi, and their biological functions have remained poorly understood. Verticillium dahliae, a soil-borne pathogen that causes plant wilt disease, secretes hundreds of proteins during the process of pathogenic infection, including the AA1 member PevD1. In this study, we found that the pevd1 transcript was present in all of the hosts studied (cotton, Arabidopsis, tomato, and tobacco) and showed elevated expression throughout the infection process. Furthermore, pevd1 knockout mutants displayed attenuated pathogenicity compared with the wild-type (WT) strain and complemented strains in hosts. A partner protein of PevD1, pathogenesis-related protein 5 (PR5)-like protein GhPR5, was isolated from cotton (Gossypium hirsutum) plants by co-purification assays, and the PevD1-GhPR5 interaction was determined to be localized in the C-terminus (PevD1b, amino acids residues 113-155) by pull-down and yeast two-hybrid techniques. Re-introduction of the pevd1b gene into a pevd1 knockout mutant resulted in restoration of the virulence phenotype to WT levels. In addition, PevD1b, which is similar to PevD1, decreased the antifungal activity of GhPR5 in vitro. Our findings reveal an infection strategy in which V. dahliae secretes PevD1 to inhibit GhPR5 antifungal activity in order to overcome the host defence system.

Oxaliplatin is widely used as a key drug in the treatment of colorectal cancer. However, its administration is associated with the dose-limiting adverse effect, peripheral neuropathy. Platinum accumulation in the dorsal root ganglion (DRG) is the major mechanism responsible for oxaliplatin-induced neuropathy. Some drug transporters have been identified as platinum complex transporters in kidney or tumor cells, but not yet in DRG. In the present study, we investigated oxaliplatin transporters and their contribution to peripheral neuropathy. We identified 12 platinum transporters expressed in DRG with real-time PCR, and their transiently overexpressing cells were established. After exposure to oxaliplatin, the accumulation of platinum in these overexpressing cells was evaluated using a coupled plasma mass spectrometer. Octn1/2- and Mate1-expressing cells showed the intracellular accumulation of oxaliplatin. In an animal study, peripheral neuropathy developed after the administration of oxaliplatin (4 mg/kg, intravenously, twice a week) to siRNA-injected rats (0.5 nmol, intrathecally, once a week) was demonstrated with the von Frey test. The knockdown of Octn1 in DRG ameliorated peripheral neuropathy, and decreased platinum accumulation in DRG, whereas the knockdown of Octn2 did not. Mate1 siRNA-injected rats developed more severe neuropathy than control rats. These results indicate that Octn1 and Mate1 are involved in platinum accumulation at DRG and oxaliplatin-induced peripheral neuropathy.

T1 colorectal polyps with at least 1 risk factor for metastasis to lymph node should be treated surgically and are considered endoscopically unresectable. Optical analysis, based on the Narrow-Band Imaging International Colorectal Endoscopic (NICE) classification system, is used to identify neoplasias with invasion of the submucosa that require endoscopic treatment. We assessed the accuracy of the NICE classification, along with other morphologic characteristics, in identifying invasive polyps that are endoscopically unresectable (have at least 1 risk factor for metastasis to lymph node).

Aging results in increased activation of inflammatory glial cells and decreased neuronal viability following spinal cord injury (SCI). Metabolism and transport of glucose is also decreased with age, although the influence of age on glucose transporter (GLUT) expression or glucose uptake in SCI is currently unknown. We therefore performed [18F]Fluorodeoxyglucose (FDG) PET imaging of young (3 month) and middle-aged (12 month) rats. Glucose uptake in middle-aged rats was decreased compared to young rats at baseline, followed by increased uptake 14 days post contusion SCI. qRT-PCR and protein analysis revealed an association between 14 day glucose uptake and 14 day post-injury inflammation. Further, gene expression analysis of neuron-specific GLUT3 and non-specific GLUT4 (present on glial cells) revealed an inverse relationship between GLUT3/4 gene expression and glucose uptake patterns. Protein expression revealed increased GLUT3 in 3 month rats only, consistent with age related decreases in glucose uptake, and increased GLUT4 in 12 month rats only, consistent with age related increases in inflammatory activity and glucose uptake. Inconsistencies between gene and protein suggest an influence of age-related impairment of translation and/or protein degradation. Overall, our findings show that age alters glucose uptake and GLUT3/4 expression profiles before and after SCI, which may be dependent on level of inflammatory response, and may suggest a therapeutic avenue in addressing glucose uptake in the aging population.

Excessive risk-taking is common in multiple psychiatric conditions, including substance use disorders. The risky decision-making task (RDT) models addiction-relevant risk-taking in rats by measuring preference for a small food reward vs. a large food reward associated with systematically increasing risk of shock. Here, we examined the relationship between risk-taking in the RDT and multiple addiction-relevant phenotypes. Risk-taking was associated with elevated impulsive action, but not impulsive choice or habit formation. Furthermore, risk-taking predicted locomotor sensitivity to first-time nicotine exposure and resilience to nicotine-evoked anxiety. These data demonstrate that risk preference in the RDT predicts other traits associated with substance use disorder, and may have utility for identification of neurobiological and genetic biomarkers that engender addiction vulnerability.

Recent reports have demonstrated that lipopolysaccharide (LPS)-induced depressive-like behaviour is mediated via NMDA receptor. In this study, we further investigated the role of GluN2 A subunit of NMDA receptor in synaptic processes in the prefrontal cortex (PFC) and hippocampus of GluN2 A knockout (KO) mice in LPS-induced depressive-like behavior. Our data suggest that LPS-treated mice, lacking GluN2 A subunit, did not exhibit depressive-like behaviour. This was accompanied by unaltered levels of IL-6 and significant changes in neuroplasticity markers and glutamate receptor subunits composition in PFC and hippocampus. In particular, an immune challenge in GluN2 A KO mice resulted in unchanged PSA-NCAM levels and proBDNF increase in both brain structures as well as in increase in BDNF levels in hippocampus. Furthermore, the absence of GluN2 A resulted in increased levels of all NCAM isoforms in PFC upon LPS which was followed with a decrease in GluN1 and GluN2B subunits. The levels of AMPA receptor subunits (GluA1, GluA3, and GluA4) in the hippocampus of GluN2 A mice were unaltered upon the treatment and abundantly present in the PFC of KO mice. These results indicate that the GluN2 A subunit is critical in neuroinflammation-related depression, that its absence abolishes LPS-induced depressive phenotype, sustains PSA-NCAM levels, increases proBDNF signalling in the PFC and hippocampus and potentiates synaptic stabilization through NCAM in the PFC upon an immune challenge.

Glutamate is the most abundant excitatory neurotransmitter in the human brain, but in vivo imaging of acute fluctuations in glutamatergic levels has not been well established. The purpose of this study was to examine acute changes in glutamate after stimulation with N-acetylcysteine (NAC) using a simultaneous positron emission tomography/magnetic resonance spectroscopy (PET/MRS) approach. Ten healthy adult males were examined in two scanning sessions, and 5g NAC was administered 1 h prior to one of the scan sessions. Simultaneous PET/MR data were acquired using an integrated 3T PET/MR scanner. Glutamate (Glu), glutamine (Gln), and glutamate + glutamine (Glx) levels were assessed from MRS data collected from the basal ganglia with PRESS and from the left prefrontal cortex with PRESS and MEGAPRESS, and mGluR5 binding (BPND) was assessed from PET data collected with [18F]PSS232. NAC administration was associated with a significant reduction in Glx and Gln in the basal ganglia spectra, and in Glx in the frontal MEGAPRESS spectra (p < 0.05); no differences in [18F]PSS232 BPND were observed with NAC, although a correlation between pre-/post-treatment Glx and baseline BPnd was found. The MRS-visible Glx signal is sensitive to acute fluctuations in glutamate. The change in Glx was mostly driven by a change in Gln, lending weight to the notion that Gln can provide a proxy marker for neurotransmitter/synaptic glutamate. [18F]PSS232 binding is not sensitive to acute glutamate shifts independently, but was associated with the extent of glutamate liberation upon NAC stimulation.

Classical theories suggest placebo analgesia and nocebo hyperalgesia are based on expectation and conditioned experience. Whereas the neural mechanism of how expectation modulates placebo and nocebo effects during pain anticipation have been extensively studied, little is known about how experience may change brain networks to produce placebo and nocebo responses. We investigated the neural pathways of direct and observational conditioning for conscious and nonconscious conditioned placebo/nocebo effects using magnetoencephalography and a face visual cue conditioning model. We found that both direct and observational conditioning produced conscious conditioned placebo and nocebo effects and a nonconscious conditioned nocebo effect. Alpha band brain connectivity changes before and after conditioning could predict the magnitude of conditioned placebo and nocebo effects. Particularly, the connectivity between the rostral anterior cingulate cortex and middle temporal gyrus was an important indicator for the manipulation of placebo and nocebo effects. Our study suggests that conditioning can mediate our pain experience by encoding experience and modulating brain networks.

Chronic low back pain (CLBP) has major public health implications, and underlying mechanisms are still unclear. Sensorimotor incongruence (SMI)-an ongoing mismatch between top-down motor output and predicted sensory feedback-may play a role in the course of chronic nonspecific low back pain. The hypothesis of this study was that the induction of SMI causes sensory disturbances and/or pain in people with CLBP and healthy volunteers. A sample of 66 people (33 people with CLBP and 33 healthy volunteers) participated in a visual feedback experiment involving real-time images of their own lower backs-either during movement or in a static position-provided via a live video feed. Experimental SMI was induced via distorting visual feedback of the lower back during movement. There were no significant differences in sensory disturbances or pain intensity between experimental SMI and the other movement conditions in people with CLBP and healthy volunteers (P > .05). Static visual feedback had a significant effect on the intensity of sensory disturbances in people with CLBP (P = .038) and healthy volunteers (P < .001). In conclusion, experimental SMI did not affect sensory disturbances or pain in either group. Therefore, the research hypothesis was not supported. PERSPECTIVE: The results of this study show that sensorimotor incongruence does not cause additional symptoms and pain in people with chronic low back pain. The conceptual premise that sensorimotor incongruence is an underlying contributor in the course of pain in this population is not supported.

The oncogene ERG encodes an ETS family transcription factor and is implicated in blood, vascular, and bone development and in prostate, blood, and bone cancer. The ERG gene is alternatively spliced; of particular interest is its cassette exon 7b which adds 24 amino acids, in frame, to the transcriptional activation domain. Higher exon 7b inclusion rates are associated with increased cell proliferation and advanced prostate cancer. The 24 amino acids encoded by exon 7b show evolutionary conservation from humans to echinoderms, highlighting their functional importance. Throughout evolution, these 24 amino acids are encoded by a distinct short exon. Splice-switching oligonucleotides based on morpholino chemistry were designed to induce skipping of ERG exon 7b in MG63 osteosarcoma and VCaP prostate cancer cells. Induction of exon 7b skipping reduced cell proliferation and invasion, increased apoptosis in vitro, and reduced xenograft growth in vivo. We also show that ERG's exon 7b is required for the induction of tissue nonspecific alkaline phosphatase. Together, these findings show that the evolutionarily conserved cassette exon 7b is central to ERG's oncogenic properties.

Fenofibrate, a peroxisome proliferator-activated receptors α (PPARα) agonist, reduces vascular complications of diabetic patients but its protective mechanisms are not fully understood. Here we tested the hypothesis that fenofibrate improves vascular endothelial dysfunction by balancing endothelium-dependent relaxation and contractility of the aorta in diabetes mellitus (DM). In streptozotocin-induced diabetic mice, eight weeks of fenofibrate treatment (100 mg/Kg/d) improved endothelium dependent relaxation in the macro- and microvessels, increased nitric oxide (NO) levels, reduced renal damage markers and effects of the vasoconstrictor prostaglandin. Levels of superoxide dismutase and catalase were both reduced and hydrogen peroxide was increased in vehicle-treated DM, but these changes were reversed by fenofibrate treatment. Vasodilation of the aorta after fenofibrate treatment was reversed by PPARα or AMPKα inhibitors. Western blots showed that fenofibrate treatment elevated PPARα expression, induced liver kinase B1 (LKB1) translocation from the nucleus to the cytoplasm and activated AMP-activated protein kinase-α (AMPKα), thus activating endothelial NO synthase (eNOS). Also, fenofibrate treatment decreased NF-κB p65 and cyclooxygenase 2 proteins in aortas. Finally, incubation with indomethacin in vitro improved aortic contractility in diabetic mice. Overall, our results show that fenofibrate treatment in diabetic mice normalizes endothelial function by balancing vascular reactivity via increasing NO production and suppressing the vasoconstrictor prostaglandin, suggesting mechanism of action of fenofibrate in mediating diabetic vascular complications.

Polycyclic aromatic hydrocarbons are an important group of persistent organic pollutants. Using plants to remediate PAHs has been recognized as a cost-effective and environmentally friendly technique. However, the overall impact of PAHs on the regulation of plant metabolism has not yet been explored. In this study, we analyzed the alteration in the maize (Zea mays L.) metabolome on exposure to high molecular weight PAHs such as benzo[a]pyrene (BaP) and pyrene (PYR) in a hydroponic medium, individually and as a mixture (BaP + PYR) using GC-MS. The differences in the metabolites were analyzed using XCMS (an acronym for various forms (X) of chromatography-mass spectrometry), an online-based data analysis tool. A significant variation in metabolites was observed between treatment groups and the unspiked control group. The univariate, multivariate and pathway impact analysis showed there were more significant alterations in metabolic profiles between individual PAHs and the mixture of BaP and PYR. The marked changes in the metabolites of galactose metabolism and aminoacyl tRNA biosynthesis in PAHs treated maize leaves exhibit the adaptive defensive mechanisms for individual and PAHs mixture. Therefore, the metabolomics approach is essential for an understanding of the complex biochemical responses of plants to PAHs contaminants. This knowledge will shed new light in the field of phytoremediation, bio-monitoring, and environmental risk assessment.

Exposure to high levels of metals/metalloids may impair semen quality. Computer-aided sperm analysis (CASA) can be used for kinematic analysis of spermatozoa, which provides additional insights into sperm motion characteristics.

Poor quality data in preclinical trials can result from inconsistent and unstandardized experimental processes. Unpredictable pre-intervention variability generates unreliable data, biases outcomes and results in needless waste of animals and resources. We applied Define-Measure-Analyse-Improve-Control (DMAIC) quality improvement processes to pilot development of a swine model of trauma, haemorrhagic shock and coagulopathy. The goal was to reduce variability through protocol standardization and error reduction. Six male Sinclair swine were sequentially anesthetized, intubated, mechanically ventilated and instrumented, then subjected to multiple-hit injury, followed by fluid resuscitation monitoring and coagulation testing. Experimental tasks were defined and mapped. Performance measures were task performance times, subject stabilization time and number of task execution errors. Process improvement was assessed by reduced times and errors, and subject stability at target physiological values. Previously-overlooked performance errors and deficiencies were identified. 'Mistake-proofing' actions included personnel retraining, revisions of standard operating procedures and use of checklists. The quality improvement pilot trial produced a stable model with reduced protocol deviations. Data quality can be improved and animal waste minimized, if experimental planning incorporates strategies to ensure protocol adherence and reduced operator performance variation and errors. Properly designed pilot trials can be essential components of refinement and reduction strategies in animal-based research.

Early theories on face perception posit that invariant (i.e., identity) and changeable (i.e., expression) facial aspects are processed separately. However, many researchers have countered the hypothesis of parallel processes with findings of interactions between identity and emotion perception. The majority of tasks measuring interactions between identity and emotion employ a selective attention design, in which participants are instructed to attend to one dimension (e.g., identity) while the other dimension varies orthogonally (e.g., emotion), but is task irrelevant. Recently, a divided attention design (i.e., the redundancy gain paradigm) in which both identity and emotion are task relevant was employed to assess the interaction between identity and emotion. A redundancy gain is calculated by a drop in reaction time in trials in which a target from both dimensions is present in the stimulus face (e.g., "sad Person A"), compared with trials with only a single target present (e.g., "sad" or "Person A"). Redundancy gains are hypothesized to point to an interactive activation of both dimensions, and as such, could complement designs adopting a selective attention task. The initial aim of the current study was to reproduce the earlier findings with this paradigm on identity and emotion perception (Yankouskaya, Booth, & Humphreys, Attention, Perception, & Psychophysics, 74(8), 1692-1711, 2012), but our study failed to replicate the results. In a series of subtasks, multiple aspects of the design were manipulated separately in our goal to shed light on the factors that influence the redundancy gain effect in faces. A redundancy gain was eventually obtained after controlling for contingencies and stimulus presentation time.