The study of cerebral microvessels is becoming increasingly important in a wide variety of conditions, such as stroke, sepsis, traumatic brain injury and neurodegenerative diseases. However, the molecular mechanisms underlying cerebral microvascular dysfunction in these conditions are largely unknown. The molecular characterization of cerebral microvessels in experimental disease models has been hindered by the lack of a standardized method to reproducibly isolate intact cerebral microvessels with consistent cellular compositions and without the use of enzymatic digestion, which causes undesirable molecular and metabolic changes. Herein, we describe an optimized protocol for microvessel isolation from mouse brain cortex that yields microvessel fragments with consistent populations of discrete blood-brain barrier (BBB) components (endothelial cells, pericytes and astrocyte end feet) while retaining high RNA integrity and protein post-translational modifications (e.g., phosphorylation). We demonstrate that this protocol allows the quantification of changes in gene expression in a disease model (stroke) and the activation of signaling pathways in mice subjected to drug administration in vivo. We also describe the isolation of genomic DNA (gDNA) and bisulfite treatment for the assessment of DNA methylation, as well as the optimization of chromatin extraction and shearing from cortical microvessels. This optimized protocol and the described applications should improve the understanding of the molecular mechanisms governing cerebral microvascular dysfunction, which may help in the development of novel therapies for stroke and other neurologic conditions.

It is possible to raise the rate of the uptake of 131I in the thyroid gland (RAIU) by increasing the endogenous TSH level through appropriate use of methimazole (MMI) prior to 131I therapy. The purpose of this paper is to assess the impact of pre treatment with MMI on the efficacy of 131I therapy in non-toxic multinodular goitre (NMG).

Hematologic complete remission (CR) is achievable for most adults with B cell precursor acute lymphoblastic leukemia (BCP-ALL). However, minimal residual disease (MRD) in patients with hematologic CR is associated with increased risk of relapse, shorter survival, and poorer transplantation outcomes. This study explored the concept of cure in adults with Philadelphia chromosome-negative (Ph-) BCP-ALL by MRD status at first hematologic CR (CR1) to inform evaluation of the clinical and economic benefits of new agents, where the concept of cure is important but long-term data are not available. The study used modified Delphi methodology involving clinicians experienced in the treatment of adult ALL. Participants completed a questionnaire, which was followed by country-specific panel discussions to discuss results and identify consensus on concepts and definitions. Clinicians from France (n = 4), Germany (n = 4), and the UK (n = 5) took part. Participants described cure in terms of the probability of future relapse. Relapse-free survival (RFS) was the preferred outcome measure to describe cure for the three patient groups considered (patients with MRD at CR1; patients who become negative for MRD after further treatment; patients who continue to have MRD). Consensus was reached on definitions of cure: that cure would begin to be considered at 3 years' RFS and/or would be highly likely at 5 years' RFS. Participants agreed that patients with MRD should usually undergo hematopoietic stem cell transplantation to have the best chance of survival; consensus was reached that alternatives are required when transplantation is not an option. Panels agreed that patients who achieve cure have a higher mortality rate and lower health-related quality of life than the general population. This study provides quantitative and qualitative information on the concept of cure in Ph- BCP-ALL in CR by MRD status applicable to interpreting the value of new therapies.Funding: Amgen.Plain Language Summary: Plain language summary available for this article.

Although fatigue is a well-studied concept in neurological disease, cognitive fatigability (CF) is less understood. While most studies measure fatigue using subjective self-report, fewer have measured CF objectively. Given the negative impact of CF on quality-of-life, there is a need for targeted interventions. The objective of this review was to determine which procedural, behavioural and pharmacological treatments for objectively measured CF are available to people living with neurological conditions.

We developed machine learning methods to identify fibrolipidic and fibrocalcific A-lines in intravascular optical coherence tomography (IVOCT) images using a comprehensive set of handcrafted features. We incorporated features developed in previous studies (e.g., optical attenuation and A-line peaks). In addition, we included vascular lumen morphology and three-dimensional (3-D) digital edge and texture features. Classification methods were developed using expansive datasets (∼7000  images), consisting of both clinical in-vivo images and an ex-vivo dataset, which was validated using 3-D cryo-imaging/histology. Conditional random field was used to perform 3-D classification noise cleaning of classification results. We tested various multiclass approaches, classifiers, and feature selection schemes and found that a three-class support vector machine with minimal-redundancy-maximal-relevance feature selection gave the best performance. We found that inclusion of our morphological and 3-D features improved overall classification accuracy. On a held-out test set consisting of >1700 images, we obtained an overall accuracy of 81.58%, with the following (sensitivity/specificity) for each class: other (81.43/89.59), fibrolipidic (94.48/87.32), and fibrocalcific (74.82/95.28). The en-face views of classification results showed that automated classification easily captured the preponderance of a disease segment (e.g., a calcified segment had large regions of fibrocalcific classifications). Finally, we demonstrated proof-of-concept for streamlining A-line classification output with existing fibrolipidic and fibrocalcific boundary segmentation methods, to enable fully automated plaque quantification. The results suggest that our classification approach is a viable step toward fully automated IVOCT plaque classification and segmentation for live-time treatment planning and for offline assessment of drug and biologic therapeutics.

Stress-related disorders are associated with significant suffering, functional impairment, and high societal costs. Internet-based cognitive behavioral therapy (ICBT) is a promising treatment for stress-related disorders but has so far not been subjected to health economic evaluation.

Global change and biotic stress, such as tropospheric contamination and virus infection, can individually modify the quality of host plants, thereby altering the palatability of the plant for herbivorous insects. The bottom-up effects of elevated O3 and tomato yellow leaf curl virus (TYLCV) infection on tomato plants and the associated performance of Bemisia tabaci Mediterranean (MED) were determined in open-top chambers. Elevated O3 decreased eight amino acid levels and increased the salicylic acid (SA) and jasmonic acid (JA) content and the gene expression of pathogenesis-related protein (PR1) and proteinase inhibitor (PI1) in both wild-type (CM) and JA defense-deficient tomato genotype (spr2). TYLCV infection and the combination of elevated O3 and TYLCV infection increased eight amino acids levels, SA content and PR1 expression, and decreased JA content and PI1 expression in both tomato genotypes. In uninfected tomato, elevated O3 increased developmental time and decreased fecundity by 6.1 and 18.8% in the CM, respectively, and by 6.8 and 18.9% in the spr2, respectively. In TYLCV-infected tomato, elevated O3 decreased developmental time and increased fecundity by 4.6 and 14.2%, respectively, in the CM and by 4.3 and 16.8%, respectively, in the spr2. These results showed that the interactive effects of elevated O3 and TYLCV infection partially increased the amino acid content and weakened the JA-dependent defense, resulting in increased population fitness of MED on tomato plants. This study suggests that whiteflies would be more successful at TYLCV-infected plants than at uninfected plants in elevated O3 levels.

To investigate the potential protective effect of novel G protein coupled estrogen receptor (GPER1) against the neurotoxicity induced by NMDA in the mouse retina.

Tactile stimuli are integrated and processed by neuronal circuits in the deep dorsal horn of the spinal cord. Several spinal interneuron populations have been implicated in tactile information processing. However, dorsal horn projection neurons that contribute to the postsynaptic dorsal column (PSDC) pathway transmitting tactile information to the brain are poorly characterized. Here, we show that spinal neurons marked by the expression of Zic2creER mediate light touch sensitivity and textural discrimination. A subset of Zic2creER neurons are PSDC neurons that project to brainstem dorsal column nuclei, and chemogenetic activation of Zic2 PSDC neurons increases sensitivity to light touch stimuli. Zic2 neurons receive direct input from the cortex and brainstem motor nuclei and are required for corrective motor movements. These results suggest that Zic2 neurons integrate sensory input from cutaneous afferents with descending signals from the brain to promote corrective movements and transmit processed touch information back to the brain. VIDEO ABSTRACT.

The oxidative pentose phosphate pathway (oxiPPP) contributes to cell metabolism through not only the production of metabolic intermediates and reductive NADPH but also inhibition of LKB1-AMPK signaling by ribulose-5-phosphate (Ru-5-P), the product of the third oxiPPP enzyme 6-phosphogluconate dehydrogenase (6PGD). However, we found that knockdown of glucose-6-phosphate dehydrogenase (G6PD), the first oxiPPP enzyme, did not affect AMPK activation despite decreased Ru-5-P and subsequent LKB1 activation, due to enhanced activity of PP2A, the upstream phosphatase of AMPK. In contrast, knockdown of 6PGD or 6-phosphogluconolactonase (PGLS), the second oxiPPP enzyme, reduced PP2A activity. Mechanistically, knockdown of G6PD or PGLS decreased or increased 6-phosphogluconolactone level, respectively, which enhanced the inhibitory phosphorylation of PP2A by Src. Furthermore, γ-6-phosphogluconolactone, an oxiPPP byproduct with unknown function generated through intramolecular rearrangement of δ-6-phosphogluconolactone, the only substrate of PGLS, bound to Src and enhanced PP2A recruitment. Together, oxiPPP regulates AMPK homeostasis by balancing the opposing LKB1 and PP2A.

The common marmoset (marmoset; Callithrix jacchus) harbors various desired features as a non-human primate (NHP) model for neuroscience research. Recently, efforts have been made to induce neural cells in vitro from marmoset pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), which are characterized by their capacity to differentiate into all cell types from the three germ layers. Successful generation of marmoset neural cells is not only invaluable for understanding neural development and for modeling neurodegenerative and psychiatric disorders, but is also necessary for the phenotypic screening of genetically-modified marmosets. However, differences in the differentiation propensity among PSC lines hamper the applicability and the reproducibility of differentiation methods. To overcome this limitation, we evaluated the efficacy of small molecules for neural differentiation of marmoset ESCs (cjESCs) and iPSCs using multiple differentiation methods. By immunochemical and transcriptomic analyses, we confirmed that our methods using the small molecules are efficient for various differentiation protocols by either enhancing the yield of a mixture of neural cells including both neurons and glial cells, or a pure population of neurons. Collectively, our findings optimized in vitro neural differentiation methods for marmoset PSCs, which would ultimately help enhance the utility of the animal model in neuroscience.

Astrocytes take up glucose via the 45 kDa isoform of the Glucose Transporter 1 (GLUT-1), and in this work we have investigated whether histamine regulates GLUT-1 expression in rat cerebro-cortical astrocytes in primary culture. Cultured astrocytes expressed histamine H1 and H3 receptors (H1Rs and H3Rs) as evaluated by radioligand binding. Receptor functionality was confirmed by the increase in the intracellular concentration of Ca2+ (H1R) and the inhibition of forskolin-induced cAMP accumulation (H3R). Quantitative RT-PCR showed that histamine and selective H1R and H3R agonists (1 h incubation) significantly increased GLUT-1 mRNA to 153 ± 7, 163 ± 2 and 168 ± 13% of control values, respectively. In immunoblot assays, incubation (3 h) with histamine or H1R and H3R agonists increased GLUT-1 protein levels to 224 ± 12, 305 ± 11 and 193 ± 13% of control values, respectively, an action confirmed by inmunocytochemistry. The effects of H1R and H3R agonists were blocked by the selective antagonists mepyramine (H1R) and clobenpropit (H3R). The pharmacological inhibition of protein kinase C (PKC) prevented the increase in GLUT-1 protein induced by either H1R or H3R activation. Furthermore, histamine increased ERK-1/2 phosphorylation, and the effect of H1R and H3R activation on GLUT-1 protein levels was reduced or prevented, respectively, by MEK-1/2 inhibition. These results indicate that by activating H1Rs and H3Rs histamine regulates the expression of GLUT-1 by astrocytes. The effect appears to involve the phospholipase C (PLC) → diacylglycerol (DAG)/Ca2+→ PKC and PLC → DAG/Ca2+ → PKC → MAPK pathways.

Type 1 diabetes (T1D) is a multifactorial autoimmune disease that develops as a consequence of macrophage- and T cell-dependent pancreatic β-cell death. Multiple approaches for induction of anti-inflammatory/regulatory mechanisms that would attenuate T1D have been utilized, with little or no beneficial effects. To achieve prolonged stimulation of regulatory immune cells, we orally introduced microparticles (MPs) loaded with all-trans retinoic acid (ATRA) and transforming growth factor-β (TGF-β) to C57BL/6 mice treated with multiple low doses of streptozotocin (MLDS) for T1D induction. Disease incidence was significantly lower in ATRA/TGF-β MPs-treated mice, as was the degree of immune cell infiltration into the pancreatic islets. In Peyer's patches (PP), ATRA/TGF-β MPs up-regulated tolerogenic dendritic cells (tolDCs) (CD11c+CD11b-CD103+), while the proportion of mature dendritic cells was not altered. This was accompanied by reduced Th1 and Th17 proportions and up-regulation of regulatory T cells (Tregs - CD4+CD25highFoxP3+). The immune cell composition in the pancreatic lymph nodes was similar to PP. Further, the proportion of effector Tbet+CD25med cells was decreased, while the proportion of Tbet+ Treg cells that specifically inhibit Th1 response was increased. Moreover, ATRA/TGF-β MPs treatment resulted in increased Treg proliferation and frequency of CTLA-4+PD1+ and CD39+IL-10+ Tregs, suggestive of their higher suppressive capacity. Reduced pancreatic infiltration may have been a consequence of lower cell capacity for matrix degradation. In conclusion, oral application of ATRA/TGF-β MPs ameliorated T1D through potentiation of tolDCs and Tregs, inhibition of Th1 response and prevention of the immune cell entrance into the islets.

Pancreastatin (PST), a chromogranin A derived peptide has anti-insulin effects and plays a significant role in obesity-induced insulin resistance. In obesity and type 2 diabetes mellitus, both insulin and PST level are elevated, but it is not clearly understood how anti-insulin effect of PST get regulated in hyperinsulinemic state. Simultaneously we have explored pancreastatin inhibitor PSTi8 against the native PST in the same hyperinsulinemic state. In in-vitro studies, we found that PST treatment increases lipid droplets and reactive oxygen species production in 3T3L1 adipocyte cells and theses effects of PST was found synergistic with chronic-insulin treatment. Treatment of PSTi8 in 3T3L1 adipocytes attenuates PST effect on lipid droplet formation and reactive oxygen species production. We further validated these findings in epididymal white adipose tissue of C57BL/6 mice, implanted with mini-osmotic insulin pump with and without PSTi8 for 4 weeks. We found that chronic hyperinsulinemia enhanced PST levels in circulation which in turn induces expression of various pro-inflammatory cytokines and oxidative stress. In addition, it also stimulated the expression of lipogenic genes, fat mass and body weight gain through the regulation of circulating adiponectin level. The change in PST mediated inflammatory and lipogenic parameters were attenuated by PSTi8 treatment, leading to enhanced insulin sensitivity and improved glucose homeostasis. PSTi8 rescue from PST mediated insulin resistance in adipose via inhibition of MAPK and NOX3-JNK stress signalling pathway which stimulates GLUT4 expression through activation of AKT-AS160 pathway. Thus PSTi8 may be a novel therapeutic agent for the treatment of hyperinsulinemia induced obesity and inflammation mediated insulin resistance.

Homocysteine (Hcy) is an independent risk factor in the development of cardiovascular diseases (CVD). Hyperhomocysteinemia (HHcy), induces the injury of vascular endothelial cells via oxidative stress. Oxymatrine (OMT), one of the main components of Sophora flavescens, has displayed anti-inflammatory, anti-oxidant and anti-apoptotic activity. However, the effect of OMT on the Hcy-induced endothelial injury is not clearly defined yet. The aim of this study was to determine the protective effect of OMT on the Hcy-induced endothelial injury and its mechanisms involved. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro. Methyl thiazolyl tetrazolium assay (MTT), fluorescence staining, flow cytometry and western blotting were used in this study. OMT prevented the Hcy-induced toxicity and apoptosis in HUVECs. Moreover, OMT suppressed Hcy-induced increases in reactive oxygen species, lactate dehydrogenase, malondialdehyde levels and increased superoxide dismutase levels. OMT reversed the Hcy-induced decrease in the protein expression of nuclear factor erythroid-2-related factor 2 (Nrf2). In addition, OMT reversed the Hcy-induced apoptosis related biochemical changes such as decreased mitochondrial membrane potential and Bcl-2/Bax protein ratio, and increased protein expression of caspase-9 and caspase-3. Furthermore, OMT elevated the phosphorylation levels of Akt and eNOS, and the formation of nitric oxide (NO) in injured cells. These results suggest that OMT prevents Hcy-induced endothelial injury by regulating mitochondrial-dependent apoptosis and Akt-eNOS-NO signaling pathways concomitantly with accentuation of Nrf2 expression.

Children lay the foundation for later academic achievement by acquiring core mathematical abilities in the first school years. Neural reorganization processes associated with individual differences in early mathematical learning, however, are still poorly understood. To fill this research gap, we followed a sample of 5-6-year-old children longitudinally to the end of second grade in school (age 7-8 years) combining magnetic resonance imaging (MRI) with comprehensive behavioral assessments. We report significant links between the rate of neuroplastic change of cortical surface anatomy, and children's early mathematical skills. In particular, most of the behavioral variance (about 73%) of children's visuospatial abilities was explained by the change in cortical thickness in the right superior parietal cortex. Moreover, half of the behavioral variance (about 55%) of children's arithmetic abilities was explained by the change in cortical folding in the right intraparietal sulcus. Additional associations for arithmetic abilities were found for cortical thickness change of the right temporal lobe, and the left middle occipital gyrus. Visuospatial abilities were related to right precentral and supramarginal thickness, as well as right medial frontal gyrus folding plasticity. These effects were independent of other individual differences in IQ, literacy and maternal education. Our findings highlight the critical role of cortical plasticity during the acquisition of fundamental mathematical abilities.

The aim of the present study was to uncover a possible common neural organizing principle in spoken and written communication, through the coupling of perceptual and motor representations. In order to identify possible shared neural substrates for processing the basic units of spoken and written language, a sparse sampling fMRI acquisition protocol was performed on the same subjects in two experimental sessions with similar sets of letters being read and written and of phonemes being heard and orally produced. We found evidence of common premotor regions activated in spoken and written language, both in perception and in production. The location of those brain regions was confined to the left lateral and medial frontal cortices, at locations corresponding to the premotor cortex, inferior frontal cortex and supplementary motor area. Interestingly, the speaking and writing tasks also appeared to be controlled by largely overlapping networks, possibly indicating some domain general cognitive processing. Finally, the spatial distribution of individual activation peaks further showed more dorsal and more left-lateralized premotor activations in written than in spoken language.

Recent neurophysiological studies have proposed distinct roles of β and γ oscillations in implementing top-down and bottom-up processes. The present study aims to test this hypothesis in the domain of speech perception. We examined β and γ oscillations elicited to a tone contrast in a passive oddball paradigm, and their relationships with discrimination sensitivity d' and RT from two groups of healthy adults who showed high and low discrimination sensitivity to the contrast. The low-sensitivity group showed a significant reduction in β, which was further related to d'. Individual differences in RT were related to different frequency bands in the two groups, with a RT-β correlation in the low-sensitivity group, and a RT-γ relation in the high-sensitivity group. Based on these findings, we suggest that β, implicated in top-down processing, reflects individual differences in phonological representations, and that γ, involved in bottom-up processing, reflects individual differences in acoustic encoding.

Osteoarthritis (OA) microenvironment is marked by matrix metalloproteinases-13 (MMP-13) overexpression and weak acidity, making it possible to develop dual-stimuli responsive theranostic nanoprobes for OA diagnosis and therapy. However, current MMP/pH-responsive systems are not suitable for OA because of their poor biocompatibility, poor degradation and non-cartilage-targeting of the responsive probes. Here we designed a novel biocompatible cartilage-targeting and MMP-13/pH-responsive ferritin nanocages (CMFn) loaded with an anti-inflammatory drug (Hydroxychloroquine, HCQ), termed CMFn@HCQ, for OA imaging and therapy. We found that CMFn could be smartly "turned on" to emit light for OA imaging in response to the level of overexpressed MMP-13 in OA microenvironment, corresponding to the degree of OA severity. Thus the light intensity detected reflected the degree of OA severity, enabling the precise disease classification by our CMFn. CMFn could be "turned off" to stop emitting light in the normal joint. CMFn@HCQ nanocages could target the cartilage and release HCQ in the OA joint specifically under acidic pH conditions in a sustained manner, prolonging the drug retention time to 14 days to remarkably reduce synovial inflammation in the OA joints. The CMFn@HCQ nanocages represent a smart dual-stimuli responsive and cartilage-targeting nanoprobes, and hold promise for imaging-guided precision therapy for OA.

Role of avian hosts in shaping persistence, evolution, and dispersal of global low pathogenic avian influenza virus (LPAIV) H9N2 remains uncertain. Under Bayesian Markov Chain Monte Carlo framework, we used the discrete trait analysis (DTA) to reconstruct host and location switches in the evolutionary history of global H9N2 given hemagglutinin gene sequences from 18 countries/regions between 1976 and 2018. We employed generalized linear models (GLMs) to inform virus migration rates by empirical predictors. Global H9N2 isolates were mostly sampled from domestic Phasianidae in low- and middle-income countries with poor bio-security. Anatidae was inferred as the ancestral source from which the virus spread to domestic waterfowl, and later to domestic Phasianidae who have become the dominant host to sustain the virus, especially in Asia. Poultry trade was a well-supported driver to H9N2 spread across countries/regions. Strict bio-security and separation between wild and domestic poultry can be used to mitigate virus spread.