Histamine-4 receptor antagonist ameliorates Parkinson-like pathology in the striatum.

Growing evidence indicates that microglia activation and a neuroinflammatory trigger contribute to dopaminergic cell loss in Parkinson's disease (PD). Furthermore, increased density of histaminergic fibers and enhanced histamine levels have been observed in the substantia nigra of PD-postmortem brains. Histamine-induced microglial activation is mediated by the histamine-4 receptor (H4R). In the current study, gene set enrichment and pathway analyses of a PD basal ganglia RNA-sequencing dataset revealed that upregulation of H4R was in the top functional category for PD treatment targets. Interestingly, the H4R antagonist JNJ7777120 normalized the number of nigrostriatal dopaminergic fibers and striatal dopamine levels in a rotenone-induced PD rat model. These improvements were accompanied by a reduction of α-synuclein-positive inclusions in the striatum. In addition, intracerebroventricular infusion of JNJ7777120 alleviated the morphological changes in Iba-1-positive microglia and resulted in a lower tumor necrosis factor-α release from this brain region, as well as in ameliorated apomorphine-induced rotation behaviour. Finally, JNJ7777120 also restored basal ganglia function by decreasing the levels of γ-aminobutyric acid (GABA) and the 5-hydroxyindoleactic acid to serotonin (5-HIAA/5-HT) concentration ratios in the striatum of the PD model. Our results highlight H4R inhibition in microglia as a promising and specific therapeutic target to reduce or prevent neuroinflammation, and as such the development of PD pathology.

Histamine-4 receptor antagonist JNJ7777120 inhibits pro-inflammatory microglia and prevents the progression of Parkinson-like pathology and behaviour in a rat model.

The activation of microglial cells is presumed to play a key role in the pathogenesis of Parkinson's disease (PD). The activity of microglia is regulated by the histamine-4 receptor (H4R), thus providing a novel target that may prevent the progression of PD. However, this putative mechanism has so far not been validated. In our previous study, we found that mRNA expression of H4R was upregulated in PD patients. In the present study, we validated this possible mechanism using the rotenone-induced PD rat model, in which mRNA expression levels of H4R-, and microglial markers were significantly increased in the ventral midbrain. Inhibition of H4R in rotenone-induced PD rat model by infusion of the specific H4R antagonist JNJ7777120 into the lateral ventricle resulted in blockade of microglial activation. In addition, pharmacological targeting of H4R in rotenone-lesioned rats resulted in reduced apomorphine-induced rotational behaviour, prevention of dopaminergic neuron degeneration and associated decreases in striatal dopamine levels. These changes were accompanied by a reduction of Lewy body-like neuropathology. Our results provide first proof of the efficacy of an H4R antagonist in a commonly used PD rat model, and proposes the H4R as a promising target to clinically tackle microglial activation and thereby the progression of PD.

Selective inhibition of mutant isocitrate dehydrogenase 1 (IDH1) via disruption of a metal binding network by an allosteric small molecule.

Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg(2+). A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme.

Antimicrobial Resistance and Molecular Typing of Salmonella Stanley Isolated from Humans, Foods, and Environment.

Salmonella enterica serovar Stanley is an important serovar that has been increasingly identified in human salmonellosis. The present study aimed to investigate the antimicrobial resistance and molecular typing of 88 Salmonella Stanley strains isolated from humans (diarrhea patients, n = 64; and healthy carrier, n = 1), foods (aquatic products, n = 16; vegetable, n = 1; and pork, n = 1), and environment (waste water, n = 2; and river water, n = 3) in Shanghai, China from 2006 to 2012. Nearly half of the strains were resistant to sulfafurazole (43/88, 48.9%), and many were resistant to streptomycin (35/88, 39.8%), tetracycline (22/88, 25%), and nalidixic acid (19/88, 21.6%). Approximately a quarter of the strains (24/88, 27.3%) were resistant to more than three antimicrobials, and five had ACSSuT resistance type. Six clusters (A-F) were identified by pulsed-field gel electrophoresis (PFGE) with 80% similarity. Interestingly, strains in the same cluster identified by PFGE possessed similar antibiotic resistance patterns. PFGE typing also indicated that aquatic products might serve as a transmission reservoir for Salmonella Stanley infections in humans.

Trispecific antibody targeting HIV-1 and T cells activates and eliminates latently-infected cells in HIV/SHIV infections.

Agents that can simultaneously activate latent HIV, increase immune activation and enhance the killing of latently-infected cells represent promising approaches for HIV cure. Here, we develop and evaluate a trispecific antibody (Ab), N6/αCD3-αCD28, that targets three independent proteins: (1) the HIV envelope via the broadly reactive CD4-binding site Ab, N6; (2) the T cell antigen CD3; and (3) the co-stimulatory molecule CD28. We find that the trispecific significantly increases antigen-specific T-cell activation and cytokine release in both CD4+ and CD8+ T cells. Co-culturing CD4+ with autologous CD8+ T cells from ART-suppressed HIV+ donors with N6/αCD3-αCD28, results in activation of latently-infected cells and their elimination by activated CD8+ T cells. This trispecific antibody mediates CD4+ and CD8+ T-cell activation in non-human primates and is well tolerated in vivo. This HIV-directed antibody therefore merits further development as a potential intervention for the eradication of latent HIV infection.

A Polyanionic Strategy to Modify the Perovskite Grain Boundary for a Larger Switching Ratio in Flexible Woven Resistive Random-Access Memories.

The intergranular interface modification of organic-inorganic hybrid perovskites (OHP) is an important issue to regulate the flexibility, stability, and resistive switching (RS) performance of resistive random-access memories (RRAMs). A novel strategy of polymer additives for OHP intergranular interface modification is explored in this work with the polyanionic backbone to improve the distribution of cage-shaped cavity molecules at the perovskite grain boundaries. Specifically speaking, poly(1-adamantylammonium acrylate) (PADAm) is first synthesized through the acid-base reaction of polyacrylic acid with 1-adamantylamine to simultaneously realize the introduction of a cage-shaped cavity molecule and the polyanionic backbone. Herein, organic ammonium cations 1-adamantylammonium (ADNH3+) in PADAm are applied as the cage-shaped cavity molecules to tune the dielectric property by being anchored at the perovskite grain boundaries. Meanwhile, polyacrylic anions in PADAm play the role of the polyanionic backbone to produce the more uniform distribution of ADNH3+. Simultaneously, the flexibility and stability of OHP RRAM devices are also improved due to the introduction of the polyanionic backbone. Consequently, the 4% ADNH3I-modified planar device exhibits the stable nonvolatile RS behavior with an on/off ratio of ∼104, even with one-month exposure under an ambient environment. Importantly, the introduction of PADAm in the flexible fibrous crosspoint of functional fiber Al@MAPbI3:PADAm and bare Al fiber further increases the on/off ratio to 108 due to the effectively improved distribution of hollow cage-shaped ADNH3+ at the perovskite intergranular interfaces together with the application of the fibrous crosspoint device configuration. Especially, these excellent crosspoint RRAM devices can be integrated into the woven fibrous RRAM array in the thermal plastic packaging configuration. In addition, the excellent multilevel RS behavior can also be realized in the woven fibrous RRAM array, indicating potential high-density data storage. This work provides a novel strategy of polymer additives bearing the polyanionic backbone to improve the flexibility, stability, and RS performance of perovskite RRAM devices.

The Candida albicans pescadillo homolog is required for normal hypha-to-yeast morphogenesis and yeast proliferation.

A single species, Candida albicans, causes half of all invasive fungal infections in humans. Unlike other fungal pathogens, this organism switches between growth as budding yeast and as pseudohyphal and hyphal filaments in host organs and in vitro. Both cell types play a role in invasive disease: while hyphal and pseudohyphal filaments penetrate host cells and tissues, yeast cells are likely to facilitate dissemination through the bloodstream and establishment of distant foci of infection. Many regulators of the yeast-to-hypha switch have emerged from intensive investigations of this morphogenetic step, but the hypha-to-yeast switch remains poorly understood. Using a forward genetic approach, a novel putative regulator involved in the hypha-to-yeast switch was identified, the C. albicans pescadillo homolog, PES1. In eukaryotes from yeast to human, pescadillo homologs are involved in cell cycle control and ribosome biogenesis, and are essential. We find a pescadillo homolog to act in fungal morphogenesis, specifically in lateral yeast growth on filamentous cells. We also find essentiality of PES1 in C. albicans to be dependent on cell type, because hyphal cells, but not yeast cells, tolerate its loss. PES1 is therefore critical for completion of the C. albicans life cycle, in which the fungus switches between filamentous and yeast growth. Consistent with these in vitro findings, PES1 is required for C. albicans virulence in an in vivo insect model of infection.

Characterization of Reference Materials for Spinal Muscular Atrophy Genetic Testing: A Genetic Testing Reference Materials Coordination Program Collaborative Project.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder predominately caused by bi-allelic loss of the SMN1 gene. Increased copies of SMN2, a low functioning nearly identical paralog, are associated with a less severe phenotype. SMA was recently recommended for inclusion in newborn screening. Clinical laboratories must accurately measure SMN1 and SMN2 copy number to identify SMA patients and carriers, and to identify individuals likely to benefit from therapeutic interventions. Having publicly available and appropriately characterized reference materials with various combinations of SMN1 and SMN2 copy number variants is critical to assure accurate SMA clinical testing. To address this need, the CDC-based Genetic Testing Reference Materials Coordination Program, in collaboration with members of the genetic testing community and the Coriell Institute for Medical Research, has characterized 15 SMA reference materials derived from publicly available cell lines. DNA samples were distributed to four volunteer testing laboratories for genotyping using three different methods. The characterized samples had zero to four copies of SMN1 and zero to five copies SMN2. The samples also contained clinically important allele combinations (eg, zero copies SMN1, three copies SMN2), and several had markers indicative of an SMA carrier. These and other reference materials characterized by the Genetic Testing Reference Materials Coordination Program are available from the Coriell Institute and are proposed to support the quality of clinical laboratory testing.

Nontyphoidal Salmonella Gastroenteritis in Baoshan, Shanghai, China, 2010 to 2014: An Etiological Surveillance and Case-Control Study.

Nontyphoidal Salmonella (NTS) gastroenteritis is a widespread global foodborne disease. To identify the epidemiologic characteristics, sources of food contamination, and risk factors of NTS gastroenteritis, epidemiologic data and stool specimens of diarrheal patients were collected from sentinel hospitals in Baoshan, Shanghai, People's Republic of China, between 2010 and 2014. Food products from nearby farmers' markets and animal feces from live poultry markets and livestock farms were sampled to identify the pathogen; a case-control study was conducted to characterize risk factors of NTS gastroenteritis. Of 3,906 diarrheal patients examined, 266 (6.8%) were positive for Salmonella. The positive rates were higher in summer than in the other seasons. Salmonella Typhimurium (36.1%) and Salmonella Enteritidis (30.8%) were the dominant serovars in the patients. Salmonella was detected in 26.2% pork samples, 7.1 to 7.8% poultry meats, and 3.3 to 8.9% poultry feces. Salmonella Typhimurium was the major serovar in contaminated food and animal feces. Multivariate conditional logistic regression analysis indicated that consumption of pork and quickly cooked eggs increased, whereas separating kitchen knives for cooked and raw food decreased the risk of NTS gastroenteritis, independently. We believe that NTS in poultry feces contaminated the meat products in the same markets and then infected humans if these foods were not sufficiently cooked. To prevent NTS gastroenteritis, it is necessary to survey Salmonella in meats and poultry feces, to cook eggs and pork sufficiently, to separate kitchen knives for cooked and raw food, and to prohibit live poultry trade in fresh meat markets.

The c.1364C>A (p.A455E) Mutation in the CFTR Pseudogene Results in an Incorrectly Assigned Carrier Status by a Commonly Used Screening Platform.

Cystic fibrosis (CF) is one of the most common recessive conditions among whites, with an estimated carrier frequency of 1 in 25 in the United States. Population-based CF carrier screening was implemented in the United States in 2001. The number of mutations screened by each laboratory may vary; however, the 23 most common CF mutations recommended for screening by the American College of Medical Genetics and American College of Obstetricians and Gynecologists are included in all platforms. The c.1364C>A (p.A455E) mutation located in exon 10 of the CFTR gene is one of the 23 mutations. Because CFTR exon 10 and its flanking intronic regions are duplicated and transposed onto several other chromosomes of the human genome during evolution and function as unprocessed pseudogenes, variations in the CFTR pseudogenes may confound CF screening results for mutations located in exon 10 of the CFTR gene. We report an incorrectly identified carrier status for the c.1364C>A (p.A455E) mutation in a healthy individual using the Hologic InPlex CF assay. Further analysis revealed that the mutation resides in one of the CFTR pseudogenes. Because most commercial kits and laboratory-developed tests for CF carrier screening involve a short amplicon encompassing this mutation, this finding suggests that individuals with the c.1364C>A (p.A455E) mutation may require further investigation to avoid a false assignment of CF carrier status.

Biochemical Characterization of PAI-2 and Its Mutants.

To study and compare the biochemical characterization of PAI-2 and its mutants, PCR and site-directed mutagenesis methods were used to generate two PAI-2 mutants, PAI-2CD and PAI-2Q, respectively. The two mutant cDNAs were inserted into prokaryotic expression vector and expressed in a special strain of E.coli, JF1125. The expected PAI-2 mutant proteins were identified by SDS-PAGE, both covering about 14% of total bacteria proteins. The antigenicity and activity inhibiting uPA of the two mutant proteins were verified to be identical with that of wild PAI-2 by using Western blot and milk-agarose plate assay and reverse milk-agarose zymograph. The harvested recombinant bacterial cells growing in 5 L fermentor were homogenized and purified by the protocols including ammonium sulfate precipitation, Sephadex G-75 gel filtration, Q-Sepharose ion-exchange chromatography and hydrophobic interaction chromatography. The purity of the purified PAI-2CD and PAI-2Q was up to 98% and 90%, the protein yield was 18.4% and 22.1%, the specific activity was 28 640 u/mg and 14 836 u/mg, respectively. The results indicate that the biochemical characterization of PAI-2 mutants was very similar to those of the wide-type PAI-2 except that PAI-2Q can not be catalyzed by tTG.

The Protection Function of the PAI-2 from TNF-alpha is Dependent upon Its Protein Binding Domain.

The cDNA of PAI-2 mutants, PAI-2CD and PAI-2Q were inserted into eukaryotic expression vector, pcDNA3, producing pZLE-PAI2CD and pZLE-PAI2Q respectively. The PAI-2 mutant expression plasmids were transfected into HeLa cells and the transfected cells expressing mutant proteins were selected by G418, Northern blot and ELISA assay. The ELISA assay showed that the expression level of PAI-2 mutant protein was equal to that of wild type PAI-2 in transfected cells. The biochemical characteristics of PAI-2 mutant proteins were very similar to that of the wild type PAI-2. The results of crystal violet and MTT assays showed that the PAI-2 mutants did not protect cells from the cytotoxic effects of TNF-alpha. The results of this study indicate that the protection of PAI-2 from apoptosis induced by TNF-alpha is dependent upon the protein-protein interaction mediated by the CD-interhelical domain of PAI-2. The tTG-mediated interaction between PAI-2 and cytosol protein might play a leading role in protecting from apoptosis induced by TNF-alpha.

Asthmatics with exacerbation during acute respiratory illness exhibit unique transcriptional signatures within the nasal mucosa.

BACKGROUND: Acute respiratory illness is the leading cause of asthma exacerbations yet the mechanisms underlying this association remain unclear. To address the deficiencies in our understanding of the molecular events characterizing acute respiratory illness-induced asthma exacerbations, we undertook a transcriptional profiling study of the nasal mucosa over the course of acute respiratory illness amongst individuals with a history of asthma, allergic rhinitis and no underlying respiratory disease. METHODS: Transcriptional profiling experiments were performed using the Agilent Whole Human Genome 4X44K array platform. Time point-based microarray and principal component analyses were conducted to identify and distinguish acute respiratory illness-associated transcriptional profiles over the course of our study. Gene enrichment analysis was conducted to identify biological processes over-represented within each acute respiratory illness-associated profile, and gene expression was subsequently confirmed by quantitative polymerase chain reaction. RESULTS: We found that acute respiratory illness is characterized by dynamic, time-specific transcriptional profiles whose magnitudes of expression are influenced by underlying respiratory disease and the mucosal repair signature evoked during acute respiratory illness. Most strikingly, we report that people with asthma who experience acute respiratory illness-induced exacerbations are characterized by a reduced but prolonged inflammatory immune response, inadequate activation of mucosal repair, and the expression of a newly described exacerbation-specific transcriptional signature. CONCLUSION: Findings from our study represent a significant contribution towards clarifying the complex molecular interactions that typify acute respiratory illness-induced asthma exacerbations.

Human rhinovirus infection causes different DNA methylation changes in nasal epithelial cells from healthy and asthmatic subjects.

BACKGROUND: Mechanisms underlying the development of virus-induced asthma exacerbations remain unclear. To investigate if epigenetic mechanisms could be involved in virus-induced asthma exacerbations, we undertook DNA methylation profiling in asthmatic and healthy nasal epithelial cells (NECs) during Human Rhinovirus (HRV) infection in vitro. METHODS: Global and loci-specific methylation profiles were determined via Alu element and Infinium Human Methylation 450 K microarray, respectively. Principal components analysis identified the genomic loci influenced the most by disease-status and infection. Real-time PCR and pyrosequencing were used to confirm gene expression and DNA methylation, respectively. RESULTS: HRV infection significantly increased global DNA methylation in cells from asthmatic subjects only (43.6% to 44.1%, p = 0.04). Microarray analysis revealed 389 differentially methylated loci either based on disease status, or caused by virus infection. There were disease-associated DNA methylation patterns that were not affected by HRV infection as well as HRV-induced DNA methylation changes that were unique to each group. A common methylation locus stood out in response to HRV infection in both groups, where the small nucleolar RNA, H/ACA box 12 (SNORA12) is located. Further analysis indicated that a relationship existed between SNORA12 DNA methylation and gene expression in response to HRV infection. CONCLUSIONS: We describe for the first time that Human rhinovirus infection causes DNA methylation changes in airway epithelial cells that differ between asthmatic and healthy subjects. These epigenetic differences may possibly explain the mechanism by which respiratory viruses cause asthma exacerbations.

Fragment-Based Discovery of 7-Azabenzimidazoles as Potent, Highly Selective, and Orally Active CDK4/6 Inhibitors.

Herein, we describe the discovery of potent and highly selective inhibitors of both CDK4 and CDK6 via structure-guided optimization of a fragment-based screening hit. CDK6 X-ray crystallography and pharmacokinetic data steered efforts in identifying compound 6, which showed >1000-fold selectivity for CDK4 over CDKs 1 and 2 in an enzymatic assay. Furthermore, 6 demonstrated in vivo inhibition of pRb-phosphorylation and oral efficacy in a Jeko-1 mouse xenograft model.

4-(Pyrazol-4-yl)-pyrimidines as selective inhibitors of cyclin-dependent kinase 4/6.

Identification and structure-guided optimization of a series of 4-(pyrazol-4-yl)-pyrimidines as selective CDK4/6 inhibitors is reported herein. Several potency and selectivity determinants were established based on the X-ray crystallographic analysis of representative compounds bound to monomeric CDK6. Significant selectivity for CDK4/6 over CDK1 and CDK2 was demonstrated with several compounds in both enzymatic and cellular assays.

CaNAT1, a heterologous dominant selectable marker for transformation of Candida albicans and other pathogenic Candida species.

A dominant selectable marker for Candida albicans and other Candida species, which confers resistance to nourseothricin, was characterized. In a heterologous promoter system and a recyclable cassette, the marker efficiently permitted deletion and complementation of C. albicans genes. Neither growth nor filamentous development was affected in strains expressing this marker.