Immune checkpoint inhibitors such as anti-CTLA-4 antibody are widely accepted therapeutic options for many cancers, but there is still a considerable gap in achieving their full potential. We explored the potential of activating the innate and adaptive immune pathways together to improve tumor reduction and survival outcomes. We treated a mouse model of melanoma with intratumoral injections of Toll-like receptor 1/2 (TLR1/2) ligand Pam3CSK4 plus i.p. injections of anti-CTLA-4 antibody. This combination treatment enhanced antitumor immune responses both qualitatively and quantitatively over anti-CTLA-4 alone, and its efficacy depended on CD4 T cells, CD8 T cells, Fcγ receptor IV, and macrophages. Interestingly, our results suggest a unique mechanism by which TLR1/2 ligand increased Fcγ receptor IV expression on macrophages, leading to antibody-dependent macrophage-mediated depletion of regulatory T cells in the tumor microenvironment and increasing efficacy of anti-CTLA-4 antibody in the combination treatment. This mechanism could be harnessed to modulate the clinical outcome of anti-CTLA-4 antibodies and possibly other antibody-based immunotherapies.

Relevant odors signaling food, mates, or predators can be masked by unpredictable mixtures of less relevant background odors. Here, we developed a mouse behavioral paradigm to test the role played by the novelty of the background odors. During the task, mice identified target odors in previously learned background odors and were challenged by catch trials with novel background odors, a task similar to visual CAPTCHA. Female wild-type (WT) mice could accurately identify known targets in novel background odors. WT mice performance was higher than linear classifiers and the nearest neighbor classifier trained using olfactory bulb glomerular activation patterns. Performance was more consistent with an odor deconvolution method. We also used our task to investigate the performance of female Cntnap2-/- mice, which show some autism-like behaviors. Cntnap2-/- mice had glomerular activation patterns similar to WT mice and matched WT mice target detection for known background odors. However, Cntnap2-/- mice performance fell almost to chance levels in the presence of novel backgrounds. Our findings suggest that mice use a robust algorithm for detecting odors in novel environments and this computation is impaired in Cntnap2-/- mice.

L-myo-inositol phosphate synthase (MIPS; EC 5.5.1.4) is involved in abiotic stress tolerance, however its disruption and overexpression has also been associated with enhanced tolerance to pathogens. The molecular mechanism underlying the role of MIPS in growth, immunity and abiotic stress tolerance remains uncharacterized. We explore the molecular mechanism of MIPS action during growth and heat stress conditions. We raised and characterized the TaMIPS over-expressing rice transgenics which showed a reduced reproductive potential. Transcriptome analysis of overexpression transgenics revealed the activation of ET/JA dependent immune response. Pull-down analysis revealed the interaction of TaMIPS-B with ethylene related proteins. Our results suggest an essential requirement of MIPS for mediating the ethylene response and regulate the growth. A model is proposed outlining how fine tuning of MIPS regulate growth and stress tolerance of the plant.

Renal trauma is present in 0.5-5% of patients admitted for trauma. Advancements in radiologic imaging and minimal-invasive techniques have led to decreased need for surgical intervention. We used a large trauma cohort to characterise renal trauma patients, their management and outcomes.

Escalating evidence shows that ginseng possesses an antiaging potential with cognitive enhancing activity. As mountain cultivated ginseng (MCG) is cultivated without agricultural chemicals, MCG has emerged as a popular herb medicine. However, little is known about the MCG-mediated pharmacological mechanism on brain aging.

A selective, sensitive and rugged liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been developed for the simultaneous determination of doxepin (Dox) and its pharmacologically active metabolite, nordoxepin (NDox) in human plasma. The analytes and their internal standards (IS) were extracted from 500 µL of human plasma by liquid-liquid extraction using methyl tert-butyl ether. Chromatographic separation was achieved on Hypurity C8 column (100 mm × 4.6 mm, 5 µm) using a mixture of acetonitrile-methanol (95:5, v/v) and 2.0 mM ammonium formate in 93:7 (v/v) ratio. Detection was accomplished by tandem mass spectrometry in the positive ionization and multiple reaction monitoring acquisition mode. The protonated precursor to product ion transitions studied for Dox, NDox, and their corresponding ISs, propranolol and desipramine, were m/z 280.1→107.0, 266.0 →107.0, 260.1→116.1 and 267.1→72.1, respectively. A linear dynamic range of 15.0-3900 pg/mL for Dox and 5.00-1300 pg/mL for NDox was established with mean correlation coefficient (r 2) of 0.9991 and 0.9993, respectively. The extraction recovery ranged from 86.6%-90.4% and 88.0%-99.1% for Dox and NDox, respectively. The intra-batch and inter-batch precision (% CV) across quality control levels was ≤ 8.3% for both the analytes. Stability evaluated under different storage conditions showed no evidence of degradation and the % change in stability samples compared to nominal concentration ranged from 4.7% to 12.3%. The method was successfully applied to a bioequivalence study of 6 mg doxepin hydrochloride orally disintegrating tablet in 41 healthy Indian subjects under fasting and fed conditions.

We examined the role of spinally located β-adrenergic receptors in the regulation of the blood glucose level. The intrathecal (i.t.) injections with dobutamine (β1-adrenergic receptor agonist) or terbutaline (β2-adrenergic receptor agonist) caused an elevation of the blood glucose level, whereas metoprolol (β1-adrenergic receptor antagonist) or butoxamine (β2-adrenergic receptor antagonist) did not. In addition, i.t. pretreatment with pertussis toxin (PTX) attenuated the hyperglycemic effect induced by dobutamine or terbutaline. Moreover, plasma insulin level was increased by dobutamine but not by terbutaline, and PTX reduced dobutamine-induced up-regulation of the plasma insulin level. Terbutaline significantly increased plasma corticosterone level, and PTX further enhanced terbutaline-induced corticosterone level. Furthermore, intraperitoneal (i.p.) pretreatment with hexamethonium- (a preganglionic blocker) attenuated dobutamine- and terbutaline-induced hyperglycemic effects. Our results suggest that activation of spinal β1- and β2-adrenergic receptors produces hyperglycemic effects in a different manner. Spinally located PTX-sensitive G-proteins appear to be involved in hyperglycemic effect induced by terbutaline. Furthermore, dobutamine- or terbutaline-induced hyperglycemia appears to be mediated through the spinal nerves.

A functional genomics approach was used to examine the immune response for transcriptional profiling of PBMC M. bovis infected cattle and healthy control cattle to stimulation with bovine tuberculin (purified protein derivative PPD-b). Total cellular RNA was extracted from non-challenged control and M. bovis challenged MDM for all animals at intervals of 6 h post-challenge, in response to in-vitro challenge with M. bovis (multiplicity of infection 2:1) and prepared for global gene expression analysis using the Agilent Bovine (V2) Gene Expression Microarray, 8 × 60 K. The pattern of expression of these genes in PPD bovine stimulated PBMC provides the first description of an M. bovis specific signature of infection that may provide insights into the molecular basis of the host response to infection. Analysis of these mapped reads showed 2450 genes (1291 up regulated and 1158 down regulated) 462 putative natural antisense transcripts (354 up-regulated and 108 down regulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). The results provided enrichment for genes involved top ten up regulated and down regulated panel of genes, including transcription factors proliferation of T and B lymphocytes. The highest differentially-expressed genes were associated to immune and inflammatory responses, immunity, differentiation, cell growth, apoptosis, cellular trafficking and regulation of lipolysis and thermogenesis. Microarray results were confirmed in infected cattle by RT qPCR to identify potential biomarkers TLR2, CD80, NFKB1, IL8, CXCL6 and ADORA3 of bovine tuberculosis.

Chromohalobacter salexigens, a Gammaproteobacterium belonging to the family Halomonadaceae, shows a broad salinity range for growth. In order to reveal the factors influencing architecture of protein coding genes in C. salexigens, pattern of synonymous codon usage bias has been investigated. Overall codon usage analysis of the microorganism revealed that C and G ending codons are predominantly used in all the genes which are indicative of mutational bias. Multivariate statistical analysis showed that the genes are separated along the first major explanatory axis according to their expression levels and their genomic GC content at the synonymous third positions of the codons. Both NC plot and correspondence analysis on Relative Synonymous Codon Usage (RSCU) indicates that the variation in codon usage among the genes may be due to mutational bias at the DNA level and natural selection acting at the level of mRNA translation. Gene length and the hydrophobicity of the encoded protein also influence the codon usage variation of genes to some extent. A comparison of the relative synonymous codon usage between 10% each of highly and lowly expressed genes determines 23 optimal codons, which are statistically over represented in the former group of genes and may provide useful information for salt-stressed gene prediction and gene-transformation. Furthermore, genes for regulatory functions; mobile and extrachromosomal element functions; and cell envelope are observed to be highly expressed. The study could provide insight into the gene expression response of halophilic bacteria and facilitate establishment of effective strategies to develop salt-tolerant crops of agronomic value.

Previously we demonstrated that p53 mediates dopaminergic neurotoxicity via inducing mitochondrial burdens and proapoptotsis. However, little is known about the role of p53 in the excitotoxicity induced by psychostimulant, such as cocaine. Cocaine-induced kindling (convulsive) behaviors significantly increased p53 expression in the brain. Cocaine-induced p53 expression was more pronounced in hippocampus than in striatum or prefrontal cortex. Genetic depletion of p53 significantly attenuated cocaine-induced convulsive behaviors, followed by c-Fos immunoreactivity, and oxidative burdens in the hippocampus of mice. The antioxidant potentials mediated by genetic depletion of p53 were more pronounced in the mitochondrial-than cytosolic-fraction. Depletion of p53 significantly attenuated the changes in mitochondrial transmembrane potential, intramitochondrial Ca2+ level, and mitochondrial oxidative burdens induced by cocaine. Consistently, depletion of p53 significantly inhibited mitochondrial p53 translocation, and cleaved-PKCδ induced by cocaine. In addition, depletion of p53 protected from cytosolic cytochrome c release, and pro-apoptotic changes induced by cocaine. Importantly, the protective/anticonvulsant potentials by genetic depletion of p53 were comparable to those by pifithrin-μ (PFT), a p53 inhibitor. Our results suggest that depletion of p53 offers anticonvulsive and neuroprotective potentials mainly via attenuating mitochondrial oxidative burdens, mitochondrial dysfunction, and pro-apoptotic signalings against cocaine-induced convulsive neurotoxicity.

Compelling evidence indicates that oxidative stress contributes to cocaine neurotoxicity. The present study was performed to elucidate the role of the glutathione peroxidase-1 (GPx-1) in cocaine-induced kindling (convulsive) behaviors in mice. Cocaine-induced convulsive behaviors significantly increased GPx-1, p-IkB, and p-JAK2/STAT3 expression, and oxidative burdens in the hippocampus of mice. There was no significant difference in cocaine-induced p-IkB expression between non-transgenic (non-TG) and GPx-1 overexpressing transgenic (GPx-1 TG) mice, but significant differences were observed in cocaine-induced p-JAK2/STAT3 expression and oxidative stress between non-TG and GPx-1 TG mice. Cocaine-induced glial fibrillary acidic protein (GFAP)-labeled astrocytic level was significantly higher in the hippocampus of GPx-1 TG mice. Triple-labeling immunocytochemistry indicated that GPx-1-, p-STAT3-, and GFAP-immunoreactivities were co-localized in the same cells. AG490, a JAK2/STAT3 inhibitor, but not pyrrolidone dithiocarbamate, an NFκB inhibitor, significantly counteracted GPx-1-mediated protective potentials (i.e., anticonvulsant-, antioxidant-, antiapoptotic-effects). Genetic overexpression of GPx-1 significantly attenuated proliferation of Iba-1-labeled microglia induced by cocaine in mice. However, AG490 or astrocytic inhibition (by GFAP antisense oligonucleotide and α-aminoadipate) significantly increased Iba-1-labeled microglial activity and M1 phenotype microglial mRNA levels, reflecting that proinflammatory potentials were mediated by AG490 or astrocytic inhibition. This microglial activation was less pronounced in GPx-1 TG than in non-TG mice. Furthermore, either AG490 or astrocytic inhibition significantly counteracted GPx-1-mediated protective potentials. Therefore, our results suggest that astrocytic modulation between GPx-1 and JAK2/STAT3 might be one of the underlying mechanisms for protecting against convulsive neurotoxicity induced by cocaine.

A highly selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been described for the determination of asenapine (ASE) in presence of its inactive metabolites N-desmethyl asenapine (DMA) and asenapine-N-glucuronide (ASG). ASE, and ASE 13C-d3, used as internal standard (IS), were extracted from 300 µL human plasma by a simple and precise liquid-liquid extraction procedure using methyl tert-butyl ether. Baseline separation of ASE from its inactive metabolites was achieved on Chromolith Performance RP8e (100 mm × 4.6 mm) column using acetonitrile-5.0 mM ammonium acetate-10% formic acid (90:10:0.1, v/v/v) within 4.5 min. Quantitation of ASE was done on a triple quadrupole mass spectrometer equipped with electrospray ionization in the positive mode. The protonated precursor to product ion transitions monitored for ASE and ASE 13C-d3 were m/z 286.1 → 166.0 and m/z 290.0 → 166.1, respectively. The limit of detection (LOD) and limit of quantitation (LOQ) of the method were 0.0025 ng/mL and 0.050 ng/mL respectively in a linear concentration range of 0.050-20.0 ng/mL for ASE. The intra-batch and inter-batch precision (% CV) and mean relative recovery across quality control levels were ≤ 5.8% and 87.3%, respectively. Matrix effect, evaluated as IS-normalized matrix factor, ranged from 1.03 to 1.05. The stability of ASE under different storage conditions was ascertained in presence of the metabolites. The developed method is much simpler, matrix free, rapid and economical compared to the existing methods. The method was successfully used for a bioequivalence study of asenapine in healthy Indian subjects for the first time.

We demonstrated that the gene of glutathione peroxidase-1 (GPx-1), a major antioxidant enzyme, is a potential protectant against the neurotoxicity and conditioned place preference induced by cocaine. Because the sigma (σ)-1 receptor is implicated in cocaine-induced drug dependence, we investigated whether the GPx-1 gene modulates the σ-1 receptor in the behavioral sensitization induced by cocaine. Cocaine-induced behavioral sensitization was more pronounced in GPx-1 knockout (KO) than wild-type (WT) mice and was less pronounced in GPx-1 overexpressing transgenic (GPx-1 TG) than non-TG mice. Cocaine treatment significantly enhanced the oxidative burden and reduced the GSH levels in the striatum of WT, GPx-1 KO, and non-TG mice but not in that of GPx-1 TG mice. In addition, cocaine significantly increased the nuclear translocation, its DNA binding activity of nuclear factor erythroid-2-related factor 2 (Nrf2) as well as the mRNA expression of γ-glutamylcysteine (GCL). The genetic depletion of GPx-1 inhibited the Nrf2-related glutathione system, whereas the genetic overexpression of GPx-1 activated this system against behavioral sensitization. BD1047, a σ-1 receptor antagonist, and U0126, an ERK inhibitor significantly induced the Nrf2-related antioxidant potential against behavioral sensitization. Unlike BD1047, U0126 did not affect the cocaine-induced σ-1 receptor immunoreactivity, suggesting that the σ-1 receptor is an upstream molecule for ERK signaling. Importantly, BD1047 and U0126 failed to affect the σ-1 receptor immunoreactivity and ERK phosphorylation induced by cocaine in GPx-1 TG mice. Our results suggest that GPx-1 is a critical mediator for the attenuation of cocaine-induced behavioral sensitization via modulating σ-1 receptor-mediated ERK activation by the induction of the Nrf2-related system.

Myo-inositol is a ubiquitous metabolite of plants. It is synthesized by a highly conserved enzyme L-myo-inositol phosphate synthase (MIPS; EC 5.5.1.4). Myo-inositol is well characterized during abiotic stress tolerance but its role during growth and development is unclear. In this study, we demonstrate that the apical hook maintenance and hypocotyl growth depend on myo-inositol. We discovered the myo-inositol role during hook formation and its maintenance via ethylene pathway in Arabidopsis by supplementation assays and qPCR. Our results suggest an essential requirement of myo-inositol for mediating the ethylene response and its interaction with brassinosteroid to regulate the skotomorphogenesis. A model is proposed outlining how MIPS regulates apical hook formation and hypocotyl growth.

Mulberry is an important crop plant for the sericulture industry. Here, we report high-quality genome sequence of a cultivated Indian mulberry (Morus indica cv K2) obtained by combining data from four different technologies, including Illumina, single-molecule real-time sequencing, chromosome conformation capture and optical mapping, with a gene completeness of 96.5%. Based on the genome sequence, we identified 49.2% of repetitive DNA and 27,435 high-confidence protein-coding genes with >90% of them supported by transcript evidence. A comparative analysis with other plant genomes identified 4.8% of species-specific genes in the M. indica genome. Transcriptome profiling revealed tissue-specific and differential expression across multiple accessions of ~4.7% and 2-5% of protein-coding genes, respectively, implicated in diverse biological processes. Whole genome resequencing of 21 accessions/species revealed ~2.5 million single nucleotide polymorphisms and ~ 0.2 million insertions/deletions. These data and results provide a comprehensive resource to accelerate the genomics research in mulberry for its improvement.

Immune receptors expressed on TAMs are intriguing targets for tumor immunotherapy. In this study, we found inhibitory receptor LILRB4 on a variety of intratumoral immune cell types in murine tumor models and human cancers, most prominently on TAMs. LILRB4, known as gp49B in mice, is a LILRB family receptor. Human and murine LILRB4 have two extracellular domains but differ in the number of intracellular ITIMs (three versus two). We observed a high correlation in LILRB4 expression with other immune inhibitory receptors. After tumor challenge, LILRB4-/- mice and mice treated with anti-LILRB4 antibody showed reduced tumor burden and increased survival. LILRB4-/- genotype or LILRB4 blockade increased tumor immune infiltrates and the effector (Teff) to regulatory (Treg) T cell ratio and modulated phenotypes of TAMs toward less suppressive, CD4+ T cells to Th1 effector, and CD8+ T cells to less exhausted. These findings reveal that LILRB4 strongly suppresses tumor immunity in TME and that alleviating that suppression provides antitumor efficacy.

Converging evidence has demonstrated that oxidative burdens are associated with drug dependence induced by psychostimulants. Here, we investigated whether oxidative stress directly mediates conditioned place preference and behavioral sensitization (drug dependence) induced by cocaine and whether glutathione peroxidase-1 (GPx-1), a major GPx, modulates cocaine-induced psychotoxic changes in mice. Cocaine-induced drug dependence was followed by increases in c-Fos-immunoreactivity (c-Fos-IR) in the nucleus accumbens. Simultaneously, cocaine significantly increased oxidative parameters and nuclear factor κB (NFκB) activity (i.e. nuclear translocation and DNA binding activity) in the striatum (including nucleus accumbens). Genetic depletion of GPx-1 made mice susceptible to drug dependence induced by cocaine in mice, while genetic overexpression of GPx-1 protected the mice from drug dependence. Pyrrolidine dithiocarbamate (PDTC), a NFκB inhibitor, significantly attenuated the sensitivity induced by the genetic depletion of GPx-1 in mice. However, PDTC did not exhibit any additive effects against the protection afforded by the genetic overexpression of GPx-1. Our results suggest that drug dependence induced by cocaine requires oxidative stress and NFκB activation, and that the GPx-1 gene is a potential protective factor against cocaine-induced drug dependence through positive modulation of NFκB.

It has been recognized that serotonin 2A receptor (5-HT2A) agonist 2,5-dimethoxy-4-iodo-amphetamine (DOI) impairs serotonergic homeostasis. However, the mechanism of DOI-induced serotonergic behaviors remains to be explored. Moreover, little is known about therapeutic interventions against serotonin syndrome, although evidence suggests that ginseng might possess modulating effects on the serotonin system. As ginsenoside Re (GRe) is well-known as a novel antioxidant in the nervous system, we investigated whether GRe modulates 5-HT2A receptor agonist DOI-induced serotonin impairments. We proposed that protein kinase Cδ (PKCδ) mediates serotonergic impairments. Treatment with GRe or 5-HT2A receptor antagonist MDL11939 significantly attenuated DOI-induced serotonergic behaviors (i.e., overall serotonergic syndrome behaviors, head twitch response, hyperthermia) by inhibiting mitochondrial translocation of PKCδ, reducing mitochondrial glutathione peroxidase activity, mitochondrial dysfunction, and mitochondrial oxidative stress in wild-type mice. These attenuations were in line with those observed upon PKCδ inhibition (i.e., pharmacologic inhibitor rottlerin or PKCδ knockout mice). Furthermore, GRe was not further implicated in attenuation mediated by PKCδ knockout in mice. Our results suggest that PKCδ is a therapeutic target for GRe against serotonergic behaviors induced by DOI.

Ginseng is known to possess anti-aging potential. Klotho mutant mice exhibit phenotypes that resemble the phenotype of the human aging process. Similar to Klotho deficient mice, patients with chronic kidney disease (CKD) suffer vascular damage and cognitive impairment, which might upregulate the angiotensin II AT1 receptor. Since AT1 receptor expression was more pronounced than endothelin ET-1 expression in the hippocampus of aged Klotho deficient (±) mice, we focused on the AT1 receptor in this study. Ginsenoside Re (GRe), but not ginsenoside Rb1 (GRb1), significantly attenuated the increase in AT1 receptor expression in aged Klotho deficient mice. Both GRe and the AT1 receptor antagonist losartan failed to attenuate the decrease in phosphorylation of JAK2/STAT3 in aged Klotho deficient (±) mice but significantly activated nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated signaling. Both GRe and losartan attenuated the increased NADPH oxidase (NOX) activity and reactive oxygen species (ROS) in aged Klotho deficient mice. Furthermore, of all the antioxidant enzymes, GRe significantly increased glutathione peroxidase (GPx) activity. GRe significantly attenuated the reduced phosphorylation of ERK and CREB in GPx-1 knockout mice; however, genetic overexpression of GPx-1 did not significantly affect them in aged mice. Klotho-, Nrf2-, and GPx-1-immunoreactivities were co-localized in the same cells of the hippocampus in aged Klotho wild-type mice. Both the GPx inhibitor mercaptosuccinate and Nrf2 inhibitor brusatol counteracted the effects of GRe on all neurobehavioral impairments in aged Klotho deficient (±) mice. Our results suggest that GRe attenuates all alterations, such as AT1 receptor expression, NOX-, ROS-, and GPx-levels, and cognitive dysfunction in aged Klotho deficient (±) mice via upregulation of Nrf2/GPx-1/ERK/CREB signaling.

Faecal microbiota transplantation (FMT) has previously been explored as a treatment for ulcerative colitis (UC) however, biomarkers that predict and / or are associated with clinical response are poorly defined. The aim of this systematic review was to identify donor and recipient clinical, microbial and metabolomic predictive biomarkers of response to FMT in UC.