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Amitraz is an acaricide that is widely used in apiculture. Several studies have reported that in honeybees (Apis mellifera Linnaeus; Hymenoptera: Apidae), amitraz affects learning, memory, behavior, immunity, and various other physiological processes. Despite this, few studies have explored the molecular mechanisms underlying the action of amitraz on honeybees. Here, we investigated the transcriptome of honeybees after exposure to 9.4 mg/L amitraz for 10 d, a subchronic dose. Overall, 279 differentially expressed genes (DEGs) were identified (237 upregulated, 42 downregulated). Several, including Pla2, LOC725381, LOC413324, LOC724386, LOC100577456, LOC551785, and P4504c3, were validated by quantitative PCR. According to gene ontology, DEGs were mainly involved in metabolism, biosynthesis, and translation. Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that amitraz treatment affected the relaxin signaling pathway, platelet activation, and protein digestion and absorption.

The increased PD-L1 expression induces poorer prognosis in melanoma. The small molecule inhibitors of PD-1/PD-L1 pathways have been an encouraging drug development strategy because of good affinity and oral bioavailability without immunogenicity and immunotoxicities of PD-1/PD-L1 antibodies. In this study, we studied the effects of PCC0208025 (BMS202), a small molecule inhibitor of PD-L1, on PD-1/PD-L1 binding and the cytokines secretion in human CD3+ cells in vitro. We also investigated the antitumor and immunomodulatory activity of PCC0208025 and the pharmacokinetics properties in B16-F10 melanoma-bearing mice. The results showed that PCC0208025 inhibited the PD-1/PD-L1 proteins binding, and rescued PD-L1-mediated inhibition of IFN-γ production in human CD3+ T cells in vitro. Furthermore, in B16-F10 melanoma-bearing mice, PCC0208025 presented the antitumor effects, enhanced IFN-γ levels in plasma, increased the frequency of CD3+CD8+ T and CD8+IFN-γ+ T and the ratios of CD8+/Treg, and deceased the CD4+CD25+CD127low/- (Treg) number in tumor. Pharmacokinetics study found that PCC0208025 was absorbed and distributed into the tumors with much higher concentrations than those of the blockade against PD-1/PD-L1 binding. Our work suggests that PCC0208025 exhibited anti-tumor effects through inhibiting Treg expansion and increasing cytotoxic activity of tumor-infiltrating CD8+ T cells by the blockade of PD-1/PD-L1 binding, which may provide the pharmacological basis to develop small molecule inhibitors of PD-1/PD-L1 binding for PCC0208025 as a lead compound.

In recent years it has become increasingly clear that astrocytes play a much more active role in neural processes than the traditional view of them as supporting cells suggests. Although not electrically excitable, astrocytes exhibit diverse Ca2+ dynamics across spatial and temporal scales, more or less dependent on the animal's behavioral state. Ca2+ dynamics range from global elevations lasting multiple seconds encompassing the soma up to the finest processes, to short elevations restricted to so-called microdomains within fine processes. Investigations of astrocyte Ca2+ dynamics have particularly benefitted from the development of Genetically-Encoded Calcium Indicators (GECIs). GECI expression can be achieved non-invasively in a cell type-specific manner and it can be genetically targeted to subcellular domains. The GCaMP family, a group of GECIs derived from the green fluorescent protein, has experienced some of the fastest advancements during the past decade. As a consequence we are now facing the challenge of needing to compare published data obtained with different versions of GECIs. With the intention to provide some guidance, here we compared Ca2+ dynamics across scales in awake transgenic mice expressing either the well-established GCaMP3, or the increasingly popular GCaMP6f, specifically in astrocytes. We found that locomotion-induced global Ca2+ elevations in cortical astrocytes displayed only minor kinetic differences and their apparent dynamic ranges for Ca2+ sensing were not different. In contrast, Ca2+ waves in processes and microdomain Ca2+ transients were much more readily detectable with GCaMP6f. Our findings suggest that behavioral state-dependent global astrocyte Ca2+ responses can be studied with either GCaMP3 or GCaMP6f whereas the latter is more appropriate for studies of spatially restricted weak and fast Ca2+ dynamics.

Demand for the commercial use of genetically modified (GM) crops has been increasing in light of the projected growth of world population to nine billion by 2050. A prerequisite of paramount importance for regulatory submissions is the rigorous safety assessment of GM crops. One of the components of safety assessment is molecular characterization at DNA level which helps to determine the copy number, integrity and stability of a transgene; characterize the integration site within a host genome; and confirm the absence of vector DNA. Historically, molecular characterization has been carried out using Southern blot analysis coupled with Sanger sequencing. While this is a robust approach to characterize the transgenic crops, it is both time- and resource-consuming. The emergence of next-generation sequencing (NGS) technologies has provided highly sensitive and cost- and labor-effective alternative for molecular characterization compared to traditional Southern blot analysis. Herein, we have demonstrated the successful application of both whole genome sequencing and target capture sequencing approaches for the characterization of single and stacked transgenic events and compared the results and inferences with traditional method with respect to key criteria required for regulatory submissions.

PFKFB3 (6-phosphofructo-2-kinase) synthesizes fructose 2,6-bisphosphate (F2,6P2), which is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1), the rate-limiting enzyme of glycolysis. Overexpression of the PFKFB3 enzyme leads to high glycolytic metabolism, which is required for cancer cells to survive in the harsh tumor microenvironment. The objective of this study was to investigate the antitumor activity of PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), a small molecule inhibitor of PFKFB3, against gastric cancer and to explore its potential mechanisms. The effects of PFK15 on proliferation, apoptosis and cell cycle progression in gastric cancer cells were evaluated by cytotoxicity and apoptosis assays, flow cytometry, and western blotting. In addition, the invasion inhibition effects of PFK15 were measured by transwell invasion assay and western blot analysis, and a xenograft tumor model was used to verify the therapeutic effect of PFK15 in vivo. Results showed that PFK15 inhibited the proliferation, caused cell cycle arrest in G0/G1 phase by blocking the Cyclin-CDKs/Rb/E2F signaling pathway, and induced apoptosis through mitochondria in gastric cancer cells. Tumor volume and weight were also significantly reduced upon intraperitoneal injection with PFK15 at 25 mg/kg. In addition, PFK15 inhibited the invasion of gastric cancer cells by downregulating focal adhesion kinase (FAK) expression and upregulating E-cadherin expression. Taken together, our findings indicate that PFK15 is a promising anticancer drug for treating gastric cancer.

Tris-(2,3-dibromopropyl) isocyanurate (TDBP-TAZTO), an emerging brominated flame retardant, possesses the characteristics of candidate persistent organic pollutants and has displayed toxicity to fish and rodents. TDBP-TAZTO can pass through the blood brain barrier and accumulate in brain. However, the neurotoxicity of TDBP-TAZTO has not yet studied in rodents. We hypothesize that TDBP-TAZTO could induce the neurotoxicity in rat hippocampal neurons. The male adult rats were exposed to TDBP-TAZTO of 5 and 50 mg/kg by gavage, daily for 6 months. TDBP-TAZTO resulted in cognitive impairment and depression-like behaviors, which may be related with TDBP-TAZTO-induced hypothalamic-pituitary-adrenal axis hyperactivation, upregulation of inflammatory and oxidative stress markers, overexpression of pro-apoptotic proteins, downexpression of neurogenesis-related proteins in hippocampus, and hippocampal neurons damage in DG, CA1 and CA3 areas. Our findings suggested that TDBP-TAZTO induces significant hippocampal neurotoxicity, which provokes cognitive impairment and depression-like behaviors in adult rats. Therefore, this research will contribute to evaluate the neurotoxic effects of TDBP-TAZTO in human.

A PEG-based, folate mediated, active tumor targeting drug delivery system using DOX-hyd-PEG-FA nanoparticles (NPs) were prepared. DOX-hyd-PEG-FA NPs showed a significantly faster DOX release in pH 5.0 medium than in pH 7.4 medium. Compared with DOX-hyd-PEG NPs, DOX-hyd-PEG-FA NPs increased the intracellular accumulation of DOX and showed a DOX translocation from lysosomes to nucleus. The cytotoxicity of DOX-hyd-PEG-FA NPs on KB cells was much higher than that of free DOX, DOX-ami-PEG-FA NPs and DOX-hyd-PEG NPs. The cytotoxicity of DOX-hyd-PEG-FA NPs on KB cells was attenuated in the presence of exogenous folic acid. The IC50 of DOX-hyd-PEG-FA NPs and DOX-hyd-PEG NPs on A549 cells showed no significant difference. After DOX-hyd-PEG-FA NPs were intravenously administered, the amount of DOX distributed in tumor tissue was significantly increased, while the amount of DOX distributed in heart was greatly decreased as compared with free DOX. Compared with free DOX, NPs yielded improved survival rate, prolonged life span, delayed tumor growth and reduced the cardiotoxicity in tumor bearing mice model. These results indicated that the acid sensitivity, passive and active tumor targeting abilities were likely to act synergistically to enhance the drug delivery efficiency of DOX-hyd-PEG-FA NPs. Therefore, DOX-hyd-PEG-FA NPs are a promising drug delivery system for targeted cancer therapy.

The purpose of this study is to investigate the anti-angiogenic activities of NSK-01105, a novel sorafenib derivative, in in vitro, ex vivo and in vivo models, and explore the potential mechanisms. NSK-01105 significantly inhibited vascular endothelial growth factor (VEGF)-induced migration and tube formation of human umbilical vein endothelial cells at non-cytotoxic concentrations as shown by wound-healing, transwell migration and endothelial cell tube formation assays, respectively. Cell viability and invasion of LNCaP and PC-3 cells were significantly inhibited by cytotoxicity assay and matrigel invasion assay. Furthermore, NSK-01105 also inhibited ex vivo angiogenesis in matrigel plug assay. Western blot analysis showed that NSK-01105 down-regulated VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR2) and the activation of epidermal growth factor receptor (EGFR). Tumor volumes were significantly reduced by NSK-01105 at 60 mg/kg/day in both xenograft models. Immunohistochemical staining demonstrated a close association between inhibition of tumor growth and neovascularization. Collectively, our results suggest a role of NSK-01105 in treatment for human prostate tumors, and one of the potential mechanisms may be attributed to anti-angiogenic activities.

The neuroprotective activity of pyruvate has been confirmed in previous in vivo and in vitro studies. Here, we report a novel mechanism that pyruvate prevents SH-SY5Y cells from glutamate excitotoxicity by regulating death-associated protein kinase 1 (DAPK1) protein complex. Our results showed pyruvate regulated DAPK1 protein complex to protect cells by two ways. First, pyruvate induced the dissociation of DAPK1 with NMDA receptors. The disruption of the DAPK1-NMDA receptors complex resulted in a decrease in NMDA receptors phosphorylation. Then the glutamate-stimulated Ca2+ influx was inhibited and intracellular Ca2+ overload was alleviated, which blocked the release of cytochrome c and cell death. In addition, increased Bcl-xL induced by pyruvate regulated Bax/Bak dependent death by inhibiting the release of cytochrome c from the mitochondrial inter-membrane space into the cytosol. As a result, the cytochrome c-initiated caspase cascade, including caspase-3 and caspase-9, was inhibited. Second, pyruvate promoted the association between DAPK1 and Beclin-1, which resulted in autophagy activation. The autophagy inhibitor 3-methyladenine reversed the protection afforded by pyruvate. Furthermore, the attenuation of mitochondrial damage induced by pyruvate was partly reduced by 3-methyladenine. This suggested autophagy mediated pyruvate protection by preventing mitochondrial damage. Taken together, pyruvate protects cells from glutamate excitotoxicity by regulating DAPK1 complexes, both through dissociation of DAPK1 from NMDA receptors and association of DAPK1 with Beclin-1. They go forward to protect cells by attenuating Ca2+ overload and activating autophagy. Finally, a convergence of the two ways protects mitochondria from glutamate excitotoxicity, which leads to cell survival.

OBJECTIVE: This study aimed to construct a model for using in differentiating benign and malignant nodules with the artificial neural network and to increase the objective diagnostic accuracy of US. MATERIALS AND METHODS: 618 consecutive patients (528 women, 161 men) with 689 thyroid nodules (425 malignant and 264 benign nodules) were enrolled in the present study. The presence and absence of each sonographic feature was assessed for each nodule - shape, margin, echogenicity, internal composition, presence of calcifications, peripheral halo and vascularity on color Doppler. The variables meet the following criteria: important sonographic features and statistically significant difference were selected as the input layer to build the ANN for predicting the malignancy of nodules. RESULTS: Six sonographic features including shape (Taller than wide, p

Background: Sheepgrass [Leymus chinensis (Trin.) Tzvel.] is an important perennial forage grass across the Eurasian Steppe and is known for its adaptability to various environmental conditions. However, insufficient data resources in public databases for sheepgrass limited our understanding of the mechanism of environmental adaptations, gene discovery and molecular marker development. Results: The transcriptome of sheepgrass was sequenced using Roche 454 pyrosequencing technology. We assembled 952,328 high-quality reads into 87,214 unigenes, including 32,416 contigs and 54,798 singletons. There were 15,450 contigs over 500 bp in length. BLAST searches of our database against Swiss-Prot and NCBI non-redundant protein sequences (nr) databases resulted in the annotation of 54,584 (62.6%) of the unigenes. Gene Ontology (GO) analysis assigned 89,129 GO term annotations for 17,463 unigenes. We identified 11,675 core Poaceae-specific and 12,811 putative sheepgrass-specific unigenes by BLAST searches against all plant genome and transcriptome databases. A total of 2,979 specific freezing-responsive unigenes were found from this RNAseq dataset. We identified 3,818 EST-SSRs in 3,597 unigenes, and some SSRs contained unigenes that were also candidates for freezing-response genes. Characterizations of nucleotide repeats and dominant motifs of SSRs in sheepgrass were also performed. Similarity and phylogenetic analysis indicated that sheepgrass is closely related to barley and wheat. Conclusions: This research has greatly enriched sheepgrass transcriptome resources. The identified stress-related genes will help us to decipher the genetic basis of the environmental and ecological adaptations of this species and will be used to improve wheat and barley crops through hybridization or genetic transformation. The EST-SSRs reported here will be a valuable resource for future gene-phenotype studies and for the molecular breeding of sheepgrass and other Poaceae species. [ABSTRACT FROM AUTHOR]