The locus coeruleus (LC)-norepinephrine (NE) system in the brainstem plays a critical role in a variety of behaviors is an important target of pharmacological intervention to several neurological disorders. Although GABA is the major inhibitory neurotransmitter of LC neurons, the modulation of LC neuronal firing activity by local GABAergic interneurons remains poorly understood with respect to their precise location, intrinsic membrane properties and synaptic modulation. Here, we took an optogenetic approach to address these questions. Channelrhodopsin (ChR2) in a tandem with the yellow fluorescent protein (YFP) was expressed in GABAergic neurons under the control of glutamic acid decarboxylase 2 (GAD2) promoter. Immediately dorsomedial to the LC nucleus, a group of GABAergic neurons was observed. They had small soma and were densely packed in a small area, which we named the dorsomedial LC or dmLC nucleus. These GABAergic neurons showed fast firing activity, strong inward rectification and spike frequency adaptation. Lateral inhibition among these GABAergic neurons was observed. Optostimulation of the dmLC area drastically inhibited LC neuronal firing frequency, expanded the spike intervals, and reset their pacemaking activity. Analysis of the light evoked inhibitory postsynaptic currents (IPSCs) indicated that they were monosynaptic. Such light evoked IPSCs were not seen in slices where this group of GABAergic neurons was absent. Thus, an isolated group of GABAergic neurons is demonstrated in the LC area, whose location, somatic morphology and intrinsic membrane properties are clearly distinguishable from adjacent LC neurons. They interact with each and may inhibit LC neurons as well as a part of local neuronal circuitry in the LC.

Late embryogenesis abundant proteins (LEA) are stress resistance-related proteins that play crucial roles in protecting against desiccation, cold and high salinity in a variety of animals and plants. However, the expression pattern, distribution and functions of LEA proteins in the post-diapause period of Artemia sinica, and under high salinity and low temperature stresses, remain unknown. In this study, the complete cDNA sequences of the group 1 LEA (As-g1lea) and group 3 LEA (As-g3lea) genes from A. sinica were cloned. The expression patterns and location of As-G1LEA and As-G1LEA were investigated. The protein abundances of As-G1LEA, As-G3LEA and Trehalase were analyzed during different developmental stages of the embryo and under low temperature and high salinity stresses in A. sinica. The full-length cDNA of As-g1lea was 960 bp, encoding a 182 amino acid protein, and As-g3lea was 2089 bp, encoding a 364 amino acid protein. As-g1lea and As-g3lea showed their highest expressions at 0 h of embryonic development and both showed higher relative expression in embryonic, rather than adult, development stages. The abundances of As-G1LEA, As-G3LEA and trehalose were upregulated under low temperature and downregulated under high salinity stress. These two genes did not show any tissue or organ specific expression. Our results suggested that these LEA proteins might play a pivotal role in stress tolerance in A. sinica.

Like the anti-angiogenic strategy, anti-vascular mimicry is considered as a novel targeting strategy for glioma. In the present study, we used NGR as a targeting ligand and prepared NGR-modified liposomes containing combretastatin A4 (NGR-SSL-CA4) in order to evaluate their potential targeting of glioma tumor cells and vasculogenic mimicry (VM) formed by glioma cells as well as their anti-VM activity in mice with glioma tumor cells. NGR-SSL-CA4 was prepared by a thin-film hydration method. The in vitro targeting of U87-MG (human glioma tumor cells) by NGR-modified liposomes was evaluated. The in vivo targeting activity of NGR-modified liposomes was tested in U87-MG orthotopic tumor-bearing nude mice. The anti-VM activity of NGR-SSL-CA4 was also investigated in vitro and in vivo. The targeting activity of the NGR-modified liposomes was demonstrated by in vitro flow cytometry and in vivo biodistribution. The in vitro anti-VM activity of NGR-SSL-CA4 was indicated in a series of cell migration and VM channel experiments. NGR-SSL-CA4 produced very marked anti-tumor and anti-VM activity in U87-MG orthotopic tumor-bearing mice in vivo. Overall, the NGR-SSL-CA4 has great potential in the multi-targeting therapy of glioma involving U87-MG cells, and the VM formed by U87-MG cells as well as endothelial cells producing anti-U87-MG cells, and anti-VM formed by U87-MG cells as well as anti-endothelial cell activity.

Endoplasmic reticulum (ER) stress induced apoptosis plays a pivotal role in myocardial ischemia/reperfusion (I/R)-injury. Inhibiting ER stress is a major therapeutic target/strategy in treating cardiovascular diseases. Our previous studies revealed that lycopene exhibits great pharmacological potential in protecting against the I/R-injury in vitro and vivo, but whether attenuation of ER stress (and) or ER stress-induced apoptosis contributes to the effects remains unclear. In the present study, using neonatal mouse cardiomyocytes to establish an in vitro model of hypoxia/reoxygenation (H/R) to mimic myocardium I/R in vivo, we aimed to explore the hypothesis that lycopene could alleviate the ER stress and ER stress-induced apoptosis in H/R-injury. We observed that lycopene alleviated the H/R injury as revealed by improving cell viability and reducing apoptosis, suppressed reactive oxygen species (ROS) generation and improved the phosphorylated AMPK expression, attenuated ER stress as evidenced by decreasing the expression of GRP78, ATF6 mRNA, sXbp-1 mRNA, eIF2α mRNA and eIF2α phosphorylation, alleviated ER stress-induced apoptosis as manifested by reducing CHOP/GADD153 expression, the ratio of Bax/Bcl-2, caspase-12 and caspase-3 activity in H/R-treated cardiomyocytes. Thapsigargin (TG) is a potent ER stress inducer and used to elicit ER stress of cardiomyocytes. Our results showed that lycopene was able to prevent TG-induced ER stress as reflected by attenuating the protein expression of GRP78 and CHOP/GADD153 compared to TG group, significantly improve TG-caused a loss of cell viability and decrease apoptosis in TG-treated cardiomyocytes. These results suggest that the protective effects of lycopene on H/R-injury are, at least in part, through alleviating ER stress and ER stress-induced apoptosis in neonatal mouse cardiomyocytes.

To investigate the impact of Ramipril (RAM) on the expressions of insulin-like growth factor-1 (IGF-1) and renal mesangial matrix (RMM) in rats with diabetic nephropathy (DN).

Injured axons undergo a controlled, self-destruction process, known as Wallerian degeneration. However, the underlying mechanism remains elusive. Using the Drosophila wing nerve as a model, we identify the ESCRT component Vps4 as a previously unidentified essential gene for axonal integrity. Up-regulation of Vps4 remarkably delays degeneration of injured axons. We further reveal that Vps4 is required and sufficient to promote autophagic flux in axons and mammalian cells. Moreover, using both in vitro and in vivo models, we show that the function of Vps4 in maintaining axonal autophagy and suppressing Wallerian degeneration is conserved in mammals. Last, we uncover that Vps4 protein is rapidly depleted in injured mouse axons, which may underlie the injury-induced autophagic impediment and the subsequent axonal degeneration. Together, Vps4 and ESCRT may represent a novel signal transduction mechanism in axon injury and Wallerian degeneration.

Graphene oxide (GO) has attracted considerable attention for biomedical applications such as drug delivery because of its two-dimensional structure, which provides a large surface area on both sides of the nanosheet. Here, a new method for titanium (Ti) surface modification involving a GO coating and aspirin (A) loading (A/Ti-GO) was developed, and the bioactive effects on mouse osteoblastic MC3T3-E1 cells were preliminarily studied. The X-ray photoelectron spectrometry indicated new C-O-N, C-Si-O-C, and C-N=C bond formation upon GO coating. Remarkably, the torsion test results showed stable bonding between the GO coating and Ti under a torsional shear force found in clinical settings, in that, there was no tearing or falling off of GO coating from the sample surface. More importantly, through π-π stacking interactions, the release of aspirin loaded on the surface of Ti-GO could sustain for 3 days. Furthermore, the A/Ti-GO surface displayed a significantly higher proliferation rate and differentiation of MC3T3-E1 cells into osteoblasts, which was confirmed by a water-soluble tetrazolium salt-8 (WST-8) assay and alkaline phosphatase activity test. Consequently, Ti surface modification involving GO coating and aspirin loading might be a useful contribution to improve the success rate of Ti implants in patients, especially in bone conditions.

Immunotherapy based on the immune checkpoint blockade has emerged as the most promising approach for cancer therapy. However, the proportion of colorectal cancer patients who benefit from immunotherapy is small due to the immunosuppressive tumor microenvironment. Hence, combination immunotherapy is an ideal strategy to overcome this limitation. In this study, we developed a novel combination of CSF-1R (colony-stimulating factor 1 receptor) inhibitor (PLX3397), oncolytic viruses, and anti-PD-1 antibody. Our results demonstrated that the triple treatment synergistically conferred significant tumor control and prolonged the survival of mouse models of colon cancer. Approximately 43% and 82% of mice bearing the CT26 and MC38 tumor, respectively, survived long term following the triple treatment. This combination therapy reprogrammed the immunosuppressive tumor microenvironment toward a CD8+ T cell-biased anti-tumor immunity by increasing T cell infiltration in the tumor and augmenting anti-tumor CD8+ T cell function. Our results provide a robust strategy for clinical combination therapy.

The soy protein isolates (SPI) extracted from different extruded full-fat soybean flakes (FFSF), and their conformational and functional properties were characterized. Overall, the free thiol (SH) content of SPI increased when the extrusion temperature was below 80 °C and decreased at higher temperatures. Soy glycinin (11S) showed higher stability than β-conglycinin (7S) during extrusion. Results also indicated that the increase in some hydrophobic groups was due to the movement of hydrophobic groups from the interior to the surface of the SPI molecules at extrusion temperatures from 60 to 80 °C. However, the aggregation of SPI molecules occurred at extrusion temperatures of 90 and 100 °C, with decreasing levels of hydrophobic groups. The extrusion temperature negatively affected the emulsifying activity index (EAI); on the other side, it positively affected the emulsifying stability index (ESI), compared to unextruded SPI.

Long non-coding RNAs (lncRNAs) have potential applications in clinical diagnosis and targeted cancer therapies. However, the expression profile of lncRNAs in colorectal cancer (CRC) initiation is still unclear. In this study, the expression profiles of lncRNAs and mRNAs were determined by microarray at specific tumour stages in an AOM/DSS-induced primary colon cancer model. The temporal expression of lncRNAs was analysed by K-means clustering. Additionally, weighted correlation network analysis (WGCNA) and gene ontology analysis were performed to construct co-expression networks and establish functions of the identified lncRNAs and mRNAs. Our results suggested that 4307 lncRNAs and 5798 mRNAs are deregulated during CRC initiation. These differential expression genes (DEGs) exhibited a clear correlation with the differential stage of tumour initiation. WGCNA results suggested that a series of hub lncRNAs are involved in regulating cell stemness, colon inflammation, oxidative stress response and cell death at each stage. Among them, lncRNA H19 was up-regulated in colon tumours and correlated with poor patient prognosis. Collectively, we have been the first to demonstrate the temporal expression and function of lncRNAs in CRC initiation. These results provide novel diagnosis and therapy targets for CRC.

Bone marrow mesenchymal stem cells (BM‑MSCs) are pluripotent stem cells that are regarded as ideal resources for the reconstruction of tissues and organs. The Tibetan mastiff is a breed of domesticated Chinese native dog that is well‑adjusted to the high‑altitude environments of Tibet. To the best of our knowledge, the biological characterization and multi‑lineage differentiation of Tibetan mastiff BM‑MSCs have not been reported previously. Therefore, the present study aimed to investigate the biological characteristics and therapeutic potential of Tibetan mastiff BM‑MSCs. A cell culture system was constructed and cells were cultured to 23 passages in vitro. Growth curves and colony formation studies suggested that BM‑MSCs had a high self‑renewal capacity and that their proliferation rate declined with age. Karyotype analysis demonstrated that BM‑MSCs were diploid and genetically stable. Semi‑quantitative polymerase chain reaction analysis revealed that BM‑MSCs positively expressed cluster of differentiation (CD)73, CD90, CD105, CD166 and vimentin, although they were negative for the endothelial cell marker CD31. Additionally, immunofluorescence staining revealed that the cells expressed CD29, CD44, CD90, CD105 and vimentin. Flow cytometric analysis revealed that the rates of positive expression of vimentin, CD44, CD90 and CD105 were all >97%. BM‑MSCs were able to differentiate into adipocytes, osteoblasts, cartilage cells, hepatocytes and functional insulin‑secreting cells. In conclusion, Tibetan mastiff BM‑MSCs may be purified successfully using a whole bone marrow culture method. The findings of the current study suggested important potential applications of BM‑MSCs as a source for regenerative therapies.

Anaphase Promoting Complex or Cyclosome (APC/C) is a representative E3 ubiquitin ligase, triggering the transition of metaphase to anaphase by regulating degradation and ensures the exit from mitosis. Cell division cycle 20 (CDC20) and Cell division cycle 20 related protein 1 (CDH1), as co-activators of APC/C, play significant roles in the spindle assembly checkpoint, guiding ubiquitin-mediated degradation, together with CDC23. During the embryonic development of the brine shrimp, Artemia sinica, CDC20, CDH1 and CDC23 participate in cell cycle regulation, but the specific mechanisms of their activities remain unknown. Herein, the full-length cDNAs of cdc20 and cdc23 from A. sinica were cloned. Real-time PCR analyzed the expression levels of As-cdc20 and As-cdc23. The locations of CDH1, CDC20 and CDC23 showed no tissue or organ specificity. Furthermore, western blotting showed that the levels of As-CDC20, securin, cyclin B, CDK1, CDH1, CDC14B, CDC23 and geminin proteins conformed to their complicated degradation relationships during different embryo stages. Our research revealed that As-CDC20, As-CDH1 and APC mediate the mitotic progression, downstream proteins degradation and cellular differentiation in the process of embryonic development in A. sinica.

In the past 10 years, adipose-derived stem cells (ADSCs) have been applied due to their pluripotency. Experimental tissues have been frequently obtained from mammals, including rabbits, mice and humans, but rarely from broilers, Gallus gallus domesticus. In the present study, ADSCs were obtained from 20-day-old broiler embryos. Primary ADSCs were sub-cultured to passage 37 in vitro. The surface markers of ADSCs, namely CD29, CD31, CD44, CD71 and CD73, were detected by reverse transcription polymerase chain reaction and immunofluorescence assays. The result indicated that CD29, CD44, CD71 and CD73 were expressed on the surface of cells at various passages, but not CD31. The growth curve of cells at the different passages had a typical sigmoidal shape. Furthermore, ADSCs were successfully induced to differentiate into osteoblasts, adipocytes and hepatocyte-like cells. The results denote that the ADSCs isolated from broilers have similar biological properties to those of ADSCs obtained from other animals. The present study provided a theoretical and experimental foundation for the use of poultry as a source of stem cells, and laid a foundation for adipose tissue engineering and strategies in regenerative medicine.

The nuclear factor-kappa B (NF-κB) pathways play important roles in innate immune responses. IκB is the main cytoplasmic inhibitor of NF-κB. In this study, we identified the LvCactus gene from Litopenaeus vannamei, which is the first cloned IκB homologue in subphylum Crustacea. LvCactus contains six predicted ankyrin repeats, which show similarities to those of Cactus proteins from insects. LvCactus localizes in cytoplasm and interacts with LvDorsal, an L. vannamei homologue to Drosophila melanogaster Dorsal belonging to class II NF-κB family, to prevent its nuclear translocation. Contrary to that of LvDorsal, over-expression of LvCactus down-regulates the activities of shrimp antimicrobial peptides promoters, suggesting LvCactus is an inhibitor of LvDorsal. The promoter of LvCactus was predicted to contain five putative NF-κB binding motifs, among which four were proved to be bound by LvDorsal by chromatin immunoprecipitation assays. Dual-luciferase reporter assays also showed that transcription of LvCactus was promoted by LvDorsal but inhibited by LvCactus itself, indicating a feedback regulatory pathway between LvCactus and LvDorsal. Expression of LvCactus was up-regulated after Lipopolysaccharides, poly (I:C), Vibrio parahaemolyticus, and Staphylococcus aureus injections, suggesting an activation response of LvCactus to bacterial and immune stimulant challenges. Differently, the LvCactus expression levels obviously decreased during white spot syndrome virus (WSSV) infection, indicating the feedback regulatory pathway of LvCactus/LvDorsal could be modified by WSSV.

Medium spiny neurons (MSNs) in the nucleus accumbens (NAc) undergo persistent alterations in their biological and physiological characteristics upon exposure to drugs of abuse. Previous studies demonstrated that the biochemical, morphological, and intrinsic physiological properties of MSNs are heterogeneous and provided new insights into the physiological and molecular roles of individual MSNs in addictive behaviors. However, it remains unclear whether MSNs in the NAc shell (NAcSh), an important region for mediating behavioral sensitization, are electrophysiologically heterogeneous and how such heterogeneity is relevant to neuroadaptation associated with drug addiction. Here, the membrane properties, i.e., the intrinsic excitability and spike adaptation, of MSNs in the NAcSh from saline- or morphine-treated rats were investigated in vitro by whole-cell recording. In saline-treated rats, three distinct cell types were identified by their membrane properties: type I neurons showed high levels of intrinsic excitability and rapid spike adaptation; type II neurons showed moderate levels of intrinsic excitability and relatively slow spike frequency adaptation; type III neurons showed low levels of intrinsic excitability and putative strong spike adaptation. MSNs in rats undergoing withdrawal from chronic morphine treatment (10-14 days after the last injection) also exhibited the typical firing behaviors of these three types of neurons. However, the membrane properties of the MSNs were differentially altered after withdrawal. There was an enhancement in intrinsic excitability in type II MSNs and a promotion of spike adaptation in type I MSNs. The apamin-sensitive afterhyperpolarization current (I(AHP)) and the apamin-insensitive I(AHP) of the NAcSh MSNs were attenuated after chronic morphine withdrawal. These findings suggest that individual MSNs in the NAcSh manifest unique electrophysiological properties, which might contribute to psychostimulant-induced neuroadaptation.

Myeloid differentiation factor 88 (MyD88) is a universal and essential signaling protein in Toll-like receptor/interleukin-1 receptor-induced activation of nuclear factor-kappa B. In this study, two MyD88 protein variants (LvMyD88 and LvMyD88-1) were identified in Litopenaeus vannamei. The LvMyD88 cDNA is 1,848 bp in length and contains an open reading frame (ORF) of 1,428 bp, whereas the LvMyD88-1 cDNA is 1,719 bp in length and has an ORF of 1,299 bp. Both variants encode proteins with death and Toll/interleukin-1 receptor domains and share 91% sequence identity. In healthy L. vannamei, the LvMyD88 genes were highly expressed in hemocytes but at a low level in the hepatopancreas. The LvMyD88s expression was induced in hemocytes after challenge with lipopolysaccharide, CpG-ODN2006, Vibrio parahaemolyticus, Staphyloccocus aureus, and white spot syndrome virus, but not by poly I∶C. Overexpression of LvMyD88 and LvMyD88-1 in Drosophila Schneider 2 cells led to activation of antimicrobial peptide genes and wsv069 (ie1), wsv303, and wsv371. These results suggested that LvMyD88 may play a role in antibacterial and antiviral response in L. vannamei. To our knowledge, this is the first report on MyD88 in shrimp and a variant of MyD88 gene in invertebrates.

Coxsackievirus A16 (CVA16) is responsible for nearly 50% of all the confirmed hand, foot, and mouth disease (HFMD) cases in mainland China, sometimes it could also cause severe complications, and even death. To clarify the genetic characteristics and the epidemic patterns of CVA16 in mainland China, comprehensive bioinfomatics analyses were performed by using 35 CVA16 whole genome sequences from 1998 to 2011, 593 complete CVA16 VP1 sequences from 1981 to 2011, and prototype strains of human enterovirus species A (EV-A). Analysis on complete VP1 sequences revealed that subgenotypes B1a and B1b were prevalent strains and have been co-circulating in many Asian countries since 2000, especially in mainland China for at least 13 years. While the prevalence of subgenotype B1c (totally 20 strains) was much limited, only found in Malaysia from 2005 to 2007 and in France in 2010. Genotype B2 only caused epidemic in Japan and Malaysia from 1981 to 2000. Both subgenotypes B1a and B1b were potential recombinant viruses containing sequences from other EV-A donors in the 5'-untranslated region and P2, P3 non-structural protein encoding regions.

Antagonizing vascular endothelial growth factor receptor 2 (VEGFR2) to block angiogenesis has been applied toward cancer therapy for its role in promoting cancer growth and metastasis. However, most these clinical anticancer drugs have unexpected side effects. Development of novel VEGFR2 inhibitors with less toxicity remains an urgent need. In this study, we describe a novel, well-tolerated, and orally active VEGFR2 inhibitor, YLT192, which inhibits tumor angiogenesis and growth. YLT192 significantly inhibited kinase activity of VEGFR2 and suppressed proliferation, migration, invasion, and tube formation of human umbilical vascular endothelial cells (HUVEC) in vitro. In addition, it inhibited VEGF-induced phosphorylation of VEGFR2 and its downstream signaling regulator in HUVEC. Zebrafish embryonic models and alginate-encapsulated tumor cell assays indicated YLT192 also inhibited angiogenesis in vivo. Moreover, YLT192 could directly inhibit proliferation and induce apoptosis of cancer cells in vitro and in vivo. Oral administration of YLT192 at a dose of 100 mg/kg/day could markedly inhibited human tumor xenograft growth without causing obvious toxicities. It decreased microvessel densities (MVD) in tumor sections. It also shows good safety profiles in the studies with mice and rats. Taken together, these preclinical evaluations suggest that YLT192 inhibits angiogenesis and may be a promising anticancer drug candidate.

Mammalian inner cell mass cells undergo lineage-specific differentiation into germ layers of endoderm, mesoderm and ectoderm during gastrulation. It has been a long-standing challenge in developmental biology to replicate these organized germ layer patterns in culture. Here we present a method of generating organized germ layers from a single mouse embryonic stem cell cultured in a soft fibrin matrix. Spatial organization of germ layers is regulated by cortical tension of the colony, matrix dimensionality and softness, and cell-cell adhesion. Remarkably, anchorage of the embryoid colony from the 3D matrix to collagen-1-coated 2D substrates of ~1 kPa results in self-organization of all three germ layers: ectoderm on the outside layer, mesoderm in the middle and endoderm at the centre of the colony, reminiscent of generalized gastrulating chordate embryos. These results suggest that mechanical forces via cell-matrix and cell-cell interactions are crucial in spatial organization of germ layers during mammalian gastrulation. This new in vitro method could be used to gain insights on the mechanisms responsible for the regulation of germ layer formation.

FIP200, a critical autophagy initiating protein, can participate in numerous cellular functions including cancer development; however, its functional role in P. aeruginosa infection of alveolar macrophages is unknown.