Bispecific antibodies capable of redirecting the lytic potential of immune effector cells to kill tumor targets have long been recognized as a potentially potent biological therapeutic intervention. Unfortunately, efforts to produce such molecules have been limited owing to inefficient production and poor stability properties. Here, we describe a novel Fv-derived strategy based on a covalently linked bispecific diabody structure that we term dual-affinity re-targeting (DART). As a model system, we linked an Fv specific for human CD16 (FcgammaRIII) on effector cells to an Fv specific for mouse or human CD32B (FcgammaRIIB), a normal B-cell and tumor target antigen. DART proteins were produced at high levels in mammalian cells, retained the binding activity of the respective parental Fv domains as well as bispecific binding, and showed extended storage and serum stability. Functionally, the DART molecules demonstrated extremely potent, dose-dependent cytotoxicity in retargeting human PBMC against B-lymphoma cell lines as well as in mediating autologous B-cell depletion in culture. In vivo studies in mice demonstrated effective B-cell depletion that was dependent on the transgenic expression of both CD16A on the effector cells and CD32B on the B-cell targets. Furthermore, DART proteins showed potent in vivo protective activity in a human Burkitt's lymphoma cell xenograft model. Thus, DART represents a biologically potent format that provides a versatile platform for generating bispecific antibody fragments for redirected killing and, with the selection of appropriate binding partners, applications outside of tumor cell cytotoxicity.

Glutamate released by activated microglia induces excitotoxic neuronal death, which likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases, including amyotrophic lateral sclerosis and Alzheimer's disease. Although both blockade of glutamate receptors and inhibition of microglial activation are the therapeutic candidates for these neurodegenerative diseases, glutamate receptor blockers also perturbed physiological and essential glutamate signals, and inhibitors of microglial activation suppressed both neurotoxic/neuroprotective roles of microglia and hardly affected disease progression. We previously demonstrated that activated microglia release a large amount of glutamate specifically through gap junction hemichannel. Hence, blockade of gap junction hemichannel may be potentially beneficial in treatment of neurodegenerative diseases.

An increasing number of small RNAs have been discovered in mammals. However, their primary transcripts and upstream regulatory networks remain largely to be determined. Genomic analysis of small RNAs facilitates identification of their primary transcripts, and hence contributes to researches of their upstream regulatory networks. We here report a batch platform, BatchGenAna, which is specifically designed for large-scale genomic analysis of mammalian small RNAs. It can map and annotate for as many as 1000 small RNAs or 10,000 genomic loci of small RNAs at a time. It provides genomic features including RefSeq genes, mRNAs, ESTs and repeat elements in tabular and graphical results. It also allows extracting flanking sequences of submitted queries, specified genomic regions and host transcripts, which facilitates subsequent analysis such as scanning transcription factor binding sites in upstream sequences and poly(A) signals in downstream sequences. Besides small RNA fields, BatchGenAna can also be applied to other research fields, e.g. in silico analysis of target genes of transcription factors.

Triptolide (TP) is the major active principle of Tripterygium wilfordii Hook f. and very effective in treatment of autoimmune diseases. However, TP induced hepatotoxicity limited its clinical applications. Our previous study found that TP was a substrate of P-glycoprotein and its hepatobiliary clearance was markedly affected by P-gp modulation in sandwich-cultured rat hepatocytes. In this study, small interfering RNA (siRNA) and specific inhibitor tariquidar were used to investigate the impact of P-gp down regulation on TP-induced hepatotoxicity. The results showed that when the function of P-gp was inhibited by mdr1a-1 siRNA or tariquidar, the systemic and hepatic exposures of TP were significantly increased. The aggravated hepatotoxicity was evidenced with the remarkably lifted levels of serum biomarkers (ALT and AST) and pathological changes in liver. The other toxicological indicators (MDA, SOD and Bcl-2/Bax) were also significantly changed by P-gp inhibition. The data analysis showed that the increase of TP exposure in mice was quantitatively correlated to the enhanced hepatotoxicity, and the hepatic exposure was more relevant to the toxicity. P-gp mediated clearance played a significant role in TP detoxification. The risk of herb-drug interaction likely occurs when TP is concomitant with P-gp inhibitors or substrates in clinic.

We previously reported the promising effect of dioscin against hepatic ischemia/reperfusion (I/R) injury, but its effect on cerebral I/R injury remains unknown. In this work, an in vitro oxygen-glucose deprivation and reoxygenation (OGD/R) model and an in vivo middle cerebral artery occlusion (MCAO) model were used. The results indicated that dioscin clearly protected PC12 cells and primary cortical neurons against OGD/R insult and significantly prevented cerebral I/R injury. Further research demonstrated that dioscin-induced neuroprotection was accompanied by a significant inhibition in the expression and the nuclear to cytosolic translocation of HMGB-1, reflected by decreased TLR4 expression. Blockade of the TLR4/MyD88/TRAF6 signaling pathway by dioscin inhibited NF-κB and AP-1 transcriptional activities, MAPK and STAT3 phosphorylation, and pro-inflammatory cytokine responses, and upregulated the levels of anti-inflammatory factors. In addition, small interfering RNA (siRNA) and overexpressed genes of HMGB-1 and TLR4 were applied in in vitro experiments, respectively, and the results further confirmed that dioscin showed an efficient neuroprotection because of its inhibiting effects on HMGB-1/TLR4 signaling and subsequent suppressing inflammation. These findings provide new insights that will aid in elucidating the effect of dioscin against cerebral I/R injury and support the development of dioscin as a potential treatment for ischemic stroke.

Although microRNA-1 (miR-1) is a known liver cancer suppressor, the role of miR-1 in apoptosis of hepatoma cells has remained largely unknown. Our study shows that ectopic miR-1 overexpression induced apoptosis of liver hepatocellular carcinoma (HepG2) cells. Apoptosis inhibitor 5 (API-5) was found to be a potential regulator of miR-1 induced apoptosis, using a bioinformatics approach. Furthermore, an inverse relationship between miR-1 and API-5 expression was observed in human liver cancer tissues and adjacent normal liver tissues. Negative regulation of API-5 expression by miR-1 was demonstrated to promote apoptosis of HepG2 cells. Our study provides a novel regulatory mechanism of miR-1 in the apoptosis of hepatoma cells.

Phytochrome-interacting factor 3 (PIF3) activates light-responsive transcriptional network genes in coordination with the circadian clock and plant hormones to modulate plant growth and development. However, little is known of the roles PIF3 plays in the responses to abiotic stresses. In this study, the cloning and functional characterization of the ZmPIF3 gene encoding a maize PIF3 protein is reported. Subcellular localization revealed the presence of ZmPIF3 in the cell nucleus. Expression patterns revealed that ZmPIF3 is expressed strongly in leaves. This expression responds to polyethylene glycol, NaCl stress, and abscisic acid application, but not to cold stress. ZmPIF3 under the control of the ubiquitin promoter was introduced into rice. No difference in growth and development between ZmPIF3 transgenic and wild-type plants was observed under normal growth conditions. However, ZmPIF3 transgenic plants were more tolerant to dehydration and salt stresses. ZmPIF3 transgenic plants had increased relative water content, chlorophyll content, and chlorophyll fluorescence, as well as significantly enhanced cell membrane stability under stress conditions. The over-expression of ZmPIF3 increased the expression of stress-responsive genes, such as Rab16D, DREB2A, OSE2, PP2C, Rab21, BZ8 and P5CS, as detected by real-time PCR analysis. Taken together, these results improve our understanding of the role ZmPIF3 plays in abiotic stresses signaling pathways; our findings also indicate that ZmPIF3 regulates the plant response to drought and salt stresses.

Activatable cell-penetrating peptides (aCPPs) allow non-viral, low cytotoxic and selective delivery of compounds into target cells for cancer therapy. In tumour cells, up-regulation of human telomerase reverse transcriptase (hTERT) frequently occurs and is being considered as a target in cancer diagnosis and treatment. siRNA sequence that target hTERT mRNA can silence the gene and reduce hTERT protein expression to reduce cell proliferation and inhibit cell growth. In our study, we tested a matrix metalloproteinase-2 (MPP2) aCPP in delivering hTERT siRNA into hepatocellular carcinoma cells (SMMC-7721) to silence the hTERT gene. Cultured SMMC-7721 cells were transfected with a complex of aCPPs and hTERT-specific siRNA-encoding or control plasmids. Compared with cells treated with the complex of control plasmid-CPPs, cells treated with the hTERT-specific siRNA-encoding plasmid-CPP complex had a prolonged G1-phase, but a shorter G2/S-phase, indicating a G1-arrest. Treatment with the hTERT-specific siRNA resulted in a significant decrease (by 26%; P<0.05) in hTERT mRNA levels. The aCPPs tested in this study provides a non-viral delivery of siRNA into cancer cells to silence target genes in cancer therapy.

Epithelial‑mesenchymal transition (EMT) is the process by which epithelial cells depolarize and acquire a mesenchymal phenotype, and is a common early step in the process of metastasis. Patients with lung cancer frequently already have distant metastases when they are diagnosed, highlighting the requirement for early and effective interventions to control metastatic disease. Transforming growth factor‑β1 (TGF‑β1) is able to induce EMT, however the molecular mechanism of this remains unclear. In the current study, TGF‑β1 was reported to induce EMT and promote the migration of non‑small cell lung cancer (NSCLC) cells. A notable observation was that EMT induction was accompanied by the upregulation of human glioma‑associated oncogene homolog 1 (Gli1) mRNA and protein levels. Furthermore, Gli1 levels were depleted by small interfering RNA, and the Gli1 inhibitor GANT 61 attenuated the TGF‑β1‑mediated induction of EMT and cell migration. The results of the current study suggest that Gli1 regulates TGF‑β1‑induced EMT, which may provide a novel therapeutic target to inhibit metastasis in patients with NSCLC.

The liver is a vital organ with critical functions in metabolism, protein synthesis, and immune defense. Most of the liver functions are not mature at birth and many changes happen during postnatal liver development. However, it is unclear what changes occur in liver after birth, at what developmental stages they occur, and how the developmental processes are regulated. Long non-coding RNAs (lncRNAs) are involved in organ development and cell differentiation. Here, we analyzed the transcriptome of lncRNAs in mouse liver from perinatal (day -2) to adult (day 60) by RNA-Sequencing, with an attempt to understand the role of lncRNAs in liver maturation. We found around 15,000 genes expressed, including about 2,000 lncRNAs. Most lncRNAs were expressed at a lower level than coding RNAs. Both coding RNAs and lncRNAs displayed three major ontogenic patterns: enriched at neonatal, adolescent, or adult stages. Neighboring coding and non-coding RNAs showed the trend to exhibit highly correlated ontogenic expression patterns. Gene ontology (GO) analysis revealed that some lncRNAs enriched at neonatal ages have their neighbor protein coding genes also enriched at neonatal ages and associated with cell proliferation, immune activation related processes, tissue organization pathways, and hematopoiesis; other lncRNAs enriched at adolescent ages have their neighbor protein coding genes associated with different metabolic processes. These data reveal significant functional transition during postnatal liver development and imply the potential importance of lncRNAs in liver maturation.

Atractylenolide I (ATL-1) is the major sesquiterpenoid of Atractylodes macrocephala. This study was designed to investigate whether ATL-1 induced apoptosis in A549 and HCC827 cells in vitro and in vivo. In our results, ATL-1 significantly decreased the percentage of in vitro viability, in a dose-dependent manner. In addition, DAPI staining and flow cytometry tests demonstrated the induction of apoptosis by ATL-I. Western blot analysis indicated that the protein levels of caspase-3, caspase-9 and Bax were increased in A549 and HCC827 cells after ATL-I exposure; to the contrary, the expressions of Bcl-2, Bcl-XL were decreased after treatment with ATL-1. In the in vivo study, ATL-I effectively suppressed tumor growth (A549) in transplanted tumor nude mice with up-regulation of caspase-3, caspase-9, and Bax and down-regulation of Bcl-2 and Bcl-XL. In conclusion, our results demonstrated that ATL-I has significant antitumor activity in lung carcinoma cells, and the possible mechanism of action may be related to apoptosis induced by ATL-I via a mitochondria-mediated apoptosis pathway.

Apoptosis has been proven to play a crucial role in early brain injury pathogenesis and to represent a target for the treatment of subarachnoid hemorrhage (SAH). Previously, we demonstrated that astaxanthin (ATX) administration markedly reduced neuronal apoptosis in the early period after SAH. However, the underlying molecular mechanisms remain obscure. In the present study, we tried to investigate whether ATX administration is associated with the phosphatidylinositol 3-kinase-Akt (PI3K/Akt) pathway, which can play an important role in the signaling of apoptosis. Our results showed that post-SAH treatment with ATX could cause a significant increase of phosphorylated Akt and Bad levels, along with a significant decrease of cleaved caspase-3 levels in the cortex after SAH. In addition to the reduced neuronal apoptosis, treatment with ATX could also significantly reduce secondary brain injury characterized by neurological dysfunction, cerebral edema and blood-brain barrier disruption. In contrast, the PI3K/Akt inhibitor, LY294002, could partially reverse the neuroprotection of ATX in the early period after SAH by downregulating ATX-induced activation of Akt/Bad and upregulating cleaved caspase-3 levels. These results provided the evidence that ATX could attenuate apoptosis in a rat SAH model, potentially, in part, through modulating the Akt/Bad pathway.

As cuisine becomes globalized, large volumes of fresh produce are traded internationally. The potential exists for pathogens infecting fresh produce to hitchhike to new locations and perhaps to establish there. It is difficult to identify them using traditional methods if pathogens are novel, scarce, and/or unexpected. In an attempt to overcome this limitation, we used high-throughput sequencing technology as a means of detecting all RNA viruses infecting garlic (Allium sativum L.) bulbs imported into Australia from China, the USA, Mexico, Argentina and Spain, and those growing in Australia. Bulbs tested were grown over multiple vegetative generations and all were stably infected with one or more viruses, including two species not previously recorded in Australia. Present in various combinations from 10 garlic bulbs were 41 virus isolates representing potyviruses (Onion yellow dwarf virus, Leek yellow stripe virus), carlaviruses (Shallot latent virus, Garlic common latent virus) and allexiviruses (Garlic virus A, B, C, D, and X), for which 19 complete and 22 partial genome sequences were obtained, including the first complete genome sequences of two isolates of GarVD. The most genetically distinct isolates of GarVA and GarVX described so far were identified from Mexico and Argentina, and possible scenarios explaining this are presented. The complete genome sequence of an isolate of the potexvirus Asparagus virus 3 (AV3) was obtained in Australia from wild garlic (A. vineale L.), a naturalized weed. This is first time AV3 has been identified from wild garlic and the first time it has been identified beyond China and Japan. The need for routine generic diagnosis and appropriate legislation to address the risks to primary production and wild plant communities from pathogens spread through the international trade in fresh produce is discussed.

The fruit of Psoralea corylifolia L. (Fabaceae) (PC), known as "Bo-Gol-Zhee" in Korea has been used as traditional medicine. Ethanol and aqueous extracts of PC have an anti-hyperglycemic effect by increasing plasma insulin levels and decreasing blood glucose and total plasma cholesterol levels in type 2 diabetic rats. In this study, we purified six compounds from PC and investigated their anti-diabetic effect. Among the purified compounds, bavachin most potently accumulated lipids during adipocyte differentiation. Intracellular lipid accumulation was measured by Oil Red-O (ORO) cell staining to investigate the effect of compounds on adipogenesis. Consistently, bavachin activated gene expression of adipogenic transcriptional factors, proliferator-activated receptorγ (PPARγ) and CCAAT/enhancer binding protein-α (C/EBPα). Bavachin also increased adiponectin expression and secretion in adipocytes. Moreover, bavachin increased insulin-induced glucose uptake by differentiated adipocytes and myoblasts. In differentiated adipocytes, we found that bavachin enhanced glucose uptake via glucose transporter 4 (GLUT4) translocation by activating the Akt and 5'AMP-activated protein kinase (AMPK) pathway in the presence or absence of insulin. These results suggest that bavachin from Psoralea corylifolia might have therapeutic potential for type 2 diabetes by activating insulin signaling pathways.

Tropical evergreen perennials undergo recurrent flush growth, and their terminal buds alternate between growth and dormancy. In sharp contrast to the intensive studies on bud development in temperate deciduous trees, there is little information about bud development regulation in tropical trees. In this study, litchi (Litchi chinensis Sonn.) was used as a model tropical perennial for morphological characterization and transcriptomic analysis of bud development. Litchi buds are naked with apical meristem embraced by rudimentary leaves, which are brown at dormant stage (Stage I). They swell and turn greenish as buds break (Stage II), and as growth accelerates, the rudimentary leaves elongate and open exposing the inner leaf primodia. With the outgrowth of the needle-like leaflets, bud growth reaches a maximum (Stage III). When leaflets expand, bud growth cease with the abortion of the rudimentary leaves at upper positions (Stage IV). Then buds turn brown and reenter dormant status. Budbreak occurs again when new leaves become hard green. Buds at four stages (Stage I to IV) were collected for respiration measurements and in-depth RNA sequencing. Respiration rate was the lowest at Stage I and highest at Stage II, decreasing toward growth cessation. RNA sequencing obtained over 5 Gb data from each of the bud samples and de novo assembly generated a total of 59,999 unigenes, 40,119 of which were annotated. Pair-wise comparison of gene expression between stages, gene profiling across stages, GO/KEGG enrichment analysis, and the expression patterns of 17 major genes highlighted by principal component (PC) analysis displayed significant changes in stress resistance, hormone signal pathways, circadian rhythm, photosynthesis, cell division, carbohydrate metabolism, programmed cell death during bud development, which might be under epigenetic control involving chromatin methylation. The qPCR results of 8 selected unigenes with high PC scores agreed with the RPKM values obtained from RNA-seq. Three Short Vegetative Phase (SVP) genes, namely LcSVP1, LcSVP2, and LcSVP3 displayed different expression patterns, suggesting their differential roles in bud development regulation. The study brought an understanding about biological processes associated with the phase transitions, molecular regulation of bud development, as well as cyclic bud growth as a strategy to survive tropical conditions.

Proper regulation of neuronal gene expression is crucial for the development and differentiation of the central nervous system. The transcriptional repressor REST (repressor element-1 silencing transcription factor) is a key regulator in differentiation of pluripotent stem cells to neuronal progenitors and mature neurons. Dysregulated REST activity has been implicated in various diseases, among which the most deadly is glioblastoma multiforme (GBM). Here we have developed an expression-based REST signature (EXPREST), a device providing quantitative measurements of REST activity for GBM tumors. EXPREST robustly quantifies REST activity (REST score) using gene expression profiles in absence of clinic-pathologic assessments of REST. Molecular characterization of REST activity identified global alterations at the DNA, RNA, protein and microRNA levels, suggesting a widespread role of REST in GBM tumorigenesis. Although originally aimed to capture REST activity, REST score was found to be a prognostic factor for overall survival. Further, cell lines with enhanced REST activity was found to be more sensitive to IGF1R, VEGFR and ABL inhibitors. In contrast, cell lines with low REST score were more sensitive to cytotoxic drugs including Mitomycin, Camptothecin and Cisplatin. Together, our work suggests that therapeutic targeting of REST provides a promising opportunity for GBM treatment.

Heart transplantation is an uncommon treatment for unresectable and non-metastatic primary cardiac sarcomas, and the role of it is unclear. This study aims to offer a survival analysis of it.

Environmental noise exposure is associated with adverse effects on human health including hearing loss, heart disease, and changes in stress-related hormone levels. Alteration in DNA methylation in response to environmental exposures is a well-known phenomenon and it is implicated in many human diseases. Understanding how environmental noise exposures affect DNA methylation patterns may help to elucidate the link between noise and adverse effects on health. In this pilot study we examined the effects of environmental noise exposure on DNA methylation of genes related to brain function and investigated whether these changes are related with metabolic health. We exposed four groups of male Wistar rats to moderate intensity noise (70-75dB with 20-4000Hz) at night for three days as short-term exposure, and for three weeks as long-term exposure. Noise exposure was limited to 45dB during the daytime. Control groups were exposed to only 45dB, day and night. We measured DNA methylation in the Bdnf, Comt, Crhr1, Mc2r, and Snca genes in tissue from four brain regions of the rats (hippocampus, frontal lobe, medulla oblongata, and inferior colliculus). Further, we measured blood pressure and body weight after long-term noise exposure. We found that environmental noise exposure is associated with gene-specific DNA methylation changes in specific regions of the brain. Changes in DNA methylation are significantly associated with changes in body weight (between Bdnf DNA methylation and Δ body weight: r=0.59, p=0.018; and between LINE-1 ORF DNA methylation and Δ body weight: =-0.80, p=0.0004). We also observed that noise exposure decreased blood pressure (p=0.038 for SBP, p=0.017 for DBP and p 0. 017 for MAP) and decreased body weight (β=-26g, p=0.008). In conclusion, environmental noise exposures can induce changes in DNA methylation in the brain, which may be associated with adverse effects upon metabolic health through modulation of response to stress-related hormones.

β-TrCP and SKP2 are two well-studied F-box proteins, which often act as oncogenes. Whether and how they communicate with each other is unknown. Here we report that FBXW2, a poorly characterized F-box, is a substrate of β-TrCP1 and an E3 ligase for SKP2. While β-TrCP1 promotes FBXW2 ubiquitylation and shortens its half-life, FBXW2 does the same to SKP2. FBXW2 has tumour suppressor activity against lung cancer cells and blocks oncogenic function of both β-TrCP1 and SKP2. The levels of β-TrCP1-FBXW2-SKP2 are inversely correlated during cell cycle with FBXW2 and β-TrCP/SKP2 being high or low, respectively, in arrested cells, whereas the opposite is true in proliferating cells. Consistently, FBXW2 predicts a better patient survival, whereas β-TrCP1 and SKP2 predict a worse survival. Finally, the gain- and loss-of-function mutations of FBXW2 are found in various human cancers. Collectively, our data show that the β-TrCP-FBXW2-SKP2 axis forms an oncogene-tumour suppressor-oncogene cascade to control cancer cell growth with FBXW2 acting as a tumour suppressor by promoting SKP2 degradation.

Traditional Chinese medicinal herbs Cortex Moutan and Radix Salviae Milthiorrhizaeare are prescribed together for their putative cardioprotective effects in clinical practice. However, the rationale of the combined use remains unclear. The present study was designed to investigate the cardioprotective effects of paeonol and danshensu (representative active ingredient of Cortex Moutan and Radix Salviae Milthiorrhizae, respectively) on isoproterenol-induced myocardial infarction in rats and its underlying mechanisms.