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Background Bacillus subtilis has been widely used as a host for heterologous protein expression in food industry. B. subtilis ATCC6051 is an alternative expression host for the production of industrial enzymes, and exhibits favorable growth properties compared to B. subtilis 168. Extracellular expression of pullulanase from recombinant B. subtilis is still limited due to the issues on promoters of B. subtilis expression system. This study was undertaken to develop a new, high-level expression system in B. subtilis ATCC6051. Results To further optimize B. subtilis ATCC6051 as a expression host, eight extracellular proteases (aprE, nprE, nprB, epr, mpr, bpr, vpr and wprA), the sigma factor F (spoIIAC) and a surfactin (srfAC) were deleted, yielding the mutant B. subtilis ATCC6051∆10. ATCC6051∆10 showed rapid growth and produced much more extracellular protein compared to the widetype strain ATCC6051, due to the inactivation of multiple proteases. Using this mutant as the host, eleven plasmids equipped with single promoters were constructed for recombinant expression of pullulanase (PUL) from Bacillus naganoensis. The plasmid containing the PspovG promoter produced the highest extracellular PUL activity, which achieved 412.9 U/mL. Subsequently, sixteen dual-promoter plasmids were constructed and evaluated using this same method. The plasmid containing the dual promoter PamyL–PspovG produced the maximum extracellular PUL activity (625.5 U/mL) and showed the highest expression level (the dry cell weight of 18.7 g/L). Conclusions Taken together, we constructed an effective B. subtilis expression system by deleting multiple proteases and screening strong promoters. The dual-promoter PamyL–PspovG system was found to support superior expression of extracellular proteins in B. subtilis ATCC6051. Electronic supplementary material The online version of this article (10.1186/s12934-018-1011-y) contains supplementary material, which is available to authorized users.

Malus halliana is an iron (Fe)-efficient apple rootstock growing in calcareous soil that shows obvious 'greenness' traits during Fe deficiency. Recent studies have shown that exogenous sugars can be involved in abiotic stress. To identify the key regulatory steps of chlorophyll (Chl) biosynthesis in M. halliana under Fe deficiency and to verify whether exogenous sucrose (Suc) is involved in Fe deficiency stress, we determined the contents of the Chl precursor and the expression of several Chl biosynthetic genes in M. halliana. The results showed that Fe deficiency caused a significant increase in the contents of protoporphyrin IX (Proto IX), Mg-protoporphyrin IX (Mg-Proto IX) and protochlorophyllide (Pchlide) in M. halliana compared to the Fe-sensitive rootstock Malus hupehensis. Quantitative real-time PCR (RT-qPCR) also showed that the expression of protoporphyrinogen oxidase (PPOX), which synthesizes Proto IX, was upregulated in M. halliana and downregulated in M. hupehensis under Fe deficiency. Exogenous Suc application prominently enhanced the contents of porphobilinogen (PBG) and the subsequent precursor, whereas it decreased the level of δ-aminolaevulinic acid (ALA), suggesting that the transformation from ALA to PBG was catalyzed in M. halliana. Additionally, the transcript level of δ-aminolevulinate acid dehydratase (ALAD) was noticeably upregulated after exogenous Suc treatment. This result, combined with the precursor contents, indicated that Suc accelerated the steps of Chl biosynthesis by modulating the ALAD gene. Therefore, we conclude that PPOX is the key regulatory gene of M. halliana in response to Fe deficiency. Exogenous Suc enhances M. halliana tolerance to Fe deficiency stress by regulating Chl biosynthesis.

The bacterial diseases of tilapia caused by Streptococcus agalactiae have resulted in the high mortality and huge economic loss in the tilapia industry. Matrix metalloproteinase-9 (MMP-9) may play an important role in fighting infection. However, the role of MMP-9 in Nile tilapia against S. agalactiae is still unclear. In this work, MMP-9 cDNA of Nile tilapia (NtMMP-9) has been cloned and characterized. NtMMP-9 has 2043 bp and encodes a putative protein of 680 amino acids. NtMMP-9 contains the conserved domains interacting with decorin and inhibitors via binding forces compared to those in other teleosts. Quantitative real-time-polymerase chain reaction (qPCR) analysis reveals that NtMMP-9 distinctly upregulated following S. agalactiae infection in a tissue- and time-dependent response pattern, and the tissues, including liver, spleen, and intestines, are the major organs against a S. agalactiae infection. Besides, the proteolytic activity of NtMMP-9 is also confirmed by heterologous expression and zymography, which proves the active function of NtMMP-9 interacting with other factors. The findings indicate that NtMMP-9 was involved in immune responses against the bacterial challenge at the transcriptional level. Further work will focus on the molecular mechanisms of NtMMP-9 to respond and modulate the signaling pathways in Nile tilapia against S. agalactiae invasion and the development of NtMMP-9-related predictive biomarkers or vaccines for preventing bacterial infection in the tilapia industry.

Cathepsin B (CatB) has been widely known for its hydrolytic ability and involvement in the innate immunity. However, the mechanism of CatB from teleosts participating in immunoregulation remains poorly understood; and the sequence of CatB from Nile tilapia (NtCatB) has not been cloned and characterized. In this study, the coding sequence of NtCatB was cloned, and then characterized by bioinformatic analysis and heterologous expression. The deduced amino acid sequence (330-aa) of NtCatB contains the representative features of CatB. Quantitative real-time PCR revealed the extensive mRNA expression of NtCatB in six tissues of healthy Nile tilapia, and its transcription level was significantly up-regulated after Streptococcus agalactiae challenge. NtCatB may interact with some immunological function proteins and take part in the regulatory pathway. These results suggest that NtCatB is likely to be involved in the immune reaction. The mature region (residues 79-328, mNtCatB) of NtCatB was cloned and transferred to pET-28a for expressing the recombinant protein. The purified recombinant mNtCatB was verified with the activity of 992.34 U mg-1 min-1 under the optimal condition using a substrate hydrolyzing assay. The recombinant cystatin-A1-like can effectively inhibit the activity of the recombinant mNtCatB, and their binding form was predicted by molecular docking. Our results contribute to elucidating the immunological functions of NtCatB.

Background Although increased accumulation of neutrophils has been noted in chronic rhinosinusitis (CRS), the function and regulation of neutrophils in CRS are largely unknown. IL-36 family cytokines may play an important role in neutrophilic inflammation. Objective This study sought to investigate the expression and function of IL-36 cytokines in CRS. Methods Quantitative RT-PCR, immunohistochemistry, immunofluorescence, and ELISA were used to investigate the expression of IL-36 cytokines and IL-36 receptor (IL-36R) in sinonasal mucosa. The expression of IL-36R on neutrophils in polyps and blood was measured by flow cytometry. Purified blood neutrophils were cultured to investigate the regulation of IL-36R expression. The cleavage of IL-36γ was detected by Western blotting. Dispersed nasal polyp cells were treated with IL-36γ with or without elastase inhibitor and dexamethasone. Results Neutrophil infiltration and expression of IL-36 cytokines and IL-36R were upregulated in both CRS with and without nasal polyps. IL-36γ was the most abundant isoform and mainly expressed by epithelial cells in CRS. Neutrophils were the principal IL-36R + cell type in polyps. IL-36R expression was almost absent in blood neutrophils and upregulated by IL-6, IL-1β, and Dermatophagoides pteronyssinus group 1. Elastase activity was increased in polyps and degraded full-length IL-36γ. Consistently, the levels of cleaved IL-36γ were increased in polyps. Full-length IL-36γ promoted the production of matrix metalloproteinase 9; IL-17A; and chemokine (C-X-C motif) ligands 1, 2, and 8 from dispersed nasal polyp cells, which was abolished by elastase inhibitor. The proinflammatory effect of IL-36γ was not suppressed by dexamethasone. Conclusions Increased production and activation of IL-36γ may act on neutrophils and further exaggerate neutrophilic inflammation in CRS.

The early onset breast cancer patients (age ≤ 40) often display higher incidence of axillary lymph node metastasis, and poorer five-year survival than the late-onset patients. To identify the genes and molecules associated with poor prognosis of early onset breast cancer, we examined gene expression profiles from paired breast normal/tumor tissues, and coupled with Gene Ontology and public data base analysis. Our data showed that the expression of GAS7b gene was lower in the early onset breast cancer patients as compared to the elder patients. We found that GAS7 was associated with CYFIP1 and WAVE2 complex to suppress breast cancer metastasis via blocking CYFIP1 and Rac1 protein interaction, actin polymerization, and β1-integrin/FAK/Src signaling. We further demonstrated that p53 directly regulated GAS7 gene expression, which was inversely correlated with p53 mutations in breast cancer specimens. Our study uncover a novel regulatory mechanism of p53 in early onset breast cancer progression through GAS7–CYFIP1-mediated signaling pathways.

Plants have evolved an array of adaptive responses to low Pi availability, a process modulated by various external stimuli and endogenous growth regulatory signals. Little is known about how these signaling processes interact to produce an integrated response. Arabidopsis thaliana PHOSPHATE STARVATION RESPONSE1 (PHR1) encodes a conserved MYB-type transcription factor that is essential for programming Pi starvation-induced gene expression and downstream Pi starvation responses (PSRs). Here, we show that loss-of-function mutations in FHY3 and FAR1, encoding two positive regulators of phytochrome signaling, and in EIN3, encoding a master regulator of ethylene responses, cause attenuated PHR1 expression, whereas mutation in HY5, encoding another positive regulator of light signaling, causes increased PHR1 expression. FHY3, FAR1, HY5, and EIN3 directly bind to the PHR1 promoter through distinct cis-elements. FHY3, FAR1, and EIN3 activate, while HY5 represses, PHR1 expression. FHY3 directly interacts with EIN3, and HY5 suppresses the transcriptional activation activity of FHY3 and EIN3 on PHR1 Finally, both light and ethylene promote FHY3 protein accumulation, and ethylene blocks the light-promoted stabilization of HY5. Our results suggest that light and ethylene coordinately regulate PHR1 expression and PSRs through signaling convergence at the PHR1 promoter.

Cathepsin L (CatL) has been widely known for its involvement in the innate immunity. However, it still remains poorly understand how CatL modulates the immune system of teleosts. Moreover, the CatL of Nile tilapia (NtCatL) has not been cloned or characterized. In this study, the gene encoding NtCatL was cloned, and was characterized by bioinformatics analysis, heterologous expression and protease activity assay. The coding sequence of NtCatL is 1017 bp in length and encodes 338 amino acid residues with a predicted molecular weight of 38.487 kDa and a theoretical isoelectric point of 5.79. NtCatL possesses the features of a typical cathepsin L, including one signal peptide, one propeptide region, and one papain family cysteine protease domain containing four active site residues (Gln 135 , Cys 141 , His 281 , and Asn 305 ). The prediction of protein-protein interaction shows that NtCatL may interact with some functional proteins for realizing an immune function. Real-time quantitative PCR revealed the widespread transcriptional expression of NtCatL in six tissues of healthy Nile tilapia, and the NtCatL mRNA is significantly up-regulated after Streptococcus agalactiae challenge. These results suggest that NtCatL is likely to be involved in the immune reaction of Nile tilapia. Recombinant proteins from the mature domain (residues 117–337) of NtCatL were obtained by heterologous expression using pET28a and Rosetta (DE3) competent cells. A protein product with the high purity was obtained by using TALON Superflow purification rather than adopting HisTrap HP columns. The protease activity of the recombinant protein was verified by using a substrate hydrolyzing assay. This work has cloned and characterized the CatL from Nile tilapia for the first time, and contributes to elucidating the immunological functions of CatL.

Summary Previous studies on erythropoiesis revealed that microRNAs (miRNAs) play a critical role in erythroid differentiation. Given the abundance of identified miRNAs and the limited understanding of erythroid miRNAs, additional examination is required. Here, two sets of erythroid differentiation miRNome data were analysed to screen for novel erythroid-inhibiting miRNAs. MIR200A was selected based on its pattern of downregulated expression in the miRNome datasets during induction of erythroid differentiation. Overexpression of MIR200A in K562 and TF-1 cells confirmed its inhibitory role in erythroid differentiation. Further in vivo study indicated that overexpression of mir200a inhibited primitive erythropoiesis of zebrafish. Transcriptome analyses after MIR200A overexpression in TF-1 cells indicated a significant role in regulating erythroid function and revealed potential regulation networks. Additionally, bioinformatics and experimental analyses confirmed that PDCD4 (programmed cell death 4) and THRB (thyroid hormone receptor, beta) are both targets of MIR200A-3p. Gain- and loss-of-function studies of PDCD4 and THRB revealed that the two targets were capable of promoting erythroid gene expression. Overall, our results revealed that microRNA 200a inhibits erythroid differentiation by targeting PDCD4 and THRB.

Walnut is cultivated all over the world for its precious woody nut and edible oil. Walnut anthracnose caused by Colletotrichum spp. is a disastrous disease in the walnut plantation. In China, this disease is difficult to control. The efficacies of the traditional preventing and controlling measures are inadequate. It causes serious losses every year. In the interaction systems of ‘antagonistic organism-pathogen’, the response mechanisms of the pathogen to the stress of antagonistic organisms were largely unknown. To understand the response mechanisms of walnut anthracnose pathogen, Colletotrichum gloeosporioides sensu stricto (s.s.) TS-09R was treated with Bacillus amyloliquefaciens, and RNAseq gene expression analyses were performed. A total of 610 genes expressed differentially (DEGs). Among these genes, 387 genes were up-regulated and 223 genes were down-regulated. Gene ontology (GO) analysis showed that 409 DEGs involved in the categories of biological process, and cellular component. The KEGG pathway enrichment analysis showed that all up-regulated DEGs were enriched in amino acid metabolism, biosynthesis of other secondary metabolites, and xenobiotics biodegradation and metabolism. Further analyses indicated that C. gloeosporioides s.s. TS-09R up-expressed significantly some DEGs associated with cell membrane synthesis and, or maintaining stabilization, antioxidant and anti-stress to build the cell membrane, maintain the normal physiological function of cells. At the same time, it improved detoxification efficiency and reduced its sensitivity to the exogenous antimicrobial substance (EAS). It also improved the activity of the efflux pump translocating EAS out of the cells, reduced accumulation, and minimized injuries to cells. C. gloeosporioides s.s. TS-09R also up-expressed significantly genes associated with sporulation to promote spore production for survival in adversity. C. gloeosporioides s.s. TS-09R recruited numerous genes to respond to or resist the biocontrol of B. amyloliquefaciens. The response model was a holistic and complex network involving multiple genes and multiple signaling pathways. Additionally, the glutathione-S-transferases (GST) activities of C. gloeosporioides s.s. TS-09R, TS-09 cultured for 5 days and 9 days were calculated, and the GST activity had a similar change trend to the GST-related gene expression. The result indicated that the hypothetical response strategies were reliable. In this paper, we provided new insights into the onset of the interaction system of ‘C. gloeosporioides s.s. – B. amyloliquefaciens’. The results will contribute to exploring comprehensively the mechanism of B. amyloliquefaciens according to the response mechanisms of C. gloeosporioides s.s.

Bacillus subtilis is widely used for the large-scale industrial production of proteins. In this study, the transcriptomes of B. subtilis 168 and B. megaterium DSM319 cells grown in stationary phase were analyzed to expand the repertoire of highly-active promoters for high-level protein expression based on the transcriptomes of these Bacillus strains. 24 genes with the highest expression levels among 2048 highly expressed gene families were chosen to examine promoter activity. The activities of four promoters with the beta-galactosidase (bgaB) gene as a reporter were stronger than those of the well-characterized strong promoter P43. The expression level of recombinant Pro-transglutaminase (pro-MTG) from Streptomyces mobaraensis achieved 87.6 U/mL and 70.7 U/mL under the control of two constitutive promoter PsodA and PydzA, respectively, compared to the promoter P43. Our study provides a basis for further studies on the Bacillus transcriptome by identifying strong promoters for industrial uses.

The Asian tiger mosquito, Aedes albopictus, is a competent vector for the majority of arboviruses. The mosquito innate immune response is a primary determinant for arthropod-borne virus transmission, and the midgut is the first barrier to pathogen transmission. Mosquito antiviral immunity is primarily mediated by the small interfering RNA pathway. However, the roles that the P-element induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway play in antiviral immunity in Ae. albopictus and its midgut still need further exploration. This study aimed to explore the profiles of both viral-derived and host-originated piRNAs in the whole body and midgut infected with Dengue virus 2 (DENV-2) in Ae. albopictus, and to elucidate gene expression profile differences of the PIWI protein family between adult females and their midguts. A deep sequencing-based method was used to identify and analyze small non-coding RNAs, especially the piRNA profiles in DENV-2-infected Ae. albopictus and its midgut. The top-ranked, differentially-expressed piRNAs were further validated using Stem-loop qRT-PCR. Bioinformatics analyses and reverse-transcription PCR (RT-PCR) methods were used to detect PIWI protein family members, and their expression profiles. DENV-2 derived piRNAs (vpiRNA, 24–30 nts) were observed in both infected Ae. albopictus and its midgut; however, only vpiRNA in the whole-body library had a weak preference for adenine at position 10 (10A) in the sense molecules as a feature of secondary piRNA. These vpiRNAs were not equally distributed, instead they were derived from a few specific regions of the genome, especially several hot spots, and displayed an obvious positive strand bias. We refer to the differentially expressed host piRNAs after DENV infection as virus-induced host endogenous piRNAs (vepiRNAs). However, we found that vepiRNAs were abundant in mosquito whole-body tissue, but deficient in the midgut. A total of eleven PIWI family genes were identified in Ae. albopictus; however, only AalPiwi5–7 and AalAgo3(1–2) were readily detected in the midgut. The characteristics of piRNAs in DENV-2-infected Ae. albopictus adult females were similar to those previously described for flavivirus infections but were not observed in the midgut. The reduced levels of vepiRNAs and incomplete expression of PIWI pathway genes in midgut samples from DENV-2-infected Ae. albopictus suggests that viral regulation of host piRNAs may not be an important factor in the midgut.

veA belongs to the velvet regulatory system that regulates the development and secondary metabolism of many fungi. To identify the function of veA in Aspergillus cristatus, veA deletion mutants were constructed by homologous recombination via Agrobacterium tumefaciens-mediated transformation. Deletion of veA led to increased conidial production and reduced sexual sporulation. The regulatory role of veA in A. cristatus was not light-dependent, and this differed from its role in other Aspergilli. Furthermore, veA deletion mutants were more sensitive to environmental stressors, including salt, osmotic pressure, temperature and pH. In contrast, deletion of veA resulted in increased resistance to oxidative stress. veA also affected aerial vegetative growth. Transcriptomic analysis of the veA-null mutant and wild type indicated that most asexual and sexual development genes were upregulated and downregulated, respectively. These findings confirmed that veA has a positive effect on sexual development but represses conidial formation. Overall, these results suggested that the veA gene plays a critical role in maintaining a developmental balance between asexual and sexual sporulation and is involved in vegetative growth and environmental stress response in A. cristatus.

Summary Objective Intervertebral disc (IVD) degeneration is associated with a malfunction of the nucleus pulposus (NP). Alginate culturing provides a favorable microenvironment for the phenotypic maintenance of chondrocyte-like NP cells. However, NP cells are recently evidenced to present heterogeneous populations, including progenitors, fibroblastic cells and primitive NP cells. The aim of this study is to profile the phenotypic changes of distinct human NP cells populations and describe the dynamic expression of chondroitin sulfate glycosaminoglycans (CS-GAGs) in extended alginate encapsulation. Method Non-degenerated (ND-NPC) and degenerated (D-NPC) NP cells were expanded in monolayers, and subject to 28-day culture in alginate after serial passaging. CS-GAG compositional expression in monolayer-/alginate-cultured NP cells was evaluated by carbohydrate electrophoresis. Cellular phenotypic changes were assessed by immunologic detection and gene expression analysis. Results Relative to D-NPC, ND-NPC displayed remarkably higher expression levels of chondroitin-4-sulfate GAGs over the 28-day culture. Compared with monolayer culture, ND-NPC showed increased NP marker expression of KRT18 , KRT19 , and CDH2 , as well as chondrocyte markers SOX9 and MIA in alginate culture. In contrast, expression of fibroblastic marker COL1A1 , COL3A1 , and FN1 were reduced. Interestingly, ND-NPC showed a loss of Tie2+ but gain in KRT19+/CD24+ population during alginate culture. In contrast, D-NPC showed more consistent expression levels of NP surface markers during culture. Conclusion We demonstrate for the first time that extended alginate culture selectively enriches the committed NP cells and favors chondroitin-4-sulfate proteoglycan production. These findings suggest its validity as a model to investigate IVD cell function.

Plants have evolved a repertoire of strategies collectively termed the shade-avoidance syndrome to avoid shade from canopy and compete for light with their neighbors. However, the signaling mechanism governing the adaptive changes of adult plant architecture to shade is not well understood. Here, we show that in Arabidopsis, compared with the wild type, several PHYTOCHROME-INTERACTING FACTORS (PIFS) overexpressors all display constitutive shade-avoidance syndrome under normal high red to far-red light ratio conditions but are less sensitive to the simulated shade, whereas the MIR156 overexpressors exhibit an opposite phenotype. The simulated shade induces rapid accumulation of PIF proteins, reduced expression of multiple MIR156 genes, and concomitant elevated expression of the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) family genes. Moreover, in vivo and in vitro assays indicate that PIFs bind to the promoters of several MIR156 genes directly and repress their expression. Our results establish a direct functional link between the phytochrome-PIFs and miR156-SPL regulatory modules in mediating shade-avoidance syndrome., Plants employ developmental strategies to avoid shade and compete with neighbors for light. Here, Xie et al. show that phytochrome-interacting factors, which are regulated in a light-dependent manner, directly repress MIR156 genes and promote the expression of SPL genes to enhance shade-avoidance responses.

Background: The Wnt signaling pathway controls the development of thymic epithelial cells by regulating the expression of FoxN1. Thymoma is a type of malignant tumor arising from the thymic epithelial cells. To determine whether Wnt4 and FoxN1 are involved in the pathogenesis of thymoma, this study determined the mRNA and protein levels of Wnt4 and Foxn1 in thymoma, and analyzed the effect of thymoma cell apoptosis and tumor growth in nude mice after Wnt4 and FoxN1 downregulation. Methods: Wnt4 and FoxN1 mRNA and protein levels in thymoma tissues were analyzed by RT-qPCR and immunohistochemistry, respectively. Thymoma cells were cultured and transfected with siRNA targeting the Wnt4, JNK , and FoxN1 genes. Apoptosis of thymoma cells were analyzed after Wnt4 and FoxN1 downregulation. In addition, thymoma cells were inoculated into nude mice and tumor growth was analyzed. Results: The rates of expression of Wnt4 and FoxN1 protein were 64.3% and 58.9%, while the levels of mRNA expression were 2.56±0.04 and 1.83±0.11, respectively. With increasing malignancy of thymoma, the rates of positivity for Wnt4 and FoxN1 mRNA and protein expression gradually increased. Upon interfering with Wnt4, JNK , and FoxN1 gene expression by using siRNA technology, the inhibition rates were 56.7%, 72.6%, and 63.2%, respectively. The expression of FoxN1 mRNA and protein was decreased after Wnt4 and JNK downregulation. After downregulation of Wnt4 and FoxN1 gene expression, the apoptosis rate of thymoma cells increased and the tumor volume decreased in nude mice. Conclusions: High expression of Wnt4 and FoxN1 may play an important role in the generation and development of thymoma. The FoxN1 gene produced a marked downstream effect through the regulation of Wnt4 . Determining the positivity for both Wnt4 and FoxN1 can help us to evaluate the level of malignancy of thymoma.

The majority of breast cancer models for drug discovery are based on orthotopic or subcutaneous tumours. Therapeutic responses of metastases, especially microscopic metastases, are likely to differ from these tumours due to distinct cancer-microenvironment crosstalk in distant organs. Here, to recapitulate such differences, we established an ex vivo bone metastasis model, termed bone-in-culture array or BICA, by fragmenting mouse bones preloaded with breast cancer cells via intra-iliac artery injection. Cancer cells in BICA maintain features of in vivo bone micrometastases regarding the microenvironmental niche, gene expression profile, metastatic growth kinetics and therapeutic responses. Through a proof-of-principle drug screening using BICA, we found that danusertib, an inhibitor of the Aurora kinase family, preferentially inhibits bone micrometastases. In contrast, certain histone methyltransferase inhibitors stimulate metastatic outgrowth of indolent cancer cells, specifically in the bone. Thus, BICA can be used to investigate mechanisms involved in bone colonization and to rapidly test drug efficacies on bone micrometastases., The bone microenvironment may alter therapeutic responses of disseminated breast cancer cells. Here the authors establish an ex vivo bone metastasis model, termed BICA, to delineate the effects of bone microenvironment and to rapidly discover anti-metastasis drugs.

Chewing insects cause severe yield losses in crop production worldwide. Crop plants counteract chewing insects by transcriptionally promoting a repertoire of defense gene products that are either toxic to, or attractive to the natural enemies of, pest insects. However, the complexity of the transcriptional reprogramming in plant defense response against chewing insects is still not well understood. In this study, the genome-wide early responses in maize seedlings to Asian corn borer (ACB, Ostrinia furnacalis) and also to jasmonic acid(JA), the pivotal phytohormone controlling plant defense response against herbivory, were transcriptionally profiled by RNA-Seq. Clustering of differentially expressed genes (DEGs) along with functional enrichment analysis revealed important biological processes regulated in response to ACB infestation and/or jasmonic acid. Moreover, DEGs with distinct expression patterns were differentially enriched with diverse families of cis-elements on their promoters. Multiple inventories of differentially expressed transcription factors (DETFs) in each DEG group were also analyzed. A transient expression assay using transfected maize protoplastswas established to examine the potential roles of DETFs in maize defense response and JA signaling, and this was used to show that ZmNAC60, an ACB- and JA-inducible DETF, represented a novel positive regulator of JA and defense pathway genes. This study provided a comprehensive transcriptional picture for the early dynamics of maize defense responses and JA signaling, and the identification of DETFs offered potential targets for further functional genomics investigation of master regulators in maize defense responses against herbivory.

Purpose: The proinflammatory cytokine interleukin-32 (IL-32) is a novel tumor marker highly expressed in various human carcinomas, including gastric cancer. However, its effects on prognosis of patients with gastric cancer and cancer metastasis are virtually unknown at present. The main aim of this study was to explore the clinical significance of IL-32 in gastric cancer and further elucidate the molecular mechanisms underlying IL-32–mediated migration and invasion. Experimental Design: Gastric cancer cells with ectopic expression or silencing of IL-32 were examined to identify downstream molecules and establish their effects on cell motility, invasion, and lung metastasis in vivo. Results: IL-32 was significantly upregulated in gastric cancer and positively correlated with aggressiveness of cancer and poor prognosis. Ectopic expression of IL-32 induced elongated morphology and increased cell migration and invasion via induction of IL-8, VEGF, matrix metalloproteinase 2 (MMP2), and MMP9 expression via phosphor-AKT/phospho-glycogen synthase kinase 3β/active β-catenin as well as hypoxia-inducible factor 1α (HIF-1α) signaling pathways. Conversely, depletion of IL-32 in gastric cancer cells reversed these effects and decreased lung colonization in vivo. Examination of gene expression datasets in oncomine and staining of gastric cancer specimens demonstrated the clinical significance of IL-32 and its downstream molecules by providing information on their coexpression patterns. Conclusions: IL-32 contributes to gastric cancer progression by increasing the metastatic potential resulting from AKT, β-catenin, and HIF-1α activation. Our results clearly suggest that IL-32 is an important mediator for gastric cancer metastasis and independent prognostic predictor of gastric cancer. Clin Cancer Res; 20(9); 2276–88. ©2014 AACR.

Background How to find an effective gene locus resistant to atherosclerosis has become a hotspot of today's medicine. Membrane attack complex (MAC) has proved to be related with the occurrence and development of atherosclerosis. Complement regulatory protein CD59 is a key regulator of complement MAC assembly. So this study aimed at discussing the effects of CD59 gene on occurrence and development of atherosclerosis and relative mechanism. Methods Apolipoprotein E knockout (ApoE (−/−)) mice were randomly divided into four groups: control group, empty plasmid-treated group, 0.5 ml CD59-treated group and 1.0 ml CD59-treated group. At the end of the 12th week, CD59 mRNA levels in whole blood were determined by RT-PCR and CD59 protein expressions were detected by western blot. The biochemical indexes in blood serum were detected. The paraffin sections of aortic root of mice were made and the degrees of atherosclerotic plaques formation were observed by hematoxylin/eosin (HE) staining. The expressions of cell apoptosis-related proteins (Bcl-2 and Fas) and plaque stability related protein (MMP-2) were detected by immunohistochemistry. Then the cell apoptosis levels were detected by TUNEL, the expression of Cyclin D 1 and the mRNA level of cyclin dependent protein kinase 4 (CDK4) were detected by immunofluorescence and in situ hybridization, respectively. Results Atherosclerotic mouse model was successfully established. CD59 gene was overexpressed in blood cells and tissue cells after liposome transfection. CD59 could reduce blood lipid levels, promote the expression of anti-apoptotic Bcl-2 protein and inhibit pro-apoptotic Fas proteins, so finally lead to degradation of apoptosis levels of endothelial cells. In addition, Cyclin D 1 protein and CDK4 mRNA levels were restrained by CD59 so as to inhibit the proliferation of smooth muscle cells. CD59 could inhibit the formation of atherosclerotic vulnerable plaque by suppressing the MMP-2 expression, which was further confirmed by HE staining. The anti-atherosclerotic effects were enhanced with the increase of CD59 gene dose. Conclusions CD59 could lower blood lipid levels, positively regulate cell cycle, maintain the stability of cell proliferation and apoptosis of aorta cells, slow down the development of atherosclerotic vulnerable plaque, and finally inhibit the progress of atherosclerosis. So CD59 gene might be a new genetic locus for the therapy of atherosclerosis.

Trastuzumab is an iconic rationally designed targeted therapy for HER2-positive breast cancers. However, the low response rate and development of resistance call for novel approaches for the treatment of patients. Here, we report that concurrent targeting of tumor cells and activation of T cells in the tumor microenvironment results in a synergistic inhibitory effect on tumor growth and overcomes resistance in two distinct PTEN loss–mediated trastuzumab-resistant mammary tumor mouse models. In vivo combination treatment with HER2/Neu antibody and Akt inhibitor triciribine effectively inhibited tumor growth in both models via inhibiting PI3K/AKT and mitogen-activated protein kinase signaling accompanied by increased T-cell infiltration in the tumor microenvironment. We showed that both CD8+ and CD4+ T cells were essential to the optimal antitumor effect of this combination treatment in an IFN-γ–dependent manner. Importantly, the antitumor activities of HER2/Neu antibody and triciribine combination treatment were further improved when coinhibitory receptor cytotoxic T-lymphocyte–associated antigen 4 was blocked to enhance the T-cell response. Our data indicate that multitargeted combinatorial therapies targeting tumor cells and concomitantly enhancing T-cell response in the tumor microenvironment could cooperate to exert maximal therapeutic activity, suggesting a promising clinical strategy for treating trastuzumab-resistant breast cancers and other advanced malignancies. Cancer Res; 72(17); 4417–28. ©2012 AACR.

Prior studies have demonstrated that combined treatment of testosterone with a progestin induces a more rapid and greater suppression of spermatogenesis than testosterone treatment alone. We hypothesized that the suppressive effects of the combination of testosterone undecanoate (TU) injections plus oral levonorgestrel (LNG) on spermatogenesis may be mediated through a greater perturbation of testicular gene expression than TU alone. To test this hypothesis, we performed open testicular biopsy on 12 different adult healthy subjects: 1) four healthy men as controls; 2) four men 2 wk after TU treatment; and 3) four men 2 wk after TU + LNG administration. RNA isolated from biopsies was used for DNA microarray using the Affymetrix Human Genome U133 Plus 2.0 oligonucleotide microarrays. Gene expression with ≥2-fold changes (P < 0.05) compared with control was analyzed using the National Institutes of Health Database for Annotation, Visualization, and Integrated Discovery 2008 resource. The TU treatmen...

To identify differentially expressed genes (DEGs) and further analyze potential biological processes and pathways involved in isoflurane-induced anesthesia. Microarray data (ID: GSE64617) from rat brains treated with exposure to either isoflurane in oxygen (2 %) (test group) or oxygen alone (control group) for 15 min were downloaded from Gene Expression Omnibus. Data pre-processing was performed using the Affy package, followed by DEG screening using the limma package. Protein–protein interactions (PPIs) among DEGs were obtained from STRING (Search Tool for the Retrieval of Interacting Genes/Proteins), and then visualized by constructing a network using Cytoscape. Functional and pathway enrichment analyses were further implemented to identify the biological processes and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways enriched by DEGs. A total of 240 DEGs were identified between the test and control groups, including 128 up-regulated DEGs and 112 down-regulated DEGs in the test group, of which 17 DEGs with a connectivity degree >4 were identified as hub genes (e.g., Pik3r1 and Pik3r2) in the constructed PPI network. Additionally, Slc17a7 and Camk4 interacted with Syn1, and Pik3r1 interacted with Pik3r2. Enrichment analysis further revealed the significantly enriched biological processes of ‘synaptic transmission’, ‘cell–cell signaling’ and ‘transmission of nerve impulse’ (e.g., Slc17a7, Camk4, Syn1, Gria1, Prkcg and Lphn1), as well as KEGG pathways including ‘focal adhesion’, ‘Fc γ R-mediated phagocytosis’ (e.g., Prkcg, Pik3r1 and Pik3r2), and ‘regulation of actin cytoskeleton’ (e.g., Pik3r1 and Pik3r2). The identified DEGs significantly enriched in biological processes and KEGG pathways might be implicated in isoflurane-induced anesthesia.

The Qira black sheep and the Hetian sheep are two local breeds in the Northwest of China, which are characterized by high-fecundity and low-fecundity breed respectively. The elucidation of mRNA expression profiles in the ovaries among different sheep breeds representing fecundity extremes will helpful for identification and utilization of major prolificacy genes in sheep. In the present study, we performed RNA-seq technology to compare the difference in ovarian mRNA expression profiles between Qira black sheep and Hetian sheep. From the Qira black sheep and the Hetian sheep libraries, we obtained a total of 11,747,582 and 11,879,968 sequencing reads, respectively. After aligning to the reference sequences, the two libraries included 16,763 and 16,814 genes respectively. A total of 1,252 genes were significantly differentially expressed at Hetian sheep compared with Qira black sheep. Eight differentially expressed genes were randomly selected for validation by real-time RT-PCR. This study provides a basic data for future research of the sheep reproduction.

FAR-RED ELONGATED HYPOCOTYLS3 (FHY3) and FAR-RED-IMPAIRED RESPONSE1 (FAR1), initially identified as crucial components of phytochrome A (phyA)-mediated far-red (FR) light signaling in Arabidopsis thaliana, are the founding members of the FAR1-related sequence (FRS) family of transcription factors present in most angiosperms. These proteins share extensive similarity with the Mutator-like transposases, indicative of their evolutionary history of 'molecular domestication'. Here we review emerging multifaceted roles of FHY3/FAR1 in diverse developmental and physiological processes, including UV-B signaling, circadian clock entrainment, flowering, chloroplast biogenesis, chlorophyll biosynthesis, programmed cell death, reactive oxygen species (ROS) homeostasis, abscisic acid (ABA) signaling, and branching. The domestication of FHY3/FAR1 may enable angiosperms to better integrate various endogenous and exogenous signals for coordinated regulation of growth and development, thus enhancing their fitness and adaptation.

The beta-catenin pathway plays a critical role in the pathogenesis of certain types of cancers. To gain insight into mechanisms by which altered receptor tyrosine kinases regulate cytoplasmic beta-catenin accumulation, the effect of an oncogenic receptor originated from Nantes (RON) variant on beta-catenin accumulation and the role of beta-catenin in RON-mediated tumorigenic activities were studied. In NIH3T3 cells harboring oncogenic variant RONDelta160, increased beta-catenin accumulation with tyrosine phosphorylation and nuclear translocation was observed. Overexpression of RONDelta160 also resulted in increased expression of beta-catenin target genes c-myc and cyclin D1. By analyzing cellular proteins that regulate beta-catenin stabilities, it was found that RONDelta160 activates the protein disheveled (DVL) and inactivates glycogen synthase kinase-3beta by Ser-9 residue phosphorylation. These effects were channeled by RONDelta160-activated PI 3-kinase-AKT pathways that are sensitive to specific inhibitors, such as wortmannin, but not to other chemical inhibitors. Silencing RONDelta160 expression by specific small interfering RNA blocked not only beta-catenin expression but also c-myc and cyclin D1 expression, suggesting that RON expression is required for the activation of the beta-catenin signaling pathway. Moreover, it was found that knockdown of the beta-catenin gene expression by small interfering RNA techniques reduces significantly the RONDelta160-mediated NIH3T3 cell proliferation, focus-forming activities and anchorage-independent growth. Thus, the oncogenic RON variant regulates beta-catenin stabilities through activation of DVL and inactivation of glycogen synthase kinase-3beta. The activated beta-catenin cascade is one of the pathways involved in tumorigenic activities mediated by the oncogenic RON variant.

In the present study, both zebrafish and medaka vitellogenin genes have been isolated and used as a biomarker to compare the two small aquarium fish in response to estrogen treatment and thus to evaluate the two fish models in development of a biomonitoring system for environmental estrogens. The isolated zebrafish vitellogenin gene, zvtg1, is the most abundantly expressed vitellogenin gene in zebrafish and its complete protein sequence of 1360 amino acids was deduced from a genomic and a cDNA clone. The isolated medaka vitellogenin (mvtg1) genomic clone covers 1053 amino acids in the N-terminal. Both zebrafish zvtg1 and medaka mvtg1 are specifically expressed in female liver and their expression can be induced by 17beta-estradiol (E2) in male fish both by intramuscular injection and immersion treatment. A real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for quantification of vitellogenin mRNA level in both control fish and E2-treated fish. The lowest-observed-effect concentrations of E2 for the induction of vitellogenin mRNAs were observed at 1 microg/l for zebrafish and 0.1 microg/l for medaka in a 2-day exposure experiment. Further kinetics studies of the two fish models indicated that medaka was able to respond much faster to E2 treatment than zebrafish, while the zebrafish can attain a much higher level of vitellogenin mRNAs than medaka after a long-term E2 treatment. The implication of these observations may be that the medaka system is better in monitoring acute treatment while the zebrafish system is better in monitoring chronic exposure.

The cysteine protease cathepsin B (Cat B) is important in the progression of tumor cells, however, the function and molecular mechanisms of Cat B in hepatocellular carcinoma (HCC) remain to be elucidated. Our previous study demonstrated that integrin αvβ3 regulated the biological behavior of HCC. The present study demonstrated that Cat B was also important in cell proliferation and apoptosis in HCC. Notably, Cat B was observed to activate the phosphoinositide 3‑kinase (PI3K)/Akt signaling pathway to promote HCC proliferation. Furthermore, inhibition of integrin αvβ3 significantly prevented Cat B‑induced activation of PI3K/Akt and the progression of HCC. Thus, the results of the present study suggested the presence of a Cat B/integrin αvβ3/PI3K/Akt axis in the regulation of the progression of HCC.

Tuberculosis (TB) is a leading cause of global mortality due to infectious diseases. Expression of cyclooxygenase-2 (COX-2) acts as an important influencing factor favoring bacillary survival during TB infection. In this study, we investigated the Mycobacterium tuberculosis proteins recognized by sera from TB patient collected before and after anti-TB therapy by dynamic immunoproteomics and identified a novel immune-regulating protein 3-hydroxyacyl-l-thioester dehydratase Y (HtdY), which could induce COX-2 expression in mouse macrophages. Signaling perturbation data showed that the activation of p38, ERK 1/2 and JNK 1/2 MAPK as well as NF-κB played critical role in this immune response. Taken together, our findings indicated that mycobacterial HtdY might contribute to the persistence of the TB infection by inducing COX-2 expression through MAPK-NF-κB signaling pathway.

BACKGROUND The Eph family of receptor tyrosine kinases and their ephrin ligands are membrane-bound cell-signaling proteins and they play critical regulatory roles in embryonic development and carcinogenesis. Eph receptors require direct cell to cell interaction for activation and they are divided into EphA and EphB receptor classes, depending on their preferential binding affinity for EphrinA or EphrinB ligands. Eph receptors have been documented in breast cancer, but the Ephrin ligands have not been thoroughly investigated. MATERIALS AND METHODS We conducted a systematic assessment of EphrinB1 expression in a set of 75 formalin-fixed paraffin-embedded breast cancers, using immunohistochemical staining (IHC) with a specific antibody, and we examined the relationship between EphrinB1 expression, histopathological parameters, and the expression of estrogen (ER), progesterone (PR), and HER-2 receptors. RESULTS High level expression of EphrinB1 is positively associated with lymph node metastasis (P < 0.0001) and with the presence of HER-2 receptor (P=0.041) and it is more often detected in triple-negative breast carcinomas (P=0.038). No relation was apparent between EphrinB1 expression level and other histopathological parameters, but enhanced EphrinB1 expression is associated with shorter overall survival (P=0.015). CONCLUSION Our analysis demonstrates that EphrinB1 expression is related to the metastasis of breast cancer and its enhanced expression confers a poor prognosis, suggesting that EphrinB1 may be a relevant therapeutic target in breast cancers.

Previously, we identified macrophage-stimulating protein (MSP) as being expressed during hamster lung injury induced by nitrosamine carcinogens. Transient, generalized epithelial-cell hyperplasia during the preneoplastic period, and eventually nonneuroendocrine (non-NE) lung tumors, are known to develop in these nitrosamine-treated hamsters. We wished to test the hypothesis that MSP and its tyrosine kinase receptor, RON, might represent an autocrine/paracrine system involved in the pathogenesis of human nonneuroendocrine lung tumors, the non-small-cell carcinomas (NSCLCs). We found that this occurred in a paracrine fashion in three of eight primary human NSCLCs that expressed messenger RNA (mRNA) for MSP at high levels in histologically normal lung adjacent to the tumor, but not in the primary tumor, together with mRNA for RON in both normal and tumor tissue. MSP and RON could also constitute an autocrine/paracrine system in human NSCLC cell lines: five of 16 cell lines (squamous and adenosquamous) expressed both MSP and RON; and an additional five of 16 cell lines expressed RON without detectable MSP. Although three cases of primary squamous-cell carcinomas expressed MSP (two of three in the tumor and one of three in nonneoplastic lung), mRNA for RON was not detectable in these cases. RON was functional in all tested RON mRNA-positive cell lines, with exogenous MSP inducing RON-mediated tyrosine phosphorylation. Treatment of a RON-positive adenosquamous carcinoma cell line with MSP additionally resulted in increased motility in a cell-migration assay, suggesting that MSP might promote cell migration of some NSCLCs. In conclusion, MSP and RON might represent an autocrine/paracrine system involved in the pathogenesis of lung cancer, although the nature of the biologic responses in different cell types might vary considerably.

T-bet and GATA-3 are known to regulate cytokine expression in T lymphocytes, and cytokines have been implicated in endometrial regulation and implantation. Previous work showed that female steroid hormones modulate the expression of T-bet in endometrial epithelial cells, suggesting a mechanism for local immune regulation in the human endometrium. We hypothesized that stromal cells are involved in immune regulation, as they have been shown to exert paracrine effects on other endometrial cells and compartments and also secrete cytokines. The objective of this study was to examine the modulation of the gene expression of T-bet and GATA-3, and of the cytokines interferon γ (IFN-γ) and interleukin 4 (IL-4), by female steroid hormones, in human endometrial stromal cells (HESC) in long-term cultures (30 days) mimicking the normal menstrual cycle. T-bet and GATA-3 messenger RNA (mRNA) expression was detected by real-time polymerase chain reaction, and intracellular protein production was demonstrated by immunoblotting. In addition, secretion of IL-4 and IL-15 was measured by enzyme-linked immunosorbent assay. T-bet and IL-4 mRNA expression increased and GATA-3 decreased under decidualization conditions; IFN-γ was not detected. Secretion of IL-15 increased during decidualization, and IL-15 upregulated T-bet gene expression. In conclusion, gene expression of T-bet and GATA-3 by endometrial stromal cells is under hormonal conditions mimicking decidualization, and the results are consistent with an autocrine regulatory mechanism of IL-15 secretion and T-bet expression.

Macrophage stimulating protein (MSP) is a chemotactic factor for murine peritoneal macrophages. The receptor for human MSP was recently identified as the ron gene product, a transmembrane protein tyrosine kinase cloned from a human keratinocyte cDNA library. Here we report that MSP induced proliferation of murine primary keratinocytes and established keratinocyte cell lines in a concentration-dependent manner. The growth efficacy of MSP was comparable to that of epidermal growth factor and keratinocyte growth factor. In three of four cell lines tested in a chemotaxis chamber, MSP also stimulated migration of keratinocytes on a collagen type IV substratum. The action of MSP was mediated by specific binding of MSP to the STK gene product, a murine homologue of the RON MSP receptor. Binding of MSP to keratinocyte STK induced phosphorylation of the 150 kDa STK beta chain. Herbimycin A, a protein tyrosine kinase inhibitor, blocked MSP-mediated phosphorylation of the STK receptor as well as proliferation of keratinocytes, suggesting the importance of tyrosine kinase activity for transduction of the message delivered by MSP. Previously, the only known target cell for MSP was the resident peritoneal macrophage. These studies establish the keratinocyte as a new target cell for MSP. The action of MSP on keratinocytes may have implications for tissue repair, wound healing, and tumor growth.

IL-4 causes hematopoietic cells to proliferate and express a series of genes, including CD23. We examined whether IL-4-mediated growth, as measured by 4PS phosphorylation, and gene induction were similarly controlled. Studies of M12.4.1 cells expressing human IL-4R truncation mutants indicated that the region between amino acids 557–657 is necessary for full gene expression, which correlated with Stat6 DNA binding activity. This region was not required for 4PS phosphorylation. Tyrosine-to-phenylalanine mutations in the interval between amino acids 557–657 revealed that as long as one tyrosine remained unmutated, CD23 was fully induced. When all three tyrosines were mutated, the receptor was unable to induce CD23. The results indicate that growth regulation and gene expression are principally controlled by distinct regions of IL-4R.

OBJECTIVE To observe the effects of salidroside on carbohydrate metabolism and differentiation of 3T3-L1 adipocytes. METHODS 3H-glucose uptaking rate in adipocytes treated by salidroside was detected. Adipocytes treated by salidroside in differentiation were stained by oil red O and analyzed by spectrophotography quantitatively. The expression of PPAR-gamma and C/EBP-alpha mRNA relating to differentiation of adipocytes was detected by reverse transcription PCR. RESULTS 3H-glucose uptaking rate in the salidroside group was 110.4%, higher than that in the blank control group (P < 0.01). Salidroside suppressed the differentiation of 3T3-L1 pre-adipocytes and down-regulated the expression of PPAR-gamma and C/EBP-alpha mRNA (P < 0.01, vs control group). CONCLUSION Salidroside promotes the 3H-glucose uptaking, suppresses the differentiation and down-regulates the expression of PPAR-gamma and C/EBP-alpha mRNA in 3T3-L1 adipocytes.

Mesenchymal stem cells (MSCs) from adult bone marrow maintain their self-renewal ability and the ability to differentiate into osteoblast. Thus, adult bone marrow MSCs play a key role in the regeneration of bone tissue. Previous studies indicated that TLR4 is expressed in MSCs and is critical in regulating the fate decision of MSCs. However, the exact functional role and underlying mechanisms of how TLR4 regulate bone marrow MSC proliferation and differentiation are unclear. Here, we found that activated TLR4 by its ligand LPS promoted the proliferation and osteogenic differentiation of MSCs in vitro. TLR4 activation by LPS also increased cytokine IL-6 and IL-1β production in MSCs. In addition, LPS treatment has no effect on inducing cell death of MSCs. Deletion of TLR4 expression in MSCs completely eliminated the effects of LPS on MSC proliferation, osteogenic differentiation and cytokine production. We also found that the mRNA and protein expression of Wnt3a and Wnt5a, two important factors in regulating MSC fate decision, was upregulated in a TLR4-dependent manner. Silencing Wnt3a with specific siRNA remarkably inhibited TLR4-induced MSC proliferation, while Wnt5a specific siRNA treatment significantly antagonized TLR4-induced MSC osteogenic differentiation. These results together suggested that TLR4 regulates bone marrow MSC proliferation and osteogenic differentiation through Wnt3a and Wnt5a signaling. These finding provide new data to understand the role and the molecular mechanisms of TLR4 in regulating bone marrow MSC functions. These data also provide new insight in developing new therapy in bone regeneration using MSCs by modulating TLR4 and Wnt signaling activity.

Insulin-like growth factors (Igfs) are implicated in a wide variety of physiological roles in teleost gonadal development and reproduction. In the present study, igf3 mRNA expression in the tilapia ovary was found to be higher than in the testis from 5 to 40 days after hatching (dah) but was lower than that in testis from 50 to 70 dah. Consistently, Igf3 protein signal was detected in the somatic cells of XX and XY gonads from 10 dah until adulthood by immunohistochemistry, using a specific Igf3 polyclonal antibody. Incubation of ovarian and testicular cells in primary culture with recombinant Igf3 significantly increased nr5a1, foxl2, dmrt1, cyp19a1a, cyp11a1, cyp11b2, hsd3b2 , and cyp17a1 expression in a time- and dose-dependent manner. Promoter analysis using luciferase assays in HEK293 cells revealed that igf3 promoter activity was directly activated by Nr5a1 (Sf1) and further enhanced by Foxl2, Nr0b1a (Dax1), and Nr0b1b (Dax2) but repressed by Dmrt1 and estrogen receptor (Esr1, Esr2a, or Esr2b) along with 17beta-estradiol treatment. In addition, igf3 promoter activity was increased slightly by forskolin treatment alone but synergistically up-regulated by transfection with nr5a1. These in vitro results correlated well with the expression profile of igf3 during early gonad differentiation. Our results indicated that igf3 is involved in fish gonad steroidogenesis because of its ability to regulate the expression of foxl2, dmrt1, and nr5a1 and steroidogenic enzymes. The expression of igf3 is in turn regulated by transcription factors Foxl2, Dmrt1, and Nr5a1, as well as by 17beta-estradiol treatment.

Although ginseng and related herbs have a long history of utility for various health benefits, their application in cancer therapy and underlying mechanisms of action are not fully understood. Our recent work has shown that 20(S)-25-methoxyl-dammarane-3β, 12β, 20-triol (25-OCH(3)-PPD), a newly identified ginsenoside from Panax notoginseng, exerts activities against a variety of cancer cells in vitro and in vivo. This study was designed to investigate its anti-breast cancer activity and the underlying mechanisms of action. We observed that 25-OCH(3)-PPD decreased the survival of breast cancer cells by induction of apoptosis and G1 phase arrest and inhibited the growth of breast cancer xenografts in vivo. We further demonstrated that, in a dose- and time-dependent manner, 25-OCH(3)-PPD inhibited MDM2 expression at both transcriptional and post-translational levels in human breast cancer cells with various p53 statuses (wild type and mutant). Moreover, 25-OCH(3)-PPD inhibited in vitro cell migration, reduced the expression of epithelial-to-mesenchymal transition (EMT) markers, and prevented in vivo metastasis of breast cancer. In summary, 25-OCH(3)-PPD is a potential therapeutic and anti-metastatic agent for human breast cancer through down-regulating MDM2. Further preclinical and clinical development of this agent is warranted.

JKA97, a benzylidene analog of harmine, has been found to be a promising drug candidate for human cancer therapy, although the underlying molecular mechanisms have not been fully demonstrated. In this study, we evaluated the effects of JKA97 on human breast cancer cells in vitro and in vivo. JKA97 inhibited the growth and proliferation of MCF7 (p53 wild-type), MCF7 (p53 knockdown), and MDA-MB-468 (p53 mutant) cells in a dose-dependent manner. Treatment with JKA97 arrested breast cancer cells in G1 phase and induced apoptosis. JKA97 also significantly suppressed the growth of MCF7 and MDA-MB-468 xenograft tumors. It regulated the expression levels of G1 phase regulators, such as p21, p27, cyclinE, and cylinD1. JKA97 activated p21 transcription, independent of p53, but had little effect on p21 protein stability/degradation. In summary, our results suggest that JKA97 inhibits human breast cancer cell growth through activating p21, independent of p53, which provides a basis for developing this compound as a novel drug for human breast cancer therapy.

Using GeneChip to analyze the changes in genes expression of brain potassium, sodium and calcium channels after chronic treatment with nicotine.Animals were treated with nicotine at the doses of 2.4 mg/kg sc. twice a day for 14 days. RNA was extracted from the whole brain samples and converted to double-stranded cDNA and then to biotinylated cRNA. The biotinylated cRNA was fragmented, and hybridized to GeneChip (Affymetrix Rat Neurobiology U34). The chips were scanned with a probe array scanner, and the data were analyzed with the Affymetrix Microarray Analysis Suite (MAS). The GeneChip data were confirmed u sing RT-PCR.After treatment with chronic nicotine, transcripts of potassium, sodium and calcium channels showed altered expression. K+ channel: outward rectifier K+ channel and Ca2(+)-activated K+ channel were down-regulated, other voltage-dependent K+ channel including Kv2.3r were up-regulated. Voltage-dependent Na+ channel: beta2 subunit was increased, alpha subunit and beta1 subunit were decreased. Beta3 subunit of Ca2+ channel was up-regulated.Chronic exposure to nicotine not only desensitized nicotinic receptors, but also effected genes expression, of important ion channels, such as sodium channels, potassium channels and calcium channels.

The present study described the effects of iptakalim and pinacidil, which belong to two different chemical structures of potassium channel opener (KCO), on mRNA expression for subunits of ATP-sensitive potassium channel (KATP), including SUR1, SUR2, Kir6.1, and Kir6.2.Reverse transcription-polymerase chain reaction (RT-PCR) using primers specific for these four subunit genes was performed on total RNA from rat tissues of heart, aortic smooth muscle and tail artery smooth muscle, after iptakalim and pinacidil administration for one week.Compared with the normal control group without drug administration, in heart tissue, iptakalim and pinacidil showed no significant effects on mRNA levels of the four subunits of KATP. In endothelium-free aortic smooth muscle, SUR2 mRNA level was up-regulated significantly in pinacidil group, while no subunits were changed by iptakalim treatment. In endothelium--free tail artery smooth muscle, Kir6.1 and Kir6.2 mRNA levels were reduced significantly in iptakalim group, and SUR2 and Kir6. 1 in pinacidil group.The gene expression patterns of KATP were different among tissues of heart, large artery and small artery. Iptakalim selectively down-regulated the mRNA levels of Kir6.1 and Kir6.2 in smooth muscle of small arteries, and the regulation effect of iptakalim on KATP was different from that of pinacidil in cardiovascular system.

MicroRNAs (miRNAs) are 21–24 nucleotide riboregulators, which selectively repress gene expression through transcript cleavage and/or translational inhibition. It was thought that most plant miRNAs act through target transcript cleavage due to the high degree of complementarity between miRNAs and their targets. However, recent studies have suggested widespread translational inhibition by miRNAs in plants. The mechanisms underlining translational inhibition by plant miRNAs are largely unknown, but existing evidence has indicated that plants and animals share some mechanistic similarity of translational inhibition. Translational inhibition by miRNAs has been shown to regulate floral patterning, floral timing, and stress responses. This chapter covers recent progress on plant miRNA-mediated translational control.

To analyze the expression of midkine (MK) gene in acute leukemia patients, and explore the relationship between the gene and leukemia.The MK gene expression levels were detected by real-time quantitative RT-PCR (RQ-RT-PCR) in bone marrow (BM) of 181 acute leukemia (AL) patients and 31 normal controls.MK gene was expressed in all AL patients, normal controls and AL patients in complete remission (CR). Compared with that in control group and CR group, MK gene expression was significantly increased in AL patients (P0.01 and P0.05, respectively). No statistical difference was found between CR group and control group. The expression of MK showed a notable increase in all B-ALL subtypes (including pro-B-ALL, common-B-ALL and pre-B-ALL) as well as in adult and childhood B-ALL patients (P0.01). Moreover, the gene expression in B-ALL was also significantly higher than that in TALL, HAL and FAB subtypes of AML (P0.01). In addition, M2 patients showed significantly increased in MK expression compared with that in normal controls (P0.01) and in other FAB subtypes of AML (P0.05). Median MK expression level in M3 patients was also significantly higher than that in normal controls (P0.05), but there was no statistical difference between M3 and other AL subtypes excepting for M2 and B-ALL. MK expression in CD34 positive patients was significantly higher than that in CD34 negative ones (P0.01) and within M2 patients, MK expression was higher in patients with t (8 ;21) than in those without the translocation (P0.05).MK gene expression is increased with different levels in B-ALL, M2 and M3 patients, which provides novel insights into the leukemogenesis of acute leukemia.

To explore the basis of metastasis, we compared the human breast cancer lines MCF-7 and MDA-MB453, which have low invasive ability, with their sublines MCF7-I4 and MDA-MB453-I4 with high invasive ability for gene expression and signaling pathways. We previously showed that the I4 lines had dramatically elevated levels of Twist compared with their parental lines. In this study, we observed significantly increased STAT3 Tyr(705) phosphorylation, but not the STAT3 protein levels, in the I4 lines. Activation of STAT3 by interleukin-6 or expression of activated Src induced Twist expression at protein and mRNA levels. Inhibiting STAT3 by a small molecule inhibitor, JSI-124, STAT3 small hairpin RNAs, or dominant negative STAT3 resulted in significant reduction of Twist protein and mRNA expression. STAT3 directly bound to the second proximal STAT3-binding site on the human Twist promoter and activated its transcriptional activity. Inhibition of STAT3 reduced migration, invasion, and colony formation of the I4 cells. Ectopic expression of Twist significantly rescued those phenotypes. Ten normal and 46 tumor specimens of breast tissues were examined for activation of STAT3 and expression of Twist. There was a strong correlation between Tyr(705) p-STAT3 and Twist level in the late stage tumor tissues. Our results indicate that activated STAT3 transcriptionally induces Twist, which plays an important role in promoting migration, invasion, and anchorage-independent growth. Together with our previous observation that Twist transcriptionally induces AKT2 to mediate Twist-promoted oncogenic functions, we conclude that STAT3, Twist, and AKT2 form a functional signaling axis to regulate pivotal oncogenic properties of cancer cells.

Discovering gene co-regulatory relationships is one of most important research in DNA microarray data analysis. The problem of gene specific co-regulation discovery is to, for a particular gene of interest (called target gene), identify the condition subsets where strong gene co-regulations of the target gene are observed and its co-regulated genes in these condition subsets. The co-regulations are local in the sense that they occur in some subsets of full experimental conditions. The study on this problem can contribute to better understanding and characterizing the target gene during the biological activity involved. In this paper, we propose an innovative method for finding gene specific co-regulations using genetic algorithm (GA). A sliding window is used to delimit the allowed length of conditions in which gene co-regulations occur and anad hocGA, called the progressive GA, is performed in each window position to find those condition subsets having high fitness. It is called progressive because the initial population for the GA in a window position inherits the top-ranked individuals obtained in its preceding window position, enabling the GA to achieve a better accuracy than the non-progressive algorithm.kNN Lookup Table is utilized to substantially speed up fitness evaluation in the GA. Experimental results with a real-life gene expression data demonstrate the efficiency and effectiveness of our technique in discovering gene specific co-regulations.

The problem of gene specific co-regulation discovery is that, for a particular gene of interest, identify its closely coregulated genes and the associated subsets of experimental conditions in which such co-regulations occur. The coregulations are local in the sense that they occur in some subsets of full experimental conditions. In this paper, we propose an innovative method for finding gene specific coregulations using genetic algorithm (GA). Two novel ad hoc GAs, the single-stage and two-stage progressive GA, are proposed. They are called progressive because the initial population for the GA in a window position inherits the top-ranked individuals obtained in the preceding window position, enabling them to achieve better accuracy than the non-progressive algorithm. Experimental results with reallife gene expression data demonstrate the efficiency and effectiveness of our technique in discovering gene specific coregulations.

Two groups of rats were used to examine the effect of pioglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, on rat hearts using an in vivo model of ischemia-reperfusion (I/R) to elucidate potential mechanisms. One group was the 30-min reperfusion group, which was further subdivided into sham (n = 5), vehicle (n = 6) and pioglitazone (3 mg·kg–1, n = 7) treatment groups with 30 min ischemia followed by 30 min reperfusion to detect data related to cardiac function and the area of myocardial infarction. The other group was the 120-min reperfusion group, subdivided into sham (n = 5), vehicle (n = 6), and pioglitazone 0.3 mg·kg–1 (n = 6), 1 mg·kg–1 (n = 7) and 3 mg·kg–1 (n = 6) treatment groups. Immunohistochemistry, in situ hybridization, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and DNA agarose gel electrophoresis were performed to detect apoptosis and expressions of Bax, Bcl-2, caspase 3, MMP-2 and PPARγ protein, and MMP-2 and PPARγ mRNA. We found that, after acute treatment with pioglitazone, the ratio of necrosis to area at risk decreased by 28% (p < 0.01) and that of necrosis to left ventricle was reduced by 32% (p < 0.01), compared with the vehicle group. Heart rate and +dp/dtmax, representing the cardiac systolic function, as well as –dp/dtmax, the indicator of cardiac diastolic function, improved significantly at 1 and 30 min after reperfusion (p < 0.05–0.01). Furthermore, myocardial apoptosis was significantly suppressed by acute treatment with pioglitazone as evidenced by the decreased number of TUNEL-positive myocytes and DNA ladder, enhanced Bcl-2 protein expression, reduced Bax and caspase 3 protein expression in a dose-dependent manner compared with vehicle-treated rats. In addition, acute treatment with pioglitazone dose-dependently increased PPARγ expression and decreased MMP-2 expression at protein and mRNA levels. Our findings demonstrate that a PPARγ agonist may protect the heart from I/R injury. The protective effect is likely to occur by reducing cardiomyocyte apoptosis and inhibiting MMP-2.