A multiple phenotype imputation method for genetic diversity and core collection in Taiwanese vegetable soybean.

Establishment of vegetable soybean (edamame) [Glycine max (L.) Merr.] germplasms has been highly valued in Asia and the United States owing to the increasing market demand for edamame. The idea of core collection (CC) is to shorten the breeding program so as to improve the availability of germplasm resources. However, multidimensional phenotypes typically are highly correlated and have different levels of missing rate, often failing to capture the underlying pattern of germplasms and select CC precisely. These are commonly observed on correlated samples. To overcome such scenario, we introduced the "multiple imputation" (MI) method to iteratively impute missing phenotypes for 46 morphological traits and jointly analyzed high-dimensional imputed missing phenotypes (EC impu ) to explore population structure and relatedness among 200 Taiwanese vegetable soybean accessions. An advanced maximization strategy with a heuristic algorithm and PowerCore was used to evaluate the morphological diversity among the EC impu . In total, 36 accessions (denoted as CC impu ) were efficiently selected representing high diversity and the entire coverage of the EC impu . Only 4 (8.7%) traits showed slightly significant differences between the CC impu and EC impu . Compared to the EC impu , 96% traits retained all characteristics or had a slight diversity loss in the CC impu . The CC impu exhibited a small percentage of significant mean difference (4.51%), and large coincidence rate (98.1%), variable rate (138.76%), and coverage (close to 100%), indicating the representativeness of the EC impu . We noted that the CC impu outperformed the CC raw in evaluation properties, suggesting that the multiple phenotype imputation method has the potential to deal with missing phenotypes in correlated samples efficiently and reliably without re-phenotyping accessions. Our results illustrated a significant role of imputed missing phenotypes in support of the MI-based framework for plant-breeding programs.

Fully automated identification and quantification of five polar pesticides in groundwater by isotope dilution-online solid phase extraction coupled with high-performance liquid chromatography-quadrupole Orbitrap high-resolution mass spectrometry.

A fully automated method for identification and quantification of five polar pesticides in groundwater by isotope dilution-online solid-phase extraction (SPE) coupled with high-performance liquid chromatography-quadrupole Orbitrap high-resolution mass spectrometry was developed. After one step of filtration, an aliquot of a 7.5-ml water sample was automatedly preconcentrated and purified on a turbulent Cyclone SPE column. The analytes were eluted in backflush mode, then separated on an analytical column and acquired by full MS/dd-MS2 scan in negative and positive ions mode. The major parameters for sample loading, cleanup, and elution were optimized in detail. Preconcentration and ionization efficiency were highly improved by using 0.1% acid solution in the mobile phase. The method provided good linearity of calibration coefficients (R2 > 0.995), sensitive method limits of detection (0.5-10.0 ng/L), accurate mass spectra (within 5 ppm error), satisfactory matrix spiking recoveries (98.4% to 109%), and high precision (intraday/interday relative standard deviations 1.57-8.90%). The method was successfully applied to analyze large batch groundwater of National Groundwater Monitoring Project and suspect screening of potential pesticides in groundwater. The study provided a practical alternative for a simple, robust, sensitive, and accurate identification and qualification of five polar pesticides in groundwater.

Solvothermal Synthesis of Humic Acid-Supported CeO2 Nanosheets Composite as High Performance Adsorbent for Congo Red Removal.

Surface properties and structures of materials are essential for their adsorption of pollutants in water. Humic acids (HA)-supported CeO2 nanosheet composites are synthesised by solvothermal method. The size of CeO2 nanosheets are approximately 100-500 nm. The obtained composite exhibits superior adsorption ability for Congo Red (CR) in water, which can be attributed to its unique structure and highly dispersed CeO2 nanosheet. The composite's adsorption behaviour of CR follows a pseudo-second-order mode and Langmuir adsorption model well, and the maximum adsorptive capacity for CR achieves 260 mg g-1. The presence of CeO2 nanosheets enhances surface area and enriches the mesoporous structure of the composites, thereby promoting CR adsorption capacity.

[Acupoint catgut embedding improves dysmenorrhea by suppressing activation of NLRP3 inflammasomes in uterine tissues in primary dysmenorrhea rats].

OBJECTIVE: To observe the effect of acupoint catgut embedding on the expression of nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome, interleukin (IL)-1ß and IL-18 in the uterine tissue of primary dysmenorrhea (PD) rats, so as to explore its underlying mechanisms in improving PD. METHODS: Forty female SD rats were randomly divided into control, model, acupoint catgut embedding and medication groups (n＝10 in each group). The PD model was established by subcutaneous injection of Estradiol Benzoate (0.5 mg/rat on the 1st and 10thday, and 0.2 mg/rat from 2nd to 9thday) and Oxytocin (2 U/rat, i．p.). The catgut embedding was applied to bilateral "Sanyinjiao" (SP6) and "Guanyuan" (CV4) on the 1st and 5th day after modeling. Rats of the medication group were treated by intragastric perfusion of Fenbid (0.8 mL/rat, 125 mg/100 mL) once daily for 10 days. The body writhing times in 30 min were recorded. The histopathological changes of the uterine were observed by H．E. staining. Western blot was used to detect the expression of NLRP 3, caspase-1, IL-1ß and IL-18 in uterine tissues. RESULTS: The body writhing times were notably more in the model group than in the control group (P<0.01), and obviously fewer in both medication and catgut embedding groups than in the model group (P<0.05, P<0.01). After modeling, the rats' endometrium was extensively exfoliated and got swelling, the histopathological score and the expression levels of NLRP3, caspase-1, IL-1ß and IL-18 proteins in the uterus tissue were evidently increased in the model group relevant to the control group (P<0.01). Following the treatment, the degree of endometrial exfoliation and edema of the uterus tissue was lightened, the pathological score was significantly reduced (P<0.01), and the expression levels of caspase-1, IL-1ß and IL-18 protein in uterus tissue were markedly decreased in both acupoint catgut embedding and medication groups (P<0.01, P<0.05). The NLRP3 protein expression was significantly decreased in the acupoint catgut embedding group compared with that in the model group (P<0.01). The therapeutic effect of acupoint catgut embedding was significantly superior to that of medication in reducing writhing times and down-regulating expression of NLPR3 protein (P<0.01). No significant differences were found between catgut embedding and medication in histopathological score, and expression levels of caspase-1, IL-1ß and IL-18 proteins (P>0.05). CONCLUSION: The acupoint catgut embedding can significantly alleviate the symptoms and pathological damage in PD rats, which may be related to its effect in inhibiting the activation of NLRP3 inflammasome in the uterine tissue.

Intermittent hypobaric hypoxia preconditioning induced brain ischemic tolerance by up-regulating glial glutamate transporter-1 in rats.

Several studies showed that the up-regulation of glial glutamate transporter-1 (GLT-1) participates in the acquisition of brain ischemic tolerance induced by cerebral ischemic preconditioning or ceftriaxone pretreatment in rats. To explore whether GLT-1 plays a role in the acquisition of brain ischemic tolerance induced by intermittent hypobaric hypoxia (IH) preconditioning (mimicking 5,000 m high-altitude, 6 h per day, once daily for 28 days), immunohistochemistry and western blot were used to observe the changes in the expression of GLT-1 protein in hippocampal CA1 subfield during the induction of brain ischemic tolerance by IH preconditioning, and the effect of dihydrokainate (DHK), an inhibitor of GLT-1, on the acquisition of brain ischemic tolerance in rats. The basal expression of GLT-1 protein in hippocampal CA1 subfield was significantly up-regulated by IH preconditioning, and at the same time astrocytes were activated by IH preconditioning, which appeared normal soma and aplenty slender processes. The GLT-1 expression was decreased at 7 days after 8-min global brain ischemia. When the rats were pretreated with the IH preconditioning before the global brain ischemia, the down-regulation of GLT-1 protein was prevented clearly. Neuropathological evaluation by thionin staining showed that 200 nmol DHK blocked the protective role of IH preconditioning against delayed neuronal death induced normally by 8-min global brain ischemia. Taken together, the up-regulation of GLT-1 protein participates in the acquisition of brain ischemic tolerance induced by IH preconditioning in rats.

Bacterial LPS up-regulated TLR3 expression is critical for antiviral response in human monocytes: evidence for negative regulation by CYLD.

In the host immune system, the leukocytes are often exposed to multiple pathogens including bacteria and viruses. The principal challenge for the host is to efficiently detect the invading pathogen and mount a rapid defensive response. Leukocytes recognize invading pathogens by directly interacting with pathogen-associated molecular patterns via Toll-like receptors (TLRs) expressed on the leukocyte surfaces. In this study, we provide direct evidence that bacterial LPS enhances the host antiviral response by up-regulating TLR3 expression in human peripheral blood monocytes and monocytic cell lines, THP1 cells. Moreover, LPS induces TLR3 expression via a TLR4-MyD88-IRAK-TRAF6-NF-κB-dependent signaling pathway. Interestingly, CYLD, an important deubiquitinase, acts as a negative regulator of TLR3 induction by LPS. Our study thus provides new insights into a novel role for bacterial infection in enhancing host antiviral response; furthermore, it identifies CYLD for the first time as a critical negative regulator of bacterial LPS-induced response.

Cell type-specific release of matrix-metallo-proteinase-9 by bacterial chemoattractant in human blood phagocytic leukocytes.

Stimulation of phagocytic leukocytes with bacterial chemoattractant resulted in the release of matrix metal-loproteinases (MMPs). Little is known about the mechanisms of bacterial chemoattractant regulation of MMP in phagocytic leukocytes. We report here that the mechanisms of the bacterial chemotactic peptidefMLP-induced MMP -9 release in monocytes appeared to be different from fMLP-stimulated MMP-9 release in neutrophils. In freshly prepared peripheral blood monocytes, fMLP induces MMP-9 release, starting at 8 h after stimulation. These functions of fMLP is accompanied by an increase in TNFα expression, and mediated through the phosphorylation of ERK1/2 in monocytes. However, neutrophil preparations that responded to fMLP with MMP-9 release did not require activation of ERK1/2 and TNFα expression. These results suggest a different role of fMLP in MMP-9 expression in neutrophils and monocytes, and the signal molecules involved in mediating this effect in human blood monocytes stimulated by bacterial chemoattractant.

Synergistic activation of NF-{kappa}B by bacterial chemoattractant and TNF{alpha} is mediated by p38 MAPK-dependent RelA acetylation.

In the host immune system, leukocytes are often exposed to multiple inflammation inducers. NF-κB is of considerable importance in leukocyte function because of its ability to activate the transcription of many proinflammatory immediate-early genes. Tremendous efforts have been made toward understanding how NF-κB is activated by various inducers. However, most research on NF-κB regulation has been focused on understanding how NF-κB is activated by a single inducer. This is unlike the situation in the human immune system where multiple inflammation inducers, including both exogenous and endogenous mediators, are present concurrently. We now present evidence that the formylated peptide f-Met-Leu-Phe (fMLP), a bacterial chemoattractant, synergizes with TNFα to induce NF-κB activation and the resultant inflammatory response in vitro and in vivo. The mechanism of synergistic activation of NF-κB by bacterial fMLP and TNFα may be involved in the induction of RelA acetylation, which is regulated by p38 MAPK. Thus, this study provides direct evidence for the synergistic induction of NF-κB-dependent inflammatory responses by both exogenous and endogenous inducers. The ability of fMLP to synergize with TNFα and activate NF-κB represents a novel and potentially important mechanism through which bacterial fMLP not only attracts leukocytes but also directly contributes to inflammation by synergizing with the endogenous mediator TNFα.

Synergistic activation of leukocytes by bacterial chemoattractants: potential drug targets.

Accumulating evidence demonstrates that bacterial chemoattractants not only attract leukocytes (chemotaxis) but also contribute directly to inflammation by activation of leukocytes to produce a variety of pro-inflammatory cytokines. Recent studies have shown that mixtures of the bacterial chemoattractant fMLP (N-formyl-Met-Leu-Phe) and other bacterial products/components such as LPS (lipopolysaccharide) behave synergistically in activating leukocytes. These results suggest that inflammatory responses are induced by multiple inducers that operate synergistically through multiple signaling pathways. This synergy is likely to play a significant role in the induction of host defense to bacterial infections and in the pathogenesis of inflammatory disorders. These results also demonstrate that the control of inflammation is likely best understood at the level of synergistic regulation of intracellular signaling. The use of pharmacological inhibitors to modulate synergistic molecules is therefore an attractive possibility for the treatment of inflammatory disease. In this review, we will provide a brief summary of recent studies on the regulation of leukocyte functioning by bacterial chemoattractants.

Lipopolysaccharide-driven Th2 cytokine production in macrophages is regulated by both MyD88 and TRAM.

Gram-negative bacterial lipopolysaccharide (LPS) activates macrophages by interacting with Toll-like receptor 4 (TLR4) and triggers the production of various pro-inflammatory Th1 type (type 1) cytokines such as IFNgamma, TNFalpha, and IL8. Though some recent studies cited macrophages as potential sources for Th2 type (type 2) cytokines, little however is known about the intracellular events that lead to LPS-induced type 2 cytokines in macrophages. To understand the mechanisms by which LPS induces type 2 cytokine gene expression, macrophages were stimulated with LPS, and the expression of IL-4 and IL-5 genes were examined. LPS, acting through TLR4, activates both type 1 and type 2 cytokine production both in vitro and in vivo by using macrophages from C3H/HeJ or C3H/HeOuJ mice. Although the baseline level of both TNFalpha and IL-4 protein was very low, TNFalpha was released rapidly after stimulation (within 4 h); however, IL-4 was released after 48 h LPS stimulation in secreted form. Silencing of myeloid differentiation protein (MyD88) and TRIF-related adaptor molecule (TRAM), using small interfering RNA abolished IL-4 induction induced by LPS whereas silencing of TRAM has no effect on TNFalpha induction, thereby indicating that LPS-induced TNFalpha is MyD88-dependent but IL-4 is required both MyD88 and TRAM. These findings suggest a novel function of LPS and the signaling pathways in the induction of IL-4 gene expression.

An atypical protein kinase C (PKC zeta) plays a critical role in lipopolysaccharide-activated NF-kappa B in human peripheral blood monocytes and macrophages.

We have reported that the bacterial LPS induces the activation of NF-kappaB and inflammatory cytokine gene expression and that this requires the activity of small GTPase, RhoA. In this study, we show that an atypical protein kinase C isozyme, PKCzeta, associates functionally with RhoA and that PKCzeta acts as a signaling component downstream of RhoA. Stimulation of monocytes and macrophages with LPS resulted in PKCzeta activation and that inhibition of PKCzeta activity blocks both LPS-stimulated activation of NF-kappaB and IL-1beta gene expression. Our results also indicate that transforming growth factor beta-activated kinase 1 acts as a signaling component downstream of PKCzeta in cytokine gene transcription stimulated by LPS in human peripheral blood monocytes and macrophages. The specificity of this response suggests an important role for the Rho GTPase/PKCzeta/transforming growth factor beta-activated kinase 1/NF-kappaB pathway in host defense and in proinflammatory cytokine synthesis induced by bacterial LPS.

Synergistic induction of inflammation by bacterial products lipopolysaccharide and fMLP: an important microbial pathogenic mechanism.

A wide variety of stimuli have been shown to induce inflammation, but bacteria products/components are considered the major inducers during bacterial infections. We previously demonstrated that bacterial products/components such as LPS, a glycolipid component of the bacterial outer membrane, and formylated peptides (fMLP), a bacterial-derived peptide, induced proinflammatory cytokine gene expression in human peripheral blood monocytes. We now present evidence that mixtures of bacterial products/components LPS and fMLP behave synergistically in the induction of inflammation in vitro and in vivo. Furthermore, our results indicate that the TLR4 and the IKKbeta-IkappaBalpha signaling pathways are involved in the synergistic induction of inflammatory cytokines. The mechanism of synergistic activation of NF-kappaB is depended on nuclear translocation of p65 and phosphorylation of p65 at both Ser536 and Ser276 sites. These results demonstrate an important role for bacterial products/components from lysed bacteria in the pathogenesis of infectious diseases. We believe that this synergistic induction of inflammation by bacterial products LPS and fMLP represents an important pathogenic mechanism during bacterial infection, which may suggest novel therapeutic strategies or targets to minimize host injury following bacterial infection.

A novel protein kinase C (PKCepsilon) is required for fMet-Leu-Phe-induced activation of NF-kappaB in human peripheral blood monocytes.

We have reported that the chemoattractant, fMet-Leu-Phe (fMLP), induces the activation of NF-kappaB in human peripheral blood monocytes and that this requires the activity of small GTPase, RhoA (Huang, S., Chen, L.-Y., Zuraw, B. L., Ye, R. D., and Pan, Z. K. (2001) J. Biol. Chem. 276, 40977-40981). Here we showed that the novel protein kinase C isozyme, PKCepsilon, associates functionally with RhoA in fMLP-stimulated monocytes and that PKCepsilon acted as a signaling component downstream of the GTPase RhoA during fMLP-induced activation of NF-kappaB. Stimulation of monocytes with fMLP resulted in activation of both PKCepsilon and NF-kappaB. This latter activation was largely blocked by specific inhibitors of PKCepsilon by transient expression of a dominant-negative form of PKCepsilon and by PKCepsilon-specific short interfering RNA. These findings demonstrate, for the first time, that fMLP-induced activation of NF-kappaB utilizes a signaling pathway, which requires activity of PKCepsilon, and that PKCepsilon acts as a signaling component downstream of RhoA in cytokine gene transcription stimulated by a chemoattractant. The specificity of this response suggests an important role for the Rho GTPase-PKCepsilon-NF-kappaB pathway in host defense and represents a novel and potentially important mechanism through which fMLP not only attracts leukocytes but may also contribute directly to inflammation.

RhoA and Rac1 signals in fMLP-induced NF-kappaB activation in human blood monocytes.

GTPase RhoA is required for fMet-Leu-Phe (fMLP)-stimulated NF-kappaB activation in human peripheral blood monocytes. Here we have investigated different members of the Rho family of GTPases Rac1, Cdc42, and RhoA in regulating the transcription factor nuclear factor-kappaB (NF-kappaB) in human peripheral blood monocytes. Stimulation of monocytes with fMLP rapidly activated Rac1, Cdc42, and RhoA and cotransfection of the monocytic THP1 cells with dominant negative forms of Rho GTPases, we found that Rac1 and RhoA, but not Cdc42, involved fMLP-stimulated kappaB reporter gene expression. These results indicate that fMLP stimulates three members of the Rho family of GTPases Rac1, Cdc42, and RhoA activity in monocytes, and that Rac1 and RhoA, but not Cdc42, is required for fMLP-induced NF-kappaB activation. Furthermore, our data also suggest that RhoA is mediated by signals independent of Rac1 in NF-kappaB activation in human peripheral blood monocytes stimulated with bacterial products.

A Rho exchange factor mediates fMet-Leu-Phe-induced NF-kappaB activation in human peripheral blood monocytes.

We reported previously that fMLP stimulates NF-kappaB activation, and this function of fMLP requires small GTPase RhoA in human peripheral blood monocytes (Huang, S., Chen, L.-Y., Zuraw, B. L., Ye, R. D., and Pan, Z. K. (2001) J. Biol. Chem. 276, 40977-40981). Here we present evidence that RhoA associates specifically with the guanine nucleotide exchange factor Lbc in human peripheral blood monocytes stimulated with fMLP and that Lbc specifically catalyzes the guanine nucleotide exchange activity of RhoA in human peripheral blood monocytes. Cotransfection of the monocytic THP1 cells with lbc with a kappaB promoter reporter plasmid results in a marked increase in NF-kappaB-mediated reporter gene expression. Finally, Lbc-enhanced NF-kappaB activation is inhibited by a RhoA inhibitor, C3 transferase from Clostridium botulinum. A dominant-negative form of RhoA (T19N) also inhibited Lbc-enhanced reporter gene expression in a kappaB-dependent manner. These results indicate that guanine nucleotide exchange factor Lbc is a novel signal transducer for RhoA-mediated NF-kappaB activation in human peripheral blood monocytes stimulated with bacterial products.

Kaposi's sarcoma-associated herpesvirus K-bZIP is a coregulator of K-Rta: physical association and promoter-dependent transcriptional repression.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus that has been implicated in the pathogenesis of Kaposi's sarcoma and B-cell neoplasms. The genomic organization of KSHV is similar to that of Epstein-Barr virus (EBV). EBV encodes two transcriptional factors, Rta and Zta, which functionally interact to transactivate EBV genes during replication and reactivation from latency. KSHV encodes a basic leucine zipper protein (K-bZIP), a homologue of EBV Zta, and K-Rta, the homologue of EBV Rta. EBV Rta and Zta are strong transcriptional transactivators. Although there is ample evidence that K-Rta is a potent transactivator, the role of K-bZIP as a transcriptional factor is much less clear. In this study, we report that K-bZIP modulates K-Rta function. We show that K-bZIP directly interacts with K-Rta in vivo and in vitro. This association is specific, requiring the basic domain (amino acids 122 to 189) of K-bZIP and a specific region (amino acids 499 to 550) of K-Rta, and can be detected with K-bZIP and K-Rta endogenously expressed in BCBL-1 cells treated with tetradecanoyl phorbol acetate. The functional relevance of this association was revealed by the observation that K-bZIP represses the transactivation of the ORF57 promoter by K-Rta in a dose-dependent manner. K-bZIP lacking the interaction domain fails to repress K-Rta-mediated transactivation; this finding attests to the specificity of the repression. Interestingly, this repression is not observed for the promoter of polyadenylated nuclear (PAN) RNA, another target of K-Rta; thus, repression is promoter dependent. Finally, we provide evidence that the modulation of K-Rta by K-bZIP also occurs in vivo during reactivation of the viral genome in BCBL-1 cells. When K-bZIP is overexpressed in BCBL-1 cells, the level of expression of ORF57 but not PAN RNA is repressed. These data support the model that one function of K-bZIP is to modulate the activity of the transcriptional transactivator K-Rta.

Involvement of protein tyrosine kinase in Toll-like receptor 4-mediated NF-kappa B activation in human peripheral blood monocytes.

Bacterial lipopolysaccharide (LPS) is a powerful activator of the innate immune system. Exposure to LPS induces an inflammatory reaction in the lung mediated primarily by human blood monocytes and alveolar macrophages, which release an array of inflammatory chemokines and cytokines including IL-8, TNF-alpha, IL-1beta, and IL-6. The signaling mechanisms utilized by LPS to stimulate the release of cytokines and chemokines are still incompletely understood. Pretreatment with the protein tyrosine kinase-specific inhibitors genistein and herbimycin A effectively blocked LPS-induced NF-kappaB activation as well as IL-8 gene expression in human peripheral blood monocytes. However, when genistein was added 2 min after the addition of LPS, no inhibition was observed. Utilizing a coimmunoprecipitation assay, we further showed that LPS-stimulated tyrosine phosphorylation of Toll-like receptor 4 (TLR4) may be involved in downstream signaling events induced by LPS. These findings provide evidence that LPS-induced NF-kappaB activation and IL-8 gene expression use a signaling pathway requiring protein tyrosine kinase and that such regulation may occur through tyrosine phosphorylation of TLR4.

IL-1 receptor-associated kinase and low molecular weight GTPase RhoA signal molecules are required for bacterial lipopolysaccharide-induced cytokine gene transcription.

Proinflammatory cytokines such as IL-1, TNF, IL-6, and IL-8 are produced by leukocytes in response to bacteria or bacterial components. A great deal has been learned during the past few years about the synthesis and release of proinflammatory cytokines by leukocytes; however, relatively little is known about the intracellular events that lead to leukocyte proinflammatory cytokine gene transcription. This study examined the signal transduction pathway of IL-8 induction by bacterial LPS. Stimulation of monocytes with LPS rapidly activated RhoA, and pretreatment of monocytes with a RhoA inhibitor, C3 transferase exoenzyme, effectively blocked LPS-induced IL-8 gene expression. Overexpression of dominant negative RhoA (T19N) or IL-1R-associated kinase completely inhibited LPS-stimulated reporter gene expression. Induction of IL-8 was also inhibited by dominant negative IkappaB kinase and myeloid differentiation protein (MyD88). These results indicate that RhoA and IL-1R-associated kinase are novel signal transducers for LPS-induced Toll-like receptor 4-mediated proinflammatory cytokine synthesis in human monocytes.