A Carbonic Anhydrase, ZmCA4, Contributes to Photosynthetic Efficiency and Modulates CO2 Signaling Gene Expression by Interacting with Aquaporin ZmPIP2;6 in Maize.

Carbonic anhydrase (CA) catalyzes the reversible CO2 hydration reaction that produces bicarbonate for phosphoenolpyruvate carboxylase (PEPC). This is the initial step for transmitting the CO2 signal in C4 photosynthesis. However, it remains unknown whether the maize (Zea mays L.) CA gene, ZmCA4, plays a role in the maize photosynthesis process. In our study, we found that ZmCA4 was relatively highly expressed in leaves and localized in the chloroplast and the plasma membrane of mesophyll protoplasts. Knock-out of ZmCA4 reduced CA activity, while overexpression of ZmCA4 increased rubisco activity, as well as the quantum yield and relative electron transport rate in photosystem II. Overexpression of ZmCA4 enhanced maize yield-related traits. Moreover, ZmCA4 interacted with aquaporin ZmPIP2;6 in bimolecular fluorescence complementation and co-immunoprecipitation experiments. The double-knock-out mutant for ZmPIP2;6 and ZmCA4 genes showed reductions in its growth, CA and PEPC activities, assimilation rate and photosystem activity. RNA-Seq analysis revealed that the expression of other ZmCAs, ZmPIPs, as well as CO2 signaling pathway homologous genes, and photosynthetic-related genes was all altered in the double-knock-out mutant compared with the wild type. Altogether, our study's findings point to a critical role of ZmCA4 in determining photosynthetic capacity and modulating CO2 signaling regulation via its interaction with ZmPIP2;6, thus providing insight into the potential genetic value of ZmCA4 for maize yield improvement.

Genome-wide investigation of the LARP gene family: focus on functional identification and transcriptome profiling of ZmLARP6c1 in maize pollen.

BACKGROUND: The La-related proteins (LARPs) are a superfamily of RNA-binding proteins associated with regulation of gene expression. Evidence points to an important role for post-transcriptional control of gene expression in germinating pollen tubes, which could be aided by RNA-binding proteins. RESULTS: In this study, a genome-wide investigation of the LARP proteins in eight plant species was performed. The LARP proteins were classified into three families based on a phylogenetic analysis. The gene structure, conserved motifs, cis-acting elements in the promoter, and gene expression profiles were investigated to provide a comprehensive overview of the evolutionary history and potential functions of ZmLARP genes in maize. Moreover, ZmLARP6c1 was specifically expressed in pollen and ZmLARP6c1 was localized to the nucleus and cytoplasm in maize protoplasts. Overexpression of ZmLARP6c1 enhanced the percentage pollen germination compared with that of wild-type pollen. In addition, transcriptome profiling analysis revealed that differentially expressed genes included PABP homologous genes and genes involved in jasmonic acid and abscisic acid biosynthesis, metabolism, signaling pathways and response in a Zmlarp6c1::Ds mutant and ZmLARP6c1-overexpression line compared with the corresponding wild type. CONCLUSIONS: The findings provide a basis for further evolutionary and functional analyses, and provide insight into the critical regulatory function of ZmLARP6c1 in maize pollen germination.

Microplastics in the Atmosphere and Water Bodies of Coastal Agglomerations: A Mini-Review.

Microplastics are ubiquitously in various environments from the equator to the poles. Coastal agglomerations act as both a source and sink connecting the global microplastic cycles of oceans and continents. While the problem of microplastics is particularly severe and complex in the coastal zones, where both inland and marine pollution are concentrated, the present study aimed to provide hot topics and trends of coastal urban microplastic studies and to review the researches on microplastic pollution in the atmosphere and water bodies in coastal agglomerations in terms of characteristics, behavior, and health threat of microplastics. The results of the bibliometric analysis showed an increase in the annual output of microplastic research. Research hot topics and clusters were analyzed using the VOSviewer. Characteristics of microplastics varied in abundance, size, and polymer type in different environments and countries. Furthermore, coastal cities are taken as a system to sort out the input, output, and internal transmission pathways of microplastics. The health threat of microplastics to urban residents was briefly reviewed and the exposure and health risks of microplastics to infants and young children were of particular concern. Detailed and comprehensive studies on intervention and reduction in the transmission of microplastics between the atmosphere and water bodies, whether microplastics are harmful to infants and young children, and measures to reduce the risk of microplastic exposure are needed.

Challenges, Coping and Resources: A Thematic Analysis of Foreign Teachers' Experience of Cultural Adaptation in China.

The aim of this study was to explore foreign teachers' experience of cultural and life adaptation in China, including the difficulties, emotional stress, and challenges they confronted, as well as the useful coping strategies and resources that facilitated their cultural adaptation. Ten foreign professors who taught in China were recruited from three Chinese universities. Semi-structured interviews on the participants' experience of cross-cultural adaptation in China were conducted. Thematic analysis of the transcribed dialogs was performed, and five core themes emerged: multiple challenges, mixed negative emotions, active coping and insulation, rich supportive resources, and personal traits. In the cross-cultural adaptation process in China, the main challenges for foreign teachers were language barriers; a lack of supportive relationships; and conflicts in perceptions related to teaching, privacy, and boundaries. Useful coping strategies included seeking interpersonal interactions that provided emotional and instrumental support, developing localized teaching models, and maintaining connections with the culture of origin. Under circumstances with convenient living facilities and supportive interpersonal relationships, these strategies promoted the adaptation of foreign teachers with functional personality traits. The potential correlations among these different themes and the influence of their interactions on foreign teachers' adaptation experience were also discussed. The themes that emerged in our study can help clinicians and university administrators develop therapy methods and support systems for foreign teachers who work in Chinese universities.

Interleukin-6 regulation of AMP-activated protein kinase. Potential role in the systemic response to exercise and prevention of the metabolic syndrome.

Interleukin (IL)-6 is a pleiotropic hormone that has both proinflammatory and anti-inflammatory actions. AMP-activated protein kinase (AMPK) is a fuel-sensing enzyme that among its other actions responds to decreases in cellular energy state by enhancing processes that generate ATP and inhibiting others that consume ATP but are not acutely necessary for survival. IL-6 is synthesized and released from skeletal muscle in large amounts during exercise, and in rodents, the resultant increase in its concentration correlates temporally with increases in AMPK activity in multiple tissues. That IL-6 may be responsible in great measure for these increases in AMPK is suggested by the fact it increases AMPK activity both in muscle and adipose tissue in vivo and in incubated muscles and cultured adipocytes. In addition, we have found that AMPK activity is diminished in muscle and adipose tissue of 3-month-old IL-6 knockout (KO) mice at rest and that the absolute increases in AMPK activity in these tissues caused by exercise is diminished compared with control mice. Except for an impaired ability to exercise and to oxidize fatty acids, the IL-6 KO mouse appears normal at 3 months of age. On the other hand, by age 9 months, it manifests many of the abnormalities of the metabolic syndrome including obesity, dyslipidemia, and impaired glucose tolerance. This, plus the association of decreased AMPK activity with similar abnormalities in a number of other rodents, suggests that a decrease in AMPK activity may be a causal factor. Whether increases in IL-6, by virtue of their effects on AMPK, contribute to the reported ability of exercise to diminish the prevalence of type 2 diabetes, coronary heart disease, and other disorders associated with the metabolic syndrome remains to be determined.

A novel platform for accelerated pharmacodynamic profiling for lead optimization of anticancer drug candidates.

Oncology drug discovery is, by definition, a target-rich enterprise. High-throughput screening (HTS) laboratories have supported a wide array of molecularly targeted and chemical genomic approaches for anticancer lead generation, and the number of hits emerging from such campaigns has increased dramatically. Although automation of HTS processes has eliminated primary screening as a bottleneck, the demands on secondary screening in appropriate cell-based assays have increased concomitantly with the numbers of hits delivered to therapeutic area laboratories. The authors describe herein the implementation of a novel platform using off-the-shelf solutions that have allowed them to efficiently characterize hundreds of HTS hits using a palette of Western blot-based pharmacodynamic assays. The platform employs a combination of a flatbed bufferless SDS-PAGE system, a dry ultra-rapid electroblotting apparatus, and a highly sensitive and quantitative infrared imaging system. Cumulatively, this platform has significantly reduced the cycle time for HTS hit evaluation. In addition, the routine use of this platform has resulted in higher quality data that have allowed the development of structure-activity databases that have tangibly improved lead optimization. The authors describe in detail the application of this platform, designated the Accelerated Pharmaco-Dynamic Profiler (APDP), to the annotation of inhibitors of 2 attractive oncology targets, BRAF kinase and Hsp90.

AMPK, the metabolic syndrome and cancer.

The fuel-sensing enzyme 5'-AMP-activated protein kinase (AMPK) has a major role in the regulation of cellular lipid and protein metabolism in response to stimuli such as exercise, changes in fuel availability and the adipocyte-derived hormones leptin and adiponectin. Recent studies indicate that abnormalities in cellular lipid metabolism are involved in the pathogenesis of the metabolic syndrome, possibly because of dysregulation of AMPK and malonyl-CoA, a closely related molecule. As we discuss in this article, several findings also point to a link between AMPK and the growth and/or survival of some cancer cells. Thus, it has been demonstrated recently that the tumor suppressor LKB1 is a kinase that has a major role in phosphorylating and activating AMPK, and that another tumor suppressor, tuberous sclerosis complex 2, is phosphorylated and activated by AMPK. In addition, other studies indicate that mammalian homolog of target of rapamycin (mTOR), which has been implicated in the pathogenesis of insulin resistance and many types of cancer, is inhibited by AMPK.

Regulation of RKIP binding to the N-region of the Raf-1 kinase.

The Raf kinase inhibitory protein (RKIP) binds to Raf-1 interfering with binding of the MEK substrate and potentially also Raf-1 activation. In response to mitogen stimulation RKIP dissociates from Raf-1 and later re-associates. Here, using a combination of mutational approaches, biochemical studies, peptide arrays and plasmon surface resonance (BIAcore), we fine map and characterize a minimal 24 amino acid long RKIP binding domain in the Raf-1 N-region, which consists of constitutive elements at both flanks and a center element that is regulated by phosphorylation and enhances the re-binding of RKIP to Raf-1 in the later phase of mitogen stimulation.

Wound Healing Effect of an in Situ Forming Hydrogel Loading Curcumin-Phospholipid Complex.

Wound is one of most commonly clinical diseases. Curcumin is known to improve wound healing but its bioavailability and therapeutic efficiency are very low. Here we firstly report the application of phospholipid complexes to wound healing. A curcumin-phospholipid complex (CPC) was prepared with a solvent-evaporation method. The X-ray diffraction showed that the CPC was amorphous, which was further demonstrated with the electron microscopy. Furthermore, the CPC was loaded into a poloxamer in situ forming hydrogel (ISG). The CPC ISG showed higher erosion rates than the curcumin ISG due to the amorphous structure of CPC, which could lead to increase in curcumin dissolution. The CPC ISG showed higher wound healing effect than the control on the rat skin wound model especially in the early phase. The epidermal recovery was highly improved by the CPC ISG compared to the control. The CPC ISG is a promising formulation as an effective wound healing dressing.

14-3-3 facilitates insulin-stimulated intracellular trafficking of insulin receptor substrate 1.

The appearance of a complex between tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and PI3K in a high-speed pellet fraction (HSP) is thought to be a key event in insulin action. Conversely, the disappearance of the IRS-1/PI3K complex from this fraction has been linked to insulin desensitization. The present study examines the role of 14-3-3, a specific phospho-serine binding protein, in mediating the disappearance of IRS-1 from the HSP after insulin treatment. An in vitro pull-down assay using recombinant 14-3-3 revealed that insulin enhances the association of 14-3-3 with IRS-1 in cultured adipocytes and that this is completely inhibited by wortmannin. An association of IRS-1 and 14-3-3 was also observed and was maximal after stimulation by insulin, when endogenous proteins were immunoprecipitated. Epidermal growth factor (EGF), 12-O-tetradecanoylphorbol-13-acetate, and okadaic acid, other agents that cause serine/threonine phosphorylation of IRS-1, also stimulated IRS binding to 14-3-3. The enhancement of IRS-1 binding to 14-3-3 by insulin was accompanied by movement of IRS-1 and the p85 subunit of PI3K from the HSP to the cytosol. In keeping with a key role of 14-3-3 in mediating this redistribution of IRS-1, the complexes of IRS-1 and 14-3-3 were found in the cytosol but not in the HSP of insulin-treated cells. In addition, colocalization of IRS-1 and 14-3-3 was observed in the cytoplasm after insulin treatment by confocal microscopy. Finally, the addition of a phosphorylated 14-3-3 binding peptide to an adipocyte homogenate (to remove 14-3-3 from IRS-1) increased the abundance of IRS-1/PI3K complexes in the HSP and decreased their abundance in the cytosol. These findings strongly suggest that 14-3-3 participates in the intracellular trafficking of IRS-1 by promoting the displacement of serine-phosphorylated IRS-1 from particular structures. They also suggest that 14-3-3 proteins could play an integral role in the process of insulin desensitization.

Characterization of Ser338 phosphorylation for Raf-1 activation.

Raf kinases are essential for regulating cell proliferation, survival, and tumorigenesis. However, the mechanisms by which Raf is activated are still incompletely understood. Phosphorylation plays a critical role in Raf activation in response to mitogens. The present study characterizes phosphorylation of Ser338, a crucial event for Raf-1 activation. Here we report that mutation of Lys375 to Met diminishes phosphorylation of Ser338 on both wild type Raf-1 in cells treated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA) and a constitutively active mutant in which Tyr340/Tyr341 are replaced by 2 aspartic acids, a conserved substitution present in natural B-Raf. The loss of Ser338 phosphorylation in these Raf mutants is not engendered by a mutation-induced conformational change, inasmuch as mutation of another site (Ser471 to Ala) in the activation segment also abolishes Ser338 phosphorylation, whereas both the kinase-dead mutants of Raf-1 are phosphorylated well by active Pak1. Furthermore, our data demonstrate that EGF-stimulated phosphorylation of Ser338 is inhibited by Sorafenib, a Raf kinase inhibitor, but not by the MEK inhibitor U0126. Interestingly, a kinase-dead mutation and Sorafenib also markedly reduce phosphorylation of Ser445 on B-Raf, a site equivalent to Raf-1 Ser338. Finally, our data reveal that Ser338 is phosphorylated on inactive Raf-1 by an active mutant of Raf-1 when they are dimerized in cells and that artificial dimerization of Raf-1 causes Ser338 phosphorylation, accompanied by activation of ERK1/2. Altogether, our data suggest that Ser338 on Raf-1 is autophosphorylated in response to mitogens.

MEK inhibition and phosphorylation of serine 4 on B23 are two coincident events in mitosis.

Previous studies have shown that activation of the Raf/MEK/ERK pathway is necessary for G2/M transition. However, as for the activation state of MEK in mitosis the conclusion is not consistent. Here we show that MEK is inhibited in mitosis. In addition, we identify a multifunctional protein named B23 that strongly cross-reacts with a phospho-MEK antibody in mitotic cells. Sequence homology between the N-terminus surrounding Ser 4 of B23 and the Raf phosphorylation site on MEK suggests a mechanism for cross-reaction of the antibody. Thus, mutation of Ser 4 to alanine abolishes cross-reactivity between B23 and the phospho-MEK antibody. Our findings may explain the discrepancy of results obtained with the use of phospho-MEK antibody regarding the activation state of MEK in mitosis.

AMP-activated protein kinase activators can inhibit the growth of prostate cancer cells by multiple mechanisms.

Prostate cancer cells require high rates of de novo fatty acid synthesis and protein synthesis for their rapid growth. We report here that the growth of these cells is markedly diminished by incubation with activators of AMP-activated protein kinase (AMPK), a fuel-sensing enzyme that has been shown to diminish both of these processes in intact tissues. Inhibition of cell growth was observed when AMPK was activated by either 5-aminoimidazole-4-carboxamide riboside (AICAR) or the thiazolidinedione rosiglitazone. Thus, a 90% inhibition of the growth of androgen-independent (DU145, PC3) and androgen-sensitive (LNCaP) cells was achieved after 4 days of exposure to one or both of these agents. Where studied, this was associated with a decrease in the concentration of malonyl CoA, an intermediate of de novo fatty acid synthesis, and an increase in expression of the cell cycle inhibitor p21. In addition, AICAR inhibited two key enzymes involved in protein synthesis, mTOR and p70S6K, and blocked the ability of the androgen R1881 to increase cell growth and the expression of two enzymes for de novo fatty acid synthesis, acetyl CoA carboxylase and fatty acid synthase, in the LNCaP cells. The results suggest that AMPK is a potential target for the treatment of prostate cancer.

Phosphorylation of 338SSYY341 regulates specific interaction between Raf-1 and MEK1.

The present study characterizes the interaction between the Raf-1 kinase domain and MEK1 and examines whether the magnitude of their interaction correlates to the ability of Raf to phosphorylate MEK1. Here we show that the minimal domain required for the Raf kinase activity starts from tryptophan 342. Maximal binding of the Raf kinase domain to MEK1 and its kinase activity are achieved upon phosphorylation of the region (338)SSYY(341) in response to 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA), or mutation of Y340Y341 to aspartic acids. Conversely, the TPA-stimulated MEK binding and kinase activity are diminished when this region is deleted or Ser(338) and Ser(339) are mutated to alanines. We also show that the integrity of the Raf ATP-binding site is necessary for the interaction between Raf-1 and MEK1. Furthermore, two MEK-binding sites are identified; the first is localized between amino acids 325 and 349, and the second is within the region between amino acids 350 and 648. Separately, the binding of each site to MEK1 is weak, but in a cis context, they give rise to a much stronger association, which can be further stimulated by TPA. Finally, we find that tryptophan 342, which is conserved among the Raf family and other protein kinases, is essential for the Ser(338) phosphorylation of the full-length Raf and its binding to MEK1. Taken together, our results indicate that the phosphorylation of Ser(338) and Tyr(341) on Raf exerts an important effect on reconfiguring the two MEK-binding sites. As a result, these two sites coordinate to form a high affinity MEK-binding epitope, leading to a marked increase in Raf kinase activity.

Metabolic and hormonal interactions between muscle and adipose tissue.

From the perspective of a muscle physiologist, adipose tissue has long been perceived predominantly as a fuel reservoir that provides muscle and other tissues with NEFA when exogenous nutrients are insufficient for their energy needs. Recently, studies have established that adipose tissue is also an endocrine organ. Among the hormones it releases are adiponectin and leptin, both of which can activate AMP-activated protein kinase and increase fatty acid oxidation in skeletal muscle and probably other tissues. Deficiencies of leptin or leptin receptor, adiponectin and IL-6 are associated with obesity, insulin resistance and a propensity to type 2 diabetes. In addition, a lack of adiponectin has been linked to atherosclerosis. Whether this pathology reflects a deficient activation of AMP-activated protein kinase in peripheral tissues remains to be determined. Finally, recent studies have suggested that skeletal muscle may also function as an endocrine organ when it releases the cytokine IL-6 into the circulation during sustained exercise. Interestingly, one of the apparent effects of IL-6 is to stimulate lipolysis, causing the release of NEFA from the adipocyte. Thus, hormonal communications exist between the adipocyte and muscle that could enable them to talk to each other. The physiological relevance of this cross talk clearly warrants further study.