Gene cloning and functional analysis of yellow green leaf3 (ygl3) gene during the whole-plant growth stage in rice

Chlorophyll is an important photosynthetic pigment in the process of photosynthesis in plants and photosynthetic bacteria. Genes involved in chlorophyll biosynthesis in Arabidopsis and photosynthetic bacteria have been well documented. In rice, however, these genes have not been fully annotated. In this paper, a yellow-green leaf gene, yellow green leaf3 (ygl3) was cloned and analyzed. ygl3 encodes magnesium chelation ChlD (D) subunit, a key enzyme for chlorophyll synthesis, resulting in a yellow-green leaf phenotype in all growth stages in rice. Expression content of ygl3 is highest in the leaf blades, followed by the leaf sheaths, while there is virtually no expression of the gene in the stems and seeds. The sub-cellular structure and protein content of the photosynthetic system of the ygl3 mutant were revealed by transmission electron microscopy, BN-PAGE, and western blotting. The results show that the mutation of the ygl3 gene indirectly leads to a decrease in the protein content of the photosynthetic system and severely obstructs the formation of granum thylakoids.     

Comparative phylogeography of two sympatric beeches in subtropical China: Species‐specific geographic mosaic of lineages

In subtropical China, large‐scale phylogeographic comparisons among multiple sympatric plants with similar ecological preferences are scarce, making generalizations about common response to historical events necessarily tentative. A phylogeographic comparison of two sympatric Chinese beeches (Fagus lucida and F. longipetiolata, 21 and 28 populations, respectively) was conducted to test whether they have responded to historical events in a concerted fashion and to determine whether their phylogeographic structure is exclusively due to Quaternary events or it is also associated with pre‐Quaternary events. Twenty‐three haplotypes were recovered for F. lucida and F. longipetiolata (14 each one and five shared). Both species exhibited a species‐specific mosaic distribution of haplotypes, with many of them being range‐restricted and even private to populations. The two beeches had comparable total haplotype diversity but F. lucida had much higher within‐population diversity than F. longipetiolata. Molecular dating showed that the time to most recent common ancestor of all haplotypes was 6.36 Ma, with most haplotypes differentiating during the Quaternary. [Correction added on 14 October 2013, after first online publication: the time unit has been corrected to ‘6.36’.] Our results support a late Miocene origin and southwards colonization of Chinese beeches when the aridity in Central Asia intensified and the monsoon climate began to dominate the East Asia. During the Quaternary, long‐term isolation in subtropical mountains of China coupled with limited gene flow would have lead to the current species‐specific mosaic distribution of lineages.     

Secretory/releasing proteome-based identification of plasma biomarkers in HBV-associated hepatocellular carcinoma

For successful therapy, hepatocellular carcinoma (HCC) must be detected at an early stage. Herein, we used a proteomic approach to analyze the secretory/releasing proteome of HCC tissues to identify plasma biomarkers. Serum-free conditioned media (CM) were collected from primary cultures of cancerous tissues and surrounding noncancerous tissues. Proteomic analysis of the CM proteins permitted the identification of 1365 proteins. The enriched molecular functions and biological processes of the CM proteins, such as hydrolase activity and catabolic processes, were consistent with the liver being the most important metabolic organ. Moreover, 19% of the proteins were characterized as extracellular or membrane-bound. For validation, secretory proteins involved in transforming growth factor-β signaling pathways were validated in plasma samples. Alphafetoprotein (AFP), metalloproteinase (MMP)1, osteopontin (OPN), and pregnancy-specific beta-1-glycoprotein (PSG)9 were significantly increased in HCC patients. The overall performance of MMP1 and OPN in the diagnosis of HCC remained greater than that of AFP. In addition, this study represents the first report of MMP1 as a biomarker with a higher sensitivity and specificity than AFP. Thus, this study provides a valuable resource of the HCC secretome with the potential to investigate serological biomarkers. MMP1 and OPN could be used as novel biomarkers for the early detection of HCC and to improve the sensitivity of biomarkers compared with AFP.     

δ-Opioid Receptor Activation Modified MicroRNA Expression in the Rat Kidney under Prolonged Hypoxia

Hypoxic/ischemic injury to kidney is a frequently encountered clinical problem with limited therapeutic options. Since microRNAs are differentially involved in hypoxic/ischemic events and δ-opioid receptor (DOR) activation is known to protect against hypoxic/ischemic injury, we speculated on the involvement of DOR activation in altering the microRNA (miRNA) expression in kidney under hypoxic condition. We selected 31 miRNAs based on microarray data for quantitative PCR analysis. Among them, 14 miRNAs were significantly altered after prolonged hypoxia, DOR activation or a combination of both. We found that 1) DOR activation alters miRNA expression profiles in normoxic conditions; 2) hypoxia differentially alters miRNA expression depending on the duration of hypoxia; and 3) DOR activation can modify hypoxia-induced changes in miRNA expression. For example, 10-day hypoxia reduced the level of miR-212 by over 70%, while DOR activation could mimic such reduction even in normoxic kidney. In contrast, the same stress increased miR-29a by >100%, which was reversed following DOR activation. These first data suggest that hypoxia comprehensively modifies the miRNA profile within the kidney, which can be mimicked or modified by DOR activation. Ascertaining the targeted pathways that regulate the diverse cellular and molecular functions of miRNA may provide new insights into potential therapies for hypoxic/ischemic injury of the kidney.     

Assessing the Impact of the National Smoking Ban in Indoor Public Places in China: Evidence from Quit Smoking Related Online Searches

Background

Despite the tremendous economic and health costs imposed on China by tobacco use, China lacks a proactive and systematic tobacco control surveillance and evaluation system, hampering research progress on tobacco-focused surveillance and evaluation studies.

Methods

This paper uses online search query analyses to investigate changes in online search behavior among Chinese Internet users in response to the adoption of the national indoor public place smoking ban. Baidu Index and Google Trends were used to examine the volume of search queries containing three key search terms “Smoking Ban(s),” “Quit Smoking,” and “Electronic Cigarette(s),” along with the news coverage on the smoking ban, for the period 2009–2011.

Findings

Our results show that the announcement and adoption of the indoor public place smoking ban in China generated significant increases in news coverage on smoking bans. There was a strong positive correlation between the media coverage of smoking bans and the volume of “Smoking Ban(s)” and “Quit Smoking” related search queries. The volume of search queries related to “Electronic Cigarette(s)” was also correlated with the smoking ban news coverage.

Interpretation

To the extent it altered smoking-related online searches, our analyses suggest that the smoking ban had a significant effect, at least in the short run, on Chinese Internet users’ smoking-related behaviors. This research introduces a novel analytic tool, which could serve as an alternative tobacco control evaluation and behavior surveillance tool in the absence of timely or comprehensive population surveillance system. This research also highlights the importance of a comprehensive approach to tobacco control in China.     

Surgical Treatment for Patients with Krukenberg Tumor of Stomach Origin: Clinical Outcome and Prognostic Factors Analysis

Krukenberg tumor originated from stomach in female patients is common in clinical practice, but it is still uncertain whether surgical resection of ovarian metastases could improve the outcome. Some studies suggested that a certain group of patients could benefit from the resection of ovarian metastases. However, conclusions were different between studies and there was no data to illustrate if certain molecular markers were associated with patients’ survival. In this study, we analyzed the effects of resection of ovarian metastases, and investigated prognostic factors in 133 patients with ovarian metastases originated from stomach. Furthermore, we examined the expression of some cancer stem cells (CSCs) markers or related molecules in 64 ovarian metastases specimens and analyzed the correlation between these molecules and patients’ survival. We found that the median overall survival (mOS) of all 133 patients was 16 months, and “gastrectomy” and “without ascites” were two independent prognostic factors associated with longer survival. The mOS of the patients with gastrectomy was longer than that of patients had not undergone gastrectomy (19 vs. 9 months, p = 0.048). Patients without ascites survived longer than those with ascites (mOS: 21 vs. 13 months, p = 0.008). We also found that Sox2, CD44 or CD133 positive expression in ovarian metastases were risk factors correlated with poor survival, and Sox2 expression was an independent prognostic indicator. These results suggested that ovarian metastasectomy might help to prolong the survivor of some patients with Krukenberg tumor originated from stomach. Patients without ascites, and with resected or resectable primary gastric cancer lesion could get benefit from and be potential candidate for surgical treatment. The expression of Sox2 might serve as a prognostic indicator for predicting patients’ survival and be helpful for selecting patients in future.     

Homeostatic Properties and Phenotypic Maturation of Murine CD4+ Pre-Thymic Emigrants in the Thymus

After a tightly regulated developmental program in the thymus, “mature” single positive (SP) thymocytes leave the thymus and enter the periphery. These newly arrived recent thymic emigrants (RTEs) are phenotypically and functionally immature, and will complete a dynamic maturation in the peripheral lymphoid organs before being licensed to be resident naïve T cells. To study the early events occurring in the RTE maturation process, we identified the phenotype of CD4⁺ pre-RTEs, a population of CD4⁺ SP thymocytes that have acquired the thymus egress capability. Compared to peripheral naïve T cells, CD4⁺ pre-RTEs displayed superior survival capability in lymphoreplete mice and faster proliferation under lymphopenic condition. The differences in Bcl2/Bim expression and/or heightened IL-7 signaling pathway may account for the pre-RTEs’ better responsiveness to homeostatic signals. Qa2, the expression of which indicates the phenotypic maturation of SPs and RTEs, was found to be upregulated in CD4⁺ pre-RTEs in thymic perivascular space. Migratory dendritic cells that surround this region contribute to Qa2 expression in pre-RTEs. The dendritic cell-driven Qa2 induction of CD4⁺ pre-RTEs is independent of MHC class II and Aire molecules.     

Soluble CD40 Ligand Stimulates CD40-Dependent Activation of the β2 Integrin Mac-1 and Protein Kinase C Zeda (PKCζ) in Neutrophils: Implications for Neutrophil-Platelet Interactions and Neutrophil Oxidative Burst

Recent work has revealed an essential involvement of soluble CD40L (sCD40L) in inflammation and vascular disease. Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined. We aimed to determine the impact and the mechanisms of sCD40L on neutrophils. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte recruitment to the sites of injury. Our data show that CD40L contributes to neutrophil firm adhesion to and transmigration across activated surface-adherent platelets, possibly through two potential mechanisms. One involves the direct interaction of ligand-receptor (CD40L-CD40), i.e., platelet surface CD40L interaction with neutrophil CD40; another involves an indirect mechanism, i.e. soluble CD40L stimulates activation of the leukocyte-specific β2 integrin Mac-1 in neutrophils and thereby further promotes neutrophil adhesion and migration. Activation of the integrin Mac-1 is known to be critical for mediating neutrophil adhesion and migration. sCD40L activated Mac-1 in neutrophils and enhanced neutrophil-platelet interactions in wild-type neutrophils, but failed to elicit such responses in CD40-deficient neutrophils. Furthermore, our data show that the protein kinase C zeta (PKCζ) is critically required for sCD40L-induced Mac-1 activation and neutrophil adhesive function. sCD40L strongly stimulated the focal clustering of Mac-1 (CD11b) and the colocalization of Mac-1 with PKCζ in wild-type neutrophils, but had minimal effect in CD40-deficient neutrophils. Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion. Moreover, sCD40L strongly stimulates neutrophil oxidative burst via CD40-dependent activation of PI3K/NF-KB, but independent of Mac-1 and PKCζ. These findings may contribute to a better understanding of the underlying mechanisms by which sCD40L/CD40 pathway contributes to inflammation and vascular diseases.     

The D Domain of LRRC4 anchors ERK1/2 in the cytoplasm and competitively inhibits MEK/ERK activation in glioma cells

Background

As a well-characterized key player in various signal transduction networks, extracellular-signal-regulated kinase (ERK1/2) has been widely implicated in the development of many malignancies. We previously found that Leucine-rich repeat containing 4 (LRRC4) was a tumor suppressor and a negative regulator of the ERK/MAPK pathway in glioma tumorigenesis. However, the precise molecular role of LRRC4 in ERK signal transmission is unclear.

Methods

The interaction between LRRC4 and ERK1/2 was assessed by co-immunoprecipitation and GST pull-down assays in vivo and in vitro. We also investigated the interaction of LRRC4 and ERK1/2 and the role of the D domain in ERK activation in glioma cells.

Results

Here, we showed that LRRC4 and ERK1/2 interact via the D domain and CD domain, respectively. Following EGF stimuli, the D domain of LRRC4 anchors ERK1/2 in the cytoplasm and abrogates ERK1/2 activation and nuclear translocation. In glioblastoma cells, ectopic LRRC4 expression competitively inhibited the interaction of endogenous mitogen-activated protein kinase (MEK) and ERK1/2. Mutation of the D domain decreased the LRRC4-mediated inhibition of MAPK signaling and its anti-proliferation and anti-invasion roles.

Conclusions

Our results demonstrated that the D domain of LRRC4 anchors ERK1/2 in the cytoplasm and competitively inhibits MEK/ERK activation in glioma cells. These findings identify a new mechanism underlying glioblastoma progression and suggest a novel therapeutic strategy by restoring the activity of LRRC4 to decrease MAPK cascade activation.

     

Does Marriage Make Us Healthier? Inter-Country Comparative Evidence from China,Japan, and Korea

Objectives

This study focuses on East Asian countries and investigates the difference in the marriage premium on the health-marriage protection effect (MPE) between younger and older generations and the intra-couple education concordance effect (ECE) on the health of married individuals. This study used inter-country comparative data from China, Japan, and Korea.

Methods

This study focused on individuals (n = 7,938) in China, Japan, and Korea who were sampled from the 2010 East Asian Social Survey. To investigate MPE and ECE, four health indicators were utilized: a physical and mental components summary (PCS and MCS), self-rated health status (D_self), and happiness level (D_happy). Ordinary least squares regression was conducted by country- and gender-specific subsamples.

Results

We found that the MPE on PCS, MCS, and D_self was more significant for the older generation than for the younger generation in both China and Japan, whereas the results were inconclusive in Korea. With regard to the ECE on happiness (D_happy), for both men and women, couples tend to be happier when both the husband and the wife are well educated (“higher balanced marriage”) compared to couples with a lower level of educational achievement (“lower balanced marriage”). Significant benefits from a “higher balanced marriage” on MCS and D_self were observed for women only. In contrast, no statistically significant differences in health status were observed between “higher balanced marriage” couples and couples with different levels of educational achievements (“upward marriage” or “downward marriage”).

Conclusions

This study found that (1) the MPE was more significant for the older generation, and (2) the health gap, particularly the happiness gap, between higher- and lower-balanced married couples was significant. The inter-country comparative findings are useful to explain how the role of marriage (and therefore of family) on health has been diluted due to the progress of industrialization and modernization.