Cloning and characterization of the dehydration-responsive element-binding protein 2A gene in Eruca vesicaria subsp sativa.

Eruca vesicaria subsp sativa is one of the most tolerant Cruciferae species to drought, and dehydration-responsive element-binding protein 2A (DREB2A) is involved in responses to salinity, heat, and particularly drought. In this study, a gene encoding EvDREB2A was cloned and characterized in E. vesicaria subsp sativa. The full-length EvDREB2A cDNA sequence contained a 388-bp 5'-untranslated region (UTR), a 348-bp 3'-UTR, and a 1002-bp open reading frame that encoded 334 amino acid residues. The theoretical isoelectric point of the EvDREB2A protein was 4.80 and the molecular weight was 37.64 kDa. The genomic sequence of EvDREB2A contained no introns. Analysis using SMART indicated that EvDREB2A contains a conserved AP2 domain, similar to other plant DREBs. Phylogenetic analysis revealed that EvDREB2A and DREB2As from Brassica rapa, Eutrema salsugineum, Arabidopsis thaliana, Arabidopsis lyrata, and Arachis hypogaea formed a small subgroup, which clustered with DREB2Bs from A. lyrata, A. thaliana, Camelina sativa, and B. rapa to form a larger subgroup. EvDREB2A is most closely related to B. rapa DREB2A, followed by DREB2As from E. salsugineum, A. thaliana, A. hypogaea, and A. lyrata. A quantitative real-time polymerase chain reaction indicated that EvDREB2A expression was highest in the leaves, followed by the roots and hypocotyls, and was lowest in the flower buds. EvDREB2A could be used to improve drought tolerance in crops.

Isolation and characterization of microsatellite loci in Quercus fabri (Fagaceae).

Quercus fabri is a pioneer species of secondary succession in evergreen broadleaved forests in China. In this study, we isolated and developed 12 polymorphic and 2 monomorphic microsatellite loci for Q. fabri using the biotin-streptavidin capture method. We characterized 12 polymorphic loci in 52 individuals from two populations. The number of alleles per locus ranged from 3 to 23. The observed and expected heterozygosities per locus were 0.033-0.773 and 0.138-0.924, respectively. These microsatellite loci will facilitate the studies on genetic variation, mating system, and gene flow of Q. fabri.

Th17/Treg cell expression in children with primary nephritic syndrome and the effects of ox-LDL on Th17/Treg cells.

To investigate the role of T-helper cells/Treg (Th17/Treg) and morbidity factors related to primary nephritic syndrome (PNS) in children, as well as the influence of ox-low density lipoprotein (ox-LDL) on Th17/Treg expression in children with PNS. To clarify the pathogenesis of PNS in children, 50 children with PNS treated in our hospital were enrolled in the study group. Additionally, 20 healthy children who came to our hospital for physical examination during the same period were enrolled in the control group. Th17 and Treg cells in children belonging to the two groups were detected by flow cytometry; the numbers of Th17/Treg cells in peripheral blood mononuclear cells at different concentrations of ox-LDL were detected simultaneously. Ox-LDL can affect the number of Th17/Treg cells in peripheral blood mononuclear cells, and both cell types decreased with increasing concentration of ox-LDL, with the numbers being significantly lower in the control group. However, the decrease in the number of Th17 cells was statistically insignificant (P > 0.05), whereas the decrease in Treg cells was more obvious and statistically significant (P < 0.05). The effect of ox-LDL the number of Treg cells was stronger than that on Th17 cells. We concluded that the imbalance of Th17/Treg cells influenced by high and low ox-LDL concentrations in children with PNS might be the immunological basis of the disease.

Genetic diversity and structure of native and non-native populations of the endangered plant Pinus dabeshanensis.

Owing to a severe decline in its abundance, Pinus dabeshanensis has been listed as an endangered species by the International Union for the Conservation of Nature. Although several restoration events have been undertaken since the 1960s, the natural population genetic structure of this species remains to be investigated. Herein, we examined the level of genetic diversity and structure of two native and two non-native populations using 10 microsatellite loci. A relatively high level of genetic variation (HO = 0.586 ± 0.039) and a low level of population differentiation (FST = 0.016 ± 0.011) were revealed. For forensic investigation, an assignment test was performed. To better understand the genetic differentiation between the native and non-native populations, the individuals in the transplanted and cultivated populations may have derived from populations that were not surveyed in this study. In light of our results, we discuss the real problems faced by all four populations and provide useful information for management decision-making.

Isolation and characterization of microsatellite markers of sea cucumber Stichopus horrens.

Curry fish (Stichopus horrens) is a tropical holothurian species and is widely distributed in the India-West Pacific. In the present study, 9 polymorphic microsatellite loci were isolated and characterized for S. horrens. These loci were tested in 30 individuals from Hainan Island in China. The number of alleles ranged from 2 to 5. The polymorphism information content ranged from 0.348-0.584. The levels of observed and expected heterozygosities varied from 0.1500-0.8000 and from 0.2014-0.5000, respectively. Most loci were in Hardy-Weinberg equilibrium, except HCS1-27 and HCS2-7, after sequential Bonferroni's correction, and no significant linkage disequilibrium was detected for any pairwise combination of loci. These polymorphic microsatellite loci will be useful for studying population structure and conservation strategy design for S. horrens.

Effects of forkhead box C2 on carcinogenesis and lymphatic metastasis in endometrial carcinoma.

We investigated the distribution of endometrial lymphatic vessels and expression of forkhead box C2 (FOXC2) in normal endometrium during menstrual cycle and in endometrial adenocarcinoma. Full-thickness uterine samples and endometrial adenocarcinoma samples were collected for immunohistochemical analysis using D2-40 and FOXC2 mouse monoclonal antibodies. The lymphatic vessel density (LVD) of the endometrium was significantly reduced compared with the myometrium during the cycle. Intra-tumoral LVD was significantly decreased in both stages of endometrioid adenocarcinoma compared with normal endometrium and myometrium. Intra-tumoral LVD significantly decreased from stage IA to stage IIIC. Peri-tumoral LVD for stage IA and stage IIIC tumors was significantly increased compared with normal endometrial LVD, but decreased compared with normal myometrial LVD. Stage IIIC showed increased peri-tumoral LVD when compared with stage IA. The positive rate of FOXC2 was 73.3% in proliferative endometrium and 80% in secretory endometrium. Secretory endometrium showed significantly increased FOXC2 expression compared with proliferative endometrium. Endometrioid adenocarcinoma showed significantly increased FOXC2 expression compared with normal endometrium, both in the epithelium and stroma. FOXC2 expression in the stroma significantly increased when pelvic and/or para-aotic lymph nodes were involved. FOXC2 was immunolocalized in low-risk endometrial carcinoma in endometrioid adenocarcinoma, but not in normal endometrium. Endometrial lymphatic vessels were located in normal endometrium and myometrium across the menstrual cycle and in intra-and peri-endometrioid adenocarcinoma, and increased in endometrial adenocarcinoma. Peri-tumoral lymphatics were associated with increased lymphatic metastasis. FOXC2 may be associated with the genesis of endometrial carcinoma and lymphangiogensis in endometrial adenocarcinoma in intra- and peri-tumoral lymphatics.

Isolation and characterization of polymorphic microsatellite loci in the sea cucumber Holothuria scabra.

The sea cucumber Holothuria scabra is an endangered species. In this study, nine new polymorphic microsatellite loci were developed and tested in 30 individuals. The number of alleles ranged from 2 to 5, and the observed and expected heterozygosities ranged from 0.1200 to 0.7391 and from 0.2408 to 0.5983, respectively. No loci significantly deviated from the Hardy-Weinberg equilibrium af-ter a Bonferroni correction, and no significant linkage disequilibrium was found between pairs of loci. These polymorphic microsatellite loci will be useful in studying the genetic diversity of H. scabra and its conservation.

Aberrant gene expression profiles in hepatocellular carcinoma detected by microdissection.

The aim of this study was to identify genomic aberrations in hepatocellular carcinoma (HCC) by using laser capture microdissection (LCM) combined with microarray analysis. Samples were procured by LCM from HCC and patient-matched normal liver tissue surgically resected from 4 patients. RNA was isolated from the samples and reverse transcribed into cDNA. After 2-cycle linear amplification and 2-color fluorescent labeling, the cRNA was hybridized onto a whole genome microarray. All genes expressed in the normal and HCC samples were counted and analyzed. Differentially expressed genes were identified and the top 10 up and downregulated genes (totally 20 genes) were further evaluated. LCM was able to accurately capture 50-200 cells from HCC and control tissues. The microarray spectrum showed satisfactory detection of HCC-enriched genes. A total of 1361 differentially expressed genes were identified, among which, 607 were upregulated and 754 were downregulated. Among the top 20 up and downregulated genes, 4 genes had not been documented in the literature as being differentially expressed in any tumors. Thus, LCM is an effective approach for identifying aberrantly expressed genes in HCC, and may lead to the discovery of biomarkers for diagnostic and therapeutic applications.

Development and characterization of new microsatellites for walnut (Juglans regia).

The expressed sequence tag (EST) database represents a potentially valuable resource for the development of simple sequence repeat (SSR) markers for use in evolutionary studies. EST-SSRs reveal polymorphisms not only within the source taxon, but in related taxa as well. In this paper, we describe a case study in which the publicly available walnut (Juglans regia) EST database was used to develop SSR markers for use in the genetic analysis of the widespread Juglans nigra and Carya cathayensis and an endangered species Annamocarya sinensis. A total of 7262 unigenes, including 1911 contigs and 5351 singletons, were obtained from 13,559 ESTs retrieved from the NCBI database. The 7262 unigenes were further reduced to 706 EST-SSR sequences containing 805 SSR loci. Then, 309 EST-SSR primers were randomly designed, and 77 were identified with five high across-species transferability cross-species: namely, J. regia, J. nigra, C. cathayensis, Carya dabieshanensis, and A. sinensis. Thirteen highly polymorphic EST-SSRs were further used for genetic analyses in these above five species.

Adipose tissue-derived stem cells promote pancreatic cancer cell proliferation and invasion.

To explore the effects of adipose tissue-derived stem cells (ADSCs) on the proliferation and invasion of pancreatic cancer cells in vitro and the possible mechanism involved, ADSCs were cocultured with pancreatic cancer cells, and a cell counting kit (CCK-8) was used to detect the proliferation of pancreatic cancer cells. ELISA was used to determine the concentration of stromal cell-derived factor-1 (SDF-1) in the supernatants. RT-PCR was performed to detect the expression of the chemokine receptor CXCR4 in pancreatic cancer cells and ADSCs. An in vitro invasion assay was used to measure invasion of pancreatic cancer cells. SDF-1 was detected in the supernatants of ADSCs, but not in pancreatic cancer cells. Higher CXCR4 mRNA levels were detected in the pancreatic cancer cell lines compared with ADSCs (109.3 ± 10.7 and 97.6 ± 7.6 vs 18.3 ± 1.7, respectively; P<0.01). In addition, conditioned medium from ADSCs promoted the proliferation and invasion of pancreatic cancer cells, and AMD3100, a CXCR4 antagonist, significantly downregulated these growth-promoting effects. We conclude that ADSCs can promote the proliferation and invasion of pancreatic cancer cells, which may involve the SDF-1/CXCR4 axis.

Adipose tissue-derived stem cells promote pancreatic cancer cell proliferation and invasion

To explore the effects of adipose tissue-derived stem cells (ADSCs) on the proliferation and invasion of pancreatic cancer cells in vitro and the possible mechanism involved, ADSCs were cocultured with pancreatic cancer cells, and a cell counting kit (CCK-8) was used to detect the proliferation of pancreatic cancer cells. ELISA was used to determine the concentration of stromal cell-derived factor-1 (SDF-1) in the supernatants. RT-PCR was performed to detect the expression of the chemokine receptor CXCR4 in pancreatic cancer cells and ADSCs. An in vitro invasion assay was used to measure invasion of pancreatic cancer cells. SDF-1 was detected in the supernatants of ADSCs, but not in pancreatic cancer cells. Higher CXCR4 mRNA levels were detected in the pancreatic cancer cell lines compared with ADSCs (109.3±10.7 and 97.6±7.6 vs 18.3±1.7, respectively; P<0.01). In addition, conditioned medium from ADSCs promoted the proliferation and invasion of pancreatic cancer cells, and AMD3100, a CXCR4 antagonist, significantly downregulated these growth-promoting effects. We conclude that ADSCs can promote the proliferation and invasion of pancreatic cancer cells, which may involve the SDF-1/CXCR4 axis.

Conservation genetics of Annamocarya sinensis (Dode) Leroy, an endangered endemic species.

The endangered perennial plant Annamocarya sinensis (Dode) Leroy is a tertiary relict tree restricted to southeastern China and northern Vietnam. To explore endangerment mechanisms, develop protection strategies, and guide reintroduction efforts for this species, we investigated genetic diversity and population structure by surveying 70 individuals from three distinct populations using 12 polymorphic microsatellite markers. We found high genetic diversity for A. sinensis as indicated by high allelic diversity (allelic number = 4.667 ± 0.436, effective number of alleles = 2.913 ± 0.249), excess heterozygosity (observed heterozygosity = 0.586 ± 0.039, expected heterozygosity = 0.582 ± 0.029), and low fixation index (-0.028 ± 0.057). Our research revealed low genetic differentiation (FST = 0.066 ± 0.011) and no correlation between genetic distance and geographic distance. Analysis of molecular variance attributed 87% of the variance to differences within the population, whereas 13% was distributed among populations. The protection strategy should aim to protect as many populations as possible. Promoting sexual reproduction among various genotypes and establishing an outcrossing program are advisable for A. sinensis.

Alteration of Phascolosoma esculenta heat shock protein 90 expression under heavy metal exposure and thermal stress.

The full-length complementary DNA (cDNA) of heat shock protein 90 was cloned from Phascolosoma esculenta (PeHSP90) using expressed sequence tag and rapid amplification of cDNA end approaches. The cDNA of PeHSP90 was 2521 bp including a 5'-untranslated region of 110 bp, a 3'-untranslated region of 230 bp, and an open reading frame of 2181 bp. All of the characteristic motifs of the HSP90 family were completely conserved in the deduced amino acid of PeHSP90. The expression of PeHSP90 was induced by 3 heavy metals or elevated temperature, under which Zn²âº displayed effects were more toxic than those of Cd²âº and Cu²âº. The polyclonal antibodies generated from the recombinant product of PeHSP90 were specifically identified not only in the recombinant product but also in the native protein from hemocytes. These results strongly suggested that PeHSP90 was involved in heavy metal challenge and thermal stress regulation in P. esculenta.