Osmosensor-mediated control of Ca2+ spiking in pollen germination.

Higher plants survive terrestrial water deficiency and fluctuation by arresting cellular activities (dehydration) and resuscitating processes (rehydration). However, how plants monitor water availability during rehydration is unknown. Although increases in hypo-osmolarity-induced cytosolic Ca2+ concentration (HOSCA) have long been postulated to be the mechanism for sensing hypo-osmolarity in rehydration1,2, the molecular basis remains unknown. Because osmolarity triggers membrane tension and the osmosensing specificity of osmosensing channels can only be determined in vivo3-5, these channels have been classified as a subtype of mechanosensors. Here we identify bona fide cell surface hypo-osmosensors in Arabidopsis and find that pollen Ca2+ spiking is controlled directly by water through these hypo-osmosensors-that is, Ca2+ spiking is the second messenger for water status. We developed a functional expression screen in Escherichia coli for hypo-osmosensitive channels and identified OSCA2.1, a member of the hyperosmolarity-gated calcium-permeable channel (OSCA) family of proteins6. We screened single and high-order OSCA mutants, and observed that the osca2.1/osca2.2 double-knockout mutant was impaired in pollen germination and HOSCA. OSCA2.1 and OSCA2.2 function as hypo-osmosensitive Ca2+-permeable channels in planta and in HEK293 cells. Decreasing osmolarity of the medium enhanced pollen Ca2+ oscillations, which were mediated by OSCA2.1 and OSCA2.2 and required for germination. OSCA2.1 and OSCA2.2 convert extracellular water status into Ca2+ spiking in pollen and may serve as essential hypo-osmosensors for tracking rehydration in plants.

UGT89AC1-mediated quercetin glucosylation is induced upon herbivore damage and enhances Camellia sinensis resistance to insect feeding.

Quercetin is a key flavonol in tea plants (Camellia sinensis (L.) O. Kuntze) with various health benefits, and it often occurs in the form of glucosides. The roles of quercetin and its glucosylated forms in plant defense are generally not well-studied, and remain unknown in the defense of tea. Here, we found higher contents of quercetin glucosides and a decline of the aglucone upon Ectropis grisescens (E. grisescens) infestation of tea. Nine UGTs were strongly induced, among which UGT89AC1 exhibited the highest activity toward quercetin in vitro and in vivo. The mass of E. grisescens larvae that fed on plants with repressed UGT89AC1 or varieties with lower levels of UGT89AC1 was significantly lower than that of larvae fed on controls. Artificial diet supplemented with quercetin glucoside also reduced the larval growth rate, whereas artificial diet supplemented with free quercetin had no significant effect on larval growth. UGT89AC1 was located in both the cytoplasm and nucleus, and its expression was modulated by JA, JA-ILE, and MeJA. These findings demonstrate that quercetin glucosylation serves a defensive role in tea against herbivory. Our results also provide novel insights into the ecological relevance of flavonoid glycosides under biotic stress in plants.

Anti-EBV antibodies: Roles in diagnosis, pathogenesis, and antiviral therapy.

Epstein-Barr virus (EBV) infection is prevalent in global population and associated with multiple malignancies and autoimmune diseases. During the infection, EBV-harbored or infected cell-expressing antigen could elicit a variety of antibodies with significant role in viral host response and pathogenesis. These antibodies have been extensively evaluated and found to be valuable in predicting disease diagnosis and prognosis, exploring disease mechanisms, and developing antiviral agents. In this review, we discuss the versatile roles of EBV antibodies as important biomarkers for EBV-related diseases, potential driving factors of autoimmunity, and promising therapeutic agents for viral infection and pathogenesis.

Epigenetic Regulation of MAP3K8 in EBV-Associated Gastric Carcinoma.

Super-enhancers (SEs) regulate gene expressions, which are critical for cell type-identity and tumorigenesis. Although genome wide H3K27ac profiling have revealed the presence of SE-associated genes in gastric cancer (GC), their roles remain unclear. In this study, ChIP-seq and HiChIP-seq experiments revealed mitogen-activated protein kinase 8 (MAP3K8) to be an SE-associated gene with chromosome interactions in Epstein-Barr virus-associated gastric carcinoma (EBVaGC) cells. CRISPRi mediated repression of the MAP3K8 SEs attenuated MAP3K8 expression and EBVaGC cell proliferation. The results were validated by treating EBVaGC cells with bromodomain and the extra-terminal motif (BET) inhibitor, OTX015. Further, functional analysis of MAP3K8 in EBVaGC revealed that silencing MAP3K8 could inhibit the cell proliferation, colony formation, and migration of EBVaGC cells. RNA-seq and pathway analysis indicated that knocking down MAP3K8 obstructed the notch signaling pathway and epithelial-mesenchymal transition (EMT) in EBVaGC cells. Further, analysis of the cancer genome atlas (TCGA) and GSE51575 databases exhibited augmented MAP3K8 expression in gastric cancer and it was found to be inversely correlated with the disease-free progression of GC. Moreover, Spearman's correlation revealed that MAP3K8 expression was positively correlated with the expressions of notch pathway and EMT related genes, such as, Notch1, Notch2, C-terminal binding protein 2 (CTBP2), alpha smooth muscle actin isotype 2 (ACTA2), transforming growth factor beta receptor 1 (TGFßR1), and snail family transcriptional repressors 1/2 (SNAI1/SNAI2) in GC. Taken together, we are the first to functionally interrogate the mechanism of SE-mediated regulation of MAP3K8 in EBVaGC cell lines.

OSCA1 is an osmotic specific sensor: a method to distinguish Ca2+ -mediated osmotic and ionic perception.

Genetic mutants defective in stimulus-induced Ca2+ increases have been gradually isolated, allowing the identification of cell-surface sensors/receptors, such as the osmosensor OSCA1. However, determining the Ca2+ -signaling specificity to various stimuli in these mutants remains a challenge. For instance, less is known about the exact selectivity between osmotic and ionic stresses in the osca1 mutant. Here, we have developed a method to distinguish the osmotic and ionic effects by analyzing Ca2+ increases, and demonstrated that osca1 is impaired primarily in Ca2+ increases induced by the osmotic but not ionic stress. We recorded Ca2+ increases induced by sorbitol (osmotic effect, OE) and NaCl/CaCl2 (OE + ionic effect, IE) in Arabidopsis wild-type and osca1 seedlings. We assumed the NaCl/CaCl2 total effect (TE) = OE + IE, then developed procedures for Ca2+ imaging, image analysis and mathematic fitting/modeling, and found osca1 defects mainly in OE. The osmotic specificity of osca1 suggests that osmotic and ionic perceptions are independent. The precise estimation of these two stress effects is applicable not only to new Ca2+ -signaling mutants with distinct stimulus specificity but also the complex Ca2+ signaling crosstalk among multiple concurrent stresses that occur naturally, and will enable us to specifically fine tune multiple signal pathways to improve crop yields.

Association between ALT/AST and Muscle Mass in Patients with Type 2 Diabetes Mellitus.

Objective: The alanine aminotransferase/aspartate aminotransferase (ALT/AST) ratio is thought to be related to metabolic disorders and insulin resistance. Type 2 diabetes mellitus (T2DM) is a high-risk population for low muscle mass. This study was performed to evaluate the association between ALT/AST and muscle mass in subjects with T2DM. Method: This cross-sectional study enrolled 1068 subjects (566 males and 502 females) with T2DM. General information, medical history, and blood samples were collected. Skeletal muscle index (SMI) was detected using dual-energy X-ray absorptiometry. Logistic regression analysis was utilized to determine the correlation of ALT/AST and low muscle mass in subjects with T2DM. Multiple linear regression analysis was utilized to evaluate the association between ALT/AST, SMI and other metabolic characteristics. Result: Of all subjects, 115 men (20.3%) and 71 women (14.1%) presented low muscle mass. ALT/AST was related to an increased risk for low muscle mass in both genders. Multiple linear regression analysis displayed that SMI was negatively associated with ALT/AST, age, glycosylated hemoglobin (HbA1c), and high-density lipoprotein cholesterol (HDL) in male group. While in female group, SMI was positively associated with systolic blood pressure (SBP) and negatively associated with ALT/AST and age. Furthermore, ALT/AST was associated with age and BMI in both genders. Conclusion: ALT/AST was negatively associated with muscle mass in subjects with T2DM.

Comparison of therapeutic effects of continuous epidural nerve block combined with drugs on postherpetic neuralgia.

PURPOSE: To compare the therapeutic effects of continuous epidural block combined with drugs and oral drugs alone on postherpetic neuralgia (PHN). METHODS: Ninety-six PHN patients meeting the standard were selected and divided into group A and group B. Patients in group A had epidural block combined with oral administration of gabapentin and oxycodone-acetaminophen, and patients in group B received oral gabapentin and oxycodone-acetaminophen. Visual analogue scale (VAS) and Wisconsin brief pain inventory scores were used to evaluate the patients in group A and group B for 6 times (before treatment, 1 d, 3 d, 7 d, 15 d and 30 d after treatment) respectively, and the complications and adverse reactions of the two treatment methods, as well as the number of cases requiring remedial measures were observed. RESULTS: There were significant differences in VAS and Wisconsin brief pain inventory scores at 1 d, 3 d, 7 d, 15 d and 30 d after treatment between the two groups (p < 0.05). Moreover, the scores before and after treatment decreased with the time of treatment, and there was a significant difference between the two groups at different time points (p < 0.05). No significant adverse reactions were observed in group A except for 1 patient with catheter detachment. Compared with group A, the adverse reactions of group B were more varied and obvious. CONCLUSION: Both treatments have certain effects on PHN, but epidural block combined with drug therapy is more effective, especially for patients with severe pain, early use can quickly relieve pain.

NR4A1 counteracts JNK activation incurred by ER stress or ROS in pancreatic ß-cells for protection.

Sustained hyperglycaemia and hyperlipidaemia incur endoplasmic reticulum stress (ER stress) and reactive oxygen species (ROS) overproduction in pancreatic ß-cells. ER stress or ROS causes c-Jun N-terminal kinase (JNK) activation, and the activated JNK triggers apoptosis in different cells. Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an inducible multi-stress response factor. The aim of this study was to explore the role of NR4A1 in counteracting JNK activation induced by ER stress or ROS and the related mechanism. qPCR, Western blotting, dual-luciferase reporter and ChIP assays were applied to detect gene expression or regulation by NR4A1. Immunofluorescence was used to detect a specific protein expression in ß-cells. Our data showed that NR4A1 reduced the phosphorylated JNK (p-JNK) in MIN6 cells encountering ER stress or ROS and reduced MKK4 protein in a proteasome-dependent manner. We found that NR4A1 increased the expression of cbl-b (an E3 ligase); knocking down cbl-b expression increased MKK4 and p-JNK levels under ER stress or ROS conditions. We elucidated that NR4A1 enhanced the transactivation of cbl-b promoter by physical association. We further confirmed that cbl-b expression in ß-cells was reduced in NR4A1-knockout mice compared with WT mice. NR4A1 down-regulates JNK activation by ER stress or ROS in ß-cells via enhancing cbl-b expression.

A Negative Regulator of Carotenogenesis in Blakeslea trispora.

As an ideal carotenoid producer, Blakeslea trispora has gained much attention due to its large biomass and high production of ß-carotene and lycopene. However, carotenogenesis regulation in B. trispora still needs to be clarified, as few investigations have been conducted at the molecular level in B. trispora In this study, a gene homologous to carotenogenesis regulatory gene (crgA) was cloned from the mating type (-) of B. trispora, and the deduced CrgA protein was analyzed for its primary structure and domains. To clarify the crgA-mediated regulation in B. trispora, we used the strategies of gene knockout and complementation to investigate the effect of crgA expression on the phenotype of B. trispora In contrast to the wild-type strain, the crgA null mutant (ΔcrgA) was defective in sporulation but accumulated much more ß-carotene (31.2% improvement at the end) accompanied by enhanced transcription of three structural genes (hmgR, carB, and carRA) for carotenoids throughout the culture time. When the wild-type copy of crgA was complemented into the crgA null mutant, sporulation, transcription of structural genes, and carotenoid production were restored to those of the wild-type strain. A gas chromatography-mass spectrometry (GC-MS)-based metabolomic approach and multivariate statistical analyses were performed to investigate the intracellular metabolite profiles. The reduced levels of tricarboxylic acid (TCA) cycle components and some amino acids and enhanced levels of glycolysis intermediates and fatty acids indicate that more metabolic flux was driven into the mevalonate (MVA) pathway; thus, the increase of precursors and fat content contributes to the accumulation of carotenoids.IMPORTANCE The zygomycete Blakeslea trispora is an important strain for the production of carotenoids on a large scale. However, the regulation mechanism of carotenoid biosynthesis is still not well understood in this filamentous fungus. In the present study, we sought to investigate how crgA influences the expression of structural genes for carotenoids, carotenoid biosynthesis, and other anabolic phenotypes. This will lead to a better understanding of the global regulation mechanism of carotenoid biosynthesis and facilitate engineering this strain in the future for enhanced production of carotenoids.

Prediction effects of serum CA125 decrease ratio during different cycles in relapsed ovarian cancer.

AIM: To investigate the prognostic value of serum cancer antigen 125 (CA125) levels during chemotherapy in relapsed epithelial ovarian cancer (EOC) and to identify cut-off values that distinguish patients who relapse beyond 12 months from those who relapse within 12 months. METHODS: About 93 relapsed EOC patients who received cytoreductive surgery and adjuvant chemotherapy at Obstetrics and Gynecology Hospital of Fudan University between January 2003 and March 2015 were selected. Univariate regression analysis was used to determine the significant prognostic factors. The Kaplan-Meier method was used to calculate the overall survival (OS) rate. RESULTS: The CA125 decrease ratio of more than 97.6% after the fourth chemotherapy cycle was significantly associated with relapse time (P = 0.044). The sensitivity was 70.0%, and the specificity was 76.9%. Moreover, in all relapsed patients, the group with the CA125 decrease ratio after the fourth chemotherapy cycle of more than 97.6% had a significantly better OS than any other group (P = 0.0019). CONCLUSION: The CA125 decrease ratio of less than 97.6% after the fourth chemotherapy cycle can be a predictive factor for relapse within 12 months. Patients without a significant decrease in CA125 after four cycles of chemotherapy should have a more frequent follow-up and more active re-examination.

NR4A1 retards adipocyte differentiation or maturation via enhancing GATA2 and p53 expression.

Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an orphan nuclear receptor with diverse functions. It has been reported that NR4A1, as a transcriptional activator, is implicated in glucose and lipid metabolism. The aim of this study was to investigate the regulatory role of NR4A1 in adipogenesis and explore the underlying mechanisms. Quantitative real-time PCR and Western blotting were used to analyse the expression of genes involved in synthesis and mobilization of fats in vivo and in vitro. Dual-luciferase reporter assay was conducted to study the regulatory mechanisms of NR4A1. Our data from in vivo study confirmed that NR4A1 knockout (KO) mice fed with high-fat diet were more prone to obesity, and gene expression levels of PPARγ and FAS were increased in KO mice compared to controls; our data from in vitro study showed that NR4A1 overexpression in 3T3-L1 pre-adipocytes inhibited adipogenesis. Moreover, NR4A1 enhanced GATA binding protein 2 (GATA2) expression, which in turn inhibited peroxisome proliferator-activated receptor γ (PPARγ); NR4A1 inhibited sterol regulatory element binding transcription factor 1 (SREBP1) and its downstream gene fatty acid synthase (FAS) by up-regulating p53. NR4A1 inhibits the differentiation and lipid accumulation of adipocytes by enhancing the expression of GATA2 and p53.

Cytochrome P450 (CYP) epoxygenases as potential targets in the management of impaired diabetic wound healing.

Epoxyeicosatrienoic acids (EETs) are the epoxidation products of arachidonic acid catalyzed by cytochrome P450 (CYP) epoxygenases, which possess multiple biological activities. In the present study, we aimed to explore the role and effects of CYP epoxygenases/EETs in wound healing in ob/ob mice. Full-thickness skin dorsal wounds were made on ob/ob mice and C57BL/6 control mice. The mRNA and protein expression of CYP epoxygenases were determined in granulation tissues of wounds. Effects of EETs on wound healing were evaluated. Inflammation and angiogenesis in wounds were also observed. Compared with C57BL/6 mice, the mRNA and protein expression of CYP2C65 and CYP2J6 in the granulation tissues in ob/ob mice were significantly reduced. 11,12-EET treatment significantly improved wound healing in ob/ob mice, whereas 14,15-EEZE, an EET antagonist, showed the opposite effect. 11,12-EET treatment decreased neutrophil and macrophage infiltration to the wound sites, resulting in reduced production of inflammatory cytokines, decreased MMP-9 expression, and increased collagen accumulation in the granulation tissues of ob/ob mice. In addition, 11,12-EET increased angiogenesis in the granulation tissues of wounds in ob/ob mice. These findings indicate that reduced expression of CYP epoxygenases may contribute to impaired diabetic wound healing, and exogenous EETs may improve diabetic wound healing by modulating inflammation and angiogenesis.

GLP-1/GLP-1R Signaling in Regulation of Adipocyte Differentiation and Lipogenesis.

BACKGROUND/AIMS: The aim of this study was to determine the direct role of liraglutide (LG) in adipogenesis and lipid metabolism. METHODS: Lipid accumulation was evaluated by oil red O staining, quantitative real-time PCR (qPCR) was performed to determine glucagon-like peptide 1 receptor (GLP-1R), fatty acid synthase (FASN) and adipose triglyceride lipase (ATGL) expression in 3T3-L1 preadipocytes, differentiated adipocytes and in adipose tissues from mice. The effects of LG on 3T3-L1 adipogenesis and lipid metabolism were analyzed with qPCR, Western Blotting, oil red O staining, immunohistochemistry (IHC) and immunofluorescence (IF). All measurements were performed at least three times. RESULTS: LG increased the expression of differentiation marker genes and lipid accumulation during preadipocyte differentiation. In differentiated adipocytes, LG decreased FASN expression, and simultaneously led to CREB phosphorylation and ERK1/2 activation which were abolished by a GLP-1R antagonist, exendin (9-39). LG induced-FASN down-regulation was partially reversed by PKA and ERK1/2 inhibitors. Consistent with above in vitro findings, LG treatment significantly reduced FASN expression in visceral adipose tissues of ob/ob mice, and reduced body weight gain. CONCLUSION: LG promotes preadipocytes differentiation, and inhibits FASN expression in adipocytes. LG induced down-regulation of FASN is at least partially mediated by PKA and MAPK signaling pathways.

The Orphan Nuclear Receptor NR4A1 Protects Pancreatic ß-Cells from Endoplasmic Reticulum (ER) Stress-mediated Apoptosis.

The role of NR4A1 in apoptosis is controversial. Pancreatic ß-cells often face endoplasmic reticulum (ER) stress under adverse conditions such as high free fatty acid (FFA) concentrations and sustained hyperglycemia. Severe ER stress results in ß-cell apoptosis. The aim of this study was to analyze the role of NR4A1 in ER stress-mediated ß-cell apoptosis and to characterize the related mechanisms. We confirmed that upon treatment with the ER stress inducers thapsigargin (TG) or palmitic acid (PA), the mRNA and protein levels of NR4A1 rapidly increased in both MIN6 cells and mouse islets. NR4A1 overexpression in MIN6 cells conferred resistance to cell loss induced by TG or PA, as assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and TUNEL assays indicated that NR4A1 overexpression also protected against ER stress-induced apoptosis. This conclusion was further confirmed by experiments exploiting siRNA to knockdown NR4A1 expression in MIN6 cells or exploiting NR4A1 knock-out mice. NR4A1 overexpression in MIN6 cells reduced C/EBP homologous protein (CHOP) expression and Caspase3 activation induced by TG or PA. NR4A1 overexpression in MIN6 cells or mouse islets resulted in Survivin up-regulation. A critical regulatory element was identified in Survivin promoter (-1872 bp to -1866 bp) with a putative NR4A1 binding site; ChIP assays demonstrated that NR4A1 physically associates with the Survivin promoter. In conclusion, NR4A1 protects pancreatic ß-cells against ER stress-mediated apoptosis by up-regulating Survivin expression and down-regulating CHOP expression, which we termed as "positive and negative regulation."

The Effect of Growth Hormone on Lipid Accumulation or Maturation in Adipocytes.

BACKGROUND: Adipogenesis of adipocytes includes two stages: initiation and maturation. Growth hormone (GH) secretion is decreased in obese subjects and GH levels are inversely correlated with abdominal fat mass. The effects of growth hormone (GH) on lipids accumulation or maturation of adipocytes remains elusive. METHODS: In the present study, effect of GH on lipid accumulation in vitro and in vivo was examined. cDNA microarray, quantitative real time-PCR (qPCR) and western blotting was used to analyze the expression of genes related to adipocyte lipid accumulation or degradation in pre- or mature 3T3-F442A adipocytes treated with GH and in epididymal adipose tissue of C57BL/6 mice administrated with GH. Level of adiponectin in supernatants of cultured F442A adipocytes was determined by enzyme-linked immune-sorbent assay. RESULTS: We found that in 3T3-F442A especially 6 days post initiation of adipogenesis, GH intervention resulted in decreased expression of adipocyte maturation regulators (C/EBPα, PPARγ) and prominent genes related to lipid synthesis such as FAS and FABP, while the expression of UCP1 was markedly enhanced. cDNA microarray analysis and qPCR showed that the expression of SOCS2 and Adipor2 was increased under GH-treatment in mature 3T3-F442A adipocytes. GH treatment increased the mRNA expression of adiponectin and UCP1 in mature adipocytes. The above results were confirmed by in vivo study. CONCLUSIONS: GH potentially negatively modulates the maturation and accumulation of lipid in adipocytes.

TSH/TSHR Signaling Suppresses Fatty Acid Synthase (FASN) Expression in Adipocytes.

TSH/TSHR signaling plays a role in the regulation of lipid metabolism in adipocytes. However, the precise mechanisms are not known. In the present study, we determined the effect of TSH on fatty acid synthase (FASN) expression, and explored the underlying mechanisms. In vitro, TSH reduced FASN expression in both mRNA and protein levels in mature adipocytes and was accompanied by protein kinase A (PKA) activation, cAMP-response element binding protein (CREB) phosphorylation, as well as extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH2 -terminal kinase (JNK) activation. TSH-induced downregulation of FASN was partially abolished by inhibition of PKA and ERK, but not JNK. TSHR and FASN expression in visceral tissue was significantly increased in C57BL/6 mice with diet-induced obesity compared with control animals, whereas thyroid TSHR expression was normal. These findings suggest that activation of TSHR directly inhibits FASN expression in mature adipocytes, possibly mediated by PKA and ERK. In obese animals, this function of TSHR seems to be counteracted. The precise mechanisms need further investigation.

Expression of a chimeric human/salmon calcitonin gene integrated into the Saccharomyces cerevisiae genome using rDNA sequences as recombination sites.

Calcitonin participates in controlling homeostasis of calcium and phosphorus and plays an important role in bone metabolism. The aim of this study was to endow an industrial strain of Saccharomyces cerevisiae with the ability to express chimeric human/salmon calcitonin (hsCT) without the use of antibiotics. To do so, a homologous recombination plasmid pUC18-rDNA2-ura3-P pgk -5hsCT-rDNA1 was constructed, which contains two segments of ribosomal DNA of 1.1 kb (rDNA1) and 1.4 kb (rDNA2), to integrate the heterologous gene into host rDNA. A DNA fragment containing five copies of a chimeric human/salmon calcitonin gene (5hsCT) under the control of the promoter for phosphoglycerate kinase (P pgk ) was constructed to express 5hsCT in S. cerevisiae using ura3 as a selectable auxotrophic marker gene. After digestion by restriction endonuclease HpaI, a linear fragment, rDNA2-ura3-P pgk -5hsCT-rDNA1, was obtained and transformed into the △ura3 mutant of S. cerevisiae by the lithium acetate method. The ura3-P pgk -5hsCT sequence was introduced into the genome at rDNA sites by homologous recombination, and the recombinant strain YS-5hsCT was obtained. Southern blot analysis revealed that the 5hsCT had been integrated successfully into the genome of S. cerevisiae. The results of Western blot and ELISA confirmed that the 5hsCT protein had been expressed in the recombinant strain YS-5hsCT. The expression level reached 2.04 % of total proteins. S. cerevisiae YS-5hsCT decreased serum calcium in mice by oral administration and even 0.01 g lyophilized S. cerevisiae YS-5hsCT/kg decreased serum calcium by 0.498 mM. This work has produced a commercial yeast strain potentially useful for the treatment of osteoporosis.

Changes of lipin1ß expression in gestational diabetes mellitus.

BACKGROUND: Lipin1ß is an adipokine proposed to be associated with insulin resistance (IR). Pregnancy is a physiologic state of progressive IR. The objective of the present study was to investigate the role of lipin1ß in the development of GDM. METHODS: A total of 40 pregnant women (22 normal and 18 with GDM) who delivered healthy infants at full-term (> 37 weeks gestation) were included. The mRNA and protein levels of lipin1ß in adipose tissues were determined by real-time RT-PCR and Western-blot. Plasma glucose, lipids, insulin, and estradiol (E2) levels were measured routinely at fasting state, and HOMA-IR was calculated accordingly. RESULTS: The lipin1ß expression in both mRNA and protein levels in SAT and VAT was lower in GDM patients than controls. Lipin1ß mRNA in VAT was negatively correlated with BMI (r = -0.505, p < 0.05), FINS (r = -0.539, p < 0.05), HOMA-IR (r = -0.574, p < 0.01), TG (r = -0.471, p < 0.05), and E2 (r = -0.564, p < 0.01). Lipin1ß mRNA expression in SAT was similar with VAT. Lipin1ß mRNA was not correlated with body weight gain or blood pressure. These results indicated that the lipin1ß expression in adipose tissues is down-regulated in patients with GDM. CONCLUSIONS: Lipin1ß might play a role in the pathogenesis of insulin resistance in GDM.

Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum.

The Cre/loxP site-specific recombination system was applied to Aurantiochytrium limacinum to obtain a transformant without the antibiotic resistance marker gene. First, the enhanced green fluorescent protein gene (egfp) and chloramphenicol resistance gene (Cmr), along with the two loxP loci, were integrated into the genome of A. limacinum OUC88 using 18S rDNA sequences as the homologous recombination sites. Then plasmid pSH65, containing a zeocin resistance gene (Bler) was transferred into A. limacinum OUC_CG. After induction with galactose, repeated passage in culture and PCR-based assessment, the pSH65 plasmid was lost and A. limacinum OUC_EG host was shown to no longer have resistance to 100 mg chloramphenicol/L or 5 mg zeocin/L. Through southern blotting and fluorescence detection, egfp was found to be integrated into the genome of A. limacinum OUC_EG, and EGFP was successfully expressed in the cells. The successful application of the Cre/loxP system demonstrates an experimental basis for genetic modification of A. limacinum so as to obtain transformed strains with no antibiotic resistance marker genes.