Novel MicroRNA Reporter Uncovers Repression of Let-7 by GSK-3β

Several members of the let-7 microRNA family are downregulated in ovarian and other cancers. They are thought to act as tumor suppressors by lowering growth-promoting and anti-apoptotic proteins. In order to measure cellular let-7 levels systematically, we have developed a highly sensitive let-7 reporter assay system based on the expression of a chimeric mRNA that contains the luciferase coding region and a 3′-untranslated region (UTR) bearing two let-7-binding sites. In cells expressing the reporter construct, termed pmirGLO-let7, luciferase activity was high when let-7 was absent, while luciferase activity was low when let-7 levels were elevated. The ovarian cancer cell lines BG-1 and UCI-101 were transfected with the let-7 reporter and surveyed with a library of kinase inhibitors in order to identify pathways affecting let-7 activity. Among the inhibitors causing changes in endogenous let-7 abundance, the lowering of glycogen synthase kinase 3 (GSK-3)β function specifically increased let-7 levels and lowered luciferase activity. Similarly, silencing GSK-3β increased both mature and primary-let-7 levels in BG-1 cells, and decreased BG-1 cell survival. Further studies identified p53 as a downstream effector of the GSK-3β-mediated repression of let-7 biosynthesis. Our studies highlight GSK-3β as a novel therapeutic target in ovarian tumorigenesis.     

Association between the Presence of Autoantibodies against Adrenoreceptors and Severe Pre-Eclampsia: A Pilot Study

Background

Pre-eclampsia is the leading cause of maternal and neonatal morbidity and mortality with incompletely understood etiopathogenesis. The purpose of the current study is to determine whether there is a relationship between the presence of autoantibodies against β₁, β₂ and α₁ adrenoreceptors and severe pre-eclampsia.

Methodology/Principal Findings

Synthetic peptides corresponding to amino acid sequences of the second extracellular loops of β₁, β₂ and α₁ adrenoreceptors were synthesized as antigens to test 34 patients with severe pre-eclampsia, 36 normal pregnancy women and 40 non-pregnant controls for the presence of autoantibodies using enzyme-linked immunosorbent assay. The respective frequencies of autoantibodies against β₁, β₂ and α₁ adrenoreceptors were 50.0% (17/34), 52.9% (18/34) and 55.9% (19/34) in patients with severe pre-eclampsia, 19.4% (7/36) (p = 0.011), 19.4% (7/36) (p = 0.006) and 17.6% (6/36) (p = 0.001) in normal pregnancy women and 10% (4/40), 7.5% (3/40) and 10% (4/40) (p<0.001) in non-pregnant controls. Titers of these autoantibodies were also significantly increased in patients with severe pre-eclampsia. By logistic regression analysis, the presence of these three autoantibodies significantly increased the risk of neonatal death (odds ratio, 13.5; 95% confidence interval, 1.3–141.3; p = 0.030) and long-term neonatal hospitalization (odds ratio, 5.0; 95% confidence interval, 1.3–19.1; p = 0.018). The risk of hypertension and fetal distress were also associated with the presence of these three autoantibodies.

Conclusions/Significance

This novel pilot study demonstrated for the first time that the presence of autoantibodies against β₁, β₂ and α₁ adrenoreceptors are increased in patients with severe pre-eclampsia. Pregnant women who are positive for the three autoantibodies are at increased risks of neonatal mortality and morbidity. We posit that these autoantibodies may be involved in the pathogenesis of severe pre-eclampsia.     

Assessment of the Reporting Quality of Randomized Controlled Trials on the Treatment of Diabetes Mellitus with Traditional Chinese Medicine: A Systematic Review

Background

After the publication of the CONSORT 2010 statement, few studies have been conducted to assess the reporting quality of randomized clinical trials (RCTs) on treatment of diabetes mellitus with Traditional Chinese Medicine (TCM) published in Chinese journals.

Objective

To investigate the current situation of the reporting quality of RCTs in leading medical journals in China with the CONSORT 2010 statement as criteria.

Methods

The China National Knowledge Infrastructure (CNKI) electronic database was searched for RCTs on the treatment of diabetes mellitus with TCM published in the Journal of Traditional Chinese Medicine, Chinese Journal of Integrated Traditional & Western Medicine, and the China Journal of Chinese Materia Medica from January to December 2011. We excluded trials reported as “animal studies”, “in vitro studies”, “case studies”, or “systematic reviews”. The CONSORT checklist was applied by two independent raters to evaluate the reporting quality of all eligible trials after discussing and comprehending the items thoroughly. Each item in the checklist was graded as either “yes” or “no” depending on whether it had been reported by the authors.

Results

We identified 27 RCTs. According to the 37 items in the CONSORT checklist, the average reporting percentage was 45.0%, in which the average reporting percentage for the “title and abstract”, the “introduction”, the “methods”, the “results”, the “discussion” and the “other information” was 33.3%, 88.9%, 36.4%, 54.4%, 71.6% and 14.8%, respectively. In the Journal of Traditional Chinese Medicine, Chinese Journal of Integrated Traditional & Western Medicine, and the China Journal of Chinese Materia Medica the average reporting percentage was 42.2%, 56.8%, and 46.0%, respectively.

Conclusions

The reporting quality of RCTs in these three journals was insufficient to allow readers to assess the validity of the trials. We recommend that editors require authors to use the CONSORT statement when reporting their trial results as a condition of publication.     

Porous Allograft Bone Scaffolds: Doping with Strontium

Strontium (Sr) can promote the process of bone formation. To improve bioactivity, porous allograft bone scaffolds (ABS) were doped with Sr and the mechanical strength and bioactivity of the scaffolds were evaluated. Sr-doped ABS were prepared using the ion exchange method. The density and distribution of Sr in bone scaffolds were investigated by inductively coupled plasma optical emission spectrometry (ICP-OES), X-ray photoelectron spectroscopy (XPS), and energy-dispersive X-ray spectroscopy (EDS). Controlled release of strontium ions was measured and mechanical strength was evaluated by a compressive strength test. The bioactivity of Sr-doped ABS was investigated by a simulated body fluid (SBF) assay, cytotoxicity testing, and an in vivo implantation experiment. The Sr molar concentration [Sr/(Sr+Ca)] in ABS surpassed 5% and Sr was distributed nearly evenly. XPS analyses suggest that Sr combined with oxygen and carbonate radicals. Released Sr ions were detected in the immersion solution at higher concentration than calcium ions until day 30. The compressive strength of the Sr-doped ABS did not change significantly. The bioactivity of Sr-doped material, as measured by the in vitro SBF immersion method, was superior to that of the Sr-free freeze-dried bone and the Sr-doped material did not show cytotoxicity compared with Sr-free culture medium. The rate of bone mineral deposition for Sr-doped ABS was faster than that of the control at 4 weeks (3.28±0.23 µm/day vs. 2.60±0.20 µm/day; p<0.05). Sr can be evenly doped into porous ABS at relevant concentrations to create highly active bone substitutes.     

Toxoplasma gondii Protein Disulfide Isomerase (TgPDI) Is a Novel Vaccine Candidate against Toxoplasmosis

Toxoplasma gondii is a ubiquitous protozoan parasite that can infect all warm-blooded animals, including both mammals and birds. Protein disulfide isomerase (PDI) localises to the surface of T. gondii tachyzoites and modulates the interactions between parasite and host cells. In this study, the protective efficacy of recombinant T. gondii PDI (rTgPDI) as a vaccine candidate against T. gondii infection in BALB/c mice was evaluated. rTgPDI was expressed and purified from Escherichia coli. Five groups of animals (10 animals/group) were immunised with 10, 20, 30, 40 μg of rTgPDI per mouse or with PBS as a control group. All immunisations were performed via the nasal route at 1, 14 and 21 days. Two weeks after the last immunisation, the immune responses were evaluated by lymphoproliferative assays and by cytokine and antibody measurements. The immunised mice were challenged with tachyzoites of the virulent T. gondii RH strain on the 14th day after the last immunisation. Following the challenge, the tachyzoite loads in tissues were assessed, and animal survival time was recorded. Our results showed that the group immunised with 30 μg rTgPDI showed significantly higher levels of specific antibodies against the recombinant protein, a strong lymphoproliferative response and significantly higher levels of IgG2a, IFN-gamma (IFN-γ), IL-2 and IL-4 production compared with other doses and control groups. While no changes in IL-10 levels were detected. After being challenged with T. gondii tachyzoites, the numbers of tachyzoites in brain and liver tissues from the rTgPDI group were significantly reduced compared with those of the control group, and the survival time of the mice in the rTgPDI group was longer than that of mice in the control group. Our results showed that immunisation with rTgPDI elicited a protective immune reaction and suggested that rTgPDI might represent a promising vaccine candidate for combating toxoplasmosis.     

Identification of Critical Regions in Human SAMHD1 Required for Nuclear Localization and Vpx-Mediated Degradation

The sterile alpha motif (SAM) and HD domain-containing protein-1 (SAMHD1) inhibits the infection of resting CD4+ T cells and myeloid cells by human and related simian immunodeficiency viruses (HIV and SIV). Vpx inactivates SAMHD1 by promoting its proteasome-dependent degradation through an interaction with CRL4 (DCAF1) E3 ubiquitin ligase and the C-terminal region of SAMHD1. However, the determinants in SAMHD1 that are required for Vpx-mediated degradation have not been well characterized. SAMHD1 contains a classical nuclear localization signal (NLS), and NLS point mutants are cytoplasmic and resistant to Vpx-mediated degradation. Here, we demonstrate that NLS-mutant SAMHD1 K11A can be rescued by wild-type SAMHD1, restoring its nuclear localization; consequently, SAMHD1 K11A became sensitive to Vpx-mediated degradation in the presence of wild-type SAMHD1. Surprisingly, deletion of N-terminal regions of SAMHD1, including the classical NLS, generated mutant SAMHD1 proteins that were again sensitive to Vpx-mediated degradation. Unlike SAMHD1 K11A, these deletion mutants could be detected in the nucleus. Interestingly, NLS-defective SAMHD1 could still bind to karyopherin-β1 and other nuclear proteins. We also determined that the linker region between the SAM and HD domain and the HD domain itself is important for Vpx-mediated degradation but not Vpx interaction. Thus, SAMHD1 contains an additional nuclear targeting mechanism in addition to the classical NLS. Our data indicate that multiple regions in SAMHD1 are critical for Vpx-mediated nuclear degradation and that association with Vpx is not sufficient for Vpx-mediated degradation of SAMHD1. Since the linker region and HD domain may be involved in SAMHD1 multimerization, our results suggest that SAMHD1 multimerization may be required for Vpx-mediation degradation.     

Associations of Green Tea and Rock Tea Consumption with Risk of Impaired Fasting Glucose and Impaired Glucose Tolerance in Chinese Men and Women

Objective

To explore the associations of green tea and rock tea consumption with risk of impaired fasting glucose (IFG) and impaired glucose tolerance (IGT).

Methods

A multistage, stratified, cluster, random-sampling method was used to select a representative sample from Fujian Province in China. In total, 4808 subjects without cardiovascular disease, hypertension, cancer, or pancreatic, liver, kidney, or gastrointestinal diseases were enrolled in the study. A standard questionnaire was used to gather data on tea (green, rock, and black) consumption and other relevant factors. The assessment of impaired glucose regulation (IGR) was using 75-g oral glucose tolerance test (OGTT), the diagnostic criteria of normal glucose tolerance was according to American Diabetes Association.

Results

Green tea consumption was associated with a lower risk of IFG, while rock tea consumption was associated with a lower risk of IGT. The adjusted odds ratios for IFG for green tea consumption of <1, 1–15, 16–30, and >30 cups per week were 1.0 (reference), 0.42 (95% confidence intervals (CI) 0.27–0.65), 0.23 (95% CI, 0.12–0.46), and 0.41 (95% CI, 0.17–0.93), respectively. The adjusted odds ratios for IGT for rock tea consumption of <1, 1–15, 16–30, and >30 cups per week were 1.0 (reference), 0.69 (95% CI, 0.48–0.98), 0.59 (95% CI, 0.39–0.90), and 0.64 (95% CI, 0.43–0.97), respectively. A U-shaped association was observed, subjects who consumed 16–30 cups of green or rock tea per week having the lowest odds ratios for IFG or IGT.

Conclusions

Consumption of green or rock tea may protect against the development of type 2 diabetes mellitus in Chinese men and women, particularly in those who drink 16–30 cups per week.     

Differential Responses of Hepatic Endoplasmic Reticulum Stress and Inflammation in Diet-Induced Obese Rats with High-Fat Diet Rich in Lard Oil or Soybean Oil

Scopes

To investigate the effects of high-fat diet enriched with lard oil or soybean oil on liver endoplasmic reticulum (ER) stress and inflammation markers in diet-induced obese (DIO) rats and estimate the influence of following low-fat diet feeding.

Methods and Results

Male SD rats were fed with standard low-fat diet (LF, n = 10) and two isoenergentic high-fat diets enriched with lard (HL, n = 45) or soybean oil (HS, n = 45) respectively for 10 weeks. Then DIO rats from HL and HS were fed either high-fat diet continuously (HL/HL, HS/HS) or switched to low-fat diet (HL/LF, HS/LF) for another 8 weeks. Rats in control group were maintained with low-fat diet. Body fat, serum insulin level, HOMA-IR and ectopic lipid deposition in liver were increased in HL/HL and HS/HS compared to control, but increased to a greater extent in HL/HL compared to HS/HS. Markers of ER stress including PERK and CHOP protein expression and phosphorylation of eIF2α were significantly elevated in HL/HL group while phosphorylation of IRE1α and GRP78 protein expression were suppressed in both HL/HL and HS/HS. Besides, inflammatory signals (OPN, TLR2, TLR4 and TNF-α) expressions significantly increased in HL/HL compared to others. Switching to low-fat diet reduced liver fat deposition, HOMA-IR, mRNA expression of TLR4, TNF-α, PERK in both HL/LF and HS/LF, but only decreased protein expression of OPN, PERK and CHOP in HL/LF group. In addition, HL/LF and HS/LF exhibited decreased phosphorylation of eIF2α and increased phosphorylation of IRE1α and GRP78 protein expression when compared with HL/HL and HS/HS respectively.

Conclusions

Lard oil was more deleterious in insulin resistance and hepatic steatosis via promoting ER stress and inflammation responses in DIO rats, which may be attributed to the enrichment of saturated fatty acid. Low-fat diet was confirmed to be useful in recovering from impaired insulin sensitivity and liver fat deposition in this study.     

Evolutionary Pattern of the FAE1 Gene in Brassicaceae and Its Correlation with the Erucic Acid Trait

The fatty acid elongase 1 (FAE1) gene catalyzes the initial condensation step in the elongation pathway of VLCFA (very long chain fatty acid) biosynthesis and is thus a key gene in erucic acid biosynthesis. Based on a worldwide collection of 62 accessions representing 14 tribes, 31 genera, 51 species, 4 subspecies and 7 varieties, we conducted a phylogenetic reconstruction and correlation analysis between genetic variations in the FAE1 gene and the erucic acid trait, attempting to gain insight into the evolutionary patterns and the correlations between genetic variations in FAE1 and trait variations. The five clear, deeply diverged clades detected in the phylogenetic reconstruction are largely congruent with a previous multiple gene-derived phylogeny. The Ka/Ks ratio (<1) and overall low level of nucleotide diversity in the FAE1 gene suggest that purifying selection is the major evolutionary force acting on this gene. Sequence variations in FAE1 show a strong correlation with the content of erucic acid in seeds, suggesting a causal link between the two. Furthermore, we detected 16 mutations that were fixed between the low and high phenotypes of the FAE1 gene, which constitute candidate active sites in this gene for altering the content of erucic acid in seeds. Our findings begin to shed light on the evolutionary pattern of this important gene and represent the first step in elucidating how the sequence variations impact the production of erucic acid in plants.