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Translational control is a fundamental mechanism regulating animal germ cell development. Gonadal somatic cells provide support and microenvironment for germ cell development to ensure fertility, yet the roles of translational control in gonadal somatic compartment remain largely undefined. We found that mouse homolog of conserved fly germline stem cell factor Pumilio, PUM1, is absent in oocytes of all growing follicles after the primordial follicle stage, instead, it is highly expressed in somatic compartments of ovaries. Global loss of Pum1, not oocyte-specific loss of Pum1, led to a significant reduction in follicular number and size as well as fertility. Whole-genome identification of PUM1 targets in ovarian somatic cells revealed an enrichment of cell proliferation pathway, including 48 key regulators of cell phase transition. Consistently granulosa cells proliferation is reduced and the protein expression of the PUM-bound Cell Cycle Regulators (PCCR) were altered accordingly in mutant ovaries, and specifically in granulosa cells. Increase in negative regulator expression and decrease in positive regulators in the mutant ovaries support a coordinated translational control of somatic cell cycle program via PUM proteins. Furthermore, postnatal knockdown, but not postnatal oocyte-specific loss, of Pum1 in Pum2 knockout mice reduced follicular growth and led to similar expression alteration of PCCR genes, supporting a critical role of PUM-mediated translational control in ovarian somatic cells for mammalian female fertility. Finally, expression of human PUM protein and its regulated cell cycle targets exhibited significant correlation with ovarian cancer and prognosis for cancer survival. Hence, PUMILIO-mediated cell cycle regulation represents an important mechanism in mammalian female reproduction and human cancer biology.

N-terminal pro-B-type natriuretic peptide (NT-proBNP) may reflect early prognosis in patients with valvular heart disease (VHD).The aim of this study was to examine the association between NT-proBNP and mortality in elderly patients with VHD.A total of 5,983 elderly patients (age ≥60 years) with moderate or severe VHD underwent echocardiography and NT-proBNP measurement. VHD examined included aortic stenosis, aortic regurgitation, mitral stenosis, mitral regurgitation, tricuspid regurgitation, and multivalvular heart disease. NT-proBNP ratio was defined as measured NT-proBNP relative to the maximal normal values specific to age and sex. Disease-specific thresholds were defined on the basis of penalized splines and maximally selected rank statistics.The cohort had a mean age of 71.1 ± 7.6 years. At 1-year follow-up, 561 deaths (9.4%) had occurred. In penalized splines, relative hazards showed a monotonic increase with greater NT-proBNP ratio for death with different VHDs (p 0.001 for all) except mitral stenosis. Higher NT-proBNP ratio, categorized by disease-specific thresholds, was independently associated with mortality (overall adjusted hazard ratio: 1.99; 95% confidence interval: 1.76 to 2.24; p 0.001). Different subtypes of VHD all incurred excess mortality with elevated NT-proBNP ratio, with the strongest association detected for aortic stenosis (adjusted hazard ratio: 10.5; 95% confidence interval: 3.9 to 28.27; p 0.001). The addition of NT-proBNP ratio to the prediction algorithm including traditional risk factors improved outcome prediction (overall net reclassification index = 0.28; 95% CI: 0.24 to 0.34; p 0.001; likelihood ratio test p 0.001). Results remained consistent in patients under medical care, with normal left ventricular ejection fractions, and with primary VHD.NT-proBNP provides incremental prognostic information for mortality in various VHDs. It could aid in risk stratification as a pragmatic and versatile biomarker in elderly patients.

Sepsis is more common among males than females, and the unequal estrogen levels have been suspected to play a vital role in gender differences. Recently, trained immunity is reported to be a novel strategy for the innate immune system to fight infection. However, it has not been clarified whether β-glucan-induced trained immunity causes different responses to early sepsis between male and female mice. In this study, sepsis was induced in mice by intraperitoneal injection of Escherichia coli (E. coli). The changes of inflammatory cytokines expression, and macrophage polarization in male, female, and ovariectomized C57BL/6 mice in sepsis model were investigated. For in vitro studies, different macrophages were treated with LPS. The function of estradiol (E2) on macrophage cell lines was verified and the mechanism of E2 affecting trained immunity was explored. We demonstrated that β-glucan-induced trained immunity was more resistant to sepsis in female than male mice. Macrophage polarization toward the M1 phenotype, which exhibited enhanced trained immunity, was related to the difference in sepsis resistance between female and male mice. Moreover, ovariectomized (OVX) mice manifested serious sepsis consequences with a weaker trained immunity effect than female mice. Female bone marrow-derived macrophages (BMDMs) were also apt to be polarized to the M1 phenotype in response to trained immunity in vitro. Furthermore, E2 promoted trained immunity in macrophage cell lines J774 and RAW264.7. E2 was also verified to facilitate trained immunity in primary BMDMs from female and male mice. Mechanistically, we found that E2 inhibited the nuclear translocation of RelB, which is a member of non-canonical pathway of NFκB and contributes to macrophage polarization to change the intensity of trained immunity. This study is the first to indicate the role of E2 in the trained immunity induced by β-glucan to protect against E. coli-induced sepsis via the non-canonical NFκB pathway. These results improve our understanding of the molecular mechanisms governing trained immunity in gender differences.

Even among biomarker-negative patients undergoing elective percutaneous coronary intervention (PCI), periprocedural thrombotic and bleeding complications can lead to increased morbidity and mortality. Whether stronger platelet inhibition by an intensified oral loading strategy (ILS) before PCI impacts on outcomes among these patients in contemporary practice remains unclear.This multicenter, randomized, assessor-blinded trial tested the hypothesis that in elective PCI prasugrel 60 mg (ILS) is superior to standard loading strategy with clopidogrel 600 mg regarding a composite primary end point of all-cause death, any myocardial infarction, definite/probable stent thrombosis, stroke, or urgent vessel revascularization. After PCI, all patients were on clopidogrel 75 mg/day and aspirin. The trial was terminated prematurely because of slower-than-expected recruitment and funding discontinuation.Of 781 patients included in the final analysis, 382 were assigned to ILS and 399 to standard loading strategy. At 30 days, the primary end point occurred in 66 patients (17.3%) assigned to ILS and 74 patients (18.6%) assigned to standard loading strategy (odds ratio, 0.92 [95% CI, 0.63-1.32];In biomarker-negative stable and unstable angina patients undergoing elective PCI, the trial did not find a conclusive difference in efficacy or safety. This observation should be interpreted in the context of wide CIs and premature termination of the trial. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02548611.

Background: Acute kidney injury (AKI) is a common complication after surgery. Because of unpredictable and variable age-dependent physical decline, the incidence, risk factor of postsurgical AKI and the predictive power of estimated glomerular filtration rate prior to surgery (eGFRpreSurg) has not been fully elucidated in very elderly patients. Methods: All discharged patients aged ≥80 years without chronic kidney disease who underwent surgery prior to intensive care unit (ICU) admission from 2017 through 2018 were included. Clinical, biological and surgical data were recorded. Mean of outpatient creatinine values from the year prior to ICU admission was used as the baseline value to determine the occurrence of AKI. Postoperative AKI was diagnosed according to Kidney Disease Improving Global Outcomes criteria. Results: Among 243 very elderly postoperative patients admitted during the study period, 48 had AKI during their ICU stay. The occurrence of postsurgical AKI was associated with longer ventilation times (p

Background At present, the treatment of recurrent glioblastoma is extremely challenging. In this study, we used a novel three-dimensional non-coplanar template (3DNPT) combined with open MR to guide 125I seed implantation for recurrent glioblastoma. The aim of this study was to evaluate the feasibility, accuracy, and effectiveness of this technique. Methods Twenty-four patients of recurrent glioblastoma underwent 3DNPT with open MR-guided 125I brachytherapy from August 2017 to January 2019. Preoperative treatment plan and 3DNPT were made according to enhanced isovoxel T1-weighted MR images. 125I seeds were implanted using 3DNPT and 1.0-T open MR imaging guidance. Dosimetry verification was performed after brachytherapy based on postoperative CT/MR fusion images. Preoperative and postoperative dosimetry parameters of D90, V100, V200, conformity index (CI), external index (EI) were compared. The objective response rate (ORR) at 6 months and 1-year survival rate were calculated. Median overall survival (OS) measured from the date of brachytherapy was estimated by Kaplan-Meier method. Results There were no significant differences between preoperative and postoperative dosimetry parameters of D90, V100, V200, CI, EI (P > 0.05). The ORR at 6 months was 75.0%. The 1-year survival rate was 58.3%. Median OS was 12.9 months. One case of small amount of epidural hemorrhage occurred during the procedure. There were 3 cases of symptomatic brain edema after brachytherapy treatment, including grade three toxicity in 1 case and grade two toxicity in 2 cases. The three patients were treated with corticosteroid for 2 to 4 weeks. The clinical symptoms related to brain edema were significantly alleviated thereafter. Conclusions 3DNPT combined with open MR-guided 125I brachytherapy for circumscribed recurrent glioblastoma is feasible, effective, and with low risk of complications. Postoperative dosimetry matched the preoperative treatment plan. The described method can be used as a novel implantation technique for 125I brachytherapy in the treatment of recurrent gliomas. Trial registration The study was approved by the Institutional Review Board of Shandong Provincial Hospital Affiliated to Shandong University (NSFC:NO.2017–058), registered 1st July 2017.

Mouse PUMILIO1 (PUM1) and PUMILIO2 (PUM2) belong to the PUF (Pumilio/FBF) family, a highly conserved RNA binding protein family whose homologues play critical roles in embryonic development and germ line stem cell maintenance in invertebrates. However, their roles in mammalian embryonic development and stem cell maintenance remained largely uncharacterized. Here we report an essential requirement of the Pum gene family in early embryonic development. A loss of both Pum1 and Pum2 genes led to gastrulation failure, resulting in embryo lethality at E8.5. Pum-deficient blastocysts, however, appeared morphologically normal, from which embryonic stem cells (ESCs) could be established. Both mutant ESCs and embryos exhibited reduced growth and increased expression of endoderm markers Gata6 and Lama1, making defects in growth and differentiation the likely causes of gastrulation failure. Furthermore, ESC Gata6 transcripts could be pulled down via PUM1 immunoprecipitation and mutation of conserved PUM-binding element on 3′UTR (untranslated region) of Gata6 enhanced the expression of luciferase reporter, implicating PUM-mediated posttranscriptional regulation of Gata6 expression in stem cell development and cell lineage determination. Hence, like its invertebrate homologues, mouse PUM proteins are conserved posttranscriptional regulators essential for embryonic and stem cell development.

Low frequency magnetic fields (LF-MFs) can affect cell proliferation in a cell-type and intensity-dependent way. Previous study has reported the anti-tumor effect of LF-MFs in lung cancers. Our previous study also optimized the intensity and duration of LF-MFs to effectively inhibit the proliferation of lung cancer cells. However, the anti-tumor mechanism of LF-MFs remains unclear, which limit the clinical application of LF-MFs in anti-tumor therapy. Here, in a well-established Lewis Lung Cancer (LLC) mouse model, we found that LF-MFs inhibit tumor growth and induce an autophagic cell death in lung cancer. We also found that LF-MFs could up-regulate the expression level of miR-486, which was involved in LF-MFs activated cell autophagy. Furthermore, we found B-cell adaptor for phosphatidylinositol 3-kinase (BCAP) is a direct target of miR-486. miR-486 inhibit AKT/mTOR signaling through inhibiting expression of BCAP. Moreover, a decreased expression of miR-486 and an increased expression of BCAP were found in tumor tissues of lung cancer patients. Taken together, this study proved that LF-MFs can inhibit lung cancers through miR-486 induced autophagic cell death, which suggest a clinical application of LF-MFs in cancer treatment.

Aims The predictive value of white blood cells in triple-vessel coronary artery disease (TVD) remains unclear. This study aimed to examine the relationship between WBC counts and long-term prognosis of TVD. Methods A total of 8943 consecutive patients with triple-vessel coronary artery disease were enrolled from April 2004 to February 2011. The primary endpoint was all-cause death and the secondary endpoints were major adverse cardiovascular and cerebrovascular events (MACCEs; a composite of all-cause death, myocardial infarction or stroke). Results After a median of 7.5 years of follow-up, 7678 patients were included in the final analysis. Multivariable analysis showed that the white blood cell count was an independent predictor of death (hazard ratio: 1.04, p Conclusion Total and differential white blood cell counts are independent prognostic factors of long-term mortality and MACCE in triple-vessel coronary artery disease. A combination of monocyte, lymphocyte and eosinophil counts improved the predictive value for mortality with the SYNTAX score, and improved it slightly with SYNTAX score II.

This study aimed to elucidate the association between the adverse gastric effects of enteric-coated aspirin and the timing of its administration. The study population comprised 572 patients (age range 45–84 years) admitted to Huaiyin Hospital between August 2012 and October 2014. Patients were administered a 100 mg enteric-coated aspirin tablet once daily: before a meal (30 min before a meal), during a meal, after a meal (30 min after a meal), or before sleep, and all patients were followed up for 6–9 months to observe for adverse gastric reactions and other side effects. Gastroscopy was performed if indicated by the patient’s condition after obtaining due consent. In addition, release tests for an enteric-coated aspirin tablet were conducted using the chromatography method. Enteric-coated aspirin tablets released completely, with a release rate of >99 % under 20–120 min at pH > 5.5. Furthermore, the number of patients with recurring adverse stomach reactions was significantly lower in the before-meal and before-sleep groups than that observed in the during-meal and after-meal groups (p

Hypertriglyceridaemia is one of the most common causes of severe acute pancreatitis (SAP). However, the association between elevated triglycerides (TG) level in hospital stay and outcome in SAP patients with normal TG level at admission has not been clearly demonstrated. This retrospective study assessed the serum TG levels of patients with SAP admitted to the intensive care unit (ICU) in 2017. Variables with a statistically significant association with the incidence of in-hospital TG elevation, as determined by univariate analysis, were analysed using a logistic regression model to predictors. Of the 99 patients included in the study, TG levels were within the normal range in 59 (59.59%) patients at admission. Among patients with normal TG level when admitted to ICU, 28 (47.46%) experienced at least one episode of TG level elevation during their ICU stay. Elevated TG level in hospitalization is associated with an increased length of ICU stay, as well as increased mortality. In addition to other factors, propofol usage was independently associated with the occurrence of in-hospital-TG elevation. To conclude, we retrospectively investigated the incidence, outcome, and risk factors for in-hospital TG elevation events in SAP patients admitted to the ICU. We found a high incidence of both preexisting and in-hospital-acquired TG elevation in SAP patients admitted to the ICU. The TG elevation that occurred during the ICU stay was associated with worse outcomes and long-term hospitalization of the ICU. Propofol usage was independently associated with the TG elevation occurrence in the ICU.

Pancreatic carcinoma is a common cause of cancer deaths worldwide. Biopsy is often required for the initial diagnosis of pancreatic masses. Biopsy can be performed endoscopically or percutaneously with computed tomography (CT) and ultrasound (US) guidance. MRI offers many inherent advantages over CT and US.To prospectively evaluate the feasibility, accuracy, and safety of MRI-guided percutaneous coaxial cutting needle biopsy of pancreatic lesions using an open 1.0T high-field MR scanner.Prospective.Thirty-one patients with 31 pancreatic lesions underwent MR-guided percutaneous coaxial cutting needle biopsy.1.0T TFinal diagnosis was confirmed by surgery and clinical follow-up for at least 12 months. The accuracy, sensitivity, and specificity were calculated. Complications were recorded.There was no statistical analysis in this study.The procedure was technically successful and final biopsy samples were adequate for histopathological examination in all patients. Biopsy pathology revealed malignant pancreatic tumor in 25 patients (25/31, 80.6%), and benign pancreatic lesions were present in six patients (6/31, 19.4%). The final diagnosis was pancreatic malignancy in 27 patients and benign disease in four patients, which was confirmed by surgery and clinical follow-up. Two biopsy results were false-negative. The diagnostic accuracy in biopsies was 93.5% (29 of 31). The sensitivity to detect a malignant disease was 92.6% (25 of 27), and the specificity was 100%. All patients tolerated the procedure well; minor peripancreatic hemorrhage was found in two patients after the procedure, and none had major complications either during or after the procedure.MRI-guided percutaneous biopsy of pancreatic lesions using an open 1.0T high-field scanner has high diagnostic accuracy, which is feasible and safe for use in clinical practice.2 Technical Efficacy: Stage 2 J. MAGN. RESON. IMAGING 2018;48:382-388.

Patients with non–small cell lung cancer (NSCLC) with malignant pleural effusion (MPE) have a short median survival time and increased regulatory T cells (Treg). However, it is unclear whether some specific factors in MPE are involved in Treg recruitment in the progression of NSCLC. Here, we found that Treg population was increased in MPE and inversely correlated with patient survival (P < 0.001). Increased level of CXCL1 in MPE was associated with recruitment of Tregs (P < 0.01). Moreover, miR141 regulated expression of CXCL1 in lung cancer cells, whereas the luciferase test confirmed that CXCL1 is a target of miR141. Chemotaxis assay showed that the miR141–CXCL1–CXCR2 pathway regulates migration of Tregs into MPE. Furthermore, miR141 significantly inhibited tumor growth and metastasis in an immune-competent mouse model. This suppressive function was mediated by the CXCL1–CXCR2 pathway and recruitment of Tregs. Our study uncovered a causative link between microRNA and development of MPE. Mechanistically, decreased expressions of miR141, associated with the survival of patients with NSCLC with MPE, resulted in the increased production of CXCL1 and recruitment of Tregs to promote immune escape of tumor. Mol Cancer Ther; 13(12); 3152–62. ©2014 AACR.

Estrogen is involved in promoting lung cancer cell division and metastasis. MICA and MICB function as ligands for NKG2D, an important immunoreceptor expressed on natural killer (NK) cells. However, whether estrogen regulates MICA/B expression and affects tumor immune escape remains unknown. In this study, we measured the mRNA levels of MICA, MICB and ADAM17in non-small cell lung cancer (NSCLC) cell lines treated with estrogen. Surface expression of MICA/B on LTEP-a2 and A549 was detected using flow cytometry. We demonstrate that both mRNA and secretory protein levels of MICA/B in lung adenocarcinoma cell lines were upregulated by estradiol. Estradiol enhanced the expression of ADAM17, which was associated with the secretion of MICA/B. This secretion of MICA/B downregulated the NKG2D receptor on the surface of NK92 cells and impaired the cytotoxic activity of NK cells. Estradiol enhanced the expression of ADAM17, which was associated with the secretion of MICA/B. Furthermore, a significant correlation between the concentration of estradiol and the expression of MICA was found in tumor tissues of NSCLC patients. Therefore, we conclude that estrogen can regulate the expression and secretion of MICA/B through ADAM17, which helps lung cancer cells escape NKG2D-mediated immune surveillance.

Background and Purpose— Accumulating evidence suggests that telomere length is a maker for biological aging of the cardiovascular system. Whether stroke is associated with accelerated biological aging as measured by telomere length has not been conclusively demonstrated. Our aim was to determine whether mean leukocyte telomere length is a predictor for the development of stroke. Methods— The relative telomere length of leukocytes was determined by quantitative polymerase chain reaction in 1309 stroke patients and 1309 age- and sex-matched control subjects as well as 858 stroke patients followed prospectively for 5 years. For each measure, the study sample was divided into quartiles. The associations between the telomere length and risk of stroke as well as poststroke adverse outcomes were determined. Results— Mean telomere length was significantly shorter in stroke patients than in control subjects. Shorter telomere length levels were directly associated with a higher risk of stroke in the case/control sample. As compared with the fourth (longest) quartile, the odd ratios [OR] (and 95% confidence intervals [CI]) for ischemic stroke risk were as follows: third quartile, 1.37 (1.04–1.82); second quartile, 1.53 (1.17–2.02); and first quartile, 2.12 (1.62–2.77). Follow-up of the patients from the prospective cohort also showed that shorter telomere length levels were associated with mortality from all causes but not with recurrence of stroke. Conclusions— Shorter telomere length was associated with ischemic stroke and was a strong predictor of poststroke death.

Experimental and clinical observations suggest that E-selectin could have an important role in essential hypertension (EH), but the relationship between common E-selectin variants and EH has not been extensively studied in the Chinese population. In this study, we explored the association between two common variants in the E-selectin gene (rs5361A/C and rs5355C/T) and EH in the Uygur, Kazakh and Han populations in the Xinjiang area. A case-control study was conducted to explore the association between these two single-nucleotide polymorphisms and EH in a large sample size, including 941 EH subjects (309 Uygur, 264 Kazakh and 368 Han individuals) and 924 control subjects (300 Uygur, 275 Kazakh and 349 Han individuals). Univariate analysis showed that the rs5361 A/C polymorphism was significantly associated with EH in the Uygur (P=0.002) and Han (P=3.6 × 10(-7)) populations. The CC genotype of this SNP was present only in patients with EH in all of the three nationalities studied. Han individuals who carry the CC genotype of rs5361 were more susceptible to EH, according to the dominant models (P=1.13 × 10(-4), odds ratio=3.812, 95% confidence interval: 1.685-7.792), but there was no association of genotype with EH for the other ethnicities. No significant difference in rs5355 C/T polymorphism rate was found between the EH and control groups. Our results indicate that the common variant rs5361 is strongly associated with EH in Han individuals and weakly associated in Uygur individuals. The CC genotype of rs5361 might be an independent risk factor for EH among Uygur, Kazakh and Han individuals in the Xinjiang area.

To investigate the association of polymorphisms in four critical genes implicated in the NO-forming pathway with ischemic stroke (IS) in a Chinese Han population.DNA samples of 558 IS patients and 557 healthy controls from Chinese Han population were genotyped using the Taqman(TM) 7900HT Sequence Detection System. Six SNPs (rs841, rs1049255, rs2297518, rs1799983, rs2020744, rs4673) of the 4 related genes (eNOS, iNOS, GCH1, and CYBA) in the NO forming pathway were analyzed using the SPSS 13.0 software package for Windows.One SNP located in the intron of GCH1 (rs841) was associated with IS independent of the traditional cardiovascular risk factors in co-dominant and dominant models (P=0.003, q=0.027; P=0.00006, q=0.0108; respectively). Moreover, the combination of rs1049255 CC+CT and rs841 GA+AA genotypes was associated with significantly higher risk for IS after adjustments (OR=1.73, 95% CI: 1.27-2.35, P0.0001, q0.0001).The data suggest that genetic variants within the NO-forming pathway alter susceptibility to IS in Chinese Han population. Replication of the present results in other independent cohorts is warranted.

20-Hydroxyeicosatetraenoic acid has been shown to play an important role in cerebral vascular function. We hypothesized that polymorphisms in genes encoding 20-Hydroxyeicosatetraenoic acid synthesizing enzymes might confer susceptibility to stroke.To test the hypothesis, haplotype tagging single nucleotide polymorphisms and potential functional polymorphisms of CYP4A11 and CYP4F2 genes were genotyped in 558 ischemic stroke patients, 221 hemorrhagic stroke patients and 557 controls. The association analyses were performed at both single nucleotide polymorphism and haplotype levels. We further verified our findings in an independent cohort of 551 ischemic stroke cases and 48 hemorrhagic stroke cases and 694 unaffected controls. We identified CYP4A11 C-296T and CYP4F2 V433M were associated with significantly increased risk of ischemic stroke (CT+TT vs. CC, adjusted odds ratio: 1.50, 95% confidence interval: 1.17-1.93, Pcombined=0.001, Pcorr=0.008; V/M+M/M vs. V/V, odds ratio: 1.38, 95% confidence interval: 1.15-1.65, Pcombined=5.6x10, Pcorr=0.005, respectively). Interestingly, the effects of CYP4F2 V433M on ischemic stroke in our study was only evident in male individuals.Our results suggest that genetic variation in CYP4A11 and CYP4F2 alters susceptibility to stroke in the Han Chinese population.

Background— Recent studies on genome-wide association have identified common variants on chromosome 9p21 associated with coronary artery disease (CAD). Given that ischemic stroke and CAD share several aspects of etiology and pathogenesis, we investigated the association of variants on chromosome 9p21 with ischemic stroke and CAD in the Chinese Han population by capturing the majority of diversity in this locus using haplotype-tagging single-nucleotide polymorphisms. Methods and Results— We performed a shared control-cases study using 15 tagging single-nucleotide polymorphisms and 2 previously reported susceptibility single-nucleotide polymorphisms spanning 58 kb of the chromosome of 9p21 in a set of 558 patients with ischemic stroke, 510 patients with CAD, and 557 unaffected participants (controls) in the Chinese Han population. The association analyses were performed at both SNP and haplotype levels. We further verified our findings in an independent cohort of 442 ischemic stroke cases and 502 control subjects. In the first study, rs2383206, rs1004638, and rs10757278 in block 3 were significantly associated with CAD but not with ischemic stroke independent of traditional cardiovascular risk factors in additive model ( P =0.002 to 0.0001, q=0.026 to 0.004). Analysis from all blocks revealed that haplotype profiles of block 3 on 9p21 were significantly different between shared control and cases of CAD ( P =1.3�10 −10 , q=1.2�10 −9 ) and ischemic stroke ( P =1.7�10 −6 , q=7.7�10 −6 ). In the expanded second case-control study, block 3 on 9p21 remained associated with ischemic stroke ( P =2.6�10 −4 , q=6.3�10 −4 ). Conclusions— Our results suggest for the first time that 9p21 is a shared susceptibility locus, strongly for CAD and weakly for ischemic stroke, in a Chinese Han population.

In this study, we characterized the promoter activity of a 1.7 kb sequence in the 5′ flanking region of the mouse Deleted in Azoospermia-Like (Dazl) gene. We found the 1.7 kb sequence sufficient to drive robust germ cell-specific expression of green fluorescent protein (GFP) in adult mouse testis and lower levels of expression in adult ovary and in fetal and newborn gonads of both sexes. This expression pattern was confirmed in two independently-derived transgenic mouse lines. In adult testis, Dazl-GFP exhibited a developmentally-regulated, stage-specific expression pattern during spermatogenesis. GFP was highly expressed in spermatocyte stages, with strongest expression in pachytene spermatocytes. Weaker expression was observed in round and elongating spermatids, as well as spermatogonial cells. In the fetal gonad, GFP transcript was detected by e12.5 in both sexes; however, GFP fluorescence was only detected during later embryonic stages. In addition, we produced mouse embryonic stem cell (ESC) lines harboring the Dazl-GFP reporter and used this reporter to isolate putative germ cell populations derived from mouse ESCs following embryoid body differentiation and fluorescence activated cell sorting.

Lung cancer remains the leading cause of cancer mortality. Myeloid-derived suppressor cells (MDSCs) are potent immune-suppressive cells and present in most cancer patients. Recently, several studies have shown that curcumin inhibits the expansion of MDSCs in some cancers. However, it is not clear how curcumin modulates the suppressive function of MDSCs, and whether curcumin achieves anti-tumor effects via regulating the expansion of MDSCs in lung cancer. Here, our results showed that curcumin significantly inhibited tumor growth in a Lewis lung carcinoma (LLC) isogenic tumor model. Curcumin reduced the accumulation of MDSCs in spleen and tumor tissue in LLC isogenic model. And curcumin promoted the maturation and differentiation of MDSCs in tumor tissue. Notably, curcumin inhibited the expression level of immune suppressive factors of MDSCs, arginase-1 (Arg-1) and ROS, in purified MDSCs from tumor tissue in vivo. Expectedly, curcumin also inhibited the immunosuppressive function of isolated MDSCs from tumor tissue and spleen of tumor bearing mice in vitro. Moreover, curcumin decreased the level of IL-6 in the tumor tissue and serum from LLC-bearing mice. Taken together, curcumin indeed possesses anti-cancer effect and inhibits the accumulation and function of MDSCs. And curcumin reduces the level of IL-6 in tumor-bearing mice to impair the expansion and function of MDSCs. These results suggest that inhibition of MDSCs in tumor is requisite for controlling lung cancer.

Pumilio is a member of the highly conserved PUF family of RNA-binding proteins that function as a developmental regulator in diverse animal species. Two Pumilio genes, Pum1 and Pum2, have been identified in mammals and are found to be involved in sperm development, neuron development as well as human diseases such as neurodegeneration. Generation of animal models disrupting different parts of Pum protein could help to further dissect their physiological function. Here we described characterization and analysis of a mouse line possessing a gene trap mutation of the Pumilio1 (Pum1) gene. Mice homozygous for the mutation (Pum1XE002) cannot be recovered in the adult offspring, at birth or at different time points of embryonic development (E18, E14, E12). Careful analysis of preimplantation embryos showed that no homozygous blastocysts could be detected on day 3.5 of gestation. 96-hr in vitro culture of 1-cell embryos either by natural mating or in vitro fertilization between heterozygotes failed to uncover any homozygous blastocysts, suggesting an early loss of homozygous preimplantation embryos. The lack of Pum1 gene trap homozygotes suggests a role of Pum1 in very early embryonic development or fertilization. This novel animal model affecting the beginning of embryonic development could help to understand not only the genetic mechanism underlying preimplantation embryonic development but also the translational regulation in development and diseases.

Our previous studies showed that extremely low frequency magnetic fields (ELF-MFs) inhibited tumor growth and change proportion of splenic regulatory T cells (Treg cells). Here, we focus on the effect of ELF-MFs on lung metastatic melanoma mouse model and the regulatory mechanism of ELF-MFs on the differentiation of Treg cells. Tumor-bearing mice were exposed to sham ELF-MFs and ELF-MFs (0.4 T, 7.5 Hz) 2 h/day for 27 days. Metastatic tumor burden of lung was significantly decreased after ELF-MF treatment. Compared to the control group, expressions of matrix metalloproteinase (MMP2, MMP9) and forkhead box P3 (Foxp3) in lung nodules significantly decreased in the ELF-MF group. Moreover, in vitro, after stimulated with anti-CD3, anti-CD28 antibodies and transforming growth factor-β (TGF-β) and treated with ELF-MFs for 2 h, expression of Foxp3 in total T cells was significantly decreased. Differentiation rate of Treg cells was inhibited from 32.0% to 22.1% by ELF-MFs. Furthermore, reactive oxygen species (ROS) was increased and phospho-serine/threonine protein kinase (p-AKT) was inhibited in both T cells and Jurkat cells. ROS scavenger N-acetyl-l-cysteine reversed inhibition of AKT pathway and expression of Foxp3 from 18.6% to 26.6% in T cells. Taken together, our data show that ELF-MF exposure promoted the inhibitory effect of ROS on AKT pathway and decreased Foxp3 expression, which provides an explanation for why ELF-MF exposure can inhibit differentiation of Treg cells and enhance antitumor effect in metastatic melanoma mouse model.

Although interleukin (IL) 17A can promote angiogenesis in several tumors, there are limited clinical evidences on cancer about the correlation between serum vascular endothelial growth factor (VEGF) and IL-17F, which is the most homologous to IL-17A. In this study, serum concentration of IL-17F and VEGF from healthy (n = 28), leukoplakia (n = 15), and oral squamous cell carcinoma (OSCC) groups (n = 85) were assessed and showed that IL-17F level was remarkably downregulated from healthy group (394.3 pg/ml) to OSCC group (82.96 pg/ml). Conversely, the OSCC group had a highest level of VEGF (P 0.05) in whole groups, and there was a negative correlation between IL-17F and VEGF in serum or in the peripheral blood mononuclear cells (PBMCs) at mRNA level. Moreover, the lowest ratio of IL-17F/VEGF was found in OSCC patients (P 0.05) and lower ratio of IL-17F/VEGF correlated to higher tumor stage and lymph node metastasis. Furthermore, the serum level of IL-17F and the ratio of IL-17F/VEGF were positively associated with the numbers of CD3(+) CD4(+) T cells, which indicated that serum IL-17F could originate from PBMCs during the development of OSCC, and could be used for the diagnosis by effectively distinguishing OSCC patients from healthy individuals.

Interferon-γ (IFN-γ) is known to enhance the immunosuppressive properties of mesenchymal stem cells (MSCs). The aim of this study was to determine whether gene modification with IFN-γ-expression plasmids could boost the therapeutic effects of MSCs on DSS-induced colitis. We first reconstructed pcDNA3.1-IFNγ plasmids, transfected them to human umbilical cord derived MSCs, and detected the basic characters of MSCs including immune phenotype, cell vitality, proliferation, apoptosis and cell cycle progression after transfection. Subsequently, we analyzed the inhibition effect of IFN-γ-MSCs on T cell proliferation in vitro. Finally, we induced colitis in female C57BL/6 mice by 3 % DSS treatment and evaluated the therapeutic efficacy of IFN-γ-MSCs on colitis. Transfection with pcDNA3.1-IFNγ did not change the basic characters of MSCs. Interestingly, IFN-γ-MSCs showed more potent immunosuppressive effects on the proliferation of T cells compared to normal MSCs. Furthermore, systemic infusion with IFN-γ-MSCs more efficiently ameliorated DSS-induced mouse colitis including colitis-related ease of body weight, increase of colon length, decrease of disease activity index, and improvement of small intestine tissues structure. In addition, IFN-γ-MSCs increased the populations of Foxp3+ Tregs and Th2 cells both in mesenteric lymph node and spleen, upregulated indoleamine 2, 3-dioxygenase expression, and suppressed inflammatory cytokine production in mouse colon. Gene delivery with IFN-γ-expression plasmids enhanced the therapeutic effects of MSCs on DSS-induced mouse colitis. This study provides an effective therapeutic strategy of MSCs for inflammatory diseases.

SummaryIn a previous study, we have identified a set of conserved spermatogenic genes whose expression is restricted to testis and ovary and that are developmentally regulated. One of these genes, the transcription factorMael, has been reported to play an essential role in mouse spermatogenesis. Nevertheless, the role ofMaelin mouse oogenesis has not been defined. In order to analyse the role ofMaelin mouse oogenesis, the expression of this gene was blocked during early oogenesis in mousein vitrousing RNAi technology. In addition, the role ofMaelduring differentiation of embryonic stem cells (ESC) into germ cellsin vitrowas analysed. Results show that downregulation ofMaelby a specific short interfering RNA disrupted fetal oocyte growth and differentiation in fetal ovary explants in culture and the expression of several germ-cell markers in ESC during their differentiation. These results suggest that there is an important role forMaelin early oogenesis and during germ-cell differentiation from embryonic stem cells in mousein vitro.

It is generally believed that Drosophila melanogaster has no closely related species with which it can produce the viable and fertile hybrids that are essential for the genetic analysis of speciation. Following the recent report of molecular differentiation between a Zimbabwe, Africa, population and two United States populations, we provide evidence that strong sexual isolation exists between the D. melanogaster population in Zimbabwe and populations of other continents. In the presence of males of their own kind, females from most isofemale lines of Zimbabwe would not mate with males from elsewhere; the reciprocal mating is also significantly reduced, but to a lesser degree. The genes for sexual behaviors are apparently polymorphic in Zimbabwe and postmating reproductive isolation between this and other populations has not yet evolved. Whole chromosome substitutions indicate significant genetic contributions to male mating success by both major autosomes, whereas the X chromosome effect is too weak to measure. In addition, the relative mating success between hybrid and pure line males supports the interpretation of strong female choice. These observations suggest that we are seeing the early stages of speciation in this group and that it is driven by sexual selection. The genetic and molecular tractability of D. melanogaster offers great promise for the detailed analysis of this apparent case of incipient speciation.

Variants of the high-mobility group A1 (HMGA1) gene have been shown to be associated with insulin resistance and type 2 diabetes in individuals of European origin. We aimed to determine whether this locus confers significant susceptibility to type 2 diabetes in the Han Chinese population, and thus cross-race susceptibility to type 2 diabetes.Polymorphisms in HMGA1 were identified by direct sequencing of genomic DNA derived from 192 Chinese participants (96 patients with type 2 diabetes and 96 controls). We then genotyped the common variant IVS5-13insC (c.136-14_136-13insC) in two other independent cohorts, including a total of 2,533 cases and 2,643 ethnically matched controls.We confirmed the association of the HMGA1 variant IVS5-13insC (c.136-14_136-13insC) with type 2 diabetes with an OR of 1.34 (95% CI 1.15, 1.56, p = 0.0002 under a dominant model, and 95% CI 1.16, 1.55, p = 0.0002 under an additive model) in the Han Chinese population, corresponding to a population attributable risk fraction of 5.0%.HMGA1 is an important susceptibility locus that confers a high cross-race risk of the development of type 2 diabetes.

BackgroundRecent genome-wide association studies (GWAS) have mapped several novel loci influencing blood lipid levels in Caucasians. We sought to explore whether the genetic variants at newly identified lipid-associated loci were associated with CHD susceptibility in a Chinese Han population.Methodology/principal findingsWe conducted a two-stage case-control study in a Chinese Han population. The first-stage, consisting of 1,376 CHD cases and 1,376 sex and age- frequency matched controls, examined 5 novel lipid-associated single-nucleotide polymorphisms (SNPs) identified from GWAS among Caucasians in relation to CHD risk in Chinese. We then validated significant SNPs in the second-stage, consisting of 1,269 cases and 2,745 controls. We also tested associations between SNPs within the five novel loci and blood lipid levels in 4,121 controls. We identified two novel SNPs (rs599839 in CELSR2-PSRC1-SORT1 and rs16996148 in NCAN-CILP2) that were significantly associated with reduced CHD risk in Chinese (odds ratios (95% confidence intervals) in the dominant model 0.76 (0.61-0.90; P = 0.001), 0.67 (0.57-0.77; P = 3.4×10(-8)), respectively). Multiple linear regression analyses using dominant model showed that rs599839 was significantly associated with decreased LDL levels (P = 0.022) and rs16996148 was significantly associated with increased LDL and HDL levels (P = 2.9×10(-4) and 0.001, respectively).Conclusions/significanceWe identified two novel SNPs (rs599839 and rs16996148) at newly identified lipid-associated loci that were significantly associated with CHD susceptibility in a Chinese Han population.

OBJECTIVE Common polymorphisms within cytochrome P450 2J2 (CYP2J2) and epoxide hydrolase 2 (EPHX2), which are involved in the generation or hydrolysis of epoxyeicosatrienoic acids, may determine susceptibility to the development of cardiovascular disease. To derive a more precise estimation of their relationship, we undertook a case-control study as well as a meta-analysis to assess possible associations of coronary artery disease (CAD) risk with CYP2J2 and EPHX2 genetic variations. METHODS Associations among four single nucleotide polymorphisms in CYP2J2 and five in EPHX2 with CAD were examined in a total of 1344 cases and 1267 ethnically and geographically matched controls. To further confirm the effect of two functional variants (G-50T and R287Q) in the development of CAD, we conducted a meta-analysis including seven studies on G-50T polymorphism and six studies on R287Q polymorphism before June 2010. RESULTS No significant association between common polymorphisms within these two genes and CAD was observed in our sample, either using methods of single-locus analysis or haplotype-based analysis. In addition, no association was detected in our meta-analysis between these two functional variants and the risk of developing CAD. CONCLUSION This case-control study as well as meta-analysis suggested no association between CYP2J2 G-50T and EPHX2 R287Q and the risk of developing CAD.

Background Congenital adrenal hyperplasia owing to 17α-hydroxylase/17, 20-lyase deficiency is caused by genetic mutations in the CYP17A1 gene. To date, more than 80 different genetic lesions have been described in patients suffering from this disorder. We aimed to estimate the prevalence of CYP17A1 common mutations in Chinese Han population. Methods We first reported two female patients with 17α-hydroxylase deficiency based on their clinical features and molecular genetics, and then summarized all the mutations of CYP17A1 gene reported around the world. The most common mutations of CYP17A1 among Chinese Han were genotyped in additional 3245 healthy Chinese using Taqman-assays. Results The mutation spectrum in Asian is different from European decent. All healthy controls could detect two CYP17A1 mutations, D487-S488-F489 deletion and TAC329AA, with a prevalence of 1 in 1000 or 2 in 1000, respectively. Conclusion Our data demonstrates that these two mutations are major causes of 17α-hydroxylase deficiency in Chinese Han population.

Sex-specific traits that lead to the production of dimorphic gametes, sperm in males and eggs in females, are fundamental for sexual reproduction and accordingly widespread among animals. Yet the sex-biased genes that underlie these sex-specific traits are under strong selective pressure, and as a result of adaptive evolution they often become divergent. Indeed out of hundreds of male or female fertility genes identified in diverse organisms, only a very small number of them are implicated specifically in reproduction in more than one lineage. Few genes have exhibited a sex-biased, reproductive-specific requirement beyond a given phylum, raising the question of whether any sex-specific gametogenesis factors could be conserved and whether gametogenesis might have evolved multiple times. Here we describe a metazoan origin of a conserved human reproductive protein, BOULE, and its prevalence from primitive basal metazoans to chordates. We found that BOULE homologs are present in the genomes of representative species of each of the major lineages of metazoans and exhibit reproductive-specific expression in all species examined, with a preponderance of male-biased expression. Examination of Boule evolution within insect and mammalian lineages revealed little evidence for accelerated evolution, unlike most reproductive genes. Instead, purifying selection was the major force behind Boule evolution. Furthermore, loss of function of mammalian Boule resulted in male-specific infertility and a global arrest of sperm development remarkably similar to the phenotype in an insect boule mutation. This work demonstrates the conservation of a reproductive protein throughout eumetazoa, its predominant testis-biased expression in diverse bilaterian species, and conservation of a male gametogenic requirement in mice. This shows an ancient gametogenesis requirement for Boule among Bilateria and supports a model of a common origin of spermatogenesis., Author Summary While sexual reproduction is widespread among animals, it remains enigmatic to what extent sexual reproduction is conserved and when sex-specific gametogenesis (spermatogenesis and oogenesis) originated in animals. Here we demonstrate the presence of the reproductive-specific protein Boule throughout bilaterally-symmetric animals (Bilateria) and the conservation of its male reproductive function in mice. Examination of Boule evolution in insect and mammalian lineages, representing the Protostome and Deuterostome clades of bilateral animals, failed to detect any evidence for accelerated evolution. Instead, purifying selection is the major force behind Boule evolution. Further investigation of Boule homologs among Deuterostome species revealed reproduction-specific expression, with a strong prevalence of testis-biased expression. We further determined the function of a deuterostomian Boule homolog by inactivating Boule in mice (a representative mammal, a class of Deuterostomes). Like its counterpart in Drosophila (a representative of the opposing Protostome clade), mouse Boule is also required only for male reproduction. Loss of mouse Boule prevents sperm production, resulting in a global arrest of spermatogenesis in remarkable similarity to that of Drosophila boule mutants. Our findings are consistent with a common origin for male gametogenesis among metazoans and reveal the high conservation of a reproduction-specific protein among bilaterian animals.

Background and Purpose— The first genomewide association study of ischemic stroke in whites has identified multiple susceptibility loci. We confirmed this study by examining associations with ischemic stroke in a Chinese Han population. Methods— Twenty-five common variants were genotyped in a relatively large sample size including 1123 subjects with ischemic stroke cases (thrombosis stroke=716, lacunar infarction=407) and 557 normal control subjects. The association analyses were performed at both single nucleotide polymorphism and haplotype levels. False discovery rate q value method was applied for multiple testing corrections. Results— rs11052413, a intergenic single nucleotide polymorphism, was most significantly associated with ischemic stroke independent of traditional cardiovascular risk factors in additive (OR=1.51, 95% CI=1.19 to 1.92, P =7.4×10 −4 , q=0.018) and dominant models (OR=1.59, 95% CI=1.20 to 2.08, P =9.2×10 −4 , q=0.023). In addition, both ZNF650 rs10204475 and intergenic single nucleotide polymorphism rs10486776 were associated with ischemic stroke as well as independent of traditional cardiovascular risk factors in dominant models (OR=1.47, 95% CI=1.12 to 1.96, P =0.005, q=0.040 and OR=1.53, 95% CI=1.15 to 2.02, P =0.003, q=0.036, respectively). No significant results were found in stroke subtype analysis after multiple corrections. Conclusion— Our study confirmed previously reported associations between ischemic stroke and rs11052413, rs10486776, and ZNF 650 rs10204475 in a Chinese Han population. The mechanism whereby the genetic variants exert their effects on ischemic stroke remains to be further elucidated.

The inflammatory marker C-reactive protein (CRP) has been strongly correlated with the risk of cardiovascular disease. Some single-nucleotide polymorphisms (SNPs) have been reported to be associated with serum CRP levels. In this study, we assessed the genetic association between SNPs within the CRP gene and ischemic and hemorrhagic stroke in the Han Chinese population. This study comprises 564 ischemic stroke patients, 220 hemorrhagic stroke patients and 564 controls from the ethnic Han Chinese population in Wuhan. Four CRP SNPs, −757A>G (rs3093059), −717A>G (rs2794521), −286C>T>A (rs3091244) and +2147C>T (rs1205), were genotyped from patients using TaqMan assays. The A allele frequency for the −717A>G polymorphism was significant higher in controls than in ischemic stroke patients (P=0.037), after adjustment for traditional risk factors (odds ratio 0.28; 95% CI 0.12–0.65; P=0.003), suggesting a protective effect for this allele against ischemic stroke. Haplotype analysis showed that the H3 (G-C-C) haplotype conferred a significantly increased risk of ischemic stroke (odds ratio 1.052, 95% CI 1.001–1.106: P=0.047). Neither CRP genotypes nor haplotypes showed an association with hemorrhagic stroke. However, the frequency for haplotype H5 (A-T-C) was significantly higher in ischemic stroke than hemorrhagic stroke patients (P=0.0003). These data suggest that the CRP gene −717A allele confers a protective effect against ischemic stroke. Furthermore, the H3 haplotype (G-C-C) is an independent risk marker for ischemic stroke, whereas the H5 haplotype (A-T-C) can be used as a prognostic marker of hemorrhagic stroke.

We have entered a new era of genomics in biomedical research with the availability of genome-wide sequences and expression data, resulting in the identification of a huge number of novel reproductive genes. The challenge we are facing today is how to determine the function of those novel and known genes and their roles in normal reproductive physiology, such as gamete production, pregnancy and fertilization, and the disease physiology such as infertility, spontaneous abortion and gynecological cancers. Mouse genetics has contributed tremendously to our understanding of the genetic causes of human diseases in the past decades. The establishment of mouse mutations is an effective way to understand the function of many reproductive proteins. One of the fast-growing mouse mutagenesis technologies—gene trap mutagenesis—represents a cost-effective way to generate mutations because of the public availability of mouse embryonic stem (ES) cell lines carrying insertional mutations and the continuing expansion of those ES gene trap cell lines. We review here the gene trapping technology and in particular examine its efficacy in generating mouse mutations for reproductive research. Even with the existing gene trap cell lines, many of the genes important for reproductive function through traditional knockout and chemical mutagenesis have been trapped, demonstrating gene trapping’s efficacy in mutating genes involved in reproductive development. Comparing genes expressed in specific reproductive sub-cellular organelles and in the entire testis and ovary with gene trap lines in the International Gene Trap Consortium (IGTC) database, we could identify a significant portion of those genes as having been trapped, representing a great resource for establishing mouse models for reproductive research. Establishment and analysis of these mouse models, for example, could help with identifying genetic abnormalities underlying male infertility and other reproductive diseases.

Members of the Pumilio (also called PUF) gene family belong to a class of highly conserved developmental regulators that are present in both flies and humans. Much is known about the function of Pumilio genes in invertebrate development, in particular their role as stem cell factors required for maintenance and/or self-renewal of germline stem cells in Drosophila and Caenorhabditis elegans. It remains unknown whether Pumilio genes are also required for development in mammals; however, several lines of evidence suggest similar functions based on extensive sequence homology, similar RNA-binding properties to their invertebrate counterparts and well-documented interactions with germ cell factors required for fertility. Here we report characterization of a gene trap mutation that disrupts the mouse Pumilio-2 (Pum2) gene. Our data confirm that Pumilio-2 is expressed most abundantly in germ cells with the highest expression in undifferentiated gonocytes and spermatogonia. Furthermore, the mutation in Pum2 results in significantly smaller testes although the mutants are otherwise viable and fertile. In addition, we observed no stronger reproductive defects on a genetic background homozygous for a Pum2 null mutation and heterozygous for a Dazl mutation than Pum2 mutant alone. Thus, as in C. elegans where single members of the Pumilio gene family are dispensable for reproductive development and viability, this individual member of the Pumilio gene family in mice is also not essential for reproduction or viability. Mol. Reprod. Dev. 74: 912–921, 2007. © 2007 Wiley-Liss, Inc.

X inactivation has evolved in the soma of mammalian females so that both sexes have the same ratio of X:autosomal gene expression. The X chromosome in the germ cells of XY males is also precociously inactivated for reasons that remain unclear. Unlike X inactivation in the soma, this germline X inactivation is not restricted to mammals but has evolved independently in several animal phyla. Thus, germline X inactivation might have been the precursor of somatic X inactivation in mammals. We now propose a hypothesis for the evolution of germline X inactivation. The hypothesis predicts a redistribution of late spermatogenic genes from the X chromosome to the autosomes, leading eventually to germline X inactivation as the X chromosome becomes 'demasculinized'. Sexual antagonism could be the mechanism driving this redistribution. Recent expression and genetic studies in mammals, nematodes and Drosophila support this hypothesis, and expression data on taxa that have not evolved germline X inactivation, such as birds and butterflies, should shed further light on it.

Defects in human germ cell development are common and yet little is known of genes required for germ cell development in men and women. The pathways that develop germ cells appear to be conserved broadly, at least in outline, in organisms as diverse as flies and humans beginning with allocation of cells to the germ cell lineage, migration of these cells to the fetal gonad, mitotic proliferation and meiosis of the germ cells, and maturation into sperm and eggs. In model organisms, a few thousand genes may be required for germ cell development including meiosis. To date, however, no genes that regulate critical steps of reproduction have been shown to be functionally conserved from flies to humans. This may be due in part to strong selective pressures that are thought to drive reproductive genes to high degrees of divergence. Here, we investigated the micro- and macro-evolution of the BOULE gene, a member of the human DAZ (deleted in azoospermia) gene family, within primates, within mammals and within metazoans. We report that sequence divergence of BOULE is unexpectedly low and that rapid evolution is not detectable. We extend the evolutionary analysis of BOULE to the level of phyla and show that a human BOULE transgene can advance meiosis in infertile boule mutant flies. This is the first demonstration that a human reproductive gene can rescue reproductive defects in a fly. These studies lend strong support to the idea that BOULE may encode a key conserved switch that regulates progression of germ cells through meiosis in men.

B-cell abnormality including excessive activation and lymphopenia is a central feature of systemic lupus erythematosus (SLE). Although activation threshold, auto-reaction and death of B cells can be affected by intrinsical and/or external signaling, the underlying mechanisms are unclear. Herein, we demonstrate that co-activation of Toll-like receptor 7 (TLR7) and B-cell receptor (BCR) pathways is a core event for the survival/dead states of B cells in SLE. We found that the mortalities of CD19+CD27- and CD19+IgM+ B-cell subsets were increased in the peripheral blood mononuclear cells (PBMCs) of SLE patients. The gene microarray analysis of CD19+ B cells from active SLE patients showed that the differentially expressed genes were closely correlated to TLR7, BCR, apoptosis, necroptosis and immune pathways. We also found that co-activation of TLR7 and BCR could trigger normal B cells to take on SLE-like B-cell characters including the elevated viability, activation and proliferation in the first 3 days and necroptosis in the later days. Moreover, the necroptotic B cells exhibited mitochondrial dysfunction and hypoxia, along with the elevated expression of necroptosis-related genes, consistent with that in both SLE B-cell microarray and real-time PCR verification. Expectedly, pretreatment with the receptor-interacting protein kinase 1 (RIPK1) inhibitor Necrostatin-1, and not the apoptosis inhibitor zVAD, suppressed B-cell death. Importantly, B cells from additional SLE patients also significantly displayed high expression levels of necroptosis-related genes compared with those from healthy donors. These data indicate that co-activation of TLR7 and BCR pathways can promote B cells to hyperactivation and ultimately necroptosis. Our finding provides a new explanation on B-cell lymphopenia in active SLE patients. These data suggest that extrinsic factors may increase the intrinsical abnormality of B cells in SLE patients.

Background Genetic architecture of coronary artery disease (CAD) is still to be defined. Since low density lipoprotein receptor-related protein 6 (LRP6) gene play critical roles in Wnt signal transduction which are important for vascular development and endodermis specification, we therefore resequenced it to search for mutations in CAD patients. Methods We systemically sequenced all the exons and promoter region of LRP6 gene in a sample of 380 early onset CAD patients and 380 control subjects in Chinese. Results In total, we identified 5 patient-specific mutations including K82N (two patients), S488Y (one patient), P1066T (two patients), P1206H (two patients) and I1264V (one patient) All these mutations located at the extracellular domain of LRP6 gene. In vitro functional analysis of patient-specific mutations demonstrated that these mutations resulted in a significant reduction in both protein level transporting to cell membrane and downstream Wnt signal activity. Furthermore, we found that LRP6 novel mutations attenuated proliferation and migration of human umbilical vein endothelial cells (HUVECs) when compared with wild type (WT) LRP6. Conclusion Our results demonstrated that these loss-of-function variants might contribute to disease liability in a subset of CAD and defects in Wnt signal activation might be important contributing factors for the onset of CAD.