Plant N-acylethanolamines play a crucial role in defense and its variation in response to elevated CO2 and temperature in tomato.

The ubiquitous lipid-derived molecules N-acylethanolamines (NAEs) have multiple immune functions in mammals, but their roles and mechanisms in plant defense response during changing environment remain largely unclear. Here, we found that exogenous NAE18:0 and NAE18:2 promoted defense against the necrotrophic pathogen Botrytis cinerea but suppressed defense to the hemi-biotrophic pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 in tomato. The knocking-down and overexpression function analysis of the pathogen-responsive NAE synthetic gene PHOSPHOLIPASE Dγ (PLDγ) and hydrolytic gene FATTY ACID AMID HYDROLASE 1 (FAAH1) revealed that the NAE pathway is crucial for plant defense response. Using exogenous applications and SA-abolished NahG plants, we unveiled the antagonistic relationship between NAE and SA in plant defense response. Elevated CO2 and temperature significantly changed the NAE pathway in response to pathogens, while inhibition of the NAE pathway led to the alternation of environment-mediated defense variations against Pst DC3000 in tomato, indicating that NAE pathway is associated with plant defense variations in response to elevated CO2 and temperature. The results herein reveal a new function of NAE in plant defense, and its involvement in environment-mediated defense variation in tomato. These findings shed light on the NAE-based plant defense, which may have relevance to crop disease management in future changing climate.

GSK3 inhibitor suppresses cell growth and metabolic process in FLT3-ITD leukemia cells.

Glycogen Synthase Kinase-3 (GSK-3) was recently implicated in the dysregulated biology of acute myeloid leukemia (AML). Low concentrations of GSK-3 inhibitors, SB216763 and BIO, suppressed the proliferation of AML cells with FLT3-ITD as early as 24 h after treatment. BIO was used in subsequent assays since it exhibited higher inhibitory effects than SB216763. BIO-induced G1 cell cycle arrest by regulating the expression of cyclin D2 and p21 in MV4-11 cells, and promoted apoptosis by regulating the cleaved-caspase3 signaling pathways. In vivo assays demonstrated that BIO suppressed tumor growth, while metabolomics assay showed that BIO reduced the levels of ATP and pyruvate in MV4-11 cells suggesting that it inhibited glycolysis. BIO markedly suppressed cell growth and induced apoptosis of AML cells with FLT3-ITD by partially inhibiting glycolysis, suggesting that BIO may be a promising therapeutic candidate for AML.

Correction: The novel leucine-rich repeat receptor-like kinase MRK1 regulates resistance to multiple stresses in tomato.

[This corrects the article DOI: 10.1093/hr/uhab088.].

A novel leucine-rich repeat receptor-like kinase MRK1 regulates resistance to multiple stresses in tomato.

Leucine-rich repeat receptor-like kinases (LRR-RLKs) are ubiquitous in higher plants, which act as receptors of extracellular signals to trigger multiple physiological processes. However, the functions of the majority of LRR-RLKs remain largely unknown, especially in tomato (Solanum lycopersicum L.). Here, we found that MRK1 (Multiple resistance-associated kinase 1), encoding a novel tomato LRR-RLK, was significantly induced either by temperature stresses or bacterial pathogen attacks. Knocking out MRK1 impaired the tolerance to both cold and heat stress, accompanied with the decrease in transcripts of master regulators C-repeat binding factor 1 (CBF1) and Heat shock transcription factor a-1a (HsfA1a), respectively. Additionally, mrk1 mutants were hypersensitive to Pseudomonas syringae pv. tomato DC3000 and Ralstonia solanacearum and compromised pattern-triggered immunity (PTI) responses as evidenced by decreased reactive oxygen species production and reduced upregulation of the PTI marker genes. Moreover, bimolecular fluorescence complementation, split-luciferase assay and coimmunoprecipitation supported the existence of complex formation between the MRK1, FLS2 and Somatic embryogenesis receptor kinase (SERK3A/SERK3B) in a ligand-independent manner. This work demonstrates that tomato MRK1 as a novel positive regulator of multiple stresses, which might be a potential breeding target to improve crop stress resistance.

GALNT14 regulates ferroptosis and apoptosis of ovarian cancer through the EGFR/mTOR pathway.

Background: Chemoresistance usually occurs in ovarian cancer. We aimed to explore the mechanisms of chemoresistance. Methods: Western blotting assay was used to detect the expression of GALNT14. Further cell function experiments were performed to investigate the effect of GALNT14 in ovarian cancer. Results: GALNT14 is significantly upregulated in ovarian cancer. Downregulation of GALNT14 significantly inhibits both apoptosis and ferroptosis of ovarian cancer cells. A further mechanism assay illustrated that downregulation of GALNT14 suppresses the activity of the mTOR pathway through modifying O-glycosylation of EGFR. Finally, an additive effect promoting cell death occurs with a combination of an mTOR inhibitor and cisplatin. Conclusion: Our study might provide a promising method to overcome cisplatin resistance for patients with ovarian cancer.

The regulatory module MdBT2-MdMYB88/MdMYB124-MdNRTs regulates nitrogen usage in apple.

Less than 40% of the nitrogen (N) fertilizer applied to soil is absorbed by crops. Thus, improving the N use efficiency of crops is critical for agricultural development. However, the underlying regulation of these processes remains largely unknown, particularly in woody plants. By conducting yeast two-hybrid assays, we identified one interacting protein of MdMYB88 and MdMYB124 in apple (Malus × domestica), namely BTB and TAZ domain protein 2 (MdBT2). Ubiquitination and protein stabilization analysis revealed that MdBT2 ubiquitinates and degrades MdMYB88 and MdMYB124 via the 26S proteasome pathway. MdBT2 negatively regulates nitrogen usage as revealed by the reduced fresh weight, dry weight, N concentration, and N usage index of MdBT2 overexpression calli under low-N conditions. In contrast, MdMYB88 and MdMYB124 increase nitrate absorption, allocation, and remobilization by regulating expression of MdNRT2.4, MdNRT1.8, MdNRT1.7, and MdNRT1.5 under N limitation, thereby regulating N usage. The results obtained illustrate the mechanism of a regulatory module comprising MdBT2-MdMYB88/MdMYB124-MdNRTs, through which plants modulate N usage. These data contribute to a molecular approach to improve the N usage of fruit crops under limited N acquisition.

Knockdown of NRSF inhibits cell proliferation of ovarian cancer via activating Hippo pathway.

AIMS: Ovarian cancer is the most leading cause of deaths among gynecologic malignancies, and Neuron-restrictive silencer factor (NRSF) can be upregulated or downregulated according to the type of tumor. However, the expression and function of NRSF in ovarian cancer is still unknown. MAIN METHODS: Expression of NRSF in normal ovary and ovarian cancer cells were evaluated by quantitative PCR (qPCR). NRSF expression in normal ovary and ovarian cancer tissue samples were examined by qPCR, western blotting and immunohistochemistry (IHC). MTT, colony formation, anchorage-independent growth assay were applied to examine the effect of NRSF on ovarian cancer cell proliferation. Bromodeoxyuridine (BrdUrd) labeling and flow cytometry assay were carried out to investigate the role of NRSF on cell cycle of ovarian cancer cells. Luciferase reporter assay and western blotting, immunofluorescence labeling were devoted to explore the mechanism by which NRSF contributes to proliferation of ovarian cancer cells. KEY FINDINGS: The results demonstrated that NRSF is significantly upregulated in ovarian cancer cells and tissues and negatively related with the survival of patients with ovarian cancer, and knockout of NRSF inhibit proliferation of ovarian cancer cells. Further analysis showed that NRSF can influence G1/S transition of cell cycle via regulating the transcription of Hippo pathway. SIGNIFICANCE: Herein, our study suggest that NRSF is associated with the progression of ovarian cancer, and NRSF may be a valuable early detection marker of ovarian cancer and inhibiting NRSF expression may be an effective method to treat ovarian cancer.

Impact of Abnormal DNA Methylation of Imprinted Loci on Human Spontaneous Abortion.

Currently, there is a growing concern regarding the safety of assisted reproductive technology (ART) due to increased risk of spontaneous abortion (SA) and imprinting disorders in ART-conceived offspring. Early investigations suggested that aberrant genetic imprinting may be related to pregnancy loss; however, few studies have used human tissue specimens. Here the DNA methylation patterns of 3 imprinted genes, including maternally inherited GRB10 and the paternally inherited IGF2 and PEG3 genes, were evaluated in human chorionic villus samples by pyrosequencing and bisulfite sequencing polymerase chain reaction. The samples were divided into 4 groups: (1) SA of natural conception (NC; n = 84), (2) induced abortion of NC (n = 94), (3) SA after ART (n = 73), and (4) fetal reduction after ART (n = 86). The methylation levels and the percentages of abnormal methylation of the IGF2, GRB10, and PEG3 genes between the ART group and the NC group showed no significant difference. Both IGF2 and GRB10 genes showed higher methylation levels in the SA group compared to the non-SA group. Additionally, determining the single-nucleotide polymorphisms of 4 loci, including IGF2 rs3741205, rs3741206, rs3741211, and GRB10 rs2237457, showed that the TC+CC genotype of IGF2 rs3741211 had a 1.91-fold increased risk of SA after ART. However, there was no association between the mutant genotype of IGF2 rs3741211 and the methylation levels of IGF2 and H19, and ART might not affect the distribution of the abovementioned genotypes. It provides support for the opinion that genetic imprinting defects may be associated with SA, which might not be due to ART treatments.

Long Noncoding RNAs: Potential Regulators Involved in the Pathogenesis of Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility in women of reproductive age, and its etiology remains poorly understood. Altered activities of long noncoding RNAs (lncRNAs) have been associated with human diseases and development. However, the roles of lncRNAs are unknown in reproductive medicine. We investigated the potential role of lncRNAs in the pathogenesis of PCOS, using human granulosa cells (GCs) and the KGN cell line. We used microarrays to compare lncRNA expression profiles in GCs from seven patients with PCOS and seven matched women. GC samples were collected during 2014 to 2016 from infertile women in Guangzhou, China. Quantitative real-time polymerase chain reaction was used to measure levels of the lncRNA HCG26 in GCs from 53 patients with PCOS and 50 controls. HCG26 was knocked down with locked nucleic acid GapmeRs in KGN cells to examine its role in cell proliferation, aromatase and follicle-stimulating hormone receptor gene expression, and estradiol production. A total of 862 lncRNA transcripts and 998 messenger RNA transcripts were differentially expressed (greater than or equal to twofold change; P < 0.05) in PCOS GCs compared with those of controls. HCG26 levels were upregulated in patients with PCOS and were associated with antral follicle count. HCG26 knockdown in KGN cells inhibited cell proliferation and cell-cycle progression and increased aromatase gene expression and estradiol production. Our study reports the lncRNA profiles in GCs from patients who have PCOS and those from healthy women and suggests that dysregulated lncRNAs may play vital roles in GC proliferation and steroidogenesis, providing insights into the pathogenesis of PCOS.

[Pregnancy outcome in a woman with premature ovarian insufficiency complicated by systemic lupus erythematosus during pregnancy: a case report].

OBJECTIVE: We report a case of in vitro fertilization and embryo transfer (IVF?ET) with oocyte donation in a woman with premature ovarian insufficiency (POI) complicated by systemic lupus erythematosus (SLE) during pregnancy. The patient had a diagnosis of POI 4 years earlier and 11 weeks after successful pregnancy by IVF?ET with oocyte donation in 2003, she presented with facial edema, and further examinations confirmed the diagnosis of lupus nephritis. She received treatment with prednisone to control the activity of SLE and aspirin and low?molecular?weight heparin to improve placental blood flow with close monitoring of gravida and fetus throughout pregnancy. The condition of the patient remained unstable during pregnancy, and liver damage and placental circulation disorder occurred in late gestational weeks with suspected intrauterine growth retardation (IUGR) of the fetus. For maternal and fetal safety, the patient received elective caesarean section and delivered a premature boy at 31 weeks of gestation. She subsequently received further medications for SLE and showed good recovery of the immunological parameters and absence of SLE symptoms during the follow?up for 14 years, indicating a clinical cure of SLE. Her son shows normal growth and development. Based on the experience with this case and literature review, we believe that immunological factor is an important cause of POI and thus recommend full immunological examinations in cases of idiopathic POI.

DNA methyltransferase 3A promoter polymorphism is associated with the risk of human spontaneous abortion after assisted reproduction techniques and natural conception.

PURPOSE: The aim of this study was to explore the association of the DNA-methyltransferase (DNMT)-3A and DNMT3B promoter polymorphisms with the risk of human spontaneous abortion after assisted reproduction techniques (ARTs) and natural conception. METHODS: We collected tissues from women who underwent abortion procedures: (a) chorionic villus samples (CVS) and muscle samples (MS) from spontaneous abortions conceived by ART and natural cycle (study group), n = 152; and (b) CVS and MS from normal early pregnancy and second trimester (control group), n = 155. The single-nucleotide polymorphism (SNP) -448A > G in the DNMT3A promoter region and -149C/T polymorphism of DNMT3B were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and confirmed by sequencing. RESULTS: The allele frequency of -448A among pregnancy loss group and control group was 34.2 % vs. 16.5 %, respectively. Compared with GG carriers, the DNMT3A -448AA homozygotes had an about 16-fold increased risk of spontaneous abortion [odds ratio (OR) = 16.130, 95 % confidence interval (CI), 3.665-70.984], and AG heterozygotes had an OR of 2.027 (95 % CI, 1.247-3.293). However, the distribution of -448A > G in individuals derived from ART pregnancies was not statistically significantly compared with those derived from spontaneous pregnancies (P = 0.661). For DNMT3B, we observed genotype frequencies of 100 % (TT) in the study group and the control group. CONCLUSIONS: The DNMT3A -448A > G polymorphism may be a novel functional SNP and contribute to its genetic susceptibility to spontaneous abortion in Chinese women, and ART may not affect the distribution of -448A > G in pregnancy loss and normal pregnancy. The observed TT genotype of DMNT3B suggests that this is the predominant genotype of this population. The findings provide new insights into the etiology of human spontaneous abortion.

[Clinical outcome of in vitro fertilization or intracytoplasmic sperm injection-embryo transfer in women aged 40 years and above].

OBJECTIVE: To investigate the clinical outcomes in vitro fertilization or intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) in women aged over 40 years. METHODS: We retrospectively analyzed 1050 non-donor IVF/ICSI-ET cycles performed from January, 2007 to December, 2015 in women at the age 40 years or above, including 393 women at 40 years of age, 266 at 41 years, 158 at 42 years, 107 at 43 years, 64 at 44 years, and 65 at 45-51 years. The clinical characteristics and outcomes of the women in different age groups were compared and analyzed. The pregnancy outcome of different ovarian stimulation protocols and different numbers of embryo transferred were also compared. RESULTS: Oocyte retrieval was achieved in 1032 treatment cycles. Of the 750 embryo transfer cycles, the clinical pregnancy rate was 17.7% (113/750), and the live birth rate was 8.5% (64/750). The clinical pregnancy rate in the 5 age groups was 23.4%, 21.0%, 13.1%, 9.2%, 5.6% and 0%, and the implantation rate was 11.2%, 10.2%, 6.3%, 5.1%, 2.3% and 0%, respectively; the early spontaneous abortion rate was 31.0%, 35.9%, 42.9%, 42.9% and 100%, and the live birth rate was 11.9%, 11.8%, 2.8% and 3.9%. The clinical pregnancy rates of long protocol, short prorocol, GnRHa antagonist protocol, and ovulation induction protocol were 23.6%, 10.2%, 13.3%, and 2.3%, respectively. In the 750 transfer cycles, the clinical pregnancy rate was 3.8% with single embryo transfer, 12.6% with double embryos transfer, and 23.0% with 3 embryos transfer. CONCLUSION: In women aged 40 years or above, the clinical pregnancy rate decreased significantly with age, and the live birth rate was extremely low in women aged beyond 44 years. Assisted reproductive technique is recommended for women aged 40 years and above even when no identifiable causes of sterility are present. For women aged above 44 years of age, oocyte donation may be a better option.

[Outcome analysis of monozygotic twin pregnancy conceived by assisted reproductive techniques].

OBJECTIVE: To analyze the incidence, management, and outcomes of monozygotic twin (MZT) pregnancy conceived by assisted reproductive techniques (ART). METHODS: A retrospective analysis was performed of clinical pregnancies after in vitro fertilization and embryo transfer (IVF-ET) and introcytoplasmic sperm injection and embryo transfer (ICSI-ET) from January, 2010 to June 2015 at our center. We investigated the incidence, managements and outcomes of 94 MZT pregnancies. Comparison of the pregnancy outcomes was made between the expectantly managed MZT pregnancies, dizygotic twin (DZT) pregnancies, monozygotic (MZ)-triplet pregnancies with selective embryo reduction (SER) to 2 fetuses and 1 fetus, and non-MZ triplet pregnancies with SER to 2 fetuses. RESULTS: Ninety-four MZT pregnancies occurred in the total of 6257 clinical pregnancy cycles with an incidence of 1.5%. No significant difference was found in the incidence of MZT pregnancies between IVF and ICSI cycles or between fresh and thawed cycles (P>0.05). Of the 94 MZT pregnancies, 45 were MZT pregnancy cycles, 43 were MZ-triplet pregnancy cycles, 3 were MZ-quadruplet pregnancy cycles and 3 were ectopic pregnancies. The expectantly managed MZT was associated with a significantly greater rate of miscarriage and malformation and a lower rate of live birth and term birth (P<0.05) in comparison with DZT pregnancy cycles that did not undergo SER. Similar outcomes were found between MZ-triplet pregnancies with SER to 2 fetuses and MZ-triplet pregnancies with SER to 1 fetus (P>0.05), and between MZ-triplets with SER to 2 fetuses and non-MZ triplet pregnancies with SER to 2 fetuses (P>0.05). CONCLUSION: ART is associated with a much higher incidence of MZT pregnancies than spontaneous conception. MZT pregnancies are at high risk of adverse outcomes, and reduction of MZT in multiple pregnancies may help to improve the outcomes.

Does lower dose of long-acting triptorelin maintain pituitary suppression and produce good live birth rate in long down-regulation protocol for in-vitro fertilization?

The effects of pituitary suppression with one-third depot of long-acting gonadotropin-releasing hormone (GnRH) agonist in GnRH agonist long protocol for in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) were investigated. A retrospective cohort study was performed on 3186 cycles undergoing IVF/ICSI with GnRH agonist long protocol in a university-affiliated infertility center. The pituitary was suppressed with depot triptorelin of 1.25 mg or 1.875 mg. There was no significant difference in live birth rate between 1.25 mg triptorelin group and 1.875 mg triptorelin group (41.2% vs. 43.7%). The mean luteinizing hormone (LH) level on follicle-stimulating hormone (FSH) starting day was significantly higher in 1.25 mg triptorelin group. The mean LH level on the day of human chorionic gonadotrophin (hCG) administration was slightly but statistically higher in 1.25 mg triptorelin group. There was no significant difference in the total FSH dose between the two groups. The number of retrieved oocytes was slightly but statistically less in 1.25 mg triptorelin group than in 1.875 mg triptorelin group (12.90±5.82 vs. 13.52±6.97). There was no significant difference in clinical pregnancy rate between the two groups (50.5% vs. 54.5%). It was suggested that one-third depot triptorelin can achieve satisfactory pituitary suppression and produce good live birth rates in a long protocol for IVF/ICSI.

Detection of HPV types and neutralizing antibodies in Gansu province, China.

A total of 82 samples from patients with cervical cancer (Group 1) and 50 samples from patients with other genital diseases (Group 2) were collected in Gansu, China. All 132 samples were tested for HPV DNA with a typing kit that can detect 21 types of HPV, and also tested for neutralizing antibodies against HPV-16, -18, -58, -45, -6, and -11 using pseudovirus-based neutralization assays. The results revealed that 28% (23/82) of sera in Group 1 were positive for type-specific neutralizing antibodies with a titer range of 160-640, of which 23.2% (19/82), 2.4% (2/82), 2.4% (2/82), 1.2% (1/82), and 1.2% (1/82) were against HPV-16, -58, -6, -18, and -45, respectively. Only one serum (2%) in Group 2 was positive for neutralizing antibodies, which were against HPV-6 with a titer of 2,560. Overall, 85.4% (70/82) of samples in Group 1 were HPV DNA-positive, compared with 28% (14/50) of samples in Group 2. The seven most common types detected in Group 1 were HPV-16 (80%), HPV-52 (7.1%), HPV-66 and HPV-11 (5.7% each), and HPV-58, HPV-18, and HPV-33 (4.3% each), while the four most common types in Group 2 were HPV-16 (12%), HPV-52 and HPV-11 (6% each), and HPV-68 (4%). The concordance between HPV DNA and corresponding neutralizing antibodies was 32.9% (27/82) with a significant difference (P < 0.005). More specifically, the concordance was 42.7% (35/82) for HPV-16 in Group 1. The full-length sequences of six HPV types (HPV-16, -58, -33, -59, -11, and -68) were determined and showed 99% identities with their reported genomes.

Expression and clinical significance of vascular endothelial growth factor, cyclooxygenase-2, and Bcl-2 in borderline ovarian tumors.

OBJECTIVE: The objectives were to study the expression of vascular endothelial growth factor (VEGF), cyclooxygenase-2 (Cox-2), and Bcl-2 in borderline ovarian tumors (BOTs) and the relationship within them, and to investigate the correlation between expression of VEGF, Cox-2, and Bcl-2, and the clinicopathologic features of BOTs. METHODS: An immunohistochemical technique was used to investigate the expression of VEGF ,Cox-2, and Bcl-2 in 69 borderline, 18 benign, and 27 malignant human ovarian tumor tissues. RESULTS: Expression rate of VEGF protein (59.4%) in BOTs was higher than in benign tumors (27.8%) and was lower than in ovarian carcinomas (92.6%), and there was a significant difference between BOTs and benign ovarian tumors (p < 0.05), and carcinoma (p < 0.01). Significant correlation was observed between the positive expression rate for VEGF and clinical stage of BOTs (p < 0.05). The statistical analysis did not show a close correlation between the expression of VEGF and tissue type, and peritoneal implants in BOTs (p > 0.05). The expression rate of Cox-2 was significantly higher in ovarian carcinomas (81.5%) than in BOTs (57.9%) and in benign ovarian tumors (38.9%) (p < 0.05). Significant correlation was observed between the positive expression rate for Cox-2 and the clinical stage of BOTs (p < 0.05). The statistical analysis showed no close correlation between the expression of Cox-2 and tissue type, and peritoneal implants in BOTs (p > 0.05). There was a significant difference between the expression of Bcl-2 in ovarian carcinomas and BOTs than that in benign ovarian tumors (p < 0.05). The positive expression rate of Bcl-2 was not related to clinical stages and peritoneal implants (p > 0.05). Statistical analysis showed a positive correlation between the expression of Cox-2 and VEGF, and Bcl-2 in BOTs. CONCLUSIONS: Overexpression of VEGF, Cox-2, and Bcl-2 in BOTs may play an important role in the oncogenesis and progression of BOTs. It is feasible to detect VEGF, Cox-2, and Bcl-2 in the diagnosis and to predict the prognosis of BOTs.