Epitope delimitation: A new method for defining epitopes of human IgG-reactive antigenic peptides based on rabbit-recognized epitope motifs.

The use of precise epitope peptides as antigens is essential for accurate serological diagnosis of viral-infected individuals, but now it remains an unsolvable problem for mapping precise B cell epitopes (BCEs) recognized by human serum. To address this challenge, we propose a novel epitope delimitation (ED) method to uncover BCEs in the delineated human IgG-reactive (HR) antigenic peptides (APs). Specifically, the method based on the rationale of similarities in humoral immune responses between mammalian species consists of a pair of elements: experimentally delineated HR-AP and rabbit-recognized (RR) BCE motif and corresponding pair of sequence alignment analysis. As a result of using the ED approach, after decoding four RR-epitomes of human papillomavirus types 16/18-E6 and E7 proteins utilizing rabbit serum against each recombinant protein and sequence alignment analysis of HR-APs and RR-BCEs, 19 fine BCEs in 17 of 22 known HR-APs were defined based on each corresponding RR-BCE motifs, including the type-specificity of each delimited BCE in homologous proteins. The test with 22 known 16/20mer HR-APs demonstrated that the ED method is effective and efficient, indicating that it can be used as an alternative method to the conventional identification of fine BCEs using overlapping 8mer peptides.

PD-1 monoclonal antibodies enhance the cryoablation-induced antitumor immune response: a breast cancer murine model research.

BACKGROUND: It has been demonstrated that cryoablation (Cryo) causes specific T-cell immune responses in the body; however, it is not sufficient to prevent tumor recurrence and metastasis. In this report, we evaluated changes in the tumor immune microenvironment (TIME) in distant tumor tissues after Cryo and investigated the immunosuppressive mechanisms that limit the efficacy of Cryo. METHODS: Bilateral mammary tumor models were established in mice, and we first observed the dynamic changes in immune cells and cytokines at different time points after Cryo. Then, we confirmed that the upregulation of PD-1 and PD-L1 signaling in the contralateral tumor tissue was closely related to the immunosuppressive state in the TIME at the later stage after Cryo. Finally, we also evaluated the synergistic antitumor effects of Cryo combined with PD-1 monoclonal antibody (mAb) in the treatment of breast cancer (BC) mouse. RESULTS: We found that Cryo can stimulate the body's immune response, but it also induces immunosuppression. The elevated PD-1/PD-L1 expression in distant tumor tissues at the later stage after Cryo was closely related to the immunosuppressive state in the TIME but also created the conditions for Cryo combined with PD-1 mAb for BC mouse treatment. Cryo + PD-1 mAb could improve the immunosuppressive state of tumors and enhance the Cryo-induced immune response, thus exerting a synergistic antitumor effect. CONCLUSIONS: The PD-1/PD-L1 axis plays an important role in suppressing Cryo-induced antitumor immune responses. This study provides a theoretical basis for Cryo combined with PD-1 mAb therapy in clinical BC patients.

Three-Dimensional Shear Wave Elastography for Differentiating Benign From Malignant Thyroid Nodules.

OBJECTIVES: To prospectively evaluate the diagnostic performance of 3-dimensional (3D) shear wave elastography (SWE) for assessing thyroid nodules. METHODS: A total of 176 surgically or cytologically confirmed thyroid nodules (63 malignant and 113 benign) in 176 patients who had undergone conventional ultrasound (US), 2-dimensional (2D) SWE, and 3D SWE examinations were included in this study. Quantitative elasticity values (mean elasticity, maximum elasticity, and standard deviation of elasticity of a large region of interest and mean elasticity of a 2-mm region of interest) were measured on 2D and 3D SWE. Diagnostic performances of conventional US, 2D SWE, and 3D SWE were assessed. The role of 2D and 3D SWE in reducing unnecessary fine-needle aspiration (FNA) for nodules with low suspicion was also evaluated. RESULTS: The diagnostic performances in terms of the area under the receiver operating characteristic curve were 0.612 for conventional US, 0.836 for 2D SWE (P < .001 in comparison with conventional US), and 0.839 for 3D SWE (P < .001 in comparison with conventional US). The mean elasticity achieved the highest diagnostic performance in 2D SWE, whereas the standard deviation of elasticity achieved the highest performance in 3D SWE, although no significant difference was found between them (P > .05). Three-dimensional SWE increased the specificity in comparison with 2D SWE (88.5% versus 82.3%; P = .039). For the 37 nodules with low suspicion on conventional US imaging, 2D SWE was able to avoid unnecessary FNA in 77.1% (27 of 35) of benign nodules, and 3D SWE further increased the number to 88.6% (31 of 35). CONCLUSIONS: Three-dimensional SWE is a useful tool for predicting thyroid nodule malignancy and reducing unnecessary FNA procedures in thyroid nodules with low suspicion of malignancy.

[Establishment and characterization of A549 tumor monoclonal cell line with UCHL1 gene deletion].

The purpose of this study was to investigate the effects of ubiquitin C-terminal hydrolase-L1 (UCHL1) on non-small cell lung cancer cell line A549. UCHL1 gene knockout A549 cell line was constructed by CRISPR-CAS9 gene editing technique. The mRNA and protein levels of UCHL1 were examined by RT-PCR and Western blot, respectively. Cell proliferation and cycles were analyzed by CCK-8 method and flow cytometry, respectively. The sensitivity of A549 cells to cisplatin was detected by CCK-8 method. Migration ability of A549 cells was detected by scratch assay and Transwell test, and p-Erk expression level was assessed by Western blot. The results showed that UCHL1 gene knockout A549 cells were successfully constructed by CRISPR-CAS9 gene editing technique. After UCHL1 gene knockout, there was no significant change in cell proliferation and cell cycle ratios in A549 cells. UCHL1 gene knockout A549 cells exhibited decreased sensitivity to cisplatin and migration activity, as well as increased p-Erk expression level. These results suggest that the loss of UCHL1 gene function may reduce the sensitivity and migration ability of A549 cells, and this effect may be related to the activation of Erk1/2 signaling pathway.

Insulin upregulates GRIM-19 and protects cardiac mitochondrial morphology in type 1 diabetic rats partly through PI3K/AKT signaling pathway.

Insulin is involved in the development of diabetic heart disease and is important in the activities of mitochondrial complex I. However, the effect of insulin on cardiac mitochondrial nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) 1 subunit of retinoic-interferon-induced mortality 19 (GRIM-19) has not been characterized. The aim of this study was to investigate the effect of insulin on the mitochondrial GRIM-19 in the hearts of rats with streptozotocin (STZ)-induced type 1 diabetes. Protein changes of GRIM-19 were evaluated by western blotting and reverse transcription-quantitative polymerase chain reaction. Furthermore, the effects of insulin on mitochondrial complex I were detected in HeLa cells and H9C2 cardiac myocytes. During the development of diabetic heart disease, the cardiac function did not change within the 8 weeks, but the mitochondrial morphology was altered. The hearts from the rats with STZ-induced diabetes exhibited reduced expression of GRIM-19. Prior to the overt cardiac dilatation, mitochondrial alterations were already present. Following subcutaneous insulin injection, it was demonstrated that GRIM-19 protein was altered, as well as the mitochondrial morphology. The phosphoinositide 3-kinase inhibitor LY294002 had an effect on insulin signaling in H9C2 cardiacmyocytes, and decreased the level of GRIM-19 by half compared with that in the insulin group. The results indicate that insulin is essential for the control of cardiac mitochondrial morphology and the GRIM-19 expression partly via PI3K/AKT signaling pathways.

Initial Experience With Ultrasound Elastography for Diagnosis of Major Salivary Gland Lesions.

OBJECTIVES: The aim of this study was to evaluate the usefulness of ultrasound elastography, including conventional strain elastography, acoustic radiation force impulse (ARFI)-induced strain elastography, and point shear wave elastography (SWE) for diagnosis of major salivary gland lesions. METHODS: Forty major salivary gland lesions underwent conventional sonography, conventional strain elastography, ARFI strain elastography, and point SWE before surgery or biopsy. The diagnostic performances of the sonographic and elastographic techniques were assessed with reference to histopathologic results. RESULTS: There were 32 benign (7 Warthin tumors, 17 pleomorphic adenomas, and 8 other benign lesions) and 8 malignant (1 squamous carcinoma, 2 metastases, 2 mucoepidermoid carcinomas, 1 anaplastic carcinoma, and 2 malignant lymphomas) major salivary gland lesions on pathologic analysis. No conventional sonographic features or conventional strain elastographic scores were found to be associated with malignancy (all P > .05). The ARFI strain elastographic scores between benign and malignant lesions were statistically different (P = .032) and an ARFI strain elastographic score of 4 or greater was highly predictive of malignancy (P= .025). An ARFI strain elastographic score of greater than 3 achieved specificity of 81.3% (26 of 32) and sensitivity of 62.5% (5 of 8) in differentiating benign from malignant lesions. The shear wave velocity on point SWE did not show a significant difference in distinguishing between malignant and benign lesions (6.07 versus 4.43 m/s; P > .05). However, Warthin tumors had a trend to show lower shear wave velocities compared with pleomorphic adenomas (2.84 versus 5.27 m/s; P = .024). CONCLUSIONS: Acoustic radiation force impulse strain elastography may be potentially useful for diagnosing major salivary gland lesions, whereas conventional strain elastography and point SWE are not helpful.

[Effect of Progesterone-induced MicroRNA-152 on the Proliferation of Endometrial Epithelial Cells].

OBJECTIVE: To determine the expression of microRNA-152 induced by progesterone and its effect on the proliferation of endometrial epithelial cells (EECs). METHODS: Cultured EECs, Ishikawa were divided into four groups: control group (C group), 10(-8) mol/L estrogen treated group (E group), 10(-6) mol/L progesterone group (P group) and estrogen plus progesterone treated group (E&P group). The expression of mature microRNA-152 (microRNA-152-3p) of was detected by qRT-PCR. The estrogen treated cells were transfected with mimic-microRNA-152-3p. The estrogen and progesterone treated cells were transfected with inhibitor-microRNA-152-3p. Cell proliferations were detected by CCK-8 assay. The target gene of microRNA-152-3p proteins was predicted using microRNA target databases and validated by Western blot. RESULTS: qRT-PCR showed no difference between C and E groups (P > 0.05) in the expression of microRNA-152-3p. P group had higher expressions of microRNA-152-3p than C group (P < 0.05). E&P group had higher expressions of microRNA-152-3p than C group and P group. MicroRNA target protein prediction suggested that CDC14A is one of direct target proteins of microRNA-152-3p. The results of CCK-8 assay showed that mimic-microRNA-152-3p transfection blocked proliferations of estrogen treated cells and lowered expressions of CDC14A in these cells; while inhibitor-microRNA-152-3p promotes proliferations of estrogen and progesterone treated cells and increased expressions of CDC14A in these cells. CONCLUSION: Progesterone may suppress proliferations of EECs through inducing expressions of microRNA-152-3p. CDC14A is probably one target protein of microRNA-152-3p for its action on EECs.

Uterine NDRG2 expression is increased at implantation sites during early pregnancy in mice, and its down-regulation inhibits decidualization of mouse endometrial stromal cells.

BACKGROUND: N-myc down-regulated gene 2 (NDRG2) is a tumor suppressor involved in cell proliferation and differentiation. The aim of this study was to determine the uterine expression pattern of this gene during early pregnancy in mice. METHODS: Uterine NDRG2 mRNA and protein expression levels were determined by RT-PCR and Western blot analyses, respectively, during the peri-implantation period in mice. Immunohistochemical (IHC) analysis was performed to examine the spatial localization of NDRG2 expression in mouse uterine tissues. The in vitro decidualization model of mouse endometrial stromal cells (ESCs) was used to evaluate decidualization of ESCs following NDRG2 knock down by small interfering RNA (siRNA). Statistical significance was analyzed by one-way ANOVA using SPSS 19.0 software. RESULTS: Uterine NDRG2 gene expression was significantly up-regulated and was predominantly localized to the secondary decidual zone on days 5 and 8 of pregnancy in mice. Its increased expression was associated with artificial decidualization as well as the activation of delayed implantation. Furthermore, uterine NDRG2 expression was induced by estrogen and progesterone treatments. The in vitro decidualization of mouse ESCs was accompanied by up-regulation of NDRG2 expression, and knock down of its expression in these cells by siRNA inhibited the decidualization process. CONCLUSIONS: These results suggest that NDRG2 might play an important role in the process of decidualization during early pregnancy.

Knockdown of regulator of G-protein signalling 2 (Rgs2) leads to abnormal early mouse embryo development in vitro.

Regulator of G-protein signalling 2 (Rgs2) is involved in G-protein-mediated signalling by negatively regulating the activity of the G-protein α-subunit. In the present study, the expression patterns of Rgs2 in mouse ovarian tissues and early embryos were determined by semiquantitative reverse transcription-polymerase chain reaction, immunohistochemistry and immunofluorescent analyses. Rgs2 expression was observed in the ovarian tissues of adult female mice, with an almost equal expression levels during different stages of the oestrous cycle. Rgs2 was abundant in the cytoplasm, membrane, nuclei and spindles of intact polar bodies in mouse early embryos at different developmental stages from the zygote to blastocyst. The effect of Rgs2 knockdown on early embryonic development in vitro was examined by microinjecting Rgs2-specific short interfering (si) RNAs into mouse zygotes. Knockdown of endogenous Rgs2 expression led to abnormal embryonic development in vitro, with a considerable number of early embryos arrested at the 2- or 4-cell stage. Moreover, mRNA expression of three zygotic gene activation-related genes (i.e. Zscan4, Tcstv1 and MuERV-L) was decreased significantly in 2-cell arrested embryos. These results suggest that Rgs2 plays a critical role in early embryo development.

Relocalisation and activation of integrins induced rapidly by oestrogen via G-protein-coupled receptor 30 in mouse blastocysts.

Integrins are the dominant and final adhesion molecules in the attachment process between the blastocysts and endometrium. It is necessary for oestrogen to rapidly activate mouse blastocysts so that they attach to the endometrial epithelium. Our previous study suggested that oestrogen can rapidly induce an increase in intracellular calcium in mouse blastocysts via G-protein-coupled receptor 30 (GPR30). Thus, we deduced that integrins may be involved in GPR30 mediation of the fast effect of oestrogen on mouse blastocysts in implantation. To prove our hypothesis, we used immunofluorescence staining and in vitro coculture of mouse blastocysts and endometrial epithelial cell line (EECs), Ishikawa cells, in the present study. We found that αv and ß1 integrin clustered in mouse blastocysts, and that ß3 integrin was relocalised to the apical membrane of blastocyst cells when embryos were treated with 1 µM 17ß-estradiol (E2), 1 µM E2 conjugated to bovine serum albumin (E2-BSA) and 1 µM G-1, a specific GPR30 agonist, for 30 min respectively, whereas pretreatment with 1 µM G15, a specific GPR30 antagonist, and 5 µM 1,2-Bis(2-aminophenoxy)ethane-N,N,N'',N''-tetraacetic acid tetrakis (acetoxymethyl ester)(BAPTA/AM), a cellular Ca2+ chelator, blocked the localisation of integrins induced by oestrogen via GPR30 in mouse blastocyst cells. E2, E2-BSA and G-1 increased the fibronectin (FN)-binding activity of integrins in blastocysts, whereas G15 and BAPTA/AM blocked the activation of integrins induced by oestrogen via GPR30 in mouse blastocysts. Inhibition of integrins by Arg-Gly-Asp peptide in blastocysts resulted in their failure to adhere to EECs in vitro, even if oestrogen or G-1 was provided. Together, the results indicate the fast effect of oestrogen via the GPR30 membrane receptor further induces relocalisation and activation of integrins in mouse blastocysts, which play important roles in the adhesion of blastocysts to EECs.

[Estrogen induced rapid increase of intracellular calcium plays important role in the implantation of mouse blastocysts].

OBJECTIVE: To study the roles of the increased intracellular calcium induced rapidly by estrogen in the implantation of mouse blastocysts. METHODS: The mouse blastocysts were collected from the female mice on the pregnant day 4, divided into 3 groups: control, E2-BSA and BAPTA +E2-BSA. Immunofluorescence staining, confocal microscopy, embryo and endometrial epithenial cells co-culture and embryo transfer were used to investigate the effect of increased intracellular calcium induced by E2-BSA on the expression and localization of integrins in blastocysts and their adhesion to endometrial epithenial calls (EECs) and implantation into the endometrium. RESULTS: The increase of intracellular calcium induced rapidly by estrogen could cause the cluster and relocation of integrin av and beta3, and BAPTA might block this effect, the adhesion rate of blastocysts in contol group was 35.5%, BAPTA +E2-BSA group was 26.7% and significantly lower than 65.6% of E2-BSA group (P<0.05), and the implantation rate in BAPTA+E2-BSA group was 11.8%, which was significantly lower than 52.9% of E2-BSA group (P<0.05). CONCLUSION: The rapid increase of intracellular calcium induced by estrogen may cause the relocalization of integrin in blastocysts and their adhesion to ECCs, which is important in the process of implantation.

Increased miR-3074-5p expression promotes M1 polarization and pyroptosis of macrophages via ERα/NLRP3 pathway and induces adverse pregnancy outcomes in mice.

Decidual macrophages (dMϕs) play critical roles in regulation of immune-microhomeostasis at maternal-fetal interface during pregnancy, but the underlying molecular mechanisms are still unclear. In this study, it was found that litter size and fetal weight were significantly reduced, whereas the rate of embryo resorption was increased in miR-3074-5p knock-in (3074-KI) pregnant mice, compared to that of wild-type (WT) pregnant mice. Plasma levels of pro-inflammatory cytokines in 3074-KI pregnant mice were also significantly elevated compared to WT pregnant mice at GD7.5. The quantity of M1-Mϕs in uterine tissues of 3074-KI pregnant mice was significantly increased compared to WT pregnant mice at GD13.5. Estrogen receptor-α (ERα) was validated to be a target of miR-3074-5p. Either miR-3074-5p overexpression or ERα knockdown promoted transcriptional activity of NF-κB/p65, induced M1-polarization and pyroptosis of THP1-derived Mϕs, accompanied with increased intracellular levels of cleaved Caspase-1, cleaved IL-1ß, NLRP3, cleaved GSDMD and ASC aggregation. Furthermore, ERα could not only bind to NLRP3 or ASC directly, but also inhibit the interaction between NLRP3 and ASC. The endometrial miR-3074-5p expression level at the middle secretory stage of repeated implantation failure (RIF) patients was significantly decreased compared to that of control fertile women. These data indicated that miR-3074-5p could promote M1 polarization and pyroptosis of Mϕs via activation of NLRP3 inflammasome by targeting ERα, and the dysregulation of miR-3074-5p expression in dMϕs might damage the embryo implantation and placentation by interfering with inflammatory microenvironment at the maternal-fetal interface during early pregnancy.

Mapping of minimal motifs of B-cell epitopes on human zona pellucida glycoprotein-3.

The human zona pellucida glycoprotein-3 (hZP3) by virtue of its critical role during fertilization has been proposed as a promising candidate antigen to develop a contraceptive vaccine. In this direction, it is imperative to map minimal motifs of the B cell epitopes (BCEs) so as to avoid ZP-specific oophoritogenic T cell epitopes (TCEs) in the ZP3-based immunogens. In this study, based on known results of mapping marmoset and bonnet monkey ZP3 (mstZP3 and bmZP3), two predictable epitopes(23-30 and 301-320) on hZP3 were first confirmed and five minimal motifs within four epitopes on hZP3 were defined using serum to recombinant hZP3a(22-176) or hZP3b(177-348) as well as a biosynthetic peptide strategy. These defined minimal motifs were QPLWLL(23-28) for hZP3(23-30), MQVTDD(103-108) for hZP3(93-110), EENW(178-181) for hZP3(172-190), as well as SNSWF(306-310) and EGP(313-315) for hZP3(301-320), respectively. Furthermore, the antigenicity of two peptides for hZP3(172-187) and hZP3(301-315) and specificity of the antibody response to these peptides were also evaluated, which produced high-titer antibodies in immunized animals that were capable of reacting to ZP on human oocytes, r-hZP3b(177-348) protein, as well as r-hZP3(172-190), r-hZP3(303-310), and r-hZP3(313-320) epitope peptides fused with truncated GST188 protein.

[PP2A is involved in the inhibitory effect of progesterone on proliferation of mouse endometrial epithelial cells].

OBJECTIVE: To study the effect of protein phosphatase 2A (PP2A) in the negative regulation of progesterone on the proliferation of mouse endometrial epithelial cells (EECs). METHODS: Mouse EECs were isolated and cultured in vitro, which were divided into four groups when they grown to confluence: control group (P4) was treated with 1 micromol/L progesterone only, group A, B and C were treated respectively with 1 micromol/L progesterone and different concentrations of okadaic acid (5 nmol/L, 10 nmol/L and 20 nmol/L). After 24 h, the numbers of cells in different phases of the cell cycle were counted with flow cytometry. RESULTS: The effect of OA on mouse EECs was concentration-dependent. Compared with control group of P4, the change of cell cycle procession in group A was not obvious. Lower proportion of cells in G1 and G2/M phase and higher proportion of cells in S phase in group B, higher proportion of cells in G1 and S phase and lower proportion of cells in G2/M phase in group C were observed. CONCLUSION: Adequate dose OA inhibiting PP2A could release the inhibitory effect of progesterone on proliferation of mouse EECs obviously, this suggested that PP2A was involved in the inhibitory effect of progesterone on proliferation of EECs by influencing the process of cell cycle.

[Impact of multifactor intensive intervention on self management, risk factor control and outcome of post percutaneous transluminal coronary intervention patients].

OBJECTIVE: To test the efficacy of multifactor intensive intervention for post percutaneous transluminal coronary intervention (post-PCI) outpatients on self management, risk factor control and outcome. METHODS: A total of 263 patients with coronary heart disease (CAD) discharged from our cardiac center were randomized into usual care (4 CAD lectures focusing on the 2(nd) CAD prevention and patients-oriented outpatient visit) and intensive intervention (4 CAD lectures focusing on the 2(nd) CAD prevention, CAD outpatient visit twice a month, monthly telephone instructions on risk factor control and optimal medication). Patients were followed for 12 months and 250 patients completed follow-up. RESULTS: There were more patients achieved a LDL-C level of less than 2.6 mmol/L in intensive intervention group than in usual care group (71.2% vs. 48.3%, P < 0.01). The percentages of patients taking dietary control (55.3% vs. 26.2%, P < 0.01) and physical exercises (64.4% vs. 39.0%, P < 0.01), receiving beta-adrenergic receptor blocker (75.0% vs. 50.8%, P < 0.01) and statins (72.0% vs. 54.2%, P < 0.01) were significantly higher while cardiovascular event rate (5.9% vs. 0%, P = 0.005)was significantly lower in intensive intervention group than in usual care group. CONCLUSION: Multifactor intensive intervention is helpful on improving the second prevention for post-PCI coronary heart disease patients.

Awareness of tobacco control policies and anti-tobacco attitudes and behaviors among school personnel.

INTRODUCTION: Adolescent smoking is a serious public health concern, and the role of personnel in reducing students'tobacco use has been proven. Anti-tobacco policies are strong factors for tobacco control but most are newly implemented in China. This study aimed to examine the awareness of anti-tobacco policies among school personnel in a southern city of China, and assess its influence on personnel's anti-tobacco attitudes and behaviors towards students. METHODS: An online cross-sectional study was conducted between September 2017 and January 2018 in schools of Shanghai, China. A total of 3194 subjects from 33 schools were selected by a two-stage stratified cluster randomized sampling design. Prevalence of anti-tobacco policy awareness is presented. Crude (ORs) and adjusted odds ratios (AORs) and their 95% confidence intervals (CIs) were estimated to assess the association between policy awareness and anti-tobacco attitudes or behaviors. RESULTS: In all, 22.4% of surveyed participants knew four or five polices presented in the survey and 13.0% of personnel knew none of these policies. Most of the participants fully support prohibiting indoor (94.6%) and outdoor (86.3%) smoking in public places, bans on tobacco advertising (90.9%), and printing warning pictures on cigarette boxes (89.5%). Less than half of the personnel had taken action to stop students from smoking (45.7%), encourage students to quit smoking (42.4%) or participated in relevant educational activities held by schools (37.4%) in the previous year. The school personnel's anti-tobacco attitudes (AOR=1.28; 95% CI: 1.21-1.36) and behaviors (AOR=1.10; 95% CI: 1.03-1.17) were strengthened with increasing level of policy awareness. CONCLUSIONS: The involvement of school personnel can be an important part of intervention to improve anti-tobacco campaigns on campus. The study calls for the implementation of projects or activities to improve anti-tobacco policy awareness in the school environment as part of school tobacco control strategy.

Selective seed abortion induced by nectar robbing in the selfing plant Comastoma pulmonarium.

• Self-pollination often provides plants with the benefit of reproductive assurance; thus, it is generally assumed that species' interactions that alter floral attractiveness or rewards, such as nectar robbing, will have little effect on the seed production of selfing species. We challenge this view with experimental data from Comastoma pulmonarium, a selfing annual experiencing a high ratio of nectar robbing in the Qinghai-Tibet Plateau. • We manipulated robbing (robbed or netted) and pollination mode (hand-selfed or hand-outcrossed) in a factorial design and measured the number of developing ovules and mature seeds, together with seed weight and seed germination, in each treatment. • Robbing decreased the number of mature seeds, but not the number of developing ovules, suggesting a negative influence of robbers through indirect effects via selective seed abortion. We found no evidence for early-acting inbreeding depression, but found later-acting inbreeding depression. Our data also suggested that later-acting inbreeding depression of progeny from robbed flowers could be reduced in comparison with that from unrobbed flowers. • We suggest that nectar robbing can have both negative and positive effects on the quantity and quality, respectively, of progeny produced in selfing plants, and challenge the view that robbing has no effect on selfing species.

MNSFß Regulates TNFα Production by Interacting with RC3H1 in Human Macrophages, and Dysfunction of MNSFß in Decidual Macrophages Is Associated With Recurrent Pregnancy Loss.

Decidual macrophages (dMϕ) are the second largest population of leukocytes at the maternal-fetal interface and play critical roles in maintaining pregnancy. Our previous studies demonstrated the active involvement of monoclonal nonspecific suppressor factor-ß (MNSFß) in embryonic implantation and pregnancy success. MNSFß is a ubiquitously expressed ubiquitin-like protein that also exhibits immune regulatory potential, but its function in human dMϕ remains unknown. Here, we observed that the proportion of CD11chigh (CD11cHI) dMϕ was significantly increased in dMϕ derived from patients with recurrent pregnancy loss (RPL dMϕ) compared to those derived from normal pregnant women (Control dMϕ). The production of MNSFß and TNFα by RPL dMϕ was also significantly increased compared to that by Control dMϕ. Conditioned medium from RPL dMϕ exerted an inhibitory effect on the invasiveness of human trophoblastic HTR8/SVneo cells, and this effect could be partially reversed by a neutralizing antibody against TNFα. Bioinformatics analysis indicated a potential interaction between MNSFß and RC3H1, a suppressor of TNFα transcription. Immunoprecipitation experiments with human Mϕ differentiated from the human monocyte cell line Thp1 (Thp1-derived Mϕ) proved the binding of MNSFß to RC3H1. Specific knockdown of MNSFß in Thp1-derived Mϕ led to a marked decrease in TNFα production, which could be reversed by inhibiting RC3H1 expression. Interestingly, a significant decrease in the protein level of RC3H1 was observed in RPL dMϕ. Together, our findings indicate that aberrantly increased MNSFß expression in dMϕ may promote TNFα production via its interaction with RC3H1, and these phenomena could result in the disruption of the immune balance at the maternal-fetal interface and thus pregnancy loss.

Delayed selfing in an alpine biennial Gentianopsis paludosa (Gentianaceae) in the Qinghai-Tibetan plateau.

Delayed selfing could provide ovules with an opportunity to be fertilized as a means of "pollination assurance" before the flowers wilt. It could, thus, be regarded as an adaptation to unpredictable pollinator environments. Within the alpine biennial Gentianopsis paludosa, the showy flowers and herkogamy at the early stage of a flower's life cycle may favor outcrossing. As the flower ages, anthers contact the central stigma due to the elongation of all filaments, resulting in autonomous selfing. Flower visitors are extremely rare in a high altitude population; and examination of the mating system indicates that G. paludosa is self-pollinated under natural conditions in this population. While at the lower altitude, the bumblebee visitation rate is relatively high but possibly unreliable. Stigma receptivity is the highest on the third day of anthesis, and decreases thereafter. Pollen viability is the highest when flowers open, and gradually decreases later. Self-pollination of G. paludosa occurs at the late stage of a flower's lifecycle when stigma receptivity and pollen viability have both decreased, suggesting delayed selfing and assurance of seed production. This delayed selfing could assure seed production under the constraints of pollinator scarcity, but ensure outcrossing when pollinators were available. Such a flexible pollination mechanism is highly adaptive in the alpine environment of the Qinghai-Tibetan Plateau.