Aptamer-Based Functionalized SERS Biosensor for Rapid and Ultrasensitive Detection of Gastric Cancer-Related Biomarkers.

Background: Gastric cancer (GC) as is the second deadliest malignancy still lacks rapid, simple and economical detection and early clinical screening techniques. Surface-enhanced Raman spectroscopy (SERS) is a spectroscopic technique based on the surface plasmon resonance of precious metal nanoparticles, which can effectively detect low-abundance tumor markers. Combining SERS technology with sensors has high potential in the diagnosis and screening of GC. Methods: A novel Au/Si nano-umbrella array (Au/SiNUA) was prepared as a SERS substrate and the substrate was functionalized using the corresponding tumor marker aptamers for the detection of clinical biological samples using a one-step recognition release mechanism. Optimization of aptamer and complementary chain concentrations and detection time for optimal sensor preparation. Results: Au/SiNUA were tested to have good SERS enhancement activity. The proposed aptamer biosensor has good specificity and stability, with a low detection time of 18 min and a limit of detection (LOD) at the fM level, which is superior to most of the methods reported so far; and the accuracy of the clinical assay is comparable to that of the ELISA method. The expression levels of PDGF-B and thrombin in the serum of GC patients and healthy individuals can be effectively detected and differentiated. Conclusion: The ultrasensitive and specific aptamer biosensor is highly feasible for the diagnosis and screening of GC and has good application prospects.

First report of Fusarium falciforme causing root rot of pomegranate (Punica granatum L.) in China.

Pomegranate (Punica granatum L.) is a deciduous shrub or small tree that is native to Iran and Afghanistan. It is also a commercially important fruit tree in China and worldwide. In the summer of 2022, a serious root rot disease occurred in some pomegranate orchards in Xichuan County(32º42´ N, 111º48´ E), Henan Province, China, with an incidence of ~30%. Symptoms included leaf yellowing and wilting, root browning and rotting, and stem-base cracking, eventually leading to defoliation and death. To isolate the causal agent, small pieces (5×5 mm) of diseased root from six trees were surface-sterilized by dipping in 2% NaClO for 8 min followed by 70% ethanol for 15 s, rinsed five times with sterile water, and plated on potato dextrose agar (PDA), then incubated at 28°C in the dark for 5 days. Fifteen pure fungal isolates with the same morphological characteristics were obtained from 24 pieces of roots. All isolates produced white fluffy mycelia. Microconidia were hyaline, oval or reniform, with zero to one septa and dimensions of 7.1 to 19.9 (average 14.5 )× 3.8 to 8.0 (average 5.6) µm (n = 100). Macroconidia were sickle-shaped, one to four septate, and 20.1 to 40.8 (average 26.5) × 4.8 to 8.6 (average 6.5) µm (n = 100). Chlamydospores were spherical, single, in pairs or chains, and 5.6 to 9.8 (average 6.8) µm in diameter (n = 100). Based on the above characteristics, the pathogens were identified as Fusarium sp. (Leslie and Summerell 2006). Genomic DNA was extracted from mycelia of two representative isolates Fs1 and Fs3. The internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α) and RNA polymerase II second largest subunit (RPB2) sequences were PCR amplified using primer pairs of ITS1/ITS4, EF1/EF2, and RPB2-5f2/RPB2-7cr, RPB2-7cf/RPB2-11ar (O'Donnell et al., 2022), respectively. BLAST analysis showed that the ITS, TEF-1α and RPB2 sequences of isolates Fs1(GenBank accession nos. OK001765, OQ921726 and OQ928396) and Fs3 (GenBank accession nos. OK001771, OQ921727 and OQ928397) showed 99%-100% identity with multiple GenBank sequences of Fusarium falciforme (KY617066, MN064683, KF255514, OQ933361, KY556711 and ON331935). A phylogenetic tree based on concatenated sequences of ITS, TEF-1α and RPB2 using maximum-likelihood analysis revealed that both isolates Fs1 and Fs3 were in the same clade with F. falciforme strains. Based on the morphological and molecular characteristics, the isolates were identified as members of F. falciforme. For pathogenicity testing, conidial suspensions (1×108 spores /mL) of isolates Fs1 and Fs3 were poured onto the roots of healthy pomegranate that had been planted in pots two months previously. Ten plants were inoculated for each isolate. Control plants were drenched with sterile water. After 3 months, inoculated plants developed leaf yellowing and wilting accompanied by root browning and rotting, much like symptoms observed in field plants. The same fungi re-isolated from the experimental plants were confirmed to be F. falciforme by morphology and sequence analysis. This is the first report of F. falciforme causing root rot on pomegranate. F. falciforme is a ubiquitous soil-borne pathogen that causes root rot on multiple plants around the world (Xu F., et al. 2022; Qiu R., et al. 2023). The results of pathogen identification are essential precursors to development of effective control of the disease.

[Observation of effect of Wu Yun Liu Qi on reproductive hormones in varicocele patients].

Objective： To observe roproductive hormone levels in varicocele patients during a cycle (6 years) of Wu Yun Liu Qi, and explore whether the cycle had effect on the roproductive hormone levels. Methods： Data of roproductive hormone levels in varicocele patients from 2015 to 2020 were analyzed retrospectively. FSH、LH、T、PRL、E2 levels and T/E2 ratio were compared among the six years. According to Chinese sexagenary cycle heavenly stems and earthly branches of each year from 2015 to 2020 its yunqi characteristics were determined. Results: Totally data of 848 cases of varicocele patients were collected from 2015 to 2020. Among which, in 2015 (Yiwei year) there were 57 cases, in 2016 (Bingshen year) 83 cases, in 2017 (Dingyou year ) 133 cases, in 2018(Wuxu year) 156 cases, in 2019(Sihai year) 274 cases, and in 2020(Gengzi year) 145 cases. The levels of FSH、LH、PRL、T were not diferrent statistically from the six years except individual year. However, the level of E2 in 2016 when the Yunqi was Shao Yang Xiang Huo Si Tian and Jue Yin Feng Mu Zai Quan obviously higher than other years excpet 2018( All P< 0.05). And T/E2 ratio was lower in 2016 than other years except 2018 and 2020( All P< 0.05). Conclusions: Shi Xiang factors of Wu Yun Liu Qi had effect on roproductive hormone levels in varicocele patients, showing by higher E2 level in Yinshen year when the Shi Xiang factors may have bad effect on human fertility.

[Observation on effect of five evolutive phases and six climatic factors on pregnancy and sterility].

Objective： To verify statements about the effect of five evolutive phases and six climatic factors in 《huang di nei jing》 on pregnancy and sterility. Methods： Data of missed abortion from Nanjing Maternity and Child Health Care Hospital between 2006-2020 and Yangzhou Maternity and Child Health Care Hospital between 2012-2020 were collected. According to Chinese sexagenary cycle heavenly stems and earthly branches of each year between 2006-2020 was determined. And then based on the heavenly stems and earthly branches, evolutive phases and six climatic factors of each year were established. After that data of missed abortion in each year was compared. According to relative statements in 《huang di nei jing》 , it was observed whether the number of missed abortion cases in someone year increased significantly. Results: Totally there were 28059 cases of missed abortion from the two hospital were collected between 2006-2020 and 2012-2020 respectively. It was found that in the year of Shaoyang phase fire sitian and Jueyin wind wood in the spring (that is the year of 2010 and 2016) the number of missed abortion cases was higher obviously than the other year. When the year of Taiyang cold water in the spring (that is the year of 2009 and 2015), there was a relatively increased number of missed abortion cases. Conclusions: Five evolutive phases and six climatic factors have impacts on human pregnancy and sterility. It was an adverse condition for human reproduction in the year of Shaoyang phase fire sitian and Jueyin wind wood in the spring as well as Taiyang cold water in the spring. Until to now we can still verify statements about the effect of five evolutive phases and six climatic factors on pregnancy and sterility in 《huang di nei jing》.

Eupalinolide A induces autophagy via the ROS/ERK signaling pathway in hepatocellular carcinoma cells in vitro and in vivo.

Hepatocellular carcinoma is the most common primary malignancy of the liver. The current systemic drugs used to treat hepatocellular carcinoma result in low overall survival time. It has therefore been suggested that new small­molecule drugs should be developed for treating hepatocellular carcinoma. Eupatorium lindleyanum DC. (EL) has been used to treat numerous diseases, particularly respiratory diseases; however, to the best of our knowledge, studies have not yet fully elucidated the effect of EL on hepatocellular carcinoma. In the present study, the effect of eupalinolide A (EA), one of the extracts of EL, was evaluated on tumor growth in a xenograft model of human hepatocellular carcinoma cells, and on the proliferation and migration of hepatocellular carcinoma cell lines. Cell cycle progression and the type of cell death were then evaluated using the Cell Counting Kit 8 assay, flow cytometry, electron microscopy and western blotting. EA significantly inhibited cell proliferation and migration by arresting the cell cycle at the G1 phase and inducing autophagy in hepatocellular carcinoma cells. EA­induced autophagy was mediated by reactive oxygen species (ROS) and ERK signaling activation. Specific inhibitors of ROS, autophagy and ERK inhibited EA­induced cell death and migration. In conclusion, the present study revealed that EA may inhibit the proliferation and migration of hepatocellular carcinoma cells, highlighting its potential as a promising antitumor compound for treating hepatocellular carcinoma.

Eupalinolide B inhibits hepatic carcinoma by inducing ferroptosis and ROS-ER-JNK pathway.

Primary hepatic carcinoma is a common malignant tumor. The classic molecular targeted drug sorafenib is costly and is only effective for some patients. Therefore, it is of great clinical significance to search for new molecular targeted drugs. Eupalinolide B (EB) from Eupatorium lindleyanum DC. is used to treat chronic tracheitis in clinical practice. However, the role of EB in hepatic carcinoma is unknown. In this study, we first measure the effect of EB on tumor growth in a xenograft model and PDX model. The cell proliferation and migration are also detected in human hepatocarcinoma cell lines (SMMC-7721 and HCCLM3). Then, we investigate cell cycle, cell apoptosis, cell necrosis, cell autophagy, and ferroptosis by flow cytometry, western blot analysis and electron microscopy. The results demonstrate that EB exerts anti-proliferative activity in hepatic carcinoma by blocking cell cycle arrest at S phase and inducing ferroptosis mediated by endoplasmic reticulum (ER) stress, as well as HO-1 activation. When HO-1 is inhibited, EB-induced cell death and ER protein expression are rescued. The migration-related mechanism consists of activation of the ROS-ER-JNK signaling pathway and is not connected to ferroptosis. In summary, we first discover that EB inhibits cell proliferation and migration in hepatic carcinoma, and thus EB is a promising anti-tumor compound that can be used for hepatic carcinoma.

Microfluidic preparation of a novel phoxim nanoemulsion pesticide against Spodoptera litura.

With continuous development of pesticide dosage forms, emulsifiable concentrates using large amounts of organic solvents are gradually obsoleted. Nanoemulsions with high water content have been developed and the preparation processes also evolved, but these processes still exist some problems, such as poor controllability and high energy consumption. Microfluidic is a controllable nanoemulsion preparation system which mainly applied to pharmaceutical synthesis. In this study, the pesticide phoxim nanoemulsion was prepared by microfluidic technology. The optimized formulation of phoxim nanoemulsion was composed of Tween 80 and pesticide emulsifier 500 as surfactant, hexyl acetate as oil, and n-propanol as co-surfactant. Moreover, when the flow rates of water and oil in the microfluidic system were adjusted to 5 µL/min and 20 µL/min, phoxim nanoemulsion was obtained with a cloud point/boiling point of 109 °C, a particle size of 21.5 ± 0.8 nm and a potential value of - 18.7 ± 0.6 mV. Furthermore, the nanoemulsion had a rapid release effect in vitro which could be fitted by the Ritger-Peppas model. The feeding toxicity of the phoxim nanoemulsion was higher than that of commercial formulation while the contact killing effect was higher than that of the active ingredient. Therefore, pesticide dosage was reduced and the insecticidal effect was enhanced by using phoxim nanoemulsions. These results also confirm the potential of microfluidics as a green process to produce pesticide nanoemulsions.

Sweet-flavored peptides with biological activities from mulberry seed protein treated by multifrequency countercurrent ultrasonic technology.

To increase the reuse of food residues, multifrequency countercurrent ultrasonic treatment was used to enhance the extraction yield of defatted mulberry seed protein (DMSP), and sweet-flavored peptides from DMSP hydrolysates (DMSPHs) were obtained for the first time. Here, the DMSP yield was increased by 16.2% (p < 0.05) while the power density was halved compared with single-frequency ultrasonic treatment. According to Fick's second law, a molecular diffusion dynamics model was developed to be suitable for predicting the pretreatment conditions (R2 = 0.9785). After that, the sweet-flavored peptides were purified and the main amino acid sequences were identified, i.e., FEGGSIE, KDFPEAHSQAT, and GSQPAEGAK. Moreover, the antioxidant activities of DMSPHs prepared with tri-frequency treatment was higher than 60%. The DMSPHs retarded the growth of HepG2 cells in vitro, increased the necrotic quadrant (Q1-UL), and extended the S phase. Therefore, the sweet-flavored peptides prepared from DMSPHs using the multifrequency-ultrasonic treatment have significant biological activities.

[Effect of bilateral microsurgical subinguinal varicocelectomy on asthenozoospermia and oligozoospermia: A report of 109 cases].

OBJECTIVE: To investigate the effects of bilateral microsurgical subinguinal varicocelectomy (BMSV) in patients with asthenozoospermia or oligozoospermia. METHODS: Totally 147 patients with male infertility received BMSV from January 2018 to May 2019, of whom 109 had complete data recorded. We retrospectively analyzed the clinical data, including the total sperm count per ejeculate, sperm concentration and sperm motility before and after surgery, and the rate of natural conception achieved during the follow-up. RESULTS: BMSV achieved a total effectiveness rate of 79.00% in improving the percentage of progressively motile sperm (a rise of ≥20%) and a marked effectiveness rate of 70.00% (a rise of ≥50%) in the 100 cases of asthenozoospermia as compared with the baseline, with a mean recovery time of (110.13 ± 37.43) days. Besides, a total effectiveness rate of 74.29% (an increase of ≥20%) and a marked effectiveness rate of 71.43% (an increase of ≥50%) were attained in the improvement of sperm concentration in the 35 cases of oligozoospermia, with a mean recovery time of (117.00 ± 48.79 ) days. A natural conception rate of 30.30% was observed during the follow-up. No severe adverse events occurred postoperatively. CONCLUSIONS: BMSV is significantly effective for the treatment of asthenozoospermia and oligozoospermia.

Visnagin ameliorates myocardial ischemia/reperfusion injury through the promotion of autophagy and the inhibition of apoptosis.

Visnagin is a furanochromone and one of the main compounds of Ammi visnaga L. that had been used to treat nephrolithiasis in Ancient Egypt. Nowadays, visnagin was widely used to treat angina pectoris, urolithiasis and hypertriglyceridemia. The potential mechanisms of visnagin involved in inflammation and cardiovascular disease were also identified. But the protective effect of visnagin on myocardial ischemia/reperfusion injury has not been confirmed. Our aim was, for the first time, to investigate the potential protective effect of visnagin on cardiac function after myocardial ischemia-reperfusion injury in a rat model, and to identify its underlying mechanism involving the inhibition of apoptosis and induction of autophagy. Thirty SD rats were randomly divided into sham group, ischemia/reperfusion group (IR), ischemia/reperfusion with visnagin (IR + visnagin) group. Myocardial ischemia/Reperfusion injury model was established. Hemodynamic measurements and echocardiography were used to analyze cardiac function, TUNEL staining and caspase activity, LC3 dots were detected with immunofluorescence staining, LC3 expression was evaluated by western blot analysis, transmission electron microscopy (TEM) was used to detect autophagosomes. Compared with the sham group and visnagin group, the cardiac dysfunction, LC3II, autophagy flow in the IR+ visnagin group increased significantly (P<0.01), but the activity of caspase-3 and caspase-9 and the apoptotic in the IR + visnagin group decreased significantly (P<0.01). In conclusion, visnagin may play a protective role in ischemia/reperfusion injury by inducing autophagy and reducing apoptosis.

First Report of Neofusicoccum parvum Causing Leaf Spot on Geodorum eulophioides in China.

Geodorum eulophioides Schltr. is a critically endangered orchid listed in the International Union for Conservation of Nature (IUCN) Red List of threatened species. At present, only two natural populations were found in China. It has important scientific and ornamental values because of its uniqueness. During the summer of 2019, a black leaf spot disease occurred on G. eulophioides, in Yachang Orchid National Nature Reserve (E106°13'32″，N24°44'19″) in Guangxi province, China. More than 60% of leaves of these plants were infected. The disease symptoms initially appeared as small yellow circular spots, which enlarged into irregular brown spots (6 to 9 cm length and 3 to 5 cm width). In later stages of the disease development, the center of the spots became dark brown with a clear edge and surrounded by a yellow halo. In severe infections, the spots coalesced covering the entire leaf. Six symptomatic leaves were collected from three infected plants, surface sterilized in 75% ethanol for 15 s and 0.1% HgCl2 for 4 min, and subsequently washed three times with sterile water, then plated onto potato dextrose agar (PDA), and incubated at 28℃ for three days. Eighteen fungal cultures with similar morphological characteristics were obtained from the infected tissues. Colonies were initially white, then turned dark grey after nine days. To induce sporulation, isolates were grown on 2% water agar and incubated under UVA light at 28℃ for nine days. Three isolates were selected for morphological characterization. Conidia were hyaline, unicellular, nonseptate, ellipsoidal to fusiform, externally smooth, thin-walled, and ranged from 10.7 to 16.6 µm (avg. 13.8 µm) × 4.1 to 6.7 µm (avg. 5.1 µm) (n=50). The isolate DBL-1 was selected as a representative for molecular identification. Genomic DNA was extracted and used for PCR to amplify the rDNA internal transcribed spacer region (ITS), translation elongation factor 1-alpha gene (EF1-α), and beta-tubulin gene (TUB2), using the primer pairs ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R（Alves et al. 2008；Carbone & Kohn, 1999）, and T1/T2 (O'Donnell et al., 1997), respectively. The obtained ITS sequence (GenBank Accession No. MN918440), EF1-α sequence (MN963815), and TUB2 sequence (MN963816) showed >99% homology with several GenBank sequences of Neofusicoccum parvum (JX513636, KU997497 for ITS, KU997261, MH252401 for EF1-α, and KJ841779, MK412882 for TUB2, respectively). Based on morphological characteristics of the asexual morph and maximum likelihood analyses of a combined rDNA-ITS, EF1-α and TUB2 gene sequences, was identified as N. parvum. Pathogenicity test was performed using isolate DBL-1 by inoculating 3 leaves of G. eulophioides plants. The test was repeated three times. Each leaf was wounded using a sterile needle, and a mycelial plug (6 mm diameter) harvested from the periphery of a 3-day-old colony grown on PDA was placed on each wound. Plants were then covered with plastic bags to maintain high relative humidity of 90% and kept at 28℃ in a greenhouse under natural daylight conditions. An equal number of leaves on the same plant were inoculated using sterile PDA plugs and served as mock inoculated controls. After three days, all the inoculated leaves showed black spot symptoms resembling those observed in the field, whereas controls remained symptomless. The fungus was re-isolated from the symptomatic leaves, thus completing Koch's postulates. N. parvum has been reported to cause leaf spot disease on Myristica fragrans (Jayakumar, et al., 2011), Ginkgo biloba (Mirhosseini, et al., 2014), Vitis heyneana (Wu, et al., 2015), and Hevea brasiliensis (Liu et al., 2017), respectively. To the best of our knowledge, this is the first report of N. parvum causing leaf spot disease on G. eulophioides in China. The disease control measures and in-situ conservation method need to be strengthened to protect this rare species.

[Differential expression profiles of miRNAs in the vaginal tissue of the women with lubrication disorders].

OBJECTIVE: To explore the pathogenesis of lubrication disorder (LD), a most common type of female sexual dysfunction affecting women's physical health and conception, and find the therapeutic targets for its treatment and prevention. METHODS: We chose 3 LD patients and 3 healthy controls in Nanjing Maternal and Child Health Hospital, extracted their vaginal epithelial RNA for high-throughput miRNA sequencing, screened differentially expressed miRNAs for hierarchical cluster analysis, target gene prediction and gene ontology (GO) and KEGG pathway enrichment analyses. Finally, we verified the sequencing results by real-time fluorescent quantitative PCR. RESULTS: Totally 1 673 miRNAs were predicted by high-throughput sequencing and 64 likely to be the targets for the treatment of LD were screened, including 25 up-regulated more than 4 times and 39 down-regulated more than 4 times in the LD patients compared with the healthy controls. The neuron projection morphogenesis and AMPK signaling pathway were the most significant enrichment GO term and KEGG pathway. CONCLUSIONS: miRNAs are expressed differentially in LD patients. These miRNAs and target genes may be related to the occurrence of LD, and those that are expected to be the targets for the treatment of LD have important theoretical significance and potential application value.

[HPV infection of the external genitalia in men whose female partners have cervical HPV infection].

OBJECTIVE: Human papilloma virus (HPV) is a necessary cause of cervical cancer and is also closely related to penile cancer, oropharyngeal cancer, and anal cancer in males. However, few studies are reported on male HPV. This study aimed to investigate HPV infection of the external genitalia in men whose female partners have cervical HPV infection. METHODS: We collected the relevant data on the male outpatients whose partners had cervical HPV infection in our Department of Urology and Andrology from August to December 2016. We obtained samples with nylon swabs from the glans penis, corona, inner layer of the prepuce and penile body and detected different types of HPV infection using the Hybribio HPV typing kit, PCR and membrane hybridization. RESULTS: Valid data were collected from 140 males, which showed 83.5% of HPV infection of the external genitalia, including 60 cases of HPV6 (43.2%), 27 cases of HPV16 (19.4%), 14 cases of HPV39 (10.1%), 13 cases of HPV18 (9.4%), 13 cases of HPV58 (9.4%), and 13 cases of HPV52 (9.4%). Redundant prepuce was found in 75.5% of the males, but there was no statistically significant difference in the incidence rate of HPV infection between the normal and redundant prepuce groups (P > 0.05). CONCLUSIONS: Men who have the female partners with positive cervical HPV are at high risk of HPV infection and therefore need to be screened and treated so as to reduce HPV infection in both sexes.

Black tea increases hypertonic stress resistance in C. elegans.

Here we identified that BTE (black tea extract), within the studied concentration range, is more effective than GTE (green tea extract) in protecting C. elegans against hypertonic stress, by enhancing survival after exposure to various salts, and alleviating suffered motility loss and body shrinkage. The mechanism of such protection may be due to the ability of black tea to induce the conserved WNK/GCK signaling pathway and down-regulation of the expression levels of nlp-29. Intriguingly, black tea does not relieve hypertonicity-induced protein damage. The findings implicate the potential health benefits of black tea consumed worldwide.

GDF11/BMP11 as a novel tumor marker for liver cancer.

Growth differentiation factor 11 (GDF11), also known as bone morphogenetic protein 11, a member of the transforming growth factor-ß superfamily, has been reported to be involved in colorectal cancer. However, the roles of GDF11 in Chinese patients with liver cancer and the underlying mechanisms have remained elusive. The present study assessed the expression of GDF11 in 10 paired samples of cancerous and normal tissues from Chinese liver cancer patients. The results indicated that the expression of GDF11 was significantly lower in cancerous tissues than in normal tissues. In vitro, the expression of GDF11 was reduced in a panel of liver cancer cell lines compared with that in a normal liver cell line at the mRNA and protein level. Treatment with GDF11 reduced the viability of HepG2 for up to 72 h and GDF11 treatment reduced the viability of SMMC-7721 after 48 and 72 h. Furthermore, GDF11 activated Smad2/3 signaling in HepG2 cells. In conclusion, GDF11 has a tumor suppressor role in liver cancer, exerts its effects through Smad2/3 signaling and may serve as a novel tumor marker in liver cancer diagnosis.

MicroRNA-200a is up-regulated in aged rats with erectile dysfunction and could attenuate endothelial function via SIRT1 inhibition.

MiR-200a was shown to be upregulated in the corpus cavernosum (CC) of rats with aging-related erectile dysfunction (A-ED) in our previous study. Among its target genes, SIRT1 was also reported as a protective factor in erectile function by our groups previously. Thus, miR-200a might attenuate the erectile function in A-ED via SIRT1 inhibition. In the present study, three animal groups were included: aged rats with ED (group AE, n = 8), aged rats with normal erectile function (group AN, n = 8), and young rats as normal controls (group YN, n = 8). CCs from each group were collected for histological and molecular measurements to validate the dysregulation of miR-200a and SIRT1. After that, the cavernous endothelial cells (CECs) from CC of aged rats with normal erectile function were transfected with miR-200a in vitro. Then the expression of SIRT1 and molecules within the eNOS/NO/PKG pathway were measured to investigate whether the transfection could imitate the attenuated process of erectile function in the aged. As a result, miR-200a was upregulated while the SIRT1, the levels of eNOS and cGMP were all downregulated in the CCs from AE group. After transfection in vitro, the miR-200a was upregulated while the SIRT1 and levels of eNOS and cGMP were obviously downregulated. Finally, based on the results of our previous study, we further verify that up-regulation of miR-200a could participate in the mechanisms of A-ED via SIRT1 inhibition, and mainly attenuate endothelial function via influencing the eNOS/NO/PKGpathway.

[Relationship of abnormal sperm DNA methylation with early spontaneous abortion].

OBJECTIVE: To investigate the relationship between the abnormal sperm DNA methylation level and early spontaneous abortion. METHODS: We randomly selected 98 males who met the inclusion criteria and whose wives suffered from unexplained abortion or embryo abortion, and included another 46 normal healthy men present for pre-pregnancy check-up as controls. We examined the semen quality and sperm morphology, obtained the sperm DNA fragmentation index (DFI) by modified sperm chromatin dispersion, and measured the sperm DNA methylation level using the methylated DNA quantification kit and the colorimetric method. RESULTS: Compared with the normal controls, the men in the unexplained abortion group showed a significantly lower rate of big-halo sperm (ï¼»45.50 ± 26.27ï¼½ vs ï¼»36.49 ± 23.06ï¼½%, P = 0.038), a higher rate of abnormal-head sperm (ï¼»77.08± 12.21ï¼½ vs ï¼»81.09± 10.89ï¼½%, P = 0.049), and a lower level of sperm DNA methylation (ï¼»0.47 ± 0.33ï¼½ vs ï¼»0.36 ± 0.26ï¼½ ng/µl, P = 0.035). The sperm DNA methylation level was positively correlated with the percentage of big-halo sperm (OR=0.546, P<0.01). Multivariate regression analysis manifested that sperm head abnormality was an independent risk factor of early spontaneous abortion or embryo abortion (OR=1.032, P = 0.049), while the high methylation level was protective factor against early spontaneous abortion or embryo abortion (OR=0.244, P = 0.03). CONCLUSIONS: The abnormal level of sperm DNA methylation may be one of the important reasons for early spontaneous abortion or embryo abortion.

[Semen quality and its influencing factors in preconception males in Nanjing area].

OBJECTIVE: To investigate the semen quality and its influencing factors in preconception males in Nanjing area so as to provide some evidence for working out effective intervention measures. METHODS: Totally 687 men receiving preconceptional physical examination were enrolled in this study. A questionnaire survey was conducted among the subjects along with an analysis of their semen quality. RESULTS: The median of sperm concentration was 63.3 x 10(6)/ml (95% CI [19.88-119] x 10(6)/ml). The median of grade a sperm was 33.03% (95% CI [19.38-55.05]%), that of grade a + b sperm was 52.08% (95% CI [39.53-69.37]%), and that of teratosperm was 91.75% (95% CI [69-100]%). The median concentration of seminal plasma PMN-elastase was 195.55 ng/ml (95% CI [76.16-3330.38] ng/ml) and that of seminal plasma zinc was 7.62 µmol/L (95% CI [1.5-23, 45] µmol/L). The positive rates of Ureaplasma urealyticum (UU), Chlamydia trachomatis (CT), and Gardnerella vaginalis (GV) were 42.4%, 0.3%, and 2.4%, respectively. The median of sperm DNA fragmentation index (DFI) of those whose wives had a history of adverse pregnancy was 20.25% (95% CI [2.15-68.25]%). Multivariate logistic regression analysis suggested that mental stress (OR 1.567, 95% CI [1.081-2.27]) and sedentariness (OR 1.772, 95% CI [1.211-2.592]) were independent risk factors for asthenospermia. CONCLUSION: The sperm quality of preconception males in Nanjing area is not encouraging, and it can be improved by changing undesirable lifestyle and reducing mental stress.

[Relationship between epigenetics of sperm and embryogenesis].

Epigenetics comprises the modifications made in gene expressions without changing the DNA sequence itself. Significant epigenetic changes take place during spermatogenesis and fertilization and exert direct influences on embryogenesis. This article provides an overview of the latest researches on epigenetics of male germ cells and a brief discussion on the correlation of sperm with embryogenesis in four aspects: DNA methylation, histone modification, regulation of non-coding RNAs, and genomic imprinting.

[Protective effect of pAdxsi-ERbeta adenovirus transfection on penile vascular endothelium in ERbetaKO mice].

OBJECTIVE: To investigate the effect of the overexpression of the ERbeta gene on the penile vascular endothelium of ERbeta knockout (ERbetaKO) mice and its molecular mechanisms. METHODS: We randomly divided 12 ERbetaKO male mice into groups A (ERbetaKO + TNFalpha + pAdxsi-ERbeta) and B (ERbetaKO + TNFalpha + empty virus), the former treated by pAdxsi-ERbeta adenovirus transfection, the latter with empty virus, and meanwhile both injected intraperitoneally with TNFalpha at 6 microg per kg body weight per d for 14 days. Then we observed the erectile function of the mice by APO, determined the changes of the endothelial markers CD34 and vWF by immunohistochemical staining, and detected the expressions of the relevant molecules in the eNOS-NO pathway by RT-PCR, Western blot and immunohistochemistry. RESULTS: Compared with group B, group A showed a significantly increased number of penile erections (0.50 +/- 0.55 vs 2.17 +/- 0.41, P < 0.05), shortened erectile latency ([28.83 +/- 1.33] min vs [24.00 +/- 1.27] min, P < 0.05), enriched CD34 and vWF markers (0.67 +/- 0.52 vs 1.50 +/- 0.55 and 0.50 +/- 0.55 vs 1.33 +/- 0.52, both P < 0.05), elevated expressions of eNOS and Cam (RT-PCR: 1.38 +/- 0.03 vs 1.62 +/- 0.05 and 1.02 +/- 0.09 vs 1.42 +/- 0.05, both P < 0.05; Western blot: 1.27 +/- 0.04 vs 1.55 +/- 0.07 and 0.76 +/- 0.05 vs 0.95 +/- 0.08, both P < 0.05), and reduced expression of caveolin-1 (RT-PCR: 2.13 +/- 0.13 vs 1.72 +/- 0.08, P < 0.05; Western blot: 3.99 +/- 0.16 vs 3.40 +/- 0.14, P < 0.05). The results of RT-PCR were consistent with those of Western blot. CONCLUSION: The ERbeta gene protects the penile vascular endothelium via the eNOS-NO pathway.