Modulating the RFamide-related peptide-3/G protein-coupled receptor 147 signaling pathway with nourishing Yin-removing fire herbal mixture to alleviate precocious puberty in female rats: An experimental study.

Background: Precocious puberty (PP) involves early activation of the hypothalamic gonadotropin-releasing hormone (GnRH) generator. The RFamide-related peptide/G protein-coupled receptor 147 (RFRP3/GPR147) signaling pathway is vital in inhibiting GnRH and delaying puberty onset. The nourishing Yin-removing fire (NYRF) herbal mixture has shown promising results in treating PP. Objective: This study aimed to assess the impact of the NYRF herbal mixture on the RFRP3/GPR147 signaling pathway in the hypothalamus and its potential in alleviating PP in female rats. Materials and Methods: In a controlled experiment, 24 female Sprague-Dawley rats (11.20 ± 0.69 gr, postnatal day [PD5]) were divided into normal, model, normal saline, and NYRF groups (n = 6/each). PP was induced in the model, normal saline, and NYRF groups by subcutaneous injection of danazol at PD5. The NYRF herbal mixture or normal saline was administered from PD15. Serum sex hormone levels and hypothalamic samples were collected for mRNA and protein expression at PD30. Results: In the model group, hypothalamic GnRH and kisspeptin levels increased, while RFRP3 and GPR147 levels decreased, luteinizing hormone levels elevated, reproductive organ coefficients increased, and the vagina opened earlier compared to the normal group. Conversely, the NYRF group exhibited lower GnRH and kisspeptin levels but higher RFRP3 levels in the hypothalamus. Serum luteinizing hormone levels were reduced, reproductive organ coefficients were reduced, and the vaginal opening was delayed compared to the model and normal saline groups. Conclusion: The NYRF herbal mixture delayed sexual development in rats with PP by hypothalamic upregulating RFRP3 and downregulating GnRH and kisspeptin.

Salvia bowleyana Dunn root is a novel source of salvianolic acid B and displays antitumor effects against gastric cancer cells.

Salvianolic acid B (Sal-B) is widely used in China for the treatment of numerous diseases. Currently, Salvia miltiorrhiza Bunge is the main source of this compound, but Salvia bowleyana Dunn, a surrogate of S. miltiorrhiza Bge, may provide a novel source for obtaining more Sal-B. In the present study, a simple method for separation and purification of phenolic compounds from S. bowleyana Dunn roots was employed. Sal-B was subsequently purified and its inhibitory effect on the gastric cancer HGC-27 and AGS cell lines was investigated. Sal-B extracted from S. bowleyana Dunn displayed significant antitumor activity in proliferation and apoptosis assays. Overall, it was found that S. bowleyana Dunn has a higher Sal-B content than S. miltiorrhiza Bge and may be used as a novel source of this potential anti-gastric cancer compound.

Dynamic Changes of RFRP3/GPR147 in the Precocious Puberty Model Female Rats.

BACKGROUND: Pubertal development is a complex physiological process regulated by the neuroendocrine system and hypothalamic-pituitary-gonadal axis. Sexual precocity is a common childhood endocrine disease.The pathogenesis of sexual precocity has not been fully elucidated. RFRP3/GPRl47 signal pathway is able to inhibit the reproductive capability in avians and mammals, probably by acting on the GnRH neuron and pituitary to regulate gonadotrophin synthesis and release. However, little is known about the role of RFRP3 in puberty development and sexual precocity. OBJECTIVE: To observe the dynamic changes of RFamide related peptide 3/G proteincoupled receptor 147 (RFRP3/GPR147) in hypothalamic during puberty development and explore their role in precocious puberty based on a female rat model. METHODS: The Sprague-Dawley female rats were randomly divided into three groups, normal, vehicle, and precocious puberty model. At 5 days old, the rat model with precocious puberty was prepared by subcutaneously injecting a mixture of danazoldissolved ethanol and glycol. At different day-age (15, 25, 30, 35, and 40 days), the levels of estradiol(E2), follicle-stimulating hormone(FSH), and luteinizing hormone (LH) in the peripheral blood were detected by the enzyme-linked immunosorbent assay, the messenger ribonucleic acid (mRNA) expressions of RFRP3, gonadotropin releasing hormone and GPR147 were examined by real-time polymerase chain reaction(R-T PCR). RFRP3 positive cells were observed using Immunofluorescence confocal microscopy. RESULTS: At 25 and 30 days, the levels of sex hormones and the uterus coefficients were significantly higher in the precocious puberty model group than those in the normal and vehicle groups. The ovarian morphological development in the precocious puberty model rats was significantly earlier than those in the normal and vehicle groups. The mRNA expressions of RFRP3/GPR147 and GnRH in the precocious puberty model group gradually increased and peaked at 25 days. The different day-age and the interaction have significant statistical significance on the expression of RFRP3 mRNA, while the levels of RFRP3 mRNA in the model group and vehicle groups have no significant statistical significance. There was statistical significance between the model group and vehicle groups in different day-age on the expression of GPR147 mRNA.The expression of hypothalamic RFRP3/GPR147 mRNA and RFRP3 positive cells gradually decreased with puberty onset. At 35 days, the levels of RFRP3 mRNA and GPR147 mRNA were significantly lower in the precocious puberty model group than those in the vehicle groups. Meanwhile, the levels of LH in the precocious puberty model rats reached its peak at this age. In the vehicle group, the levels of RFRP3 mRNA and serum LH were gradually increased and LH nearly peaked at 35 day-age. Subsequently, it gradually decreased and reached the lowest level at 35 day-age. The expression of RFRP3 mRNA and LH were positively correlated. CONCLUSION: The findings suggested that RFRP3/GPR147 signaling pathway may be involved in the pathogenesis of sexual precocity by regulating puberty development and sexual maturity in rats.

Immunopotentiator Aikejia improves the therapeutic efficacy of PD-1/PD-L1 immunosuppressive pathway in CT26.WT cancer cell.

Tumors can escape immunosurveillance through immunocheckpoint such as the PD-1/PD-L1 pathway. Aikejia comes from Nocardia rubra cell-wall skeleton and can increase the number of inflammatory factors and immune cells. In this work, we showed that the levels of PD-L1 increase in CT26.WT xenograft after subcutaneous injection of Aikejia in mice, but Aikejia did not induce the expression of PD-L1 in vitro. When we treated the mice with Aikejia and blocked PD-1/PD-L1 pathway in vivo at the same time, the CT26.WT xenografts were significantly inhibited or eliminated, which was better than single treatment alone. Our results suggested that Aikejia may be an effective adjuvant for PD-1/PD-L1 immunotherapy.

Low level of Cyclin-D1 correlates with worse prognosis of clear cell renal cell carcinoma patients.

Cyclin-D1 (CCND1) belongs to the highly conserved cyclin family whose members are characterized by abundant expression during the cell cycle. As an oncogene, high level of CCND1 was observed and related to poor prognosis and tumor recurrence in many cancers. In this study, we focused on the role of CCND1 in the clinical outcome of clear cell renal cell carcinoma (ccRCC). Gene Expression Omnibus database, The Cancer Genome Atlas database, and immunohistochemical staining were used. The mRNA and protein levels of CCND1 were significantly enhanced in ccRCC tumor tissues. However, the low level of CCND1, but not high level of CCND1, was related to poor prognosis and tumor recurrence in ccRCC. Further analysis showed that CCND1 mRNA level decreased with increasing ccRCC tumor grades and the rate of recurrence in ccRCC patients. In a nomogram model, the CCND1 mRNA level was shown to help predict ccRCC patient recurrence. CCND1 is a strong determinant for prediction of recurrence. The patients with high CCND1 level appear to have a more favorable prognosis together with more frequent low-grade tumors and low rate of recurrence. This is the first study to investigate the prognostic roles of CCND1 in ccRCC and discovered that CCND1 had an unconventional positive impact on the clinical outcome of ccRCC patients.

Screening for functional IRESes using α-complementation system of ß-galactosidase in Pichia pastoris.

BACKGROUND: Pichia pastoris is becoming a promising chassis cell for metabolic engineering and synthetic biology after its whole genome and transcriptome sequenced. However, the current systems for multigene co-expression in P. pastoris are not efficient. The internal ribosome entry site (IRES) has an ability to recruit the ribosome to initiate protein synthesis by cap-independent translation manner. This study seeks to screen IRES sequences that are functional in P. pastoris, which will allow P. pastoris to express multiple proteins in a single mRNA and increase its efficacy as a platform for metabolic engineering and synthetic biology. RESULTS: In order to efficiently screen the IRES sequences, we first set out to create a screening system using LacZ gene. Due to the cryptic transcription of the LacZ gene, we established the α-complementation system of ß-galactosidase in P. pastoris with the optimum length of the α-complementing peptide at ~ 92 amino acids. The optimal α-complementing peptide was then used as the second reporter to screen IRESes in the engineered GS115 expressing the corresponding ω-peptide. A total of 34 reported IRESes were screened. After ruling out all false positive or negative IRESes, only seven IRESes were functional in P. pastoris, which were from TEV, PVY, RhPV, TRV, KSHV, crTMV viruses and the 5'-UTR of the YAP1 gene of S. cerevisiae. CONCLUSIONS: We showed here that α-complementation also works in P. pastoris and it can be used in a variety of in vivo studies. The functional IRESes screened in this study can be used to introduce multiple genes into P. pastoris via a prokaryotic-like polycistronic manner, which provided new efficient tools for metabolic engineering and synthetic biology researches in P. pastoris.

Investigation on the processing and improving the cleavage efficiency of furin cleavage sites in Pichia pastoris.

BACKGROUND: Proprotein convertase furin is responsible for the processing of a wide variety of precursors consisted of signal peptide, propeptide and mature peptide in mammal. Many precursors processed by furin have important physiological functions and can be recombinantly expressed in Pichia pastoris expression system for research, pharmaceutical and vaccine applications. However, it is not clear whether the furin cleavage sites between the propeptide and mature peptide can be properly processed in P. pastoris, bringing uncertainty for proper expression of the coding DNA sequences of furin precursors containing the propeptides and mature peptides. RESULTS: In this study, we evaluated the ability of P. pastoris to process furin cleavage sites and how to improve the cleavage efficiencies of furin cleavage sites in P. pastoris. The results showed that P. pastoris can process furin cleavage sites but the cleavage efficiencies are not high. Arg residue at position P1 or P4 in furin cleavage sites significantly affect cleavage efficiency in P. pastoris. Kex2 protease, but not YPS1, in P. pastoris is responsible for processing furin cleavage sites. Heterologous expression of furin or overexpression of Kex2 in P. pastoris effectively increased cleavage efficiencies of furin cleavage sites. CONCLUSIONS: Our investigation on the processing of furin cleavage sites provides important information for recombinant expression of furin precursors in P. pastoris. Furin or Kex2 overexpressing strains may be good choices for expressing precursors processed by furin in P. pastoris.

Estimating short-term mortality and economic benefit attributable to PM10 exposure in China based on BenMAP.

With the rapidly booming economy, China has been suffering from serious particulate matter (PM) pollution in recent years. In order to improve the air quality, Chinese government issued a new China National Ambient Air Quality Standard (No. GB3095-2012) in 2012. In this study, PM10 exposure level was simulated based on the data of 912 newly constructed monitoring sites and Voronoi Neighborhood Averaging (VNA) interpolation method. It is widely accepted that PM10 can cause short-term health effects. We calculated the short-term health benefit due to decreasing PM10 concentration to the levels of China National Ambient Air Quality Standard based on Environmental Benefits Mapping and Analysis Program (BenMAP). Our results indicated that if the daily average concentration of PM10 reduced to the daily Grade II standard (150 µg/m3), the avoided deaths for all cause, cardiovascular disease, and respiratory disease would be 82,000 (95%CI: 49,000-120,000), 56,000 (95%CI: 34,000-78,000), and 16,000 (95%CI: 10,000-22,000) in 2014, respectively. The economic benefits of avoiding deaths due to all cause for rolling back the concentration of PM10 to the level of 50 µg/m3 were estimated to be 240 billion CNY and 16 billion CNY using willingness to pay (WTP) and human capital (HC) methods, respectively, which accounted for 0.38% (95%CI: 0.11-0.64%) and 0.03% (95%CI: 0.02-0.03%) of the total annual gross domestic product (GDP) of China in 2014.

The Roles of microRNAs in Regulating the Expression of PD-1/PD-L1 Immune Checkpoint.

Engagement of programmed death-ligand 1 (PD-L1) with its receptor programmed death 1 (PD-1) on T cells has been speculated to play a major role in suppressing the immune system, which helps tumor cells evade anti-tumor immunity. With the development of whole genome sequencing technologies, microRNAs have gained more attention as an important new layer of molecular regulation. Recent studies have revealed that altered expression of microRNAs play a pivotal role in immune checkpoint and various cellular processes in cancer. In this review, we focused on the latest progress about microRNAs research which involves the regulation of PD-1/PD-L1 immune checkpoint.

Quantifying public health benefits of environmental strategy of PM2.5 air quality management in Beijing-Tianjin-Hebei region, China.

In 2013, China issued "Air Pollution Prevention and Control Action Plan (Action Plan)" to improve air quality. To assess the benefits of this program in Beijing-Tianjin-Hebei (BTH) region, where the density of population and emissions vary greatly, we simulated the air quality benefit based on BenMAP to satisfy the Action Plan. In this study, we estimate PM2.5 concentration using Voronoi spatial interpolation method on a grid with a spatial resolution of 1×1km2. Combined with the exposure-response function between PM2.5 concentration and health endpoints, health effects of PM2.5 exposure are analyzed. The economic loss is assessed by using the willingness to pay (WTP) method and human capital (HC) method. When the PM2.5 concentration falls by 25% in BTH and reached 60µg/m3 in Beijing, the avoiding deaths will be in the range of 3175 to 14051 based on different functions each year. Of the estimated mortality attributable to all causes, 3117 annual deaths were due to lung cancer, 1924 - 6318 annual deaths were due to cardiovascular, and 343 - 1697 annual deaths were due to respiratory. Based on WTP, the estimated monetary values for the avoided cases of all cause mortality, cardiovascular mortality, respiratory mortality and lung cancer ranged from 1110 to 29632, 673 to 13325, 120 to 3579, 1091 to 6574 million yuan, respectively. Based on HC, the corresponding values for the avoided cases of these four mortalities were 267 to 1178, 161 to 529, 29 to 143 and 261 million yuan, respectively.

Prescriptions from Traditional Chinese Medicine compared with salbutamol and montelukast for the treatment of pediatric asthma: a randomized controlled trial.

OBJECTIVE: To compare the effects of a series of Traditional Chinese Medicine (TCM) empirical prescriptions with salbutamol and montelukast (SM) in children with asthma. METHODS: A total of 182 children with asthma were randomized into the TCM group (n = 97) or SM group (n = 85). Patients in the TCM group were treated with a series of TCM prescriptions, whereas those in the SM group received salbutamol and montelukast; both groups received their respective treatment for 12 weeks. Asthma control, changes in scores of TCM symptom patterns, and asthma symptom control (SC) scores after treatment were compared between the two groups. RESULTS: A higher percentage of patients in the TCM group had asthma control compared with those in the SM group (91.67% and 76.83%, respectively, P = 0.006). Scores for abnormal feces (P < 0.001), hyperhidrosis (P < 0.001), and tongue appearance (P = 0.001) in the TCM group were significantly better than those in the SM group. However, the total scores of TCM symptom patterns and SC scores did not differ significantly between the two groups (P > 0.05). CONCLUSION: Compared with salbutamol and montelukast, the TCM prescriptions tested were better for symptom control in children with asthma.

Assessment of population exposure to PM2.5 for mortality in China and its public health benefit based on BenMAP.

Along with the rapid socioeconomic development, air pollution in China has become a severe problem. One component of air pollution, in particular, PM2.5 has aroused wide public concern because of its high concentration. In this study, data were collected from over 900 monitoring sites of the newly constructed PM2.5 monitoring network in China. The interpolation methods were used to simulate the PM2.5 exposure level of China especially in rural areas, thus reflecting the spatial variation of PM2.5 pollution. We calculated the health benefit caused by PM2.5 in China in 2014 based on Environmental Benefits Mapping and Analysis Program (BenMAP), assuming achievement of China National Ambient Air Quality Standard (No. GB3095-2012). By reducing the annual average concentration of PM2.5 to the annual Grade II standard (35 µg/m3), the avoided deaths for cardiovascular disease, respiratory disease and lung cancer could reach 89,000 (95% CI, 8000-170,000), 47,000 (95% CI, 3000-91,000) and 32,000 (95% CI, 6000-58,000) per year using long term health function, respectively. The attributable fractions of cardiovascular disease, respiratory disease and lung cancer to all cause were 42%, 22% and 15%, respectively. The total economic benefits for rolling back the concentration of PM2.5 to the level of 35 µg/m3 were estimated to be 260 (95%CI: (73, 440) billion RMB and 72 (95%CI: (45, 99) billion RMB using willingness to pay (WTP) and human capital (HC) methods, respectively, which account for 0.40% (95%CI: (0.11%, 0.69%) and 0.11% (95%CI: (0.07%, 0.15%) of the total annual Gross Domestic Product (GDP) of China in 2014.

Regulatory effects of stage-treatment with established Chinese herbal formulas on inflammatory mediators in pediatric asthma.

OBJECTIVE: To explore the regulatory effects of established Chinese herbal formulas on inflammatory mediators released during asthma attacks, and to elucidate the molecular mechanism of Traditional Chinese Medicine in the treatment of asthma. METHODS: Seventy-five asthmatic children were randomly divided into a Chinese medication group (45 cases) and a Western medication control group (30 cases). Patients in the Chinese medication group were treated with a series of established Chinese herbal formulas, whereas the Western medication control group received a leukotriene receptor antagonist and a bronchial relaxant. Real-time PCR was used to determine the mRNA expression levels of interleukin (IL)-4, cysteinyl leukotriene receptor 1 (CysLTR1), and interferon (IFN)-gamma in peripheral blood mononuclear cells before and after treatment. Enzyme-linked immunosorbent assay was used to measure the peripheral blood levels of IL-4, leukotriene (LTE)-4, and INF-gamma before and after treatment. RESULTS: After treatment, the mRNA expression levels of IL-4 and CysLTR1 were down-regulated (P< 0.01) and the mRNA expression levels of IFN-gamma were up-regulated (P < 0.05) in the Chinese medication and Western medication groups; no significant difference was found between the two groups. In the Chinese medication group, IL-4 blood level was decreased and it was significantly different from that in the Western medication group (P < 0.05); there was also a significant increase in IFN-gamma blood levels after treatment with Chinese medication (P < 0.05). There were no significant differences in LTE-4 blood levels between the two groups before and after treatment (P > 0.05). CONCLUSION: Chinese medication has a regulatory effect on leukotriene receptor gene expression and the imbalance of Th1/Th2 immune cells during asthma attacks in pediatric patients.