Inactivated influenza virus vaccines expressing COBRA hemagglutinin elicited broadly reactive, long-lived protective antibodies.

The influenza viruses cause seasonal respiratory illness that affect millions of people globally every year. Prophylactic vaccines are the recommended method to prevent the breakout of influenza epidemics. One of the current commercial influenza vaccines consists of inactivated viruses that are selected months prior to the start of a new influenza season. In many seasons, the vaccine effectiveness (VE) of these vaccines can be relatively low. Therefore, there is an urgent need to develop an improved, more universal influenza vaccine (UIV) that can provide broad protection against various drifted strains in all age groups. To meet this need, the computationally optimized broadly reactive antigen (COBRA) methodology was developed to design a hemagglutinin (HA) molecule as a new influenza vaccine. In this study, COBRA HA-based inactivated influenza viruses (IIV) expressing the COBRA HA from H1 or H3 influenza viruses were developed and characterized for the elicitation of immediate and long-term protective immunity in both immunologically naïve or influenza pre-immune animal models. These results were compared to animals vaccinated with IIV vaccines expressing wild-type H1 or H3 HA proteins (WT-IIV). The COBRA-IIV elicited long-lasting broadly reactive antibodies that had hemagglutination-inhibition (HAI) activity against drifted influenza variants.

COBRA HA and NA vaccination elicits long-live protective immune responses against pre-pandemic H2, H5, and H7 influenza virus subtypes.

Highly pathogenic avian influenza (HPAI) viruses remain a major threat to both the poultry industry and human public health, and these viruses continue to spread worldwide. In this study, mice were vaccinated with COBRA H2, H5, and H7 hemagglutinin (HA) and two neuraminidase (NA) proteins, N1 and N2. Vaccinated mice were fully protected against lethal challenge with H5N6 influenza virus. Sera collected after vaccination showed cross-reactive IgG antibodies against a panel of wild-type H2, H5, and H7 HA proteins, and N1 and N2 NA proteins. Mice with pre-existing immunity to H1N1 and H3N2 influenza viruses that were subsequently vaccinated with COBRA HA/NA vaccines had enhanced anti-HA stem antibodies compared to vaccinated mice without pre-existing immunity. In addition, sera collected after vaccination had hemagglutinin inhibitory activity against a panel of H2Nx, H5Nx, and H7Nx influenza viruses. These protective antibodies were maintained up for up to 4 months after vaccination.

Intention to have a third child among millennial parents with two children in eastern China: A cross-sectional survey.

AIM: This Study Aimed to Assess the Intention to Have a Third Child among Millennial Parents (25-40 years old) with Two Children in a City in Eastern China and to Explore the Influencing Factors Related to Fertility Intention. DESIGN: A cross-sectional design study. METHODS: A convenience sampling method was used to enrol participants of childbearing age who visited two tertiary hospitals in Hang zhou, a city in eastern China, from June 2021 to March 2022. We conducted a face-to-face questionnaire survey with 520 participants and calculated the prevalence of intention-related factors. Multivariate logistic regression was used to analyse the independent influencing factors of fertility intention. RESULTS: In total, 105 (20.2%) participants had the intention to have a third child. The results showed that 'employment status', 'age', 'reasons for wanting a third child', the considered 'biggest barrier to having a third child', 'views on the three-child policy', 'desired free services', 'supporting work policies' and 'assistance policies' were significant independent influencing factors of intention to have a third child (p-value < 0.05). The intention of the participants 'over 30 years old' was 2.466 times that of those '30 years old and under', and 'older age/personal health status' was considered the 'biggest barrier to having a third child'. Regarding policy and social reasons, the participants who need 'medical assistance' policy negatively affect the intention to have a third child (OR = 0.453, 95% CI = 0.247-0.830). IMPLICATIONS FOR HEALTHCARE/NURSING: Nursing plays an important role in health promotion. Nurses can help couples make wise decisions about fertility by providing professional consultation, education, evaluation and support. They can also provide corresponding nursing and guidance to improve couples' health quality and overall reproductive success. CONCLUSIONS: The general level of intention to have a third child of Millennial parents with two children is still low. The participants who are 'housewives/househusbands', 'over 30 years old', and satisfied with the state of 'medical assistance' have higher fertility intentions. PATIENT OR PUBLIC CONTRIBUTION: It is particularly meaningful for the policymakers to improve the social support system and raise universal awareness to encourage childbirth.

A detection model of testis-derived circular RNAs in serum for predicting testicular sperm retrieval rate in non-obstructive azoospermia patients.

BACKGROUND: Microdissection testicular sperm extraction is an effective method to retrieve sperm from non-obstructive azoospermia patients. However, its successful rate is less than 50%. OBJECTIVES: To identify the predictive value of circular RNAs in serum for sperm retrieval rate in non-obstructive azoospermia patients. MATERIALS AND METHODS: 180 non-obstructive azoospermia patients were recruited in this study, including 84 individuals with successful sperm retrieval and 96 individuals with failed sperm retrieval. Our study contained two phases. First, 20 patients, selected from the 180 patients, were included in screening cohort. In this cohort, the top 20 circular RNAs from our previous testicular circRNA profiles were verified between successful and failed sperm retrieval groups using real-time polymerase chain reaction. Six circular RNAs with the most significantly different expressions were selected for further verification. Second, the 180 patients were included as discovery cohort to verify the six circular RNAs. Circular RNAs were extracted from serum in each participant. Logistic regression analysis was further performed to identify the predictive value and the area under the curve analysis was used to evaluate diagnostic efficiency, sensitivity, and specificity. RESULTS: Six circular RNAs including hsa_circ_0058058, hsa_circ_0008045, hsa_circ_0084789, hsa_circ_0000550, hsa_circ_0007422, and hsa_circ_0004099 showed aberrant expressions between the successful and failed sperm retrieval group. In addition, both single-circular RNA panels and multi-circular RNA panels were finally verified to be significant in predicting sperm retrieval rate. Notably, multi-circular RNAs panels demonstrated better predictive abilities compared with single-circRNA panels, and the combined panel of six-circular RNAs (risk score = 1.094×hsa_circ_0058058+0.697×hsa_circ_0008045+0.718×hsa_circ_0084789-0.591×hsa_circ_0000550-0.435×hsa_circ_0007422-1.017×hsa_circ_0004099-1.561) exhibited the best predictive ability in the present study with an AUC of 0.977, a sensitivity of 91.7% and a specificity of 86.5%. A higher risk score indicated a higher risk of failure in sperm retrieval. DISCUSSION AND CONCLUSION: Our study was the first to report that testis-derived circular RNAs in serum have the ability to predict sperm retrieval rate in non-obstructive azoospermia patients, whether it is a single-circular RNA or a combination of multi-circular RNAs.

Discovery and Characterization of a Pan-betacoronavirus S2-binding antibody.

Three coronaviruses have spilled over from animal reservoirs into the human population and caused deadly epidemics or pandemics. The continued emergence of coronaviruses highlights the need for pan-coronavirus interventions for effective pandemic preparedness. Here, using LIBRA-seq, we report a panel of 50 coronavirus antibodies isolated from human B cells. Of these antibodies, 54043-5 was shown to bind the S2 subunit of spike proteins from alpha-, beta-, and deltacoronaviruses. A cryo-EM structure of 54043-5 bound to the pre-fusion S2 subunit of the SARS-CoV-2 spike defined an epitope at the apex of S2 that is highly conserved among betacoronaviruses. Although non-neutralizing, 54043-5 induced Fc-dependent antiviral responses, including ADCC and ADCP. In murine SARS-CoV-2 challenge studies, protection against disease was observed after introduction of Leu234Ala, Leu235Ala, and Pro329Gly (LALA-PG) substitutions in the Fc region of 54043-5. Together, these data provide new insights into the protective mechanisms of non-neutralizing antibodies and define a broadly conserved epitope within the S2 subunit.

GRPR down-regulation inhibits spermatogenesis through Ca2+ mediated by PLCß/IP3R signaling pathway in long-term formaldehyde-exposed rats.

Formaldehyde (FA), which is known as an air pollutant, has been proven to induce male infertility. However, the underlying mechanism of FA-induced male infertility remains elusive. In this study, 24 male SD rats were exposed to different levels of FA (0, 0.5, 2.46, and 5 mg/m3) for eight consecutive weeks. Through HE staining and sperm smear, we observed that FA exposure resulted in spermatogenic injury and the sperm quality decreased in rats. The qRT-PCR and Western blot analysis further revealed that GRPR was down-regulated in testicular tissues of FA-exposed rats as well as primary spermatogenic cells. Meanwhile, ZDOCK uncovered an interaction between GRPR and PLCß. In addition, the CCK8, Fluo 3-AM and Flow cytometry results showed that FA exposure suppressed the expression of GRPR, PLCß and IP3R, consequently reducing the Ca2+ concentration in spermatogenic cells, inducing apoptosis and inhibiting proliferation of spermatogenic cells. Moreover, rescue experiments confirmed that promoting GRPR could improve intracellular Ca2+ concentration by upregulating PLCß and IP3R, partially reducing the apoptosis and promoting the proliferation of FA-treated spermatogenic cells. These findings revealed that GRPR participates in spermatogenesis through Ca2+ mediated by the PLCß/IP3R signaling pathway in FA-exposed rats.

Rno_circRNA_006061 participates in apoptosis induced by formaldehyde via activating p38/ATF3 pathway.

Formaldehyde, a common indoor air pollutant, is significantly toxic to the respiratory system, whereas its mechanism is unclear. CircRNAs exert critical functions via sponging microRNAs (miRNAs). To evaluate the effect of long-term formaldehyde exposure on rno_circRNA_006061 expression profiles, the downstream targets and signaling pathways associated with rno_circRNA_006061 were predicted and validated using bioinformatics methods and dual-luciferase reporter assay. Previously, our circRNA microarray showed that rno_circRNA_006061 was up-regulated in the formaldehyde-exposed lung tissue. Subsequently, bioinformatics analysis predicted that rno_circRNA_006061 bound to rno-miR-128-3p and co-regulated the p38/ATF3 signaling pathway. Meanwhile, the expressions of rno_circRNA_006061, rno-miR-128-3p and p38 were correlated with the lung histomorphopathological injury assessment. Furthermore, TUNEL and Bax/Bcl-2 ratio results revealed that up-regulated rno_circRNA_00606 induced by formaldehyde stimulated apoptosis in the lung. After the knockdown of rno_circRNA_006061, the expression of rno-miR-128-3p increased and the expression of p38 decreased slightly, which partially restored formaldehyde-induced apoptosis in alveolar epithelial cells. In conclusion, our study hinted that the rno_circRNA_006061 might enhance p38/ATF3 pathway expression via sponging the rno-miR-128-3p, thus significantly promoting apoptosis in lung tissues, which may provide potential new targets for preventing and treating lung injury by formaldehyde inhalation.

Evaluation of Pre-Pandemic Trivalent COBRA HA Vaccine in Mice Pre-Immune to Historical H1N1 and H3N2 Influenza Viruses.

Initial exposure to influenza virus(es) during early childhood produces protective antibodies that may be recalled following future exposure to subsequent viral infections or vaccinations. Most influenza vaccine research studies use immunologically naïve animal models to assess vaccine effectiveness. However, most people have an extensive influenza immune history, with memory cells produced by viruses or vaccines representing multiple influenza viruses. In this study, we explored the effect influenza seasonal virus-induced immunity has on pre-pandemic influenza virus vaccination. The mice that were pre-immune to historical H1N1 and H3N2 seasonal influenza viruses were vaccinated with adjuvanted pre-pandemic (H2, H5, and H7) HA-based computationally optimized broadly reactive antigen (COBRA) vaccines, and were fully protected from lethal challenge, whereas the mock-vaccinated mice, with or without pre-immunity, were not protected from morbidity or mortality. Detectable antibody titers were present in the pre-immune mice vaccinated with a single dose of vaccine, but not in the immunologically naïve mice. The mice vaccinated twice with the trivalent COBRA HA vaccine had similar antibody titers regardless of their pre-immune status. Overall, seasonal pre-immunity did not interfere with the immune responses elicited by pre-pandemic COBRA HA vaccines or the protection against pre-pandemic viruses.

Homeobox B9 Promotes the Progression of Hepatocellular Carcinoma via TGF-ß1/Smad and ERK1/2 Signaling Pathways.

Objectives: Homeobox B9 (HOXB9), a homeodomain-containing transcription factor, may play a role in hepatocellular carcinoma (HCC) progression. However, the exact mechanisms underlying its action remain unclear. Materials and methods. Immunohistochemistry was used to investigate the expression of HOBX9 and its prognostic values in HCC patients. HCC cells were transfected with pBabe-HOXB9 and shHOXB9 plasmids, and MTT assay, Transwell assays, and xenograft mouse models were employed to determine the effects of HOXB9 on HCC cell proliferation, migration, and invasion in vitro and in vivo. The biological mechanisms involved in the role of HOXB9 were determined with Western blot and RT-qPCR methods. Results: HOXB9 expression was significantly increased in HCC tissues and cell lines. Patients with higher HOXB9 levels were associated with poor prognosis. Overexpression of HOXB9 in BEL-7405 cells promoted proliferation, migration, and invasion, whereas knockdown of HOXB9 in HepG2 cells significantly reduced cell proliferation, migration, and invasion abilities. Mechanically, a positive correlation was found between HOXB9 expression and transforming growth factor-ß1 (TGF-ß1) and extracellular signal-regulated kinase (ERK)1/2 pathway in HCC tissues. HOXB9 overexpression stimulated TGF-ß1/Smads signaling pathway in BEL-7405 cells. In contrast, HOXB9 knockdown inhibited the TGF-ß1/Smads signaling pathway in HepG2 cells. In addition, the treatment with TGF-ß1 inhibitor, LY364947, significantly reserved HOXB9 overexpression-induced cell proliferation, migration, and invasion abilities. Conclusions: These findings validated that HOXB9 promoted proliferation, migration, and invasion in HCC cells by stimulating the TGF-ß1/Smads and ERK1/2 signaling pathway. HOXB9 could be a promising prognostic biomarker and a potential therapeutic target in HCC.

Semen Quality Following Long-term Occupational Exposure to Formaldehyde in China.

Importance: The potential effects of long-term occupational exposure to formaldehyde (FA) on human semen quality is not clear. Objective: To assess whether long-term occupational exposure to FA is associated with semen quality. Design, Setting, and Participants: This population-based cohort study was conducted from June 1 to June 30, 2021, in Xi'an, China. Participants were adults aged 23 to 40 years who had lived in the study area for 24 months or longer. Data analysis was performed from September 1 to October 1, 2021. Exposures: Long-term occupational exposure to FA was measured using a formaldehyde detector, and the FA exposure index (FEI) was calculated as follows: FEI = final concentration of FA (mg/m3) × work time during a workday (hour) × cumulative workdays (year). Main Outcomes and Measures: Semen samples were collected by masturbation after 3 to 7 days of abstinence and were then assessed by the computer-automated semen analysis system, Baso-Papanicolaou staining, and sperm-chromatin structure assay. Results: A total of 205 men (mean [SD] age, 29.49 [3.64] years), with 124 individuals in the FA exposure group (mean [SD] FEI, 73.72 [54.86]) and 81 age-matched controls, were included in the final analysis. Long-term personal occupational exposure to FA was significantly associated with poor semen quality. Specifically, a 1-unit increase in FEI was associated with a change of -0.99% (95% CI, -1.00% to -0.98%) in total sperm motility, -0.99% (95% CI, -0.99% to -0.97%) in progressive sperm motility, -0.05% (95% CI, -0.08% to -0.02%) in curvilinear velocity, -0.07% (95% CI, -0.10% to -0.04%) in straight line velocity, -0.07% (95% CI, -0.10% to -0.04%) in time-average velocity, -0.98% (95% CI, -0.99% to -0.93%) in normal sperm morphology, -0.24% (95% CI, -0.35% to -0.11%) in seminal neutral glucosidase, -0.61% (95% CI, -0.66% to -0.56%) in seminal plasma zinc, 0.52% (95% CI, 0.15% to 1.02%) in beat cross frequency, and 0.10% (95% CI, 0.06% to 0.14%) in the DNA fragmentation index. These associations remained significant after adjusting for confounding factors. Furthermore, subgroup analysis found that high levels of oxidative stress might promote the associations between FA exposure and semen quality. Conclusions and Relevance: This study found an association between long-term occupational exposure to FA and semen quality. This deterioration was dose and time dependent and might be induced by oxidative stress.

Kinetic of the Antibody Response Following AddaVax-Adjuvanted Immunization with Recombinant Influenza Antigens.

Notwithstanding the current SARS-CoV-2 pandemic, influenza virus infection still represents a global health concern in terms of hospitalizations and possible pandemic threats. The objective of next-generation influenza vaccines is not only to increase the breadth of response but also to improve the elicitation of an effective and robust immune response, especially in high-risk populations. To achieve this second objective, the administration of adjuvanted influenza vaccines has been considered. In this regard, the monitoring and characterization of the antibody response associated with the administration of adjuvanted vaccines has been evaluated in this study in order to shed light on the kinetic, magnitude and subclass usage of antibody secreting cells (ASCs) as well as of circulating antigen-specific serum antibodies. Specifically, we utilized the DBA/2J mouse model to assess the kinetic, magnitude and IgG subclass usage of the antibody response following an intramuscular (IM) or intraperitoneal (IP) immunization regimen with AddaVax-adjuvanted bivalent H1N1 and H3N2 computationally optimized broadly reactive antigen (COBRA) influenza recombinant hemagglutinins (rHAs). While the serological evaluation revealed a homogeneous kinetic of the antibody response, the detection of the ASCs through a FluoroSpot platform revealed a different magnitude, subclass usage and kinetic of the antigen-specific IgG secreting cells peaking at day 5 and day 9 following the IP and IM immunization, respectively.

Rno_circRNA_008646 regulates formaldehyde induced lung injury through Rno-miR-224 mediated FOXI1/CFTR axis.

Formaldehyde (FA) serves as a prevailing air pollutant, which has seriously threatened public health in recent years. Of all the known health effects, lung injury is one of the most severe risks. However, little is known about the circRNAs related molecular mechanism in the development of lung injury induced by FA. This study was designed to explore the potential roles of dysregulated circRNAs as well as its mechanism in FA-induced lung injury. In the present study, 24 male SD rats were exposed to formaldehyde (control, 0.5, 2.46 and 5 mg/m3) for 8 h per day for 8 weeks to induce lung injury. We used H&E staining to evaluate the histopathological changes of lung injury indifferent groups. The expression of circRNAs in lung tissue was detected by real-time PCR. Meanwhile, circRNA/miRNA/mRNA interaction networks were predicted by bioinformatics analysis. Our study revealed that formaldehyde exposure resulted in abnormal histopathological changes in lung tissues. Moreover, the expression of rno_circRNA_008646 was significantly higher in lung tissues of formaldehyde exposure rats than in control. Bioinformatics analysis showed that one potential target miRNA/mRNA for rno_circRNA_008646 was rno-miR-224/Forkhead Box I1 (FOXI1). Besides, luciferase report gene confirmed that there was targeted binding relationship between rno_circRNA_008646 and rno-miR-224, rno-miR-224 and FOXI1. Further verification experiments indicated that the expression of rno_circRNA_008646 was negatively correlated rno-miR-224, while it was positively correlated with FOXI1. JASPAR database showed transcription factor FOXI1 located in promotor of CF Transmembrane Conductance Regulator (CFTR). Both FOXI1 and CFTR were up-regulated in lung tissues after formaldehyde exposure. In conclusion, our findings suggested that formaldehyde may induce lung injury, and this may be caused by up-regulatedrno_circRNA_008646, which medicated rno-miR-224/FOXI1/CFTR axis.

Novel H7N9 influenza immunogen design enhances mobilization of seasonal influenza T cell memory in H3N2 pre-immune mice.

Strategies that improve influenza vaccine immunogenicity are critical for the development of vaccines for pandemic preparedness. Hemagglutinin (HA)-specific CD4+ T cell epitopes support protective B cell responses against seasonal influenza. However, in the case of avian H7N9, which poses a pandemic threat, HA elicits only weak neutralizing antibody responses in infection and vaccination without adjuvant. We hypothesized that an immune-engineered H7N9 HA incorporating a broadly reactive H3N2 HA-specific memory CD4+ T cell epitope that replaces a regulatory T cell-inducing epitope at the corresponding position in H7N9 HA could harness preexisting influenza T cell immunity to increase CD4+ T cells that are needed for protective antibody development. We designed and produced a virus-like particle (VLP) vaccine that carries the epitope augmented H7N9 HA (OPT1) and immunized HLA-DR3 transgenic mice with established H3N2 immunity. OPT1-VLPs stimulated higher stem cell, central, and effector memory CD4+ T cell levels over wild type VLP immunization. In addition, activated, IL-21-producing follicular helper T cell frequencies were enhanced. This novel immunogen design strategy illustrates that site-specific modifications aimed to augment T cell epitope content enhance CD4+ T cell responses among critical subpopulations capable of aiding protective immune responses upon antigen re-encounter and that mobilization of immune memory can be used to overcome the poor immunogenicity of avian influenza viruses.

Increasing prevalence of myopia and the impact of education in primary-school students in Xi'an, north-western of China.

Purpose: The present study was performed to detect the prevalence of myopia among primary-school students in Xi'an, north-western of China. Methods: The present study was a school-based study with students aged from 6 to 13 years old. All the individuals underwent ophthalmological examination and spherical equivalent (SE) of refractive error were measured with non-cycloplegic refraction. Myopia was defined as a SE of ≤ -0.5 diopters (D), and further divided into three stratified groups based on SE: low myopia (≤ -0.5 to >-3.0 D), moderate myopia (≤ -3.0 to >-6.0 D), and high myopia (≤ -6.0 D). Relative risk factors, including age, sex, grade and ethnicity were investigated using questionnaire. Results: A total of 4,680 individuals were eligible for this survey and 4,654 (99.4% participation rate) were finally included (51.2% boys). The mean age of participants was 8.756 ± 1.727 years. The whole city-level prevalence of total myopia was 57.1% (95% CI: 55.7-58.6%). Additionally, the prevalence of low, moderate, and high myopia was 45.0% (95% CI: 43.5-46.4%), 11.1% (95% CI: 10.2-12.0%), and 1.0% (95% CI: 0.7-1.3%), respectively. Moreover, grade (education level) instead of age, sex and ethnicity was the most essential risk factor for prevalence of overall myopia (OR = 1.844, 95% CI: 1.605-2.119), and an increase of prevalence by 84.4% per grade was seen. Furthermore, similar associations of grade were significant with low myopia (OR = 1.613, 95% CI: 1.385-1.877) and moderate myopia (OR = 2.186, 95% CI: 1.693-2.823), meanwhile, prevalence of low myopia and moderate myopia demonstrated an increase of prevalence by 61.3 and 118.6% per grade, respectively. None of the factors included in the present study was significant risk factor for high myopia. Conclusions: The present study investigated a non-negligible high prevalence of myopia among primary-school students in Xi'an, north-western of China, and a gradual increasing in proportion with education level.

Circular RNA-CDR1as is involved in lung injury induced by long-term formaldehyde inhalation.

OBJECTIVE: Formaldehyde (FA) is known to induce lung injury, but the underlying molecular mechanism remains largely unclear. CDR1as is an important member of the circular RNAs (circRNAs) family and functions as miRNA sponges with gene-regulatory potential. Our earlier circRNA microarray data showed CDR1as was highly expressed in lung tissue exposed to FA. However, the mechanism of circRNA-CDR1as mediates the FA-exposed lung injury is still unclear. This study aimed to explore the role of CDR1as in lung injury. MATERIALS AND METHODS: In this study, FA was inhaled at doses of 0.5, 2.46, and 5 mg/m3, respectively. After exposure 8 weeks, lung histopathological examination, lung injury score, and IL-1ß in bronchoalveolar lavage fluid (BALF) were determined. The expressions of CDR1as, rno-miR-7b and Atg7 were detected and the potential interaction of circRNA/miRNA/mRNA was predicted by bioinformatics analysis, including drawing circRNA/miRNA/mRNA interaction network, GO and KEGG analysis. RESULTS: Our results indicated FA inhalation upregulated the expression of CDR1as in lung tissues in a dose-dependent manner while the expression of rno-miR-7b decreased and Atg7 increased. Moreover, the alteration of CDR1as was positively correlated with lung injury. DISCUSSION AND CONCLUSIONS: CircRNA/miRNA/mRNA prediction further explained the possible effect mechanisms of CDR1as. These data implicated that CDR1as might be a critical regulator involved in lung injury induced by FA.

Circular RNA FOXO3 accelerates glycolysis and improves cisplatin sensitivity in lung cancer cells via the miR-543/Foxo3 axis.

Non-small cell lung cancer (NSCLC) is the most common cause of cancer-associated mortality worldwide. Our previous study revealed that circular RNA (circRNA)-FOXO3 is highly expressed in lung cancer and inhibits cell proliferation. However, to the best of our knowledge, at present, no study has focused on the specific mechanism of circRNA-FOXO3 in drug resistance. Therefore, the present study aimed to provide novel perspectives on the role of circRNA-FOXO3 in cisplatin (DDP) resistance in NSCLC. A Cell Counting Kit-8 assay was used to determine the viability of cells overexpressed with circRNA-FOXO3 and under DDP treatment. Glycolysis was analyzed by measuring glucose consumption and lactate production. The interaction of circRNA-FOXO3, microRNA 543 (miR-543) and Foxo3 was confirmed using a dual-luciferase reporter assay. It was revealed that circRNA-FOXO3 improved cell sensitivity to DDP and repressed glycolysis in DDP-sensitive and DDP-resistant NSCLC cells. Bioinformatics analysis, luciferase reporter assays, quantitative PCR and RNA pull-down assays were employed to verify the binding of circRNA-FOXO3 to miR-543. Functionally, inhibition of miR-543 could sensitize NSCLC cells to DDP, and overexpression of miR-543 at least partially abolished the circRNA-FOXO3-induced decrease in chemoresistance. Furthermore, it was revealed that Foxo3 was a direct target of miR-543. Notably, the inhibitory action of miR-543 silencing on DDP resistance and glycolysis was reversed by overexpression of Foxo3 in DDP-sensitive and DDP-resistant NSCLC cells. In conclusion, the present study demonstrated that circRNA-FOXO3 promoted DDP sensitivity in NSCLC cells by regulating the miR-543/Foxo3 axis-mediated glycolysis balance. The present findings may provide novel perspectives for the treatment of patients with NSCLC resistant to DDP.

The expression of Beclin-1 in testicular tissues of non-obstructive azoospermia patients and its predictive value in sperm retrieval rate.

BACKGROUND: Beclin-1 is an autophagy gene and higher levels suggest mammalian testicular damage. Our study aims at exploring the role of Beclin-1 in non-obstructive azoospermia (NOA) patients and clarifying the predictive value of Beclin-1for sperm retrieval in microdissection testicular sperm extraction (micro-TESE). METHODS: In the present study, 62 NOA patients were finally recruited. Serum hormone including luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol II (E2), testosterone (T) and prolactin (PRL), as well as testicular volume were measured. Testicular histopathology was diagnosed by two independent pathologists. The expression of Beclin-1 was detected by real-time PCR in testicular tissue. RESULTS: Our study illustrated that Beclin-1 was differently expressed in three pathological types of NOA. Compared with hypospermatogenesis (HS, P=0.002) or maturation arrest (MA, P=0.049), Beclin-1 showed significantly up-regulated in Sertoli cell-only syndrome (SCOS) group. Moreover, Beclin-1 expression was obviously positive related with serum LH (rho =0.269, P=0.036), meanwhile significantly negative correlation with testicular volume (rho =-0.370, P=0.003), serum T (rho =-0.326, P=0.010), Johnsen score (rho =-0.318, P=0.012), and pathologic type (rho =-0.452, P<0.001). Furthermore, a logistic regression model demonstrated that Beclin-1 is an important predictor of failed sperm retrieval (OR =0.001, P=0.007), which exhibited a pretty AUC =78.6 (P=0.001). CONCLUSIONS: Beclin-1 may play a critical role in spermatogenesis. Elevated Beclin-1 may be obviously associated with lower chances of positive sperm retrieval.

Use of Rv0222-Rv2657c-Rv1509 Fusion Protein to Improve the Accuracy of an Antibody ELISA for Extra-Pulmonary Tuberculosis in Humans.

(1) Background: Tuberculosis (TB) in humans is a serious chronic epidemic disease caused by Mycobacterium tuberculosis (M. tb). The diagnosis of TB, especially extra-pulmonary TB (EPTB), is difficult. Isolation of M. tb from culture has a low sensitivity in patients with TB and an even lower sensitivity in cases of EPTB. Although Xpert MTB/RIF assays and serological tests are more sensitive than the above tests, they still lack sensitivity for EPTB diagnosis. (2) Methods: To improve the accuracy of TB diagnosis, a Rv0222-Rv2657c-Rv1509 fusion protein based iELISA was constructed, the diagnosis of TB, pulmonary TB (PTB) and EPTB was then evaluated. Sera of 40 TB patients including 14 with PTB, 14 with EPTB and 12 with no information about the form of TB, and five pneumonia patients were investigated. (3) Results: The sensitivity of the ELISA in TB, PTB and EPTB patients was 80% (95% CI: 64.4, 90.9%), 85.7% (95% CI: 57.2, 98.2%) and 92.8% (95% CI: 66.1, 99.8%), respectively, with a specificity of 70% (95% CI: 53.5, 83.4%). Both the sensitivity and specificity with this fusion protein were higher than for CFP10/ESAT6 (used as reference antigen) fusion protein (71.4%; 95% CI: 41.9, 91.6%, and 67.5%; 95% CI: 50.9, 81.4%), respectively, in cases of EPTB. All pneumonia patients' sera tested negative in both ELISAs. (4) Conclusion: use of these new fusion proteins as antigens in serological assays has the potential to improve the diagnosis of all forms of TB in humans, especially EPTB.

Rno_circRNA_016194 might be involved in the testicular injury induced by long-term formaldehyde exposure via rno-miR-449a-5p mediated Atg4b activation.

Although circular RNAs (circRNAs) can function as microRNAs (miRNAs) sponges to participate in spermatogenesis, little is known about the functions of circRNAs in testis exposed to formaldehyde. In this study, twenty-four male SD rats (6-8 weeks) were randomly assigned to four groups, including a control group, 0.5, 2.46, and 5 mg/m3 formaldehyde exposure groups, inhaling formaldehyde for eight consecutive weeks. The RT-qPCR was used to detect the expression of rno_circRNA_016194; the testicular injuries were observed by testicular histopathology. Our study illustrated up-regulated rno_circRNA_016194 was dose-dependent with formaldehyde. Simultaneously, the testicular histopathology showed an obvious damages in the 2.46 and 5 mg/m3 formaldehyde exposure rats. Combined with bioinformatics analysis, the rno-miR-449a-5p was predicted and verified that its expression decreased in the testis exposed to formaldehyde. Meanwhile, the testicular morphometry changes were contrary to the expression of rno_circRNA_016194 and consistent with rno-miR-449a-5p. Moreover, bioinformatics analysis also prompted the potential downstream target gene for rno_circRNA_016194/rno-miR-449a-5p was Atg4b, and Atg4b expression was up-regulated in rats exposed to formaldehyde verifying by Western blot. Collectively, the rno_circRNA_016194 might be involved in formaldehyde-induced male reproductive toxicity and become potential therapeutic targets for male occupational exposure to formaldehyde.