Synthesis and anti-HIV activity of non-nucleoside reverse-transcriptase inhibitor DB02 phosphate derivatives based on water-soluble optimization.

To improve the water solubility of anti-human immunodeficiency virus (HIV) agent DB02, an excellent non-nucleoside reverse-transcriptase inhibitor (NNRTI) obtained in our previous efforts, we designed and synthesized four phosphate derivatives of DB02 based on the molecular model of DB02 with RT. Here, the antiviral activity of these four derivatives was detected, leading to the discovery of compound P-2, which possessed a superior potency to the lead compound DB02 against wild-type HIV-1 and a variety of HIV-resistant mutant viruses significantly. Furthermore, the water solubility of P-2 was nearly 17 times higher than that of DB02, and the pharmacokinetic test in rats showed that P-2 demonstrate significantly improved oral bioavailablity of 14.6%. Our study showed that the introduction of a phosphate ester group at the end of the C-2 side chain of DB02 was beneficial to the improvement of its antiviral activity and pharmacokinetic properties, which provided a promising lead for the further development of S-DACOs type of NNRTIs.

Development of a predictive model for increasing sperm retrieval success by microdissection testicular sperm extraction in patients with nonobstructive azoospermia.

Microdissection testicular sperm extraction (micro-TESE) is widely used to treat nonobstructive azoospermia. However, a good prediction model is required to anticipate a successful sperm retrieval rate before performing micro-TESE. This retrospective study analyzed the clinical records of 200 nonobstructive azoospermia patients between January 2021 and December 2021. The backward method was used to perform binary logistic regression analysis and identify factors that predicted a successful micro-TESE sperm retrieval. The prediction model was constructed using acquired regression coefficients, and its predictive performance was assessed using the receiver operating characteristic curve. In all, 67 patients (sperm retrieval rate: 33.5%) underwent successful micro-TESE. Follicle-stimulating hormone, anti-Müllerian hormone, and inhibin B levels varied significantly between patients who underwent successful and unsuccessful micro-TESE. Binary logistic regression analysis yielded the following six predictors: anti-Müllerian hormone (odds ratio [OR] = 0.902, 95% confidence interval [CI]: 0.821-0.990), inhibin B (OR = 1.012, 95% CI: 1.001-1.024), Klinefelter's syndrome (OR = 0.022, 95% CI: 0.002-0.243), Y chromosome microdeletion (OR = 0.050, 95% CI: 0.005-0.504), cryptorchidism with orchiopexy (OR = 0.085, 95% CI: 0.008-0.929), and idiopathic nonobstructive azoospermia (OR = 0.031, 95% CI: 0.003-0.277). The prediction model had an area under the curve of 0.720 (95% CI: 0.645-0.794), sensitivity of 65.7%, specificity of 72.2%, Youden index of 0.379, and cut-off value of 0.305 overall, indicating good predictive value and accuracy. This model can assist clinicians and nonobstructive azoospermia patients in decision-making and avoiding negative micro-TESE results.

Psychological status of infertile men during the Coronavirus Disease 2019 Pandemic in China: a cross-sectional investigation.

BACKGROUND: The coronavirus disease 2019 (COVID-19) outbreak has had a widespread and profound impact on people's mental health. The factors associated with mental symptoms among men diagnosed with infertility, a disease closely related to psychological conditions, remain unclear. The aim of this study is to investigate the risk factors associated with mental symptoms among infertile Chinese men during the pandemic. RESULTS: A total of 4,098 eligible participants were recruited in this cross-sectional, nationwide study, including 2,034 (49.6%) with primary infertility and 2,064 (50.4%) with secondary infertility. The prevalence of mental health conditions was 36.3%, 39.6%, and 6.7% for anxiety, depression, and post-pandemic stress, respectively. Sexual dysfunction is associated with a higher risk with adjusted odds ratios (ORs) of 1.40 for anxiety, 1.38 for depression, and 2.32 for stress. Men receiving infertility drug therapy displayed a higher risk for anxiety (adjusted OR, 1.31) and depression (adjusted OR, 1.28) symptoms, while those receiving intrauterine insemination had a lower risk of anxiety (adjusted OR, 0.56) and depression (adjusted OR, 0.55) symptoms. CONCLUSION: The COVID-19 pandemic has had a significant psychological impact on infertile men. Several psychologically vulnerable populations were identified, including individuals with sexual dysfunction, respondents receiving infertility drug therapy, and those experiencing control measures for COVID-19. The findings provide a comprehensive profile of the mental health status of infertile Chinese men during the COVID-19 outbreak and provide potential psychological intervention strategies.

RéSUMé: CONTEXTE: L'épidémie de maladie à coronavirus 2019 (COVID-19) a eu un impact étendu et profond sur la santé mentale des gens. Les facteurs associés aux symptômes mentaux chez les hommes diagnostiqués comme infertiles, une maladie étroitement liée aux conditions psychologiques, restent flous. L'objectif de cette étude est d'étudier les facteurs de risque associés aux symptômes mentaux chez les hommes chinois infertiles pendant la pandémie. RéSULTATS: Au total, 4 098 participants admissibles ont été recrutés dans cette étude transversale à l'échelle nationale, dont 2 034 (49,6%) présentaient une infertilité primaire et 2 064 (50,4%) une infertilité secondaire. La prévalence des problèmes de santé mentale était respectivement de 36,3 %, 39,6 % et 6,7 % pour l'anxiété, la dépression, et le stress postpandémique. La dysfonction sexuelle est associée à un risque plus élevé avec des odds ratios ajustés (OR) de 1,40 pour l'anxiété, 1,38 pour la dépression et 2,32 pour le stress. Les hommes recevant un traitement médicamenteux contre l'infertilité présentaient un risque plus élevé de symptômes d'anxiété (OR ajusté, 1,31) et de dépression (OR ajusté, 1,28), alors que ceux dont le traitement consistait à faire des inséminations intra-utérines présentaient un risque plus faible de symptômes d'anxiété (OR ajusté, 0,56) et de dépression (OR ajusté, 0,55). CONCLUSIONS: La pandémie de COVID-19 a eu un impact psychologique important sur les hommes infertiles. Plusieurs populations psychologiquement vulnérables ont été identifiées, notamment les personnes souffrant de dysfonction sexuelle, les hommes recevant un traitement médicamenteux contre l'infertilité, et ceux subissant des mesures de contrôle de la COVID-19. Les résultats fournissent un profil complet de l'état de santé mentale des hommes Chinois infertiles pendant l'épidémie de COVID-19 et fournissent des stratégies potentielles d'intervention psychologique.

Identification of 6ω-cyclohexyl-2-(phenylamino carbonylmethylthio)pyrimidin-4(3H)-ones targeting the ZIKV NS5 RNA dependent RNA polymerase.

Zika virus (ZIKV), a mosquito-borne flavivirus, is a global health concern because of its association with severe neurological disorders such as neonatal microcephaly and adult Guillain-Barre syndrome. Although many efforts have been made to combat ZIKV infection, there is currently no approved vaccines or antiviral drugs available and there is an urgent need to develop effective anti-ZIKV agents. In this study, 26 acetylarylamine-S-DACOs derivatives were prepared, and eight of them were found to have inhibitory activity against Zika virus. Among these substances, 2-[(4-cyclohexyl-5-ethyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(3,5-difluorophenyl)acetamide (4w) with the best anti-ZIKV activity was selected for in-depth study of antiviral activity and mechanism of action. Here, we discovered 4w targeted on the ZIKV NS5 RNA -dependent RNA polymerase (RdRp), which exhibited good in vitro antiviral activity without cell species specificity, both at the protein level and at the RNA level can significantly inhibit ZIKV replication. Preliminary molecular docking studies showed that 4w preferentially binds to the palm region of NS5A RdRp through hydrogen bonding with residues such as LYS468, PHE466, GLU465, and GLY467. ZIKV NS5 RdRp enzyme activity experiment showed that 4w could directly inhibit ZIKV RdRp activity with EC50 = 11.38 ± 0.51 µM. In antiviral activity studies, 4w was found to inhibit ZIKV RNA replication with EC50 = 6.87 ± 1.21 µM. ZIKV-induced plaque formation was inhibited with EC50 = 7.65 ± 0.31 µM. In conclusion, our study disclosed that acetylarylamine-S-DACOs is a new active scaffolds against ZIKV, among which compound 4w was proved to be a potent novel anti-ZIKV compound target ZIKV RdRp protein. These promising results provide a future prospective for the development of ZIKV RdRp inhibitors.

Theoretical insights into the gaseous and heterogeneous reactions of halogenated phenols with ˙OH radicals: mechanism, kinetics and role of (TiO2)n clusters in degradation processes.

Halogenated phenols are highly toxic chemicals with serious health risks, and the removal of these persistent environmental pollutants remains a challenge. Based on quantum chemistry calculations, the homogeneous/heterogeneous degradation mechanism and kinetics of C6X5OH (X = F, Cl, and Br) initiated by ˙OH radicals in the gas phase and TiO2 cluster surfaces are investigated in this work. Four ˙OH-addition and one proton-coupled electron-transfer (PCET) reaction channels for each halogenated phenol were found and the ˙OH-addition channels were more favorable than the PCET pathway without TiO2 clusters. At 296 K, the calculated total rate constant for ˙OH with C6F5OH in the atmosphere well agreed with the limited experimental data of (6.88 ± 1.37) × 10-12 cm3 molecule-1 s-1. The lifetimes of C6F5OH, C6Cl5OH, and C6Br5OH were about 12.04-12.86 h at 296 K, which favored their medium-range transport in the atmosphere. In the presence of (TiO2)n clusters (n = 4, 6, 8, 12, and 16), the PCET mechanism for hydrogen transfer reaction of C6F5OH with ˙OH radicals was changed from the previous four-electron/three-center into four-electron/two-center, which results in the PCET pathway becoming more favorable than the ˙OH-addition channels. Meanwhile, the heterogeneous degradation rate constants of C6F5OH were accelerated by more than 10 orders of magnitude within 200-430 K compared with those of the naked reaction. The effects of (TiO2)n cluster (n = 4, 6, 8, 12, and 16) size on the degradation rates were analyzed at 200-430 K, and the reaction on the (TiO2)8 cluster had a faster rate. The subsequent reactions including the bond cleavage of the benzene ring and O2 addition or abstraction were studied. This work provides new insights into halogenated aromatic atmospheric chemistry and nanoscale TiO2 photocatalysis in air or wastewater management.

Identification and characterization of circular RNAs in the testicular tissue of patients with non-obstructive azoospermia.

Circular RNAs (circRNAs) are highly conserved and ubiquitously expressed noncoding RNAs that participate in multiple reproduction-related diseases. However, the expression pattern and potential functions of circRNAs in the testes of patients with non-obstructive azoospermia (NOA) remain elusive. In this study, according to a circRNA array, a total of 37 881 circRNAs were identified that were differentially expressed in the testes of NOA patients compared with normal controls, including 19 874 upregulated circRNAs and 18 007 downregulated circRNAs. Using quantitative real-time polymerase chain reaction (qRT-PCR) analysis, we confirmed that the change tendency of some specific circRNAs, including hsa_circ_0137890, hsa_circ_0136298, and hsa_circ_0007273, was consistent with the microarray data in another larger sample. The structures and characteristics of these circRNAs were confirmed by Sanger sequencing, and fluorescence in situ hybridization revealed that these circRNAs were primarily expressed in the cytoplasm. Bioinformatics analysis was used to construct the competing endogenous RNA (ceRNA) network, and numerous miRNAs that could be paired with circRNAs validated in this study were reported to be vital for spermatogenesis regulation. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses indicated that genes involved in axoneme assembly, microtubule-based processes, and cell proliferation were significantly enriched. Our data suggest that there are aberrantly expressed circRNA profiles in patients with NOA and that these circRNAs may help identify key diagnostic and therapeutic molecular biomarkers for NOA patients.

Altered microRNA profiles of testicular biopsies from patients with nonobstructive azoospermia.

Many studies have shown that microRNAs (miRNAs) play vital roles during the spermatogenesis. However, little is known about the altered miRNA profiles of testicular tissues in nonobstructive azoospermia (NOA). Using microarray technology, the miRNA expression profiles of testicular biopsies from patients with NOA and of normal testicular tissues were determined. Bioinformatics analyses were conducted to predict the enriched biological processes and functions of identified miRNAs. The microarray data were validated by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), the results of which were then validated with a larger sample size. Correlations between the miRNA expression levels and clinical characteristics were analyzed. Receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic ability of miRNAs for azoospermia. Hierarchical clustering showed that 129 miRNAs were significantly differentially expressed between the NOA and control groups. Bioinformatics analysis indicated that the differentially expressed miRNAs were involved in spermatogenesis, cell cycle, and mitotic prometaphase. In the subsequent qRT-PCR assays, the selected miRNA expression levels were consistent with the microarray results, and similar validated results were obtained with a larger sample size. Some clinical characteristics were significantly associated with the expression of certain miRNAs. In particular, we identified a combination of two miRNAs (miR-10b-3p and miR-34b-5p) that could serve as a predictive biomarker of azoospermia. This study provides altered miRNA profiles of testicular biopsies from NOA patients and examines the roles of miRNAs in spermatogenesis. These profiles may be useful for predicting and diagnosing the presence of testicular sperm in individuals with azoospermia.

Nanotechnology-assisted adipose-derived stem cell (ADSC) therapy for erectile dysfunction of cavernous nerve injury: In vivo cell tracking, optimized injection dosage, and functional evaluation.

Stem cell therapy is a potentially promising option for erectile dysfunction; however, its risk of tumorigenicity is a clinical hurdle and the risk is positively related to the number of injected cells. Our previous study showed that nanotechnology improved adipose-derived stem cell (ADSC) therapy for erectile dysfunction of cavernous nerve injury (CNI) by attracting cells in the corpus cavernosum. These results indicated the possibility of using a reduced dosage of ADSCs for intracavernous injection. In this exploratory study, we used lower dosage (2 × 105 cells) of ADSCs for intracavernous injection (ICI) and the nanotechnology approach. Intracavernous pressure and mean arterial pressure were measured at day 28 to assess erectile function. The low-dose ADSC therapy group showed favorable treatment effects, and nanotechnology further improved these effects. In vivo imaging of ICI cells revealed that the fluorescein signals of NanoShuttle-bound ADSCs (NanoADSCs) were much stronger than those of ADSCs at days 0, 1, and 3. Both immunofluorescence and Western blot analysis showed a significant increase in smooth muscle, endothelium, and nerve tissue in the ADSC group compared to that in the CNI group; further improvement was achieved with assisted nanotechnology. These findings demonstrate that nanotechnology can be used to further improve the effect of small dosage of ADSCs to improve erectile function. Abundant NanoADSCs remain in the corpus cavernosum in vivo for at least 3 days. The mechanism of erectile function improvement may be related to the regeneration of the smooth muscle, endothelium, and nerve tissues.

A clinical trial on the consistency of bilateral testicular tissue histopathology and Johnsen score: single side or bilateral side biopsy?

To evaluate and compare left and right testicular tissue histopathology and Johnsen score, and to investigate the necessity for bilateral testicular biopsy. We recruited180 patients with non-obstructiveazoospermia (NOA) on testicular biopsy who had undergonetesticular sperm aspiration (TESA). Pathological sections of testicular tissue were diagnosed by specially-assigned doctors, who evaluated pathological findings, determined the Johnsen score and confirmed for the presence or absence of sperm. Sperm positive rates for left and right testicular histopathology were 55.0% and 51.7% respectively, and the proportion of Johnsen scores≥8 for left and right testes were 53.3% and 50.0%, respectively. Cohen kappa values revealed that the identification of sperm in bilateral testicular samples was not consistent and was related to random effects; Optimized cut-off value for bilateral testicular volume was 11ml (Johnsen score ≥8), and optimized cut-off values of E2 on left and right testes were 144.5pmol/L and 133.5 pmol/L (Johnsen score≤7). However, age, serum prolactin (PRL), follicle stimulating hormone (FSH), luteinizing hormone (LH) and total testosterone (TT) levels were not accurate predictors for the existence of testicular sperm. There was nostatistical significance between left and right testicular histopathology in terms of sperm positive rates or Johnsen score; the Johnsen score were caused entirely by random effects and a score from one side could not represent the other side. Therefore, we recommend that both testes need to undergo surgery when NOA patients undergo testicular biopsy or sperm retrieval.

Ran-binding protein M is associated with human spermatogenesis and oogenesis.

The aim of the present study was to explore the underlying mechanism and diagnostic potential of Ran­binding protein M (RanBPM) in human spermatogenesis and oogenesis. RanBPM expression in human testis and ovaries was analysed using polymerase chain reaction (PCR) and western blotting, and immunofluorescence was performed on testis and ovary tissue sections during different developmental stages of spermatogenesis and oogenesis using RanBPM antibodies. Interactions with a variety of functional proteins were also investigated. RanBPM mRNA and protein expression levels were determined by PCR and western blotting in the tissue sections. Results revealed that the mRNA expression levels were highest in the testis followed by the ovary. The RanBPM protein was predominantly localized in the nucleus of germ cells, and the expression levels were highest in pachytene spermatocytes and cells surrounding spermatids in testis tissue. In ovary cells, RanBPM was localized in the nucleus and cytoplasm. In conclusion, the results suggested that RanBPM may have multiple roles in the regulation of germ cell proliferation during human spermatogenesis and oogenesis. This research may provide a novel insight into the underlying molecular mechanism of RanBPM and may have implications for the clinical diagnosis and treatment of human infertility.

[Microdissection testicular sperm extraction for patients with nonmosaic Klinefelter syndrome: A report of 143 cases].

OBJECTIVE: To investigate the clinical application and outcomes of microdissection testicular sperm extraction (micro-TESE) in patients with nonmosaic Klinefelter syndrome (KS). METHODS: A total of 143 nonmosaic KS patients underwent micro-TESE in the Center of Reproductive Medicine of Peking University Third Hospital between July 2012 and August 2016. We analyzed their clinical and follow-up data and evaluated the outcomes. RESULTS: Spermatozoa were successfully retrieved from the testicular tissue in 44.76% (64/143) of the patients, 84.4% (54/64) by unilateral and 15.6% (10/64) by bilateral micro-TESE. Seventy-five of the KS patients were followed up in the years of 2014 and 2015. Of the 34 patients with successful sperm retrieval, 73.52% (25/34) achieved clinical pregnancy and 8 boys and 8 girls were already born in 14 of the 25 cases. CONCLUSIONS: The micro-TESE is a useful method for sperm retrieval in nonmosaic KS patients, with high rates of sperm retrieval, clinical pregnancy, and birth of biological offspring.

[Impact of the CFTR chloride channel on the cytoskeleton of mouse Sertoli cells].

OBJECTIVE: To study the impact of the chloride channel dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) on the cytoskeleton of Sertoli cells in the mouse. METHODS: TM4 Sertoli cells were cultured and treated with CFTR(inh)-172 at the concentrations of 1, 5, 10 and 20 µmol/L for 48 hours. Then the cytotoxicity of CFT(inh)-172 was assessed by CCK-8 assay, the expressions of F-actin and Ac-tub in the TM4 Sertoli cells detected by immunofluorescence assay, and those of N-cadherin, vimentin and vinculin determined by qPCR. RESULTS: CFTR(inh)-172 produced cytotoxicity to the TM4 Sertoli cells at the concentration of 20 µmol/L. The expressions of F-actin and Ac-tub were decreased gradually in the TM4 Sertoli cells with the prolonging of treatment time and increasing concentration of CFTR(inh)-172 (P < 0.05). The results of qPCR showed that different concentrations of CFTR(inh)-172 worked no significant influence on the mRNA expressions of N-cadherin, vimentin and vinculin in the Sertoli cells. CONCLUSION: The CFTR chloride channel plays an important role in maintaining the normal cytoskeleton of Sertoli cells. The reduced function and expression of the CFTR chloride channel may affect the function of Sertoli cells and consequently spermatogenesis of the testis.

ABC transporters coupled with the elevated ergosterol contents contribute to the azole resistance and amphotericin B susceptibility.

Most screening approaches produce compounds that target survival genes and are likely to generate resistance over time. Simply having more drugs does not address the potential emergence of resistance caused by target mutation, drug efflux pumps over-expression, and so on. There is a great need to explore new strategies to treat fungal infections caused by drug-resistant pathogens. In this study, we found that azole-resistant Candida albicans with CaCDR1 and CaCDR2 over-expression is hypersensitive against amphotericin B (AmB) by our high throughput synergy screening (HTSS). In contrast, Δcdr1 and Δcdr2 knockout strains were resistant to AmB. Moreover, clinical isolates with increased expression of CaCDR1 and CaCDR2 demonstrated susceptibility to AmB, which can also synergize with the efflux pumps inducer fluphenazine (FPZ). Finally, the increased drug susceptibility to AmB in azole-resistant C. albicans with drug efflux pumps over-expression was consistent with the elevated expression of CaERG11 and its associated ergosterols in clinical isolates. Our data implies that the level of ergosterol contents determines the susceptibility to azoles and AmB in C. albicans. Deep understanding of the above mechanisms would offer new hope to treat drug-resistant C. albicans.