Design and synthesis of novel macrolones bridged with linkers from 11,12-positions of macrolides.

Resistance to telithromycin and off-target effects associated with the metabolic instability present serious and challenging problems for the development of novel macrolides. Herein, studies of hybrids of macrolides and quinolones (termed macrolones) bridged with linkers from 11,12-cyclic carbamate of macrolides revealed different structure-activity relationships from the previously reported macrolones bridged with linkers derived from 6-, 9- and 4''-positions of macrolides. The optimized macrolone 34 g with a longer and rigid sidechain than telithromycin had improved metabolic stability compared to telithromycin (t1/2: 110 vs 32 min), whose future has been heavily clouded by metabolic issues. Moreover, 34 g was 38-fold more potent than telithromycin against A2058/2059-mutated Mycoplasma pneumoniae (8 vs 315 µM), which may be attributed to a novel mode of action between the carboxylic acid of quinolone moiety and the bacterial ribosome. This work increases the prospect for discovery of novel and safe antibacterial agents to combat serious human infectious diseases.

Testing the dimensional structure of DSM-5 posttraumatic stress disorder symptoms in a nonclinical trauma-exposed adolescent sample.

BACKGROUND: The current study investigated the underlying dimensionality of DSM-5 posttraumatic stress disorder (PTSD) symptoms in a trauma-exposed Chinese adolescent sample using a confirmatory factor analytic (CFA) alternative model approach. METHODS: The sample consisted of 559 students (242 females and 314 males) ranging in age from 12 to 18 years (M = 15.8, SD = 1.3). Participants completed the PTSD Checklist for DSM-5, the Major Depression Disorder and Panic Disorder subscales of the Revised Children's Anxiety and Depression Scale, and the Aggressive Behavior subscale of the Youth Self-Report. RESULTS: Confirmatory factor analytic results indicated that a seven-factor model comprised of intrusion, avoidance, negative affect, anhedonia, externalizing behavior, anxious arousal, and dysphoric arousal factors emerged as the best-fitting model. Further analyses showed that the external measures of psychopathological variables including major depressive disorder, panic disorder, and aggressive behavior were differentially associated with the resultant factors. CONCLUSIONS: These findings support and extend previous findings for the newly refined seven-factor hybrid model, and carry clinical and research implications for trauma-related psychopathology.

Clinical features and "early" corticosteroid treatment outcome of pediatric mycoplasma pneumoniae pneumonia.

Background: Many children with mycoplasma pneumoniae (MP) pneumonia (MPP) developed sequelae such as bronchiolitis/bronchitis obliterans (BO). Early corticosteroid therapy might prevent disease progression. This study aimed to use "early" corticosteroid and observe the treatment outcome in patients with MPP. Methods: Patients who had pulmonary infiltrations on chest imaging within 5 days of the disease course and were suspected of having MP infection on admission were enrolled. Among them, patients whose disease course was within 10 days on admission were ultimately enrolled. We analyzed their data including the clinical features, the starting time and dose of corticosteroid therapy, and the treatment outcome. According to chest imaging, we divided patients into two groups (Group A: bronchiolitis-associated lesions or ground-glass opacities; Group B: pulmonary segmental/lobar consolidation). Results: A total of 210 patients with confirmed MPP were ultimately enrolled. There were 59 patients in Group A and 151 patients in Group B. Patients in Group A were more prone to have allergy histories, hypoxemia, wheezing sound, and wet rales on auscultation than those in Group B. Corticosteroid treatment was initiated between 5 and 10 days of disease onset in all patients and 6-7 days in most patients. Methylprednisolone was prescribed in all patients within 10 days of disease onset, and the highest prescribed dose was at least 2 mg/kg/day. In Group A, methylprednisolone >2 mg/kg/day was prescribed in 22 patients, and among them, 8 patients with diffuse bronchiolitis-associated lesions received high-dose methylprednisolone therapy. After 3 months, lung CT revealed slightly segmental ground-glass opacity in three patients. In Group B, methylprednisolone >2 mg/kg/day was prescribed in 76 patients, and among them, 20 patients with pulmonary lobar consolidation received high-dose methylprednisolone therapy. After 3 months, chest imaging revealed incomplete absorption of pulmonary lesions in seven patients. Among them, five patients with consolidation in more than one pulmonary lobe ultimately had slight BO. Conclusion: In hospitalized patients with MPP, particularly severe MPP, the ideal starting time of corticosteroid treatment might be 5-10 days, preferably 6-7 days, after disease onset. The initial dosage of corticosteroid therapy should be decided according to the severity of the disease. MPP patients with diffuse bronchiolitis-associated lesions/whole lobar consolidation on imaging might require high-dose corticosteroid therapy.

A Novel Detection Procedure for Mutations in the 23S rRNA Gene of Macrolide-Resistant Mycoplasma pneumoniae with Two Non-Overlapping Probes Amplification Assay.

Mycoplasma pneumoniae is a significant cause of community-acquired pneumonia, which is often empirically treated with macrolides (MLs), but, presently, resistance to MLs has been a matter of close clinical concern. This assay is intended to contribute to resistance detection of M. pneumoniae in clinical practice. A novel real-time PCR assay with two non-overlapping probes on the same nucleic acid strand was designed in this study. It could effectively detect all mutation types of M. pneumoniae in 23S rRNA at loci 2063 and 2064. The results were determined by the following methods: ΔCT < 0.5 for MLs-sensitive M. pneumoniae; ΔCT > 2.0 for MLs-resistant M. pneumoniae; 10 copies as a limit of detection for all types. For detection of M. pneumoniae in 92 clinical specimens, the consistency between the results of this assay and the frequently used real-time PCR results was 95.65%. The consistency of MLs resistance results between PCR sequencing and this assay was 100% in all 43 specimens. The assay could not only cover a comprehensive range of targets and have high detection sensitivity but is also directly used for detection and MLs analysis of M. pneumoniae in specimens.

Periostin Attenuates Cyclophosphamide-induced Bladder Injury by Promoting Urothelial Stem Cell Proliferation and Macrophage Polarization.

Interstitial cystitis (IC) is a bladder syndrome of unclear etiology with no generally accepted treatment. Growing evidence suggest that periostin (POSTN) is an important homeostatic component in the tissue repair and regeneration in adulthood, but its function in urinary bladder regeneration is still unknown. Here we investigate whether POSTN is involved in bladder tissue repair in a cyclophosphamide (CYP)-induced interstitial cystitis model. POSTN is primarily expressed in bladder stroma (detrusor smooth muscle and lamina propria) and upregulated in response to CYP-induced injury. POSTN deficiency resulted in more severe hematuria, aggravated edema of the bladder, and delayed umbrella cell recovery. Besides, less proliferative urothelial cells (labeled by pHH3, Ki67, and EdU) and lower expression of Krt14 (a urothelial stem cell marker) were detected in POSTN-/- mice post CYP exposure, indicating a limited urothelial regeneration. Further investigations revealed that POSTN could induce Wnt4 upregulation and activate AKT signaling, which together activates ß-catenin signaling to drive urothelial stem cell proliferation. In addition, POSTN can promote resident macrophage proliferation and polarization to a pro-regenerative (M2) phenotype, which favors urothelial regeneration. Furthermore, we generated injectable P-GelMA granular hydrogel as a biomaterial carrier to deliver recombinant POSTN into the bladder, which could increase urothelial stem cells number, decrease umbrella cells exfoliation, and hence alleviate hematuria in a CYP-induced interstitial cystitis model. In summary, our findings identify a pivotal role of POSTN in bladder urothelial regeneration and suggest that intravesical biomaterials-assisted POSTN delivery may be an efficacious treatment for interstitial cystitis.

Molecular features and antimicrobial susceptibility of Mycoplasma pneumoniae isolates from paediatric inpatients in Weihai, China: Characteristics of M. pneumoniae In Weihai.

OBJECTIVES: We analysed the molecular features and antimicrobial susceptibility of Mycoplasma pneumoniae isolates from Weihai, China, in 2019. METHODS: Pharyngeal swabs of 160 paediatric patients with pneumonia-related symptoms were collected and were subjected to culture and subsequent characteristic analysis. The characteristics of M. pneumoniae isolates were analysed by real-time PCR and genotyping. Antimicrobial susceptibility testing was performed against four antibiotics. All isolates were amplified for analysis of macrolide (ML) resistance mutations in domain V of the 23S rRNA gene and were genotyped using multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) and 'AGT' VNTR detection in the p1 gene. RESULTS: The M. pneumoniae nucleic acid and culture-positive rate of 160 specimens were 88.1% (141/160) and 51.3% (82/160), respectively. Almost all isolates were ML-resistant (81/82). Point mutation at position 2063 in 23S rRNA was identified in all ML-resistant isolates. The ML resistance rate of M. pneumoniae genotype 2 isolates was 97.6% (41/42). MLVA types 4/5/7/2 and 4/5/7/3 belonged to genotype 1, while type 3/5/6/2 belonged to genotype 2. The numbers of 'AGT' VNTR in the p1 gene from all isolates was in the range of 5-15. CONCLUSION: This is the first report that the two genotypes of M. pneumoniae were present in a relatively equivalent ratio, with genotype 2 slightly predominant, in paediatric patients in Weihai in 2019, and the overall ML resistance rate was close to 100%. The minimum inhibitory concentration (MIC) of erythromycin in M. pneumoniae with ML resistance mutation A2063T in Weihai was higher than previously reported.

First report of macrolide-resistant and -susceptible Mycoplasma pneumoniae clinical strains isolated from a single case.

OBJECTIVES: The aim of this study was to investigate the biological characteristics and effect of antibiotic treatment for different Mycoplasma pneumoniae isolates co-infecting the same patient. METHODS: Two throat swab specimens from a single patient, on the day of admission (Sp01) and discharge (Sp13), were liquid cultured and subcultured on agar medium to obtain M. pneumoniae monoclones. The 23S rRNA gene of 50 monoclones from specimens Sp01 and Sp13 were analysed. Real-time PCR assay was used for detection of mutations and genotyping. Two typical monoclones were isolated for antimicrobial susceptibility testing. RESULTS: Genotype 1 monoclones accounted for 70.8% (34/48) in Sp01 and 95.7% (44/46) in Sp13. All genotype 1 monoclones were of the 4-5-7-2 multilocus variable-number tandem-repeat analysis (MLVA) type, while all genotype 2 monoclones were 3-5-6-2 MLVA type. The genotype 1 monoclone, which harboured the A2063G mutation in 23S rRNA gene, was resistant to erythromycin and azithromycin in vitro, whilst genotype 2, which did not carry the mutation, was susceptible to macrolides. The proportion of macrolide-resistant M. pneumoniae monoclones in the specimen cultures collected rose from 70.8% to 95.7% at the time of discharge. CONCLUSION: This is the first report on the isolation of macrolide-resistant and -susceptible strains of M. pneumoniae from the same patient. After treatment, the proportion of macrolide-resistant M. pneumoniae increased, but the patient still carried viable macrolide-susceptible strains, meaning that the macrolide-susceptible strains did not disappear completely.

A multisite SNP genotyping and macrolide susceptibility gene method for Mycoplasma pneumoniae based on MALDI-TOF MS.

In this study, a multisite SNP genotyping and macrolide (ML) susceptibility gene test method for Mycoplasma pneumoniae (M. pneumoniae) was developed based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The detection limit of this method for nucleic acids was 102 -103 copies/reaction. Six SNP site-based genotyping and 3 ML susceptibility sites could be detected simultaneously based on multiplex PCR and mass probe. Using the method constructed in this study, 141 Chinese clinical isolates were divided into 8 SNP types. All the SNP test results for the ML susceptibility gene were in line with those of the 23S rRNA sequencing results. With this method, the multisite SNP genotyping and ML susceptibility determination of M. pneumoniae can be completed simultaneously in one test, which greatly reduces the workload and cost, improves the genotyping ability of M. pneumoniae and deserves clinical application.

Pathogenic Analysis of the Bronchoalveolar Lavage Fluid Samples With Pediatric Refractory Mycoplasma pneumoniae Pneumonia.

Background: We conducted a pathogenic analysis in the bronchoalveolar lavage fluid (BALF) samples from refractory Mycoplasma pneumoniae pneumonia (RMPP) children. Methods: A total of 150 BALF samples from 60 RMPP patients were analyzed to investigate pathogenic changes. The characteristics of M. pneumoniae were analyzed through culture, real-time PCR, genotyping, antimicrobial susceptibility testing and proteomics. The other pathogens were determined using culture, sequencing and nucleic acid detection. Results: In 60 RMPP cases, the bacterial co-infection rate was 5%, while that of virus was 33.3%. The poor prognosis rate was 61.7%. The DNA positive rate among the 150 samples was 98.7%, while the culture positive rate was 56.7% for M. pneumoniae. Significant differences were noticed in the positivity of M. pneumoniae culture obtained from samples with a disease course of at least 3 weeks compared with those within 3 weeks. The genotype 1 M. pneumoniae strains showed a macrolide resistant (MLr) rate of 100%, and that for genotype 2 was 90.1%. Proteomics showed that there were 57 proteins up-regulated in the MLs M. pneumoniae, half of which were membrane-associated protein with adhesion or toxicity. Conclusions: Pediatric RMPP usually presented with viral co-infection, but it caused limited effects on the progression and prognosis of RMPP. Persistent presence of viable M. pneumoniae is not necessary in the later stage of RMPP. The expression of virulence factor in the MLr M. pneumoniae was higher than that of the MLs M. pneumoniae, which was more common in the RMPP children.

Real-Time PCR and Quantitative Culture for Mycoplasma pneumoniae Load in Pharyngeal Swabs from Children at Preliminary Diagnosis and Discharge.

BACKGROUND: Extensive studies have focused on the diagnosis and treatment of Mycoplasma pneumoniae infection; however, rare studies investigated the posttreatment conditions. We analyzed the carrying status of M. pneumoniae in the respiratory tract of children before and after treatment. METHODS: Ninety-two children with M. pneumoniae pneumonia were included in this study. Clinical data were obtained from each patient, and pharyngeal swab sampling was performed at preliminary diagnosis and discharge. Real-time PCR and dilution quantitative culture were utilized to determine the DNA quantification and number of viable M. pneumoniae from samples collected upon preliminary diagnosis and discharge. RESULTS: All the 92 cases showed DNA positivity upon preliminary diagnosis, serum IgM antibody was detected in 80 patients, and positivity of M. pneumoniae culture was observed in 82 cases. Upon discharge, the M. pneumoniae nucleotide and culture positivity were detected in 87 and 49 cases, respectively. The content of viable M. pneumoniae was 10-104 CCU/mL and 10-102 CCU/mL in the preliminary diagnosis samples and discharge samples, respectively. CONCLUSIONS: Real-time PCR was rapid and effective for the qualitative diagnosis of M. pneumoniae at the early stage, but it cannot be used to evaluate the prognosis of patients with M. pneumoniae infection. Quantitative analysis for M. pneumoniae DNA could not directly reflex the viable strain content.

Antimicrobial susceptibility and genotyping of Mycoplasma pneumoniae isolates in Beijing, China, from 2014 to 2016.

Background: The presence of macrolide-resistant Myocplasma pneumoniae has been frequently reported in recent years, especially in China. In this study, we investigated the antimicrobial susceptibility and genotype against M. pneumoniae isolates from 2014 to 2016, Beijing. Methods: We investigated the activities of four antibiotics against 81 M. pneumoniae isolates in vitro. All isolates were amplification of domains II and V of the 23S rRNA gene and the L4 and L22 ribosomal protein fragments. All isolates were genotyped with duplex real-time PCR, MLVA and VNTR detection in p1 gene. Results: The macrolide resistance rate was 65.4% (53/81). Each of the macrolide-resistant M. pneumoniae isolates was resistant to erythromycin (Minimum Inhibitory Concentration, MIC, ≥256 µg/ml) and azithromycin (MIC, 2-64 µg/ml), but susceptible to tetracycline and levofloxacin in vitro. Fifty two macrolide-resistant isolates harbored the A2063G mutation, and only 1 macrolide-resistant isolates harbored the A2064G mutation in domain V of the 23S ribosomal RNA gene. The C162A, A430G, and T279C mutations in the L4 and L22 ribosomal protein genes were not responsible for macrolide resistance, but they were related to the particular genotype of M. pneumoniae. 95.7% of type 1 isolates (45/47) were macrolide-resistance, and 23.5% of the type 2 isolates (8/34) were macrolide-resistance. Type 2 M. pneumoniae macrolide-resistance rate was 50.6% higher than that of the previous reports in China. The eight macrolide-resistant type 2 M. pneumoniae isolates were belong to 3/5/6/2 and 3/5/7/2 MLVA genotypes. Conclusion: To our knowledge, this phenomenon likely resulted from a combination of genotype shifting from type1 to type 2 and antibiotic selection pressure in M. pneumoniae in China in recent years. The increase of resistance in type 2 is not due to the spread of same clone. However, the relationship between genotype shifts and macrolide resistance in M. pneumoniae needs to be further verified with more extensive surveillance data.

Antimicrobial susceptibility and molecular characteristics of Mycoplasma pneumoniae isolates across different regions of China.

Background: In China mainland, most Mycoplasma pneumoniae related studies are carried out in Beijing and Shanghai, while rare studies are performed in the other regions. In this study, we analyzed the molecular biology characteristics and antimicrobial susceptibility of clinical isolates of M. pneumoniae from 5 regions between January 2017 and December 2018. Methods: Genotyping was performed to 154 M. pneumoniae isolates from 5 cities using PCR and multiple-locus variable-number tandem repeat analysis (MLVA) method. Antimicrobial susceptibility test was performed to all the isolates against 4 antibiotics. Sequencing was performed to the amplification products of the 23S rRNA drug resistant gene. Results: Genotype I was detected in 118 M. pneumoniae isolates (76.6%), and genotype II was identified in 36 isolates (23.4%). The majority (92.2%) of the MLVA genotypes were 4-5-7-2 and 3-5-6-2, which represented the genotype I and II, respectively. The total macrolide (ML) resistance rate was 79.7%. The minimum inhibitory concentration (MIC) of the erythromycin was in a range of 128- > 256 µg/ml, while that for the azithromycin was 2-32 µg/ml. There were mutations in the 23S rRNA in each ML resistance isolate. Jilin city showed the highest prevalence of genotype I (100%) and ML resistance rate (100%), while Jinan showed the lowest prevalence of genotype I (45.5%) and ML resistance rate (54.5%). Conclusions: A large variance was identified in the M. pneumoniae genotype and ML resistance among the 5 cities. The proportion of M. pneumoniae with a genotype II genotype (3-5-6-2) showed an increased trend.

Reactivation of methylation-silenced tumor suppressor gene p16INK4a by nordihydroguaiaretic acid and its implication in G1 cell cycle arrest.

Phytoestrogens, including the two major groups isoflavones and lignans, are chemicals with weak estrogenic activity which occur naturally in many foods and herbs. Recently, several intriguing studies reported that some isoflavones can affect DNA methylation status. However, little is known about the effect of plant lignans on epigenetic modification. Using cultured T47D and RKO human cancer cells as a model, we studied the modulating effects of nordihydroguaiaretic acid (NDGA), a member of the lignan family, on the methylation status of the gene promoter region. Our results indicated that NDGA reverses p16INK4a CpG island hypermethylation, and restores its transcription and expression in both cell lines. Cytometric analysis showed that NDGA significantly affects cell cycle progression by arresting cells at the G1 phase. Consistent with the reacquisition of p16INK4a expression, we also found that NDGA induces cellular senescence in cancer cells. This is the first study demonstrating that a member of the lignan family can induce demethylation in human cancer cell lines, suggesting a novel epigenetic mechanism in the prevention or treatment of cancer.

Culture-Independent Detection and Genotyping of Mycoplasma pneumoniae in Clinical Specimens from Beijing, China.

A duplex real-time PCR assay was designed for simultaneous detection and genotyping of Mycoplasma pneumoniae (M. pneumoniae). The detection/typing performance of this duplex PCR method, targeting specific genes for M. pneumoniae type 1 (mpn 459) and type 2 (mpna 5864), was compared to that of the previously published MpP1 real-time PCR assay and the genotyping method for the adhesin P1 gene (mpn 141). A total of 1,344 throat swab specimens collected from patients in Beijing, China were tested for M. pneumoniae by bacterial culture, MpP1 real-time PCR assay, and our duplex PCR assay, and positive detection rates of 26.9%, 34.4%, and 33.7%, respectively, were obtained. The duplex PCR method demonstrated high sensitivity and accuracy for detecting and genotyping M. pneumoniae, and significant differences in genotyping ability were observed when compared to the conventional P1 gene-based method. M. pneumoniae type 1 was the predominate genotype from 2008 to 2012 in Beijing, and a shift from type 1 to type 2 began to occur in 2013. To our knowledge, this is the first reported incidence of a type shift phenomenon of M. pneumoniae clinical isolates in China. These genotyping results provide important information for understanding recent changes in epidemiological characteristics of M. pneumoniae in Beijing.

Sodium thiosulfate exposure disrupts in vitro and in vivo heart development.

It is well-known that the majority of malformations found in the human population is based on complex gene-environment interactions. As an industrial chemical sodium thiosulfate (STS) is used heavily in many industries. Nevertheless, there is little known about the effects of STS on embryo development. In the present study, we have investigated the effects of STS on cardiac development in rat cardiomyocyte H9C2 cell line and chick embryos. As determined by MTT assays, the proliferation of H9C2 cells was inhibited by STS in a dose-dependent manner. Fertilized eggs injected via the yolk sac with STS at Hamburger-Hamilton (HH) stages 6, 9 and 12 showed significantly increased cardiotoxicity at HH stage 18, including cardiomyocyte apoptosis and animal mortality. Western blot analysis showed that STS significantly affected the expression of the apoptosis-related genes bcl-2, bax, and caspase-3 in a dose-dependent manner in the H9C2 cell line and in chick embryos. Dysregulation of apoptosis was correlated with embryonic heart malformations. Thus, STS may be a potent cardiac teratogen during embryo development.

Nordihydroguaiaretic acid restores expression of silenced E-cadherin gene in human breast cancer cell lines and xenografts.

In our study we use nordihydroguaiaretic acid (NDGA), the naturally occurring lignan, to investigate whether it plays a role in the prevention and treatment of cancer by epigenetic modifications. The growth inhibitory effect of NDGA on human breast cancer cell lines was determined using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay). It substantially inhibited the growth of human breast cancer cell lines SKBR3 and MDA-MB-435 with an estimated IC50 of 31.09+/-1.6 and 38.8+/-2.1 micromol/l respectively, after 4 days incubation with different NDGA concentrations. The in-vivo anticancer activity of NDGA was evaluated by calculating the tumor growth inhibition value. NDGA substantially inhibited the growth of human breast carcinoma cells in both animal and cell-based models. We also found that a single treatment with NDGA reactivates methylation-silenced E-cadherin gene in vitro and in vivo, suggesting an intriguing concept that lignans may act as natural effective epigenetic modifiers in the prevention and treatment of cancer.