Allele-specific real-time PCR testing for minor macrolide-resistant Mycoplasma Pneumoniae.

BACKGROUND: The point mutations in 23S rRNA gene of Mycoplasma pneumoniae (M. pneumoniae) can lead to high-level resistance to macrolides. This study aimed to evaluate allele-specific real-time PCR (ASPCR) to detect the resistance-related mutations located at positions A2063G and A2064G of 23S rRNA gene. METHODS: We detected 178 pharyngeal swab specimens and calculated the proportions of resistant and sensitive quasispecies using ASPCR assays. ASPCR assays can detect down to 10 copies of 23S rRNA gene and achieved sensitivities of < 0.1% for A2063G and A2064G. We also compared the findings of ASPCR with the results of nested PCR with sequencing. RESULTS: Of 178 samples, 164 were found to have M. pneumoniae including 90.85% (149/164) samples with macrolide-resistant M. pneumoniae (MRMP) quasispecies by ASPCR, while 153 were found to be M. pneumoniae-positive including 71.90% (110/153) samples with MRMP quasispecies by nested PCR with sequencing. Of the 164 M. pneumoniae-positive samples, 61.59% (101/164) had the mixed population of wild-type and mutant M. pneumoniae, and 56.44% (57/101) of the latter contained the mutations at low frequency (≤50%). CONCLUSION: ASPCR indicated that sensitive and resistant quasispecies coexisted in most of the M. pneumoniae positive samples. The ASPCR was a highly sensitive, accurate and rapid method for detecting the macrolide resistance-associated mutations and it could provide earlier and more drug-resistant information for M. pneumoniae research and the clinical therapy.

Comparative genomic sequencing to characterize Mycoplasma pneumoniae genome, typing, and drug resistance.

To analyze the characteristics of Mycoplasma pneumoniae as well as macrolide antibiotic resistance through whole-genome sequencing and comparative genomics. Thirteen clinical strains isolated from 2003 to 2019 were selected, 10 of which were resistant to erythromycin (MIC >64 µg/mL), including 8 P1-type I and 2 P1-type II. Three were sensitive (<1 µg/mL) and P1-type II. One resistant strain had an A→G point mutation at position 2064 in region V of the 23S rRNA, the others had it at position 2063, while the three sensitive strains had no mutation here. Genome assembly and comparative genome analysis revealed a high level of genome consistency within the P1 type, and the primary differences in genome sequences concentrated in the region encoding the P1 protein. In P1-type II strains, three specific gene mutations were identified: C162A and A430G in L4 gene and T1112G mutation in the CARDS gene. Clinical information showed seven cases were diagnosed with severe pneumonia, all of which were infected with drug-resistant strains. Notably, BS610A4 and CYM219A1 exhibited a gene multi-copy phenomenon and shared a conserved functional domain with the DUF31 protein family. Clinically, the patients had severe refractory pneumonia, with pleural effusion, necessitating treatment with glucocorticoids and bronchoalveolar lavage. The primary variations between strains occur among different P1-types, while there is a high level of genomic consistency within P1-types. Three mutation loci associated with specific types were identified, and no specific genetic alterations directly related to clinical presentation were observed.IMPORTANCEMycoplasma pneumoniae is an important pathogen of community-acquired pneumonia, and macrolide resistance brings difficulties to clinical treatment. We analyzed the characteristics of M. pneumoniae as well as macrolide antibiotic resistance through whole-genome sequencing and comparative genomics. The work addressed primary variations between strains that occur among different P1-types, while there is a high level of genomic consistency within P1-types. In P1-type II strains, three specific gene mutations were identified: C162A and A430G in L4 gene and T1112G mutation in the CARDS gene. All the strains isolated from severe pneumonia cases were drug-resistant, two of which exhibited a gene multi-copy phenomenon, sharing a conserved functional domain with the DUF31 protein family. Three mutation loci associated with specific types were identified, and no specific genetic alterations directly related to clinical presentation were observed.

A single residue within the V5 region of HIV-1 envelope facilitates viral escape from the broadly neutralizing monoclonal antibody VRC01.

VRC01, a broadly neutralizing monoclonal antibody, is capable of neutralizing a diverse array of HIV-1 isolates by mimicking CD4 binding with the envelope glycoprotein gp120. Nonetheless, resistant strains have been identified. Here, we examined two genetically related and two unrelated envelope clones, derived from CRF08_BC-infected patients, with distinct VRC01 neutralization profiles. A total of 22 chimeric envelope clones was generated by interchanging the loop D and/or V5 regions between the original envelopes or by single alanine substitutions within each region. Analysis of pseudoviruses built from these mutant envelopes showed that interchanging the V5 region between the genetically related or unrelated clones completely swapped their VRC01 sensitivity profiles. Mutagenesis analysis revealed that the asparagine residue at position 460 (Asn-460), a potential N-linked glycosylation site in the V5 region, is a key factor for observed resistance in these strains, which is further supported by our structural modeling. Moreover, changes in resistance were found to positively correlate with deviations in VRC01 binding affinity. Overall, our study indicates that Asn-460 in the V5 region is a critical determinant of sensitivity to VRC01 specifically in these viral strains. The long side chain of Asn-460, and potential glycosylation, may create steric hindrance that lowers binding affinity, thereby increasing resistance to VRC01 neutralization.

Term breech presentation vaginal births in Tibet: A retrospective analysis of 451 cases.

Background: In high altitude areas, like Tibet, most fetuses in breech presentation at term are delivered vaginally owing to a variety of reasons, but this has not been published. Objective: This study aimed to provide references and evidence for the delivery of breach presentation term fetuses in high altitude areas, through comparing and analyzing the data of full-term singleton fetuses with breech or cephalic presentation in Naqu People's Hospital, Tibet. Study design: We retrospectively analyzed the clinical data of 451 breech presentation fetuses mentioned above over a period of 5 years (2016-2020). A total of 526 cephalic presentation fetuses' data within 3 months (1 June to 1 September 2020) of the same period were collected too. Statistics were compared and assembled on fetal mortality, Apgar scores, and severe neonatal complications for both planned cesarean section (CS) and vaginal delivery. In addition, we also analyzed the types of breech presentation, the second stage of labor, and damage to the maternal perineum during vaginal delivery. Results: Among the 451 cases of breech presentation fetuses, 22 cases (4.9%) elected for CS and 429 cases (95.1%) elected for vaginal delivery. Of the women who chose vaginal trial labor, 17 cases underwent emergency CSs. The perinatal and neonatal mortality rate was 4.2% in the planned vaginal delivery group and the incidence of severe neonatal complications was 11.7% in the transvaginal group, no deaths were detected in the CS group. Among the 526 cephalic control groups with planned vaginal delivery, the perinatal and neonatal mortality was 1.5% (p = 0.012), and the incidence of severe neonatal complications was 1.9%. Among vaginal breech deliveries, most of them were complete breech presentation (61.17%). Among the 364 cases, the proportion of intact perinea was 45.1%, and first degree lacerations accounted for 40.7%. Conclusion: In the Tibetan Plateau region, vaginal delivery was less safe than cephalic presentation fetuses for full-term breech presentation fetuses delivered in the lithotomy position. However, if dystocia or fetal distress can be identified in time and then encouraged to convert to cesarean, its safety will be greatly improved.

Cervical cancer screening in high-altitude areas in China: A large cross-section study of 25,173 women in northern Tibet.

Background: Cervical cancer has become a worldwide concern owing to its high incidence and mortality rates. To date, high-altitude areas of Tibet have not benefited from any large-scale cervical cancer screening programs. Therefore, we initiated a screening program to investigate the prevalence of human papilloma virus (HPV) and HPV genotype distribution to reveal cervical cancer and its precursor which lead to morbidity among women in the city of Nagqu in northern Tib3et. Methods: A total of 25,173 women were recruited to undergo HPV genotype tests between June and December 2019. Women infected with HPV 16 and/or 18 underwent colposcopy and histological examination. Women with other high-risk HPV type (hr-HPV) underwent cytological tests to determine whether to conduct further colposcopy and histological examination for diagnosis. HPV prevalence was calculated in the total population and further stratified according to various parameters, such as age group, area location (altitude level), and single or mixed infection status. The HPV genotype distribution was also investigated accordingly. Cervical lesions revealed by further colposcopic findings were also analyzed; high-grade and malignant lesion morbidities were calculated in total and in each county. Most data were collected and analyzed using descriptive and consistency check statistical methods, and a risk factor investigation for HPV infection was performed using logistic regression models. Results: The total HPV infection rate among women in Nagqu was 13.42%. Of the 25,173 women in the study, 999 (3.97%) were HPV 16/18 positive, 2,379 (9.45%) were other hr-HPV-positive, and 21,795 (86.58%) were HPV-negative. The five most common HPV genotypes, accounting for more than 60% of all HPV infections in Nagqu people, were HPV 16, 58, 31, 18, and 52. Tibetan women younger than 20 years and older than 60 years were the two age groups with the highest rates of HPV infection, 26.7% and 19.8%, respectively. Among the HPV-positive women, 2,656 (78.33%) were infected with a single strain and 732 (21.67%) were infected with multiple strains (more than two genotypes). HPV prevalence increased in high-altitude areas (positive rate highest in Nyima with an altitude of 5,000 m, 23.9%) and decreased in relatively low-altitude areas (positive rate lowest in Lhari with an altitude of 4,000 m, 6.6%). Multiple analyses showed that age, parity, age at first delivery, and altitude of residence were independent factors facilitating HPV infection in Tibetan women. High-grade and malignant cervical lesions revealed by histological findings were different among living locations, with the highest rates in Xainza, Baingoin, and Nyainrong, these being 2.019%, 1.820%, and 1.116%, respectively, among women in these areas. Conclusion: Our survey provides an overall perspective on HPV genotype infection and cervical lesions in women in northern Tibet. The data not only provide useful information for the treatment of cervical lesions but also has great value in terms of the primary and secondary prevention measures that can be taken for women living in these regions. Clinical Trial Registration: www.chictr.org.cn, indentifier ChiCTR2000035061.

Epidemiology and mechanism of drug resistance of Mycoplasma pneumoniae in Beijing, China: A multicenter study.

Mycoplasma pneumoniae (M. pneumoniae) is one of the most common causes of community-acquired respiratory tract infections (RTIs). We aimed to investigate the prevalence of M. pneumoniae infection, antibiotic resistance and genetic diversity of M. pneumoniae isolates across multiple centers in Beijing, China. P1 protein was detected by Nested PCR to analyze the occurrence of M. pneumoniae in pediatric patients with RTI. M. pneumoniae isolates were cultured and analyzed by Nested-PCR to determine their genotypes. Broth microdilution method was used to determine the minimum inhibitory concentration (MIC) of antibiotics. Out of 822 children with RTI admitted to 11 hospitals in Beijing, 341 (41.48%) were positive for M. pneumoniae by Nested PCR and 236 (69.21%) samples had mutations in 23S rRNA domain V. The highest proportion of M. pneumoniae positive samples was observed in school-age children (118/190; 62.11%) and in pediatric patients with pneumonia (220/389; 56.56%). Out of 341 M. pneumoniae positive samples, 99 (12.04%) isolates were successfully cultured and the MIC values were determined for 65 M. pneumoniae strains. Out of these, 57 (87.69%) strains were resistant to macrolides, and all 65 strains were sensitive to tetracyclines or quinolones. M. pneumoniae P1 type I and P1 type II strains were found in 57/65 (87.69%) and 8/65 (12.31%) of cultured isolates, respectively. Overall, we demonstrated a high prevalence of M. pneumoniae infection and high macrolide resistance of M. pneumoniae strains in Beijing. School-age children were more susceptible to M. pneumoniae, particularly the children with pneumonia. Thus, establishment of a systematic surveillance program to fully understand the epidemiology of M. pneumoniae is critical for the standardized use of antibiotics in China.

Meta-analysis of the accuracy of transient elastography in measuring liver stiffness to diagnose esophageal varices in cirrhosis.

BACKGROUD: To assess the diagnostic performance of transient elastography (TE) in detecting the presence and size of esophageal varices (EV) in cirrhotic patients. METHODS: We searched PubMed, Web of Science, Wiley Online Library, Science Direct, China National Knowledge Infrastructure, WeiPu, WanFang database, and Baidu Scholar to identify studies that evaluated the diagnostic accuracy of TE in liver stiffness measurement, compared with esophagogastroduodenoscopy (EGD), for the detection of the presence and degree of EV in cirrhosis. RESULTS: We included 32 studies in the presence of any EV (grade 1-3; n = 4082), 27 studies on substantial EV (grade 2-3; n = 5221) and 5 studies on large EV (grade 3). The pooled sensitivity, specificity, and diagnostic odds ratio (DOR) were 0.8 (95% CI, 0.78-0.86), 0.68 (95% CI, 0.62-0.74), and 10 (95% CI, 7-14) for any EV; 0.81 (95% CI, 0.77-0.85), 0.72 (95% CI, 0.66-0.77), and 11 (95% CI, 8-15) for substantial EV; and 0.92 (95% CI, 0.83-0.96), 0.78 (95% CI, 0.70-0.85), and 40 (95% CI, 15-107) for large EV. Subgroup analysis revealed that the heterogeneity among studies on any EV could potentially be explained by study location, proportion of Child A, and time interval between TE and EGD; for substantial EV, the proportion of Child A, etiology of cirrhosis, and the time interval between TE and EGD were important heterogeneity factors. Publication bias was found among studies evaluating diagnostic performance of TE for any EV. CONCLUSION: TE is a good tool for detecting the presence and degree of EV; however, in determination of the liver stiffness cutoff values means that TE is only cautiously used in clinical practice.

Genistein ameliorated endothelial nitric oxidase synthase uncoupling by stimulating sirtuin-1 pathway in ox-LDL-injured HUVECs.

Endothelial nitric oxidase synthase (eNOS) uncoupling plays a causal role in endothelial dysfunction in atherosclerosis. Genistein consumption has been associated with the prevention of atherosclerosis. However, the effect of genistein on eNOS uncoupling has not been reported. A model of oxidized low-density lipoprotein (ox-LDL)-induced injury on human umbilical vein endothelial cells (HUVECs) was established to evaluate the effect of genistein on eNOS uncoupling. We investigated the effect of genistein on NADPH oxidase-dependent superoxide production, NOX4 expression, BH4 synthesis and oxidation, the expression of GTP cyclohydrolase 1 (GCH1) and dihydrofolate reductase (DHFR). The results showed that genistein decreased superoxide production and NOX4 expression, enhanced the ratio of BH4/BH2, augmented the expressions of GCH1 and DHFR. Accompanied with genistein ameliorating eNOS uncoupling, genistein elevated the expression of sirtuin-1; furthermore, the effects of genistein on eNOS uncoupling were blunted with sirtuin-1 siRNA. The present study indicated that genistein ameliorated eNOS uncoupling was concerned with sirtuin-1 pathway in ox-LDL-injured HUVECs.

Modulation of Leishmania donovani infection and cell viability by testosterone in bone marrow-derived macrophages: signaling via surface binding sites.

Androgens can increase susceptibility toward numerous parasitic infections as well as modulate apoptosis of immune cells. According to the current view, androgens mediate immune cell activities not only through classical intracellular androgen receptors (AR), but also through membrane receptors on the cell surface. Here, using murine bone marrow-derived macrophages (BMMs), we examined the influence of testosterone on Leishmania donovani infection and cell viability in vitro as well as the possible mechanisms. Our data demonstrated that testosterone directly increased intramacrophage infection by L. donovani. In addition, testosterone decreased cell viability by way of apoptosis, accompanied by increased Fas, FasL, and Caspase-8 expression. However, these effects of testosterone could not be associated with the classical AR in BMMs since AR was not detectable using different experimental techniques. Instead, it was found that testosterone could bind to the surface of BMMs by the use of an impermeable testosterone-BSA-FITC in confocal laser scanning microscopy and flow cytometry. Collectively, our data indicated that the influence of testosterone on L. donovani infection and viability of BMMs was mediated through the binding sites of testosterone on cell surfaces, which provided a novel mode of direct action of testosterone on AR-free BMMs.

Effect of nicotine on Oct-4 and Rex-1 expression of mouse embryonic stem cells.

To examine the influence that nicotine exposure has on early embryo development, the present study has applied real-time RT-PCR to investigate changes in Oct-4 and Rex-1 expression in mouse embryonic stem (ES) cells exposed to nicotine alone or in the presence of tubocurarine. Oct-4 is regarded as a candidate master regulator gene for the initiation, maintenance, and differentiation of pluripotent cells. Zfp42/Rex-1, another specific gene of pluripotent cells, also plays a critical role in maintaining stem cell character and pluripotency. Results indicated that nicotine (10-1000 nM) enhanced Oct-4 and Rex-1 expression without altering beta-actin expression. This up-regulation with nicotine (100-1000 nM) was prevented with tubocurarine (10 microM), a nicotinic acetylcholine receptor (nAChRs) antagonist. We conclude that nicotine may influence Oct-4 and Rex-1 expression in ES cells through a mechanism involving the nAChRs.

Testosterone influenced the expression of Notch1, Notch2 and Jagged1 induced by lipopolysaccharide in macrophages.

Testosterone plays an extensive role in modulating macrophages functions in mammals. In this study, we incubated murine bone marrow macrophages which were positive to the surface antigen F4/80 with lipopolysaccharide and increasing amounts of testosterone. Expression of Notch family members (including Notch1, Notch2 and Jagged1) was investigated at transcription and post-transcription levels through RT-PCR and Western blotting assay followed by densitometric analyses. Results showed that testosterone influenced the lipopolysaccharide-induced expression of Notch1, Notch2 and Jagged1 in macrophages. The elevated expression of Notch1 and Notch2 induced by lipopolysaccharide was repressed by testosterone at lower levels, but enhanced at higher hormone levels. In addition, the expression of Jagged1 was enhanced by various amounts of testosterone. These results suggest that testosterone affected the expression of Notch family members in activated macrophages.

[Effect of nicotine on Oct-4 transcription of embryonic stem cells].

UNLABELLED: To clarify whether nicotine affected the early embryo development, the paper investigated the influence of nicotine on embryonic stem (ES) cells specific gene Oct-4. METHODS: ES cells were treated with nicotine (1-1000 nM) and/or 10 microM tubocurarine (a usual nicotinic acetylcholine receptors [nAChRs] blocker) for 24 h. Subsequently, they were purified to discard feeder cells and total RNA was isolated. Fgf-5 was amplified to detect the purification of ES cells, and the relative expression of Oct-4 and beta-actin to GAPDH was analyzed with RT-PCR. RESULTS: RT-PCR analysis illustrated that nicotine (10-1000 nM) significantly enhanced Oct-4 transcription, while had no effect on beta-actin transcription; meanwhile, compared with nicotine (100 nM and 1000 nM) treatment alone, tubocurarine inhibited Oct-4 transcription evidently. CONCLUSION: It is reasonable to assume that nicotine could influence the development and differentiation of ES cells, and impinge on the early embryo development.

[Ultrastructural study on pharyngeal armature of seven species of sandflies in china by scanning electron microscopy].

OBJECTIVE: To observe the ultrastructure of pharyngeal armature of 7 species of sandflies in China. METHODS: The pharyngeal armature of various sandflies were studied by scanning electron microscopy. RESULTS: The pharyngeal armature of sandfly consisted of pointed-teeth with various shape, number and arrangement among different species. CONCLUSION: Such differences may provide the morphological proof for identification of species.

Persistence of VRC01-resistant HIV-1 during antiretroviral therapy.

VRC01, a broadly neutralizing monoclonal antibody (bnmAb), can neutralize a diverse array of HIV-1 isolates by mimicking CD4 binding to the envelope glycoprotein gp120. We have previously demonstrated the presence of VRC01-resistant strains in an HIV-1 infected patient during antiretroviral therapy. Here, we report follow-up studies of two subsequent samples from the same patient. With genetic and phenotypic analysis of over 70 full-length molecular clones of the HIV-1 envelope, we show that VRC01-resistant HIV-1 continued to exist and change in its proportion of the infecting virus during treatment with a highly active antiretroviral therapy. Consistent with our previous observation, the resistant phenotype was associated with a single asparagine residue at position 460 (N460), a potential N-linked glycosylation site in the V5 region. The persistence and continuing evolution of VRC01-resistant HIV-1 in vivo presents a great challenge to our future preventative and therapeutic interventions based on VRC01.

Genistein inhibits ox-LDL-induced VCAM-1, ICAM-1 and MCP-1 expression of HUVECs through heme oxygenase-1.

BACKGROUND AND AIMS: Genistein, a principal component of soybean isoflavones, plays an important role in the prevention of atherosclerosis. However, the detailed mechanisms have not been fully investigated. The aims of this study were to evaluate the anti-atherosclerotic effect and investigate potential pharmacological mechanism of genistein. METHODS: A model of oxidized low-density lipoprotein (ox-LDL)-induced injury in on human umbilical vein endothelial cells (HUVECs) was established to evaluate the protective role of genistein. Macrophage/monocyte chemoattractant protein-1 (MCP-1), vascular cellular adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) secretion and their messenger RNA transcription were observed via enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase PCR (RT-PCR). Meanwhile, the study investigated the role of Nrf2/HO-1 pathway during the process. RESULTS: Pretreatment with genistein markedly reduced ox-LDL-induced MCP-1, VCAM-1 and ICAM-1 secretion and mRNA transcription, which was further decreased by the inducer of HO and reversed by the inhibitor of HO; additionally, the effects were accompanied with upregulating HO-1 mRNA and protein expression and markedly abolished with Nrf2 siRNA. CONCLUSIONS: Anti-inflammatory effect of genistein on endothelial cells may be associated with the activation of Nrf2/HO-1 pathway.

Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4.

The spike glycoprotein (S) of recently identified Middle East respiratory syndrome coronavirus (MERS-CoV) targets the cellular receptor, dipeptidyl peptidase 4 (DPP4). Sequence comparison and modeling analysis have revealed a putative receptor-binding domain (RBD) on the viral spike, which mediates this interaction. We report the 3.0 Å-resolution crystal structure of MERS-CoV RBD bound to the extracellular domain of human DPP4. Our results show that MERS-CoV RBD consists of a core and a receptor-binding subdomain. The receptor-binding subdomain interacts with DPP4 ß-propeller but not its intrinsic hydrolase domain. MERS-CoV RBD and related SARS-CoV RBD share a high degree of structural similarity in their core subdomains, but are notably divergent in the receptor-binding subdomain. Mutagenesis studies have identified several key residues in the receptor-binding subdomain that are critical for viral binding to DPP4 and entry into the target cell. The atomic details at the interface between MERS-CoV RBD and DPP4 provide structural understanding of the virus and receptor interaction, which can guide development of therapeutics and vaccines against MERS-CoV infection.

Allele-specific real-time PCR testing for minor HIV-1 drug resistance mutations: assay preparation and application to reveal dynamic of mutations in vivo.

BACKGROUND: It is very important for the clinical management to test for minor HIV-1 resistance mutations accurately and sensitively. The conventional genotypic assays of HIV drug resistance detection based on sequencing can only discriminate the mutations which present in more than 20% - 30%. The aim of this study was to evaluate allele-specific real-time PCR (ASPCR) to detect the resistance-related mutations located at positions 103, 184 and 215. METHODS: We developed the allele-specific PCR assay, using the most common drug resistance mutations in Chinese AIDS patients, K103N, M184V/I, T215F/Y as a model system. The standards were constructed by cloning the wild-type and mutant DNA fragments into the T-vector. We designed specific primers to discriminate mutant templates in the real-time PCR using SYBR green as a fluorescence reporter. And then we evaluated the ASPCR assay and tested 140 clinical samples using this method. RESULTS: The sensitivities of ASPCR assay were 0.04% for K103N, 0.30% for M184I, 0.40% for M184V, 0.03% for T215F and 0.02% for T215Y. The intra-assay and inter-assay coefficients of variation were less than 0.42. One hundred and forty plasma samples were tested by ASPCR and dynamic resistance curves of ten patients were obtained. CONCLUSIONS: Drug resistance emerged half a year after the start of antiretroviral therapy. The mutation of T215Y emerged 1 to 1.5 years after starting treatment and then increased rapidly. The ASPCR assay we developed was a sensitive, accurate and rapid method to detect the minor HIV-1 variants and it can provide earlier and more drug-resistance information for HIV research and AIDS antiretroviral therapy.