The crystal structure of 10H-4-methyl-2H-2-oxopyrano[5,6-b]carbazole hydrate was determined by X-ray diffraction. The crystal, 2C16H11NO2·H2O, belongs to space group P21/a with cell dimensions of a = 7.927(6)Å, b = 22.78(1)Å, c = 13.73(1)Å, β = 102.31(2)°. The final R value is 0.061 for 4651 reflections (I > 2.00 σ(I)). There are two independent coumarin molecules (A, B) and one water molecule in an asymmetric unit. Molecules A and B are linked through the N-H…O=C and the C=O…H-O(water)…H-N hydrogen bondings to form a cyclic hetero dimer.
Abstract Dengue virus (DENV) causes repeated outbreaks of disease in endemic areas, with patterns of local transmission strongly influenced by seasonality, importation via human movement, immunity, and vector control efforts. An understanding of how each of these interacts to enable endemic transmission (continual circulation of local virus strains) is largely unknown. There are times of the year when no cases are reported, often for extended periods of time, perhaps wrongly implying the successful eradication of a local strain from that area. Individuals who presented at a clinic or hospital in four communes in Nha Trang, Vietnam, were initially tested for DENV antigen presence. Enrolled positive individuals then had their corresponding household members invited to participate, and those who enrolled were tested for DENV. The presence of viral nucleic acid in all samples was confirmed using quantitative polymerase chain reaction, and positive samples were then whole-genome sequenced using an amplicon and target enrichment library preparation techniques and Illumina MiSeq sequencing technology. Generated consensus genome sequences were then analysed using phylogenetic tree reconstruction to categorise sequences into clades with a common ancestor, enabling investigations of both viral clade persistence and introductions. Hypothetical introduction dates were additionally assessed using a molecular clock model that calculated the time to the most recent common ancestor (TMRCA). We obtained 511 DENV whole-genome sequences covering four serotypes and more than ten distinct viral clades. For five of these clades, we had sufficient data to show that the same viral lineage persisted for at least several months. We noted that some clades persisted longer than others during the sampling time, and by comparison with other published sequences from elsewhere in Vietnam and around the world, we saw that at least two different viral lineages were introduced into the population during the study period (April 2017–2019). Next, by inferring the TMRCA from the construction of molecular clock phylogenies, we predicted that two of the viral lineages had been present in the study population for over a decade. We observed five viral lineages co-circulating in Nha Trang from three DENV serotypes, with two likely to have remained as uninterrupted transmission chains for a decade. This suggests clade cryptic persistence in the area, even during periods of low reported incidence.
<i>Background:</i> Among several C-terminal binding proteins (CtBPs), friend of GATA (FOG) has been implicated in the down-regulation of GATA-3-mediated Th2 cell differentiation. Here we investigated the role of CtBP2 in Th1 and Th2 cytokine expression in human T cells. <i>Methods:</i> CtBP2 was introduced into human peripheral CD4+ T cells by a lentiviral transduction system. Subsequently, the expression of Th1 and Th2 cytokine mRNA was determined by quantitative real-time RT-PCR. <i>Results:</i> CtBP2 significantly suppressed stimulation-induced expression of IL-4, IL-5 and IL-13 in human T cells. However, IFN-γ expression was not affected by the introduction of CtBP2. <i>Conclusion:</i> CtBP2 selectively down-regulates Th2 cytokines, therefore it is a potential target for the treatment of allergic diseases.
Abstract Conjunctival pneumococcal serotypes among members of a community have not been investigated well. We determined the prevalence and association of Streptococcus pneumoniae in the nasopharynx and conjunctiva among children in a community before pneumococcal conjugate vaccine introduction. In October 2016, conjunctival and nasopharyngeal swabs were collected from children (< 24 months old) and nasopharyngeal swabs from mothers in Nha Trang, Vietnam. Quantitative lytA PCR and DNA microarray were performed to detect and serotype S. pneumoniae . The association between S. pneumoniae in the nasopharynx and conjunctiva was evaluated using multivariable logistic regression model. Among 698 children, 62 (8.9%, 95% CI 6.9–11.2%) were positive for S. pneumoniae in the conjunctiva. Non-encapsulated S. pneumoniae were most commonly identified, followed by serotypes 6A, 6B, and 14. Nasopharyngeal and conjunctival detection were positively associated (aOR 47.30, 95% CI 24.07–92.97). Low birth-weight, day-care attendance, and recent eye symptoms were independently associated with S. pneumoniae detection in the conjunctiva (aOR 11.14, 95% CI 3.76–32.98, aOR 2.19, 95% CI 1.45–3.31, and aOR 3.59, 95% CI 2.21–5.84, respectively). Serotypes and genotypes in the conjunctiva and nasopharynx matched in 87% of the children. Three mothers’ nasopharyngeal pneumococcal samples had matched serotype and genotype with their child’s in the conjunctiva and nasopharynx. S. pneumoniae presence in nasopharynx and conjunctiva were strongly associated. The high concordance of serotypes suggests nasopharyngeal carriage may be a source of transmission to the conjunctiva.
Introduction. Diphtheria is a potentially life-threatening infection and remains endemic in many low- and middle-income countries (LMICs). A reliable, low-cost method for serosurveys in LMICs is warranted to estimate the accurate population immunity to control diphtheria.Hypothesis/Gap Statement. The correlation between the ELISA results against diphtheria toxoid and the gold standard diphtheria toxin neutralization test (TNT) values is poor when ELISA values are <0.1 IU ml-1, which results in inaccurate estimates of susceptibility in populations when ELISA is used for measuring antibody levels.Aim. To explore methods to accurately predict population immunity and TNT-derived anti-toxin titres from ELISA anti-toxoid results.Methodology. A total of 96 paired serum and dried blood spot (DBS) samples collected in Vietnam were used for comparison of TNT and ELISA. The diagnostic accuracy of ELISA measurement with reference to TNT was assessed by area under the receiver operating characteristic (ROC) curve (AUC) and other parameters. Optimal ELISA cut-off values corresponding to TNT cut-off values of 0.01 and 0.1 IU ml-1 were identified by ROC analysis. A method based on the multiple imputation approach was also applied to estimate TNT measurements in a dataset that only included ELISA results. These two approaches were then applied to ELISA results previously generated from 510 subjects in a serosurvey in Vietnam.Results. The ELISA results on DBS samples showed a good diagnostic performance compared to TNT. The cut-off values for ELISA measurement corresponding to the TNT cut-off values of 0.01 IU ml-1 were 0.060 IU ml-1 in serum samples, and 0.044 IU ml-1 in DBS samples. When a cut-off value of 0.06 IU ml-1 was applied to the 510 subject serosurvey data, 54 % of the population were considered susceptible (<0.01 IU ml-1). The multiple imputation-based approach estimated that 35 % of the population were susceptible. These proportions were much larger than the susceptible proportion estimated by the original ELISA measurements.Conclusion. Testing a subset of sera by TNT combined with ROC analysis or a multiple imputation approach helps to adjust ELISA thresholds or values to assess population susceptibility more accurately. DBS is an effective low-cost alternative to serum for future serological studies for diphtheria.
Background: Adoptive transfer of helper T (Th) cells conferred a late asthmatic response upon antigen challenge. A possible production of a contractile activity for bronchial smooth muscle (BSM) by activated Th cells was examined. Method: Murine Th clones were stimulated with immobilized anti-CD3 antibody in the presence or absence of anti-CD28 antibody. Culture supernatants were dialyzed and then applied to the collagen gels containing cultured human BSM cells. Results: Culture supernatants of activated but not resting murine Th clones that conferred a late asthmatic response, induced the contraction of human BSM cell-containing collagen gels. Conclusion: Activated Th cells produce a contractile activity for BSM in vitro.
IL ‐2 plays an important role in immunological and other biological functions. This cytokine directly induces the production of several cytokines, such as IL ‐5 and IL ‐13. The mechanisms of IL ‐2‐mediated cytokine synthesis are mostly unclear; however, the involvement of IL ‐2 receptor ( IL ‐2R)β has been suggested. In this study, the signaling molecule downstream of IL ‐2Rβ was investigated, employing a proteomic approach. Full‐length IL ‐2Rβ and its mutant in which the intracellular component was truncated were introduced in an IL ‐2Rα‐ and IL ‐2Rγ‐stably transfected T cell hybridoma, S1. The differential phosphorylation profiles of protein tyrosine residues in these cells upon IL ‐2 stimulation were examined by two‐dimensional gel electrophoresis. The candidate phosphoproteins of interest were re‐covered, in‐gel digested and mass spectrometry fingerprinted. Among proteins specifically phosphorylated in full‐length IL ‐2Rβ‐expressing cells in response to IL ‐2 stimulation, protein phosphatase ( PP )1β and FK 506‐binding protein 4 were identified. Particularly, PP 1β augmented IL ‐5 and IL ‐13 expression stimulated by IL ‐2 but not by anti‐ CD 3 antibody in human peripheral CD 4 + T cells upon ectopic expression. IL ‐2‐induced cytokine expression was suppressed by overexpression of PP 1 regulatory subunit 2. A PP 1 inhibitor, tautomycin, but not a PP 2A inhibitor, okadaic acid, also inhibited the IL ‐2R‐mediated responses. It was conclusively shown that PP 1 is crucially involved in IL ‐2‐mediated IL ‐5 and IL ‐13 synthesis in human T cells.
T-bet is crucially implicated in Th1 differentiation due to its strong promoting activity for IFN-γ gene transcription. However, the regulatory role of T-bet in Th2 cytokines is not fully delineated. <i>Methods:</i> The effect of T-bet on mRNA expression as well as the promoter activity of IL-4 in human T cells was investigated by employing quantitative RT-PCR and fluorescence-based promoter reporter assay procedures. <i>Results:</i> IL-4 mRNA expression as well as the transcriptional activity of 5′-flanking region in the IL-4 gene encompassing –1105 to +4 in Jurkat cells was clearly upregulated upon stimulation. The inducible mRNA expression and the promoter activity of IL-4 were significantly diminished by ectopic expression of T-bet. <i>Conclusion:</i> IL-4 gene transcription is inhibited by T-bet via the suppression of its promoter activity, independently of IFN-γ. T-bet facilitates Th1 differentiation through not only upregulation of IFN-γ, but also downregulation of IL-4 gene transcription.
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