Cyclovirobuxine D inhibits growth and progression of non­small cell lung cancer cells by suppressing the KIF11­CDC25C­CDK1­CyclinB1 G2/M phase transition regulatory network and the NFκB/JNK signaling pathway.

Lung cancer is the leading cause of cancer­related mortality worldwide. Non­small cell lung cancer (NSCLC) is the most common pathological subtype of lung cancer and is associated with low 5­year overall survival rates. Therefore, novel and effective chemotherapeutic drugs are urgently required for improving the survival outcomes of patients with lung cancer. Cyclovirobuxine D (CVB­D) is a natural steroidal alkaloid, used for the treatment of cardiovascular diseases in Traditional Chinese Medicine. Several studies have also demonstrated the antitumor effects of CVB­D. Therefore, in the present study, the therapeutic effects of CVB­D in lung cancer and the underlying mechanisms were investigated using the in vivo xenograft model of NSCLC in nude mice and in vitro experiments with the NSCLC cell lines. Bioinformatics analyses of RNA­sequencing data, and cell­based functional assays demonstrated that CVB­D treatment significantly inhibited in vitro and in vivo NSCLC cell proliferation, survival, invasion, migration, angiogenesis, epithelial­to­mesenchymal transition and G2/M phase cell cycle. CVB­D exerted its antitumor effects by inhibiting the KIF11­CDK1­CDC25C­cyclinB1 G2/M phase transition regulatory oncogenic network and the NF­κB/JNK signaling pathway. CVB­D treatment significantly reduced the sizes and weights and malignancy of xenograft NSCLC tumors in the nude mice. In conclusion, the present study demonstrated that CVB­D inhibited the growth and progression of NSCLC cells by inhibiting the KIF11­CDK1­CDC25C­CyclinB1 G2/M phase transition regulatory network and the NF­κB/JNK signaling pathway. Therefore, CVB­D is a promising drug for the treatment of NSCLC patients.

Genetic Polymorphisms of Pneumocystis jirovecii in HIV-Positive and HIV-Negative Patients in Northern China.

Pneumocystis jirovecii is an opportunistic fungus that can cause severe and potentially fatal Pneumocystis pneumonia (PCP) in immunodeficient patients. In this study, we investigated the genetic polymorphisms of P. jirovecii at eight different loci, including six nuclear genes (ITS, 26S rRNA, sod, dhps, dhfr and ß-Tub) and two mitochondrial genes (mtLSU-rRNA and cyb) in three PCP cases, including two patients with HIV infection and one without HIV infection in Shanxi Province, P.R. China. The gene targets were amplified by PCR followed by sequencing of plasmid clones. The HIV-negative patient showed a coinfection with two genotypes of P. jirovecii at six of the eight loci sequenced. Of the two HIV-positive patients, one showed a coinfection with two genotypes of P. jirovecii at the same two of the six loci as in the HIV-negative patient, while the other showed a single infection at all eight loci sequenced. None of the three drug target genes (dhfr, dhps and cyb) showed mutations known to be potentially associated with drug resistance. This is the first report of genetic polymorphisms of P. jirovecii in PCP patients in Shanxi Province, China. Our findings expand our understanding of the genetic diversity of P. jirovecii in China.

Pneumocystis jirovecii colonization and its association with pulmonary diseases: a multicenter study based on a modified loop-mediated isothermal amplification assay.

BACKGROUND: Pneumocystis jirovecii (P. jirovecii) is an opportunistic fungal pathogen and the role of its colonization in pulmonary diseases has become a popular focus in recent years. The aim of this study was to develop a modified loop-mediated isothermal amplification (LAMP) assay for detection of Pneumocystis jirovecii (P. jirovecii) DNA amongst non-HIV patients with various pulmonary diseases and use it to examine the prevalence and assess the association of P. jirovecii colonization with clinical characteristics of these diseases. METHODS: We modified the previously reported LAMP assay for P. jirovecii by adding real-time detection. This method was used to detect P. jirovecii colonization in pulmonary samples collected from 403 non-HIV patients with various pulmonary diseases enrolled from 5 hospitals in China. We determined the prevalence of P. jirovecii colonization in 7 types of pulmonary diseases and assessed the association of P. jirovecii colonization with clinical characteristics of these diseases. RESULTS: The modified LAMP assay showed no cross-reactivity with other common pulmonary microbes and was 1000 times more sensitive than that of conventional PCR. Using the modified LAMP assay, we detected P. jirovecii colonization in 281 (69.7%) of the 403 patients enrolled. P. jirovecii colonization was more common in interstitial lung diseases than in chronic obstructive pulmonary disease (COPD) (84.6% vs 64.5%, P < 0.05). Patients with acute exacerbation of COPD had a higher prevalence of P. jirovecii colonization compared to patients with stabilized COPD (67.4% vs 43.3%, P < 0.05). P. jirovecii colonization was associated with decreased pulmonary function, increased levels of 1,3-ß-D-glucan and C-reactive protein, and decreased levels of CD4+ T-cell counts (P < 0.05 for each). Approximately 70% of P. jirovecii colonized patients had confections with other fungi or bacteria. CONCLUSIONS: We developed a modified LAMP assay for detecting P. jirovecii. Our multi-center study of 403 patients supports that P. jirovecii colonization is a risk factor for the development of pulmonary diseases and highlights the need to further study the pathogenesis and transmission of P. jirovecii colonization in pulmonary diseases.

Genotyping of Pneumocystis jirovecii by Use of a New Simplified Nomenclature System Based on the Internal Transcribed Spacer Regions and 5.8S rRNA Gene of the rRNA Operon.

Genotyping based on internal transcribed spacer 1 (ITS1) and ITS2 of the rRNA operon has played an important role in understanding the transmission and epidemiology of Pneumocystis jirovecii, one of the major opportunistic pathogens in individuals with AIDS and other immunocompromised individuals. The widespread use of this typing system has resulted in several problems, including inconsistent genotype nomenclatures, difficult data transferability, and complicated interpretation of the length variation in multiple homopolymeric tracts. The aim of this study was to establish a new, simplified genotype nomenclature system for P. jirovecii based on the ITS1 and ITS2 sequences. We first analyzed the complete ITS1, 5.8S rRNA gene, and ITS2 sequences (termed ITS1-5.8S-ITS2) in 27 recent P. jirovecii isolates from China and identified 18 unique genotypes. Subsequently, we performed a comprehensive classification of more than 400 ITS1- and ITS2-related sequences from GenBank and an in-depth evaluation of the length variation of multiple homopolymeric tracts within ITS1-5.8S-ITS2. Integration of the results from these analyses led to a new, simplified genotype nomenclature system including 62 unique ITS1-5.8S-ITS2 genotypes, simply designated types 1 through 62. This new system offers several advantages over traditional ITS1- and ITS2-based typing systems, including a simpler analysis and interpretation process, a higher discriminative power, and no limitation in assigning potential new genotypes. This new system is expected to facilitate the standardization of P. jirovecii genotyping and easy data exchanges across different laboratories.