[The application of next-generation sequencing in the diagnosis of Pneumocystis pneumonia in non-HIV infected patients].

Objective: To evaluate the clinical value of next-generation sequencing in the diagnosis of Pneumocystis pneumonia in non-HIV infected patients. Methods: A retrospective study was conducted on the diagnosis and treatment of Pneumocystis pneumonia in 5 non-HIV patients in the Fourth Medical Center of the General Hospital of the PLA from September 1, 2017 to September 1, 2018. Next-generation sequencing of BALF were compared with the traditional laboratory microbiological test, and the advantages of the next-generation sequencing in the diagnosis of Pneumocystis pneumonia in non-HIV infected patients were analyzed. Results: There were 3 males and 2 females, with a mean age (48±6) years. Three patients had membranous nephropathy, a patient had tuberculous meningitis, and a patient had esophageal cancer after radiotherapy and chemotherapy. All patients had glucocorticoid medication history before. The clinical manifestations were fever, cough and dyspnea. The chest CT mainly showed bilateral lung ground glass shadows. All the results of 1, 3-ß-D-glucan test were more than 1 000 ng/L. Bronchoalveolar lavage was performed in the 5 cases, and Pneumocystis cysts were found in 1 BALF by Gomori's methenamine silver nitrate staining, and the DNAs of Pneumocystis and human herpesvirus were detected in 5 BALFs by next-generation sequencing. All patients were treated with sulfamethoxazole/trimethoprim (orally, 1.44 g, q8 h) for 23 to 72 days (median 33 days), and with ganciclovir(Ⅳ, 250 mg q12 h) for 6 to 22 days (median 15 days). The chest CT manifestations and symptoms were improved after treatment, without death. Conclusions: The next-generation sequencing of BALF is more specific and sensitive in the diagnosis of Pneumocystis pneumoniae in non-HIV patients. It is faster, more comprehensive and more accurate than the traditional laboratory test, and could be widely used as a PCP diagnosis technique.

HOTAIR alleviates ox-LDL-induced inflammatory response in Raw264.7 cells via inhibiting NF-κB pathway.

OBJECTIVE: To investigate the possible role of hox transcript antisense intergenic RNA (HOTAIR) in the pathogenesis of atherosclerosis and its underlying mechanism. PATIENTS AND METHODS: The expression of HOTAIR in peripheral blood lymphocytes of atherosclerosis (AS) and healthy controls was detected by quantitative Real-time-polymerase chain reaction (qRT-PCR). In vitro AS model was established by ox-LDL induction in Raw264.7 cells. Viability of Raw264.7 cells after ox-LDL induction was detected by cell counting kit-8 (CCK-8) assay. Levels of TC (total cholesterol), TG (triglyceride), LDL-C (low density lipoprotein cholesterol) and HDL-C (high density lipoprotein cholesterol) in Raw264.7 cells were detected by enzyme-linked immunosorbent assay (ELISA). Overexpression plasmid of HOTAIR was constructed. Levels of TG, TC, LDL-C, and HDL were detected again after HOTAIR overexpression by ELISA. CD68+ cells and CD168+ cells in Raw264.7 cells were detected by flow cytometry. Protein expressions of pro-inflammatory and anti-inflammatory genes were detected by Western blot. Lipid metabolism in Raw264.7 cells was evaluated by oil red O staining and Western blot, respectively. Finally, rescue experiments were conducted to explore the specific mechanism of HOTAIR in regulating AS development. RESULTS: HOTAIR was lowly expressed in peripheral blood lymphocytes of AS patients and Raw264.7 cells induced by ox-LDL. Overexpression of HOTAIR upregulated adipose genes (PPARα and CPT-1) and downregulated lipogenesis genes (SREBP-1c and ACS). Besides, overexpression of HOTAIR decreased expressions of pro-inflammatory cytokines (TNF-α and IL-1ß), but increased expressions of anti-inflammatory cytokines (IL-4 and IL-10). In the in vitro AS model, FXR1 was remarkably downregulated in Raw264.7 cells. HOTAIR reduced inflammatory response via promoting FXR1 expression in Raw264.7 cells. Rescue experiments showed that the effect of HOTAIR on nuclear factor-kappa B (NF-κB) pathway was reversed by FXR1 knockdown. CONCLUSIONS: We found that TAIR was lowly expressed in AS patients. Overexpression of HOTAIR can reduce the lipid accumulation and inhibit inflammatory response by suppressing FXR1 via NF-κB pathway.

Downregulated long non-coding RNA TRPM2-AS inhibits cisplatin resistance of non-small cell lung cancer cells via activation of p53- p66shc pathway.

OBJECTIVE: Non-small cell lung cancer (NSCLC), as an ordinary malignant tumor, presents with high death rate and poor prognosis. Few literatures have explored the association between NSCLC development and lncRNAs expression. This study focuses on the important role of a novel lncRNA TRPM2-AS in the development of chemo-resistance in NSCLC. MATERIALS AND METHODS: The expression level of lncRNA TRPM2-AS was identified by using qRT-PCR assay. The apoptosis rate and the alteration of the cell cycle were detected by the flow cytometric analysis. Cell Counting Kit-8 assay (CCK8) was utilized for detecting chemo-sensitivity of the cisplatin-resistant A549/DDP cells. The p53 and p66shc protein levels were detected by Western blotting assay. RESULTS: A549/DDP cells presented remarkably higher expression of lncRNA TRPM2-AS than paired A549 cells. Moreover, re-sensitization to cisplatin was seen in A549/DDP cells after lncRNA TRPM2-AS knockdown. On the contrary, the sensitivity of lncRNA TRPM2-AS-overexpressed A549 cells to cisplatin decreased obviously when compared with the control. Furthermore, downregulated lncRNA TRPM2-AS induced cell apoptosis and altered cell cycle distribution through activating the p53-p66shc pathway. CONCLUSIONS: We suggest that lncRNA TRPM2-AS participates in the resistance of NSCLC cells to cisplatin, which may provide a new therapeutic target of NSCLC.

Effect of antisense oligonucleotide against mouse dentine matrix protein 1 on mineralization ability and calcium ions metabolism in odontoblast-like cell line MDPC-23.

AIM: To study the mineralization ability and the dynamic changes of intracellular and extracellular concentrations of calcium ions in the odontoblast-like cell line MDPC-23 affected by antisense oligonucleotide (AS-ODN) against mouse dentine matrix protein 1 (DMP1). METHODOLOGY: The expression of DMP1 in MDPC-23 cells was detected by an immunohistochemical method and its blocking outcome by the Western blot method. The alkaline phosphatase (ALP) activity, size and number of mineralized nodules, and the intracellular free ([Ca2+]if), total ([Ca2+]it) and the extracellular ([Ca2+]e) calcium ion concentrations in MDPC-23 cells in the experimental group affected with AS-ODN were compared with those in the control group (paired-samples t-test). RESULTS: Dentine matrix protein 1 was stably expressed in a stable way in MDPC-23 cells; the expression was only just detectable at 12 h and became negative after 24 h affected by AS-ODN. Compared with the control groups, ALP activity of MDPC-23 cells in the AS-ODN group was decreased (P < 0.05), and both the number and size of mineralized nodules were smaller than those in the control group. [Ca2+]if in the AS-ODN group increased and then decreased after 24 h. [Ca2+]it dropped substantially to the lowest point at 24 h (P < 0.01). [Ca2+]e increased before treatment for 24 h and then dropped, however, it was still higher than that of the control group. CONCLUSIONS: Antisense oligonucleotide against DMP1 could decrease mineralization ability and affect the intracellular and extracellular concentrations of calcium ions in MDPC-23 cells. This would indicate that DMP1 regulates the metabolism and transportation of calcium ions in odontoblasts, and thus boosts dentine mineralization.