Treatment of multidrug-resistant Klebsiella pneumoniae pneumonia in rats with the Wen Run Fei Ning formula: A preliminary study.

BACKGROUND & OBJECTIVES: To determine the effect of Wen Run Fei Ning formula (WRFNF) intervention in class I integron-mediated carbapenem-resistant Klebsiella pneumoniae. METHODS: A drug-susceptibility test and PCR amplification were used to screen for carbapenem-resistant K. pneumoniae containing class I integrons. Following nasal drip and tail vein injection to infect healthy male rats with carbapenem-resistant K. pneumoniae, three models were created: control (group A); model (group B, tail vein injection); and model-WRFNF treatment group (group C, by tail vein injection). Rats in Group C were gavaged with pre-warmed WRFNF extract. On the third, fifth, and seventh days after the experiment, the rats in groups A and B were gavaged with an equal quantity of saline and killed in batches. RESULTS: Group C showed considerably higher serum IL-6 and TNF- levels on days 3, 5, and 7 compared to group A, as well as a significant increase in peripheral blood leukocyte count and a histopathologic inflammatory cell infiltration of the lungs. As the WRFNF delivery duration was prolonged, group C's histopathologic inflammatory cell infiltration gradually improved in contrast to group B, with the biggest improvement occurring on day 7. Compared to group B, group C's serum IL-6 and TNF- levels were lower. When the trial's duration was increased to 7 days, the levels of IL-6 and TNF- in group C decreased on day 7 compared to on day 5. INTERPRETATION & CONCLUSION: WRFNF decreased inflammatory cell infiltration as well as IL-6 and TNF expression in the lung of the rats infected with carbapenem-resistant K. pneumoniae.

TRIM34 localizes to the mitochondria and mediates apoptosis through the mitochondrial pathway in HEK293T cells.

Tripartite motif 34 (TRIM34) is a member of TRIM family that can be highly induced by type I Interferon. Currently little is known about the subcellular localization and biological function of TRIM34. In the present study, confocal microscope assay showed that TRIM34 proteins were mainly distributed in the cytoplasm and part of TRIM34 proteins were localized to the mitochondria in human embryonic kidney 293T (HEK293T) cells. Western blot results demonstrated FLAG-TRIM34 could also be identified in the mitochondrial fractions of HEK293T cells transfected with the 5'FLAG-pcDNA3.1-TRIM34 vector. The CCK-8 assay further demonstrated that TRIM34 significantly decreased the viability of HEK293T cells. Nevertheless, TRIM34 had no apparent effect on the cell cycle distribution. Interestingly, flow cytometry showed that TRIM34 could obviously induce apoptosis in HEK293T cells. Moreover, we discovered that TRIM34 promoted apoptosis by inducing the loss of mitochondrial membrane potential (MMP) in HEK293T cells, leading to the release of cytochrome c from mitochondia. In short, these results demonstrate that TRIM34 proteins can localize to the mitochondria and induce apoptosis via the depolarization of MMP in HEK293T cells.

TRIM34 facilitates the formation of multinucleated giant cells by enhancing cell fusion and phagocytosis in epithelial cells.

Persistent microbial infection promotes the fusion of several kinds of somatic cells, such as macrophages and endothelial cells, leading to the formation of multinucleated giant cells (MGCs). However, the molecular mechanisms of MGCs formation are still poorly understood. By laser confocal microscope, we discovered that TRIM34 increased the efficiency of cell fusion in Human Embryonic Kidney cells (HEK293T). By means of DiD cell membrane probes, LysoTracker Deep Red or MitoTracker Deep Red staining, we also demonstrated that TRIM34 stimulated cell fusion in paraformaldehyde fixed or living HEK293T cells. Moreover, we discovered that the nuclei shapes of MGCs induced by TRIM34 were diversiform, such as horseshoe shape, ring like shape etc. Through 3D reconstruction of confocal z-stacks images, we found that TRIM34-EGFP proteins could form macromolecule aggregates in the central area of MGCs, while the nuclei were arranged in ring like shape and distributed around the plasma membrane. Cell fusion assay showed that cocultured TRIM34-EGFP+ cells and TRIM34-DsRed1+ cells could fuse to form MGCs. We speculate that the formation of MGCs can be divided into two phase: primary multinucleated cells (PMCs) and secondary multinucleated cells (SMCs). Firstly, TRIM34 induced fusion of multiple adjacent cells resulting in PMCs formation, and then PMCs were endowed with the capacity of phagocytosis and turned into SMCs. Collectively, these results suggest that TRIM34 proteins contribute to the formation of MGCs by promoting cell fusion and phagocytosis in epithelial cells.

[Tripartite motif-containing protein 34 (TRIM34) colocalized with micronuclei chromosome and hampers its movement to equatorial plate during the metaphase stage of mitosis].

Objective To examine whether tripartite motif-containing protein 34 (TRIM34) is colocalized with micronuclei and investigate the influence on the movement of micronuclei chromosome in mitosis. Methods The eukaryotic expression vector TRIM34-pEGFP-N3 was constructed, identified and then transfected into HEK293T cells. With 4', 6-diamidino-2-phenylindole 2HCI (DAPI) staining, the colocalization between TRIM34 and micronuclei was observed under a fluorescence microscope. Moreover, MitoTracker(R)Deep Red was used to identify the colocalization between the complex of TRIM34-micronulei and mitochondria under a confocal microscope. Finally, the effect of TRIM34 on the movement of micronuclei chromosome in mitosis was examined. Results DNA sequencing confirmed that the vector TRIM34-pEGFP-N3 was constructed successfully. A fluorescence microscope revealed that TRIM34 could be colocalized with micronuclei in HEK293T cells transfected with TRIM34-pEGFP-N3. In the same manner, a confocal microscope distinctly showed that TRIM34 was colocalized with micronuclei similarly in appearance. However, there was no distinguished colocalization relationship between the complex of TRIM34-micronulei and mitochondria. Interestingly, the micronuclei chromosome conjugated with TRIM34 was hardly transferred to equatorial plate during the metaphase stage of mitosis. Conclusion TRIM34 is colocalized with micronuclei chromosome and hampers its movement to equatorial plate in mitosis.

[Expressions and co-localization of HIV capsid protein p24 and TRIM22 in HEK293T cells].

OBJECTIVE: To observe the co-localization of human immunodeficiency virus (HIV) capsid protein p24 and tripartite motif containing 22 (TRIM22) in HEK293T cells. METHODS: The retroviral packaging vector pLP1 was used as the template of p24. After the amplification by PCR, the sequence of p24 was cloned into the eukaryotic expression vector pDsRed-Monomer-N1. The recombinant vector was confirmed by colony PCR, double restriction enzyme digestion and DNA sequencing. HEK293T cells were co-transfected with the vector pDsRed-Monomer-N1-p24 together with pEGFP-N3-TRIM22. After 24 hours, the co-localization of p24-DsRed-Monomer and TRIM22-EGFP was detected under a fluorescence microscope. RESULTS: Colony PCR, double restriction enzyme digestion and DNA sequencing confirmed that the eukaryotic expression vector pDsRed-Monomer-N1-p24 was constructed successfully. Fluorescence microscope showed that p24-DsRed-Monomer was co-localized with TRIM22-EGFP in HEK293T cells. CONCLUSION: HIV capsid protein p24 is co-localized with TRIM22 in HEK293T cells.

[Construction of Trim6 eukaryotic expression vector and its expression in HEK293 cells].

AIM: To construct the recombinant eukaryotic expression vector pcDNA3.1 (+)-Trim6, and observe its expression in HEK293T cells in vitro. METHODS: The total RNA was isolated from HeLa cells. After amplification with reverse transcription polymerase chain reaction (RT-PCR), the target sequences were cloned into the pcDNA3.1(+). The recombinant vector was confirmed by restriction enzyme digestion, PCR and sequencing. Then it was transfected into HEK293T cells.After 24 hours, the Trim6 expression was detected by Western blot. RESULTS: The results of the restriction enzyme digestion, PCR and sequencing confirmed the vector was constructed successfully, and it can express Trim6 protein in HEK293T cells. CONCLUSION: The vector is constructed successfully, which establishes the foundation for future research on the effect of Trim6.