Differential m6A methylomes between two major life stages allows potential regulations in Trypanosoma brucei.

N6-methyladenosine (m6A) is the most prevalent mRNA modification in higher eukaryotes. Recent studies suggest that m6A has a regulatory role in mRNA degradation and translation initiation or efficiency, involving in cell fate determination in yeast, plants, and stem cells of mammalian. Trypanosoma brucei (T. brucei) regulates gene expression through post-transcriptional fashion, which heavily relies on mRNA cis-motifs. However, internal mRNA modification in T. brucei has not been reported yet. Here we found m6A modification is abundant in T. brucei and presented a transcriptome wide methylome of m6A in both life stages of T. brucei. We identified 355 and 95 peaks in procyclic form and blood stream form trypanosomes respectively. A consensus motif of CAU was shared in both life stages of T. brucei. mRNA abundance of m6A-containing genes is higher in procyclic form and tend to be down-regulated in bloodstream form trypanosomes. Furthermore, m6A-containing transcripts harbor relative longer half-lives, and are enriched in pathways of cell morphology and movement in procyclic form trypanosomes. By m6A-containing RNA pulldown in both life stages, we identified TRRM2 as a potential m6A reader in T. brucei. Uncovering the m6A methylome and its binding proteins may provide a new post-transcriptional regulatory pathway in T. brucei.

Salinomycin triggers endoplasmic reticulum stress through ATP2A3 upregulation in PC-3 cells.

BACKGROUND: Salinomycin is a monocarboxylic polyether antibiotic and is a potential chemotherapy drug. Our previous studies showed that salinomycin inhibited cell growth and targeted CSCs in prostate cancer. However, the precise target of salinomycin action is unclear. METHODS: In this work, we analyzed and identified differentially expressed genes (DEGs) after treatment with or without salinomycin using a gene expression microarray in vitro (PC-3 cells) and in vivo (NOD/SCID mice xenograft model generated from implanted PC-3 cells). Western blotting and immunohistochemical staining were used to analyze the expression of ATP2A3 and endoplasmic reticulum (ER) stress biomarkers. Flow cytometry was used to analyze the cell cycle, apoptosis and intracellular Ca2+ concentration. RESULTS: A significantly upregulated gene, ATPase sarcoplasmatic/endoplasmatic reticulum Ca2+ transporting 3 (ATP2A3), was successfully identified. In subsequent studies, we found that ATP2A3 overexpression could trigger ER stress and exert anti-cancer effects in PC-3 and DU145 cells. ATP2A3 was slightly expressed, but the ER stress biomarkers showed strong staining in prostate cancer tissues. We also found that salinomycin could trigger ER stress, which might be related to ATP2A3-mediated Ca2+ release in PC-3 cells. Furthermore, we found that salinomycin-triggered ER stress could promote apoptosis and thus exert anti-cancer effects in prostate cancer cells. CONCLUSION: This study demonstrates that ATP2A3 might be one of the potential targets for salinomycin, which can inhibit Ca2+ release and trigger ER stress to exert anti-cancer effects.

The influence of HK2 blood group antigen on human B cell activation for ABOi-KT conditions.

BACKGROUND: It is well known that ABO blood group system incompatible kidney transplantation (ABOi-KT) is an effective strategy for end-stage renal disease. The main barrier for ABOi-KT is how to keep host B cell activation and blood group antibody titer in low levels. Moreover, the mechanism of B cell activation induced by blood group antigen was unclear in ABOi-KT. RESULTS: In this study, HK2 cells were identified to express blood group B antigen when cocultured with lymphocytes of blood group A. Optical microscope observation demonstrated that HK2 cells in coculture group gradually decreased. Furthermore, flow cytometer assay identified that T cell phenotypes (CD3+, CD3+CD4+ and CD3+CD8+) had no significant change and B cell phenotypes (CD19+ and CD138+) were all significantly enhanced (3.07 and 3.02 folds) at day 4. In addition, immunoturbidimetry analysis demonstrated that blood group B antibody was significantly increased to 2.35 fold at day 4, IgG was significantly increased to 3.60 and 2.81 folds at days 4 and 8 respectively, while IgM had no significant change at the measured time points. CONCLUSIONS: Taken together, B cells were activated and secreted blood group B antibody after treatment with HK2 expressing blood group B antigen. The results of this study maybe useful for further determination of the mechanism of B cell activation after ABO incompatible kidney endothelial cells stimulation.

Clinical and Molecular Characteristics and Antibacterial Strategies of Klebsiella pneumoniae in Pyogenic Infection.

Treatment of Klebsiella pneumoniae causing pyogenic infections is challenging. The clinical and molecular characteristics of Klebsiella pneumoniae causing pyogenic infections are poorly understood, and antibacterial treatment strategies are limited. We analyzed the clinical and molecular characteristics of K. pneumoniae from patients with pyogenic infections and used time-kill assays to reveal the bactericidal kinetics of antimicrobial agents against hypervirulent K. pneumoniae (hvKp). A total of 54 K. pneumoniae isolates were included, comprising 33 hvKp and 21 classic K. pneumoniae (cKp) isolates, and the hvKp and cKp isolates were identified using five genes (iroB, iucA, rmpA, rmpA2, and peg-344) that have been applied as hvKp strain markers. The median age of all cases was 54 years (25th and 75th percentiles, 50.5 to 70), 62.96% of individuals had diabetes, and 22.22% of isolates were sourced from individuals without underlying disease. The ratios of white blood cells/procalcitonin and C-reactive protein/procalcitonin were potential clinical markers for the identification of suppurative infection caused by hvKp and cKp. The 54 K. pneumoniae isolates were classified into 8 sequence type 11 (ST11) and 46 non-ST11 strains. ST11 strains carrying multiple drug resistance genes have a multidrug resistance phenotype, while non-ST11 strains carrying only intrinsic resistance genes are generally susceptible to antibiotics. Bactericidal kinetics revealed that hvKp isolates were not easily killed by antimicrobials at susceptible breakpoint concentrations compared with cKp. Given the varied clinical and molecular features and the catastrophic pathogenicity of K. pneumoniae, it is critical to determine the characteristics of such isolates for optimal management and effective treatment of K. pneumoniae causing pyogenic infections. IMPORTANCE Klebsiella pneumoniae may cause pyogenic infections, which are potentially life-threatening and bring great challenges for clinical management. However, the clinical and molecular characteristics of K. pneumoniae are poorly understood, and effective antibacterial treatment strategies are limited. We analyzed the clinical and molecular features of 54 isolates from patients with various pyogenic infections. We found that most patients with pyogenic infections had underlying diseases, such as diabetes. The ratio of white blood cells to procalcitonin and the ratio of C-reactive protein to procalcitonin were potential clinical markers for differentiating hypervirulent K. pneumoniae strains from classical K. pneumoniae strains that cause pyogenic infections. K. pneumoniae isolates of ST11 were generally more resistant to antibiotics than non-ST11 isolates. Most importantly, hypervirulent K. pneumoniae strains were more tolerant to antibiotics than classic K. pneumoniae isolates.

Identification of an activation-related protein in B cells in the ABO incompatible condition.

In ABO-incompatible (ABOi) kidney transplantation (KT), antibodies can mediate immunological accommodation or immune rejection, but the mechanism by which B cells are induced to produce antibodies with different functions is still unclear. Previous research established an ABOi kidney cell model and identified that haptoglobin (HP) is associated with the activation of lymphocytes. In the present study, the results of a flow cytometric assay demonstrated that HP was expressed by B cells. Moreover, dot-ELISA and ELISA analyses showed that the concentrations of total IgG, blood group B antibody, IgG1, IgG2 and IgG4 were all significantly increased in the cell model. In addition, dot-ELISA and haptoglobin level analyses showed that HP protein expression was significantly increased, while RT-qPCR assay indicated that HP was significantly reduced at the mRNA level. Furthermore, bioinformatics analysis showed that HP could interact with Smad3, and the HP-Smad3 complex was detected in a peripheral blood mononuclear cell (PBMC) protein extract by a dot-ELISA method. This research revealed that HP was involved in the process of B-cell activation by interacting with Smad3, and the results will be helpful to reveal the mechanism of B-cell activation in ABOi-KT.

[Overexpression of autophagy-related gene 3 promotes autophagy and inhibits salinomycin-induced apoptosis in breast cancer MCF-7 cells].

OBJECTIVE: To study the effects of the overexpression of autophagy-related gene 3 (ATG3) on autophagy and salinomycin-induced apoptosis in breast cancer cells and explore the underlying mechanisms. METHODS: We used the lentivirus approach to establish a breast cancer cell line with stable overexpression of ATG3. Western blotting, immunofluorescence staining and transmission electron microscopy were used to analyze the effect of ATG3 overexpression on autophagy in breast cancer MCF-7 cells. Using the AKT/mTOR agonists SC79 and MHY1485, we analyzed the effect of AKT/mTOR signal pathway activation on ATG3 overexpression-induced autophagy. Western blotting and flow cytometry were used to analyze the effect of autophagy on apoptosis of the ATG3-overexpressing cells treated with salinomycin and 3-MA (an autophagy inhibitor). RESULTS: In ATG3-overexpressing MCF-7 cells, ATG3 overexpression obviously promoted autophagy, inhibited the AKT/mTOR signaling pathway, significantly weakened salinomycin-induced apoptosis (P < 0.01), caused significant reduction of the levels of the pro-apoptotic proteins cleaved-caspase 3 (P < 0.01) and Bax (P < 0.05), and enhanced the expression of the anti-apoptotic protein Bcl-2 (P < 0.05). The inhibition of autophagy obviously weakened the inhibitory effect of ATG3 overexpression on salinomycin-induced apoptosis. CONCLUSIONS: ATG3 overexpression promotes autophagy possibly by inhibiting the AKT/mTOR signaling pathway to decrease salinomycin-induced apoptosis in MCF-7 cells, suggesting that autophagy induction might be one of the mechanisms of drug resistance in breast cancer cells.

Salinomycin-induced autophagy blocks apoptosis via the ATG3/AKT/mTOR signaling axis in PC-3 cells.

AIMS: This study evaluated the mechanism by which salinomycin-induced autophagy blocks apoptosis in PC-3 prostate cancer cells. MAIN METHODS: The anti-cancer effects of salinomycin in PC-3 cells were confirmed by flow cytometry, JC-1 staining and western blotting. Then, the autophagic effects were measured by western blotting, GFP-LC3 puncta formation assay, immunofluorescence staining and electron microscopy. Furthermore, we used lentivirus-mediated shRNA to silence ATG3, ATG5 and ATG7 expression in PC-3 cells to investigate the regulatory mechanisms of salinomycin-induced autophagy. KEY FINDINGS: Salinomycin could induce apoptosis and autophagy in PC-3 cells. Interestingly, autophagy inhibition could enhance salinomycin-induced apoptosis. We further showed that ATG3, a known critical regulator of autophagy, was downregulated and involved in the inhibition of apoptosis by salinomycin-induced autophagy via the AKT/mTOR signaling axis. SIGNIFICANCE: Our data indicated that salinomycin-induced autophagy blocks apoptosis via the ATG3/AKT/mTOR signaling axis in PC-3 cells, which provides new clues for the mechanisms of underlying the anti-cancer effects of salinomycin.

Salinomycin Exerts Anticancer Effects on PC-3 Cells and PC-3-Derived Cancer Stem Cells In Vitro and In Vivo.

Salinomycin is an antibiotic isolated from Streptomyces albus that selectively kills cancer stem cells (CSCs). However, the antitumor mechanism of salinomycin is unclear. This study investigated the chemotherapeutic efficacy of salinomycin in human prostate cancer PC-3 cells. We found that cytotoxicity of salinomycin to PC-3 cells was stronger than to nonmalignant prostate cell RWPE-1, and exposure to salinomycin induced G2/M phage arrest and apoptosis of PC-3 cells. A mechanistic study found salinomycin suppressed Wnt/ß-catenin pathway to induce apoptosis of PC-3 cells. An in vivo experiment confirmed that salinomycin suppressed tumorigenesis in a NOD/SCID mice xenograft model generated from implanted PC-3 cells by inhibiting the Wnt/ß-catenin pathway, since the total ß-catenin protein level was reduced and the downstream target c-Myc level was significantly downregulated. We also showed that salinomycin, but not paclitaxel, triggered more apoptosis in aldehyde dehydrogenase- (ALDH-) positive PC-3 cells, which were considered as the prostate cancer stem cells, suggesting that salinomycin may be a promising chemotherapeutic to target CSCs. In conclusion, this study suggests that salinomycin reduces resistance and relapse of prostate tumor by killing cancer cells as well as CSCs.