Combatting persister cells: The daunting task in post-antibiotics era.

Over the years, much attention has been drawn to antibiotic resistance bacteria, but drug inefficacy caused by a subgroup of special phenotypic variants - persisters - has been largely neglected in both scientific and clinical field. Interestingly, this subgroup of phenotypic variants displayed their power of withstanding sufficient antibiotics exposure in a mechanism different from antibiotic resistance. In this review, we summarized the clinical importance of bacterial persisters, the evolutionary link between resistance, tolerance, and persistence, redundant mechanisms of persister formation as well as methods of studying persister cells. In the light of our recent findings of membrane-less organelle aggresome and its important roles in regulating bacterial dormancy depth, we propose an alternative approach for anti-persister therapy. That is, to force a persister into a deeper dormancy state to become a VBNC (viable but non-culturable) cell that is incapable of regrowth. We hope to provide the latest insights on persister studies and call upon more research interest into this field.

MicroRNA-449a inhibits cell proliferation and migration by regulating mutant p53 in MDA-MB-468 cells.

The present study aimed to investigate the role of microRNA (miR)-449a in the proliferation, migration and apoptosis of MDA-MB-468 breast cancer cells and examine the association between miR-449a and mutant p53 in these cells. Cell proliferation, migration and invasion were examined using a crystal violet staining assay, wound healing scratch assay and Transwell assay, respectively. The expression level of miR-449a and p53 was detected by reverse transcription-quantitative PCR or western blotting. The results indicated that knockdown of mutant p53 suppressed the proliferation and migration of MDA-MB-468 cells by inhibiting the PI3K/AKT/mTOR signaling pathway. In addition, miR-449a suppressed proliferation and migration via downregulation of mutant p53 expression in MDA-MB-468 cells. Therefore, miR-449a may function as a tumor suppressor by regulating p53 expression in breast cancer cells, which may have potential implications in the treatment of patients with triple-negative breast cancer carrying mutant p53.

Autophagy-dependent cell cycle arrest in esophageal cancer cells exposed to dihydroartemisinin.

BACKGROUND: Dihydroartemisinin (DHA), a derivate of artemisinin, is an effective antimalarial agent. DHA has been shown to exert anticancer activities to numerous cancer cells in the past few years, while the exact molecular mechanisms remain to be elucidated, especially in esophageal cancer. METHODS: Crystal violet assay was conducted to determine the cell viability of human esophageal cancer cell line Eca109 treated with DHA. Tumor-bearing nude mice were employed to evaluate the anticancer effect of DHA in vivo. Soft agar and crystal violet assays were used to measure the tumorigenicity of Eca109 cells. Flow cytometry was performed to evaluate ROS or cell cycle distribution. GFP-LC3 plasmids were delivered into Eca109 cells to visualize autophagy induced by DHA under a fluorescence microscope. The mRNA and protein levels of each gene were tested by qRT-PCR and western blot, respectively. RESULTS: Our results proved that DHA significantly reduced the viability of Eca109 cells in a dose- and time-dependent manner. Further investigation showed that DHA evidently induced cell cycle arrest at the G2/M phase in Eca109 cells. Mechanistically, DHA induced intracellular ROS generation and autophagy in Eca109 cells, while blocking ROS by an antioxidant NAC obviously inhibited autophagy. Furthermore, we found that telomere shelterin component TRF2 was down-regulated in Eca109 cells exposed to DHA through autophagy-dependent degradation, which could be rescued after autophagy was blocked by ROS inhibition. Moreover, the DNA damage response (DDR) was induced obviously in DHA treated cells. To further explore whether ROS or autophagy played a vital role in DHA induced cell cycle arrest, the cell cycle distribution of Eca109 cells was evaluated after ROS or autophagy blocking, and the results showed that autophagy, but not ROS, was essential for cell cycle arrest in DHA treated cells. CONCLUSION: Taken together, DHA showed anticancer effect on esophageal cancer cells through autophagy-dependent cell cycle arrest at the G2/M phase, which unveiled a novel mechanism of DHA as a chemotherapeutic agent, and the degradation of TRF2 followed by DDR might be responsible for this cell phenotype.

The PI3K/mTOR dual inhibitor NVP-BEZ235 stimulates mutant p53 degradation to exert anti-tumor effects on triple-negative breast cancer cells.

Nearly half of human cancers harbor p53 mutations, and mutant p53 (mutp53) promotes carcinogenesis, metastasis, tumor recurrence and chemoresistance. mutp53 is observed in 30% of breast carcinomas, including triple-negative breast cancer (TNBC), and thus mutp53 is a promising target for treatment of TNBC. In this study, we investigated the effect of a phosphatidylinositide 3 kinase/mammalian target of rapamycin dual inhibitor, NVP-BEZ235 (BEZ235), on two TNBC cell lines with mutp53: MDA-MB-231 and MDA-MB-468. Cell growth, migration and colony-formation abilities were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, scratch assay, transwell and soft agar assay, revealing that BEZ235 can inhibit the growth, migration and colony-formation abilities of TNBC cells. In addition, BEZ235 caused degradation of mutp53 in these cells. We investigated the underlying mechanism by inhibiting proteasome function using MG132 and inhibiting autophagy using 3-methyladenine and shRNAs. We observed that BEZ235 may induce autophagy through repression of the Akt/mammalian target of rapamycin signaling pathway. The observed interplay between mutp53 and autophagy in TNBC cells was examined further by knockdown of ATG5 and ATG7, revealing that degradation of mutp53 induced by BEZ235 may be independent of the ubiquitin-proteasome pathway and autophagy mediated by ATG5 and ATG7. Moreover, we found evidence of positive feedback between mutp53 and autophagy in TNBC cells. In conclusion, BEZ235 may exert antitumor effects against TNBC cells by targeting mutp53, and this may have implications for the development of future therapies.

Aberrant expression of long non-coding RNAs in peripheral blood mononuclear cells isolated from patients with gouty arthritis.

Gouty arthritis (GA) is the most common inflammatory and immune-associated disease, and its prevalence and incidence exhibit yearly increases. The aim of the present study was to analyse the expression profile variation of long non-coding RNAs (lncRNAs) in GA patients and to explore the role of lncRNAs in the pathogenesis of GA. The peripheral blood mononuclear cells of GA patients and of healthy controls (HCs) were used to detect for the differentially expressed lncRNAs by microarray. The functional annotations and classifications of the differentially expressed transcripts were predicted using Gene Ontology (GO) and pathway analysis. The results were then verified by reverse transcription-quantitative (RT-q)PCR. A total of 1,815 lncRNAs and 971 mRNAs with a >2-fold difference in the levels of expression in the GA patients compared with those in the HCs were identified. According to the GO functional enrichment analysis, the differentially expressed lncRNAs were accumulated in terms including protein binding, catalytic activity and molecular transducer activity. The pathways predicted to be involved were the tumor necrosis factor signaling pathway, osteoclast differentiation, NOD-like receptor signaling pathway and NF-κB signaling pathway. The expression of six lncRNAs was measured by RT-qPCR and the results were consistent with those of the microarrays. Among these lncRNAs, AJ227913 was the most differentially expressed lncRNA in GA patients vs. HCs. The expression of several lncRNAs was significantly changed in GA patients compared with that in HCs, which suggests that these lncRNAs with differential expression levels may have an important role in the development and progression of GA.

Dihydroartemisinin inhibits HepG2.2.15 proliferation by inducing cellular senescence and autophagy.

Dihydroartemisinin (DHA) has been reported to possess anti-cancer activity against many cancers. However, the pharmacologic effect of DHA on HBV-positive hepatocellular carcinoma (HCC) remains unknown. Thus, the objective of the present study was to determine whether DHA could inhibit the proliferation of HepG2.2.15 cells and uncover the underlying mechanisms involved in the effect of DHA on HepG2.2.15 cells. We found that DHA effectively inhibited HepG2.2.15 HCC cell proliferation both in vivo and in vitro. DHA also reduced the migration and tumorigenicity capacity of HepG2.2.15 cells. Regarding the underlying mechanisms, results showed that DHA induced cellular senescence by up-regulating expression levels of proteins such as p-ATM, p-ATR, γ-H2AX, P53, and P21 involved in DNA damage response. DHA also induced autophagy (green LC3 puncta gathered together and LC3II/LC3I ratio increased through AKT-mTOR pathway suppression). Results also revealed that DHA-induced autophagy was not linked to senescence or cell death. TPP1 (telomere shelterin) overexpression could not rescue DHA-induced anticancer activity (cell proliferation). Moreover, DHA down-regulated TPP1 expression. Gene knockdown of TPP1 caused similar phenotypes and mechanisms as DHA induced phenotypes and mechanisms in HepG2.2.15 cells. These results demonstrate that DHA might inhibit HepG2.2.15 cells proliferation through inducing cellular senescence and autophagy. [BMB Reports 2019; 52(8): 520-524].

Identification of crucial miRNAs and genes in esophageal squamous cell carcinoma by miRNA-mRNA integrated analysis.

Esophageal squamous cell carcinoma (ESCC) is a malignancy that severely threatens human health and carries a high incidence rate and a low 5-year survival rate. MicroRNAs (miRNAs) are commonly accepted as a key regulatory function in human cancer, but the potential regulatory mechanisms of miRNA-mRNA related to ESCC remain poorly understood.The GSE55857, GSE43732, and GSE6188 miRNA microarray datasets and the gene expression microarray datasets GSE70409, GSE29001, and GSE20347 were downloaded from Gene Expression Omnibus databases. The differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) were obtained using GEO2R. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for DEGs were performed by Database for Annotation, Visualization and Integrated Discovery (DAVID). A protein-protein interaction (PPI) network and functional modules were established using the STRING database and were visualized by Cytoscape. Kaplan-Meier analysis was constructed based on The Cancer Genome Atlas (TCGA) database.In total, 26 DEMs and 280 DEGs that consisted of 96 upregulated and 184 downregulated genes were screened out. A functional enrichment analysis showed that the DEGs were mainly enriched in the ECM-receptor interaction and cytochrome P450 metabolic pathways. In addition, MMP9, PCNA, TOP2A, MMP1, AURKA, MCM2, IVL, CYP2E1, SPRR3, FOS, FLG, TGM1, and CYP2C9 were considered to be hub genes owing to high degrees in the PPI network. MiR-183-5p was with the highest connectivity target genes in hub genes. FOS was predicted to be a common target gene of the significant DEMs. Hsa-miR-9-3p, hsa-miR-34c-3p and FOS were related to patient prognosis and higher expression of the transcripts were associated with a poor OS in patients with ESCC.Our study revealed the miRNA-mediated hub genes regulatory network as a model for predicting the molecular mechanism of ESCC. This may provide novel insights for unraveling the pathogenesis of ESCC.

Identification and validation of key genes associated with non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) is one of the main causes of death induced by cancer globally. However, the molecular aberrations in NSCLC patients remain unclearly. In the present study, four messenger RNA microarray datasets (GSE18842, GSE40275, GSE43458, and GSE102287) were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) between NSCLC tissues and adjacent lung tissues were obtained from GEO2R and the overlapping DEGs were identified. Moreover, functional and pathway enrichment were performed by Funrich, while the protein-protein interaction (PPI) network construction were obtained from STRING and hub genes were visualized and identified by Cytoscape software. Furthermore, validation, overall survival (OS) and tumor staging analysis of selected hub genes were performed by GEPIA. A total of 367 DEGs (95 upregulated and 272 downregulated) were obtained through gene integration analysis. The PPI network consisted of 94 nodes and 1036 edges in the upregulated DEGs and 272 nodes and 464 edges in the downregulated DEGs, respectively. The PPI network identified 46 upregulated and 27 downregulated hub genes among the DEGs, and six (such as CENPE, NCAPH, MYH11, LRRK2, HSD17B6, and A2M) of that have not been identified to be associated with NSCLC so far. Moreover, the expression differences of the mentioned hub genes were consistent with that in lung adenocarcinoma and lung squamous cell carcinoma in the TCGA database. Further analysis showed that all the six hub genes were associated with tumor staging except MYH11, while only the upregulated DEG CENPE was associated with the worse OS of patients with NSCLC. In conclusion, the current study showed that CENPE, NCAPH, MYH11, LRRK2, HSD17B6, and A2M might be the key genes contributed to tumorigenesis or tumor progression in NSCLC, further functional study is needed to explore the involved mechanisms.

Quorum-sensing systems trigger catalase expression to reverse the oxyR deletion-mediated VBNC state in Salmonella typhimurium.

OxyR is an important regulatory protein that plays a key role in anti-oxygenation, and its deletion causes a special VBNC (Viable But Non-Culturable) state in many bacteria including Salmonella typhimurium. The S. typhimurium in the VBNC state can grow in LB broth but cannot grow on an LB plate unless its concentration is sufficiently high. However, the mechanism that reverses this state is not clear. In this study, conditioned media containing autoinducer collected from the wild type strain can restore the growth of low concentrations of the oxyR mutant strain on LB plates, and S. typhimurium collected from the plate has higher catalase activity than that from the broth, suggesting that a quorum-sensing system can trigger catalase expression to resuscitate the organism from the VBNC state independent of the OxyR regulon. We discovered a novel catalase (STM14_2049, Cat) whose expression is strictly concentration-dependent. The purified Cat protein has obvious catalase activity in vitro and in vivo and can restore the growth of the low concentration oxyR mutant strain. Thus, we believed Cat plays a role in VBNC resuscitation process. By understanding this mechanism, we can further understand the antioxidation and quorum-sensing systems in Salmonella typhimurium.

The special existences: nanoRNA and nanoRNase.

To adapt to a wide range of nutritional and environmental changes, cells must adjust their gene expression profiles. This process is completed by the frequent transcription and rapid degradation of mRNA. mRNA decay is initiated by a series of endo- and exoribonucleases. These enzymes leave behind 2- to 5-nt-long oligoribonucleotides termed "nanoRNAs" that are degraded by specific nanoRNases; the degradation of nanoRNA is essential because nanoRNA can mediate the priming of transcription initiation that is harmful for the cell via an unknown mechanism. Identified nanoRNases include Orn in E. coli, NrnA and NrnB in B. subtilis, and NrnC in Bartonella. Even though these nanoRNases can degrade nanoRNA specifically into mononucleotides, the biochemical features, structural features and functional mechanisms of these enzymes are different. Sequence analysis has identified homologs of these nanoRNases in different bacteria, including Gammaproteobacteria, Betaproteobacteria, Alphaproteobacteria, Firmicutes and Cyanobacteria. However, there are several bacteria, such as those belonging to the class Thermolithobacteria, that do not have homologs of these nanoRNases. In this paper, the source of nanoRNA, the features of different kinds of nanoRNases and the distribution of these enzymes in prokaryotes are described in detail.

The Detection of Hemin-Binding Proteins in Riemerella anatipestifer CH-1.

Riemerella anatipestifer (R. anatipestifer) is among the most prevalent duck pathogens, causing acute or chronic septicemia characterized by serositis. Riemerella anatipestifer can be grown on blood-enriched media, in vitro, which provides a hemin source essential for the sustainment of R. anatipestifer and activation of hemin-uptake systems. However, the genes associated with hemin uptake cannot be identified exclusively through genome sequence analysis. Here, we show that R. anatipestifer encodes outer-membrane hemin-binding proteins. Hemin-binding proteins were identified in the cytoplasm with apparent molecular mass of ~45/37/33/23/20/13 kDa, and outer membrane with apparent molecular mass of ~90/70/60/50/15 kDa by batch affinity chromatography and hemin-blotting assays. Our results indicate that these proteins are involved in hemin acquisition. Finally, hemin-binding assay further showed that R. anatipestifer can bind hemin and this capability is increased in iron limited medium, indicating the hemin-uptake system of R. anatipestifer was regulated by iron.

[Structural features and functional mechanism of TonB in some Gram-negative bacteria-A review].

TonB systems of gram-negative bacteria play an important role in transportation of nutriment from outside environments. TonB systems consist of plasma membrane proteins ExbB-ExbD and periplasmic protein TonB, which provide the energy to TonB-dependent receptors to transport substrates. These substrates include iron, hemin, vitamin B12, carbohydrate and some transition metal elements. The energy supporting function of TonB relies on its special structure which contains N-terminal domain for fixation, flexible periplasmic linker Pro-rich domain and C-terminal domain for contacting receptors. The precise mechanism of TonB system is not fully understood though its structural was studied a lot. To provide insights into direction for further research of TonB, we reviewed the TonB-dependent substrates uptake, structural features, functional mechanism and expression regulation of TonB.

TonB Energy Transduction Systems of Riemerella anatipestifer Are Required for Iron and Hemin Utilization.

Riemerella anatipestifer (R. anatipestifer) is one of the most important pathogens in ducks. The bacteria causes acute or chronic septicemia characterized by fibrinous pericarditis and meningitis. The R. anatipestifer genome encodes multiple iron/hemin-uptake systems that facilitate adaptation to iron-limited host environments. These systems include several TonB-dependent transporters and three TonB proteins responsible for energy transduction. These three tonB genes are present in all the R. anatipestifer genomes sequenced so far. Two of these genes are contained within the exbB-exbD-tonB1 and exbB-exbD-exbD-tonB2 operons. The third, tonB3, forms a monocistronic transcription unit. The inability to recover derivatives deleted for this gene suggests its product is essential for R. anatipestifer growth. Here, we show that deletion of tonB1 had no effect on hemin uptake of R. anatipestifer, though disruption of tonB2 strongly decreases hemin uptake, and disruption of both tonB1 and tonB2 abolishes the transport of exogenously added hemin. The ability of R. anatipestifer to grow on iron-depleted medium is decreased by tonB2 but not tonB1 disruption. When expressed in an E. coli model strain, the TonB1 complex, TonB2 complex, and TonB3 protein from R. anatipestifer cannot energize heterologous hemin transporters. Further, only the TonB1 complex can energize a R. anatipestifer hemin transporter when co-expressed in an E. coli model strain.