Understanding Solvothermal Growth of Metal-Organic Framework Colloids for CO2 Capture Applications.

A quantitative study of the synthesis of metal-organic framework (MOF) colloids via a solvothermal growth process was demonstrated using electrospray-differential mobility analysis (ES-DMA), a gas-phase electrophoresis approach. HKUST-1, a copper-based MOF (Cu-MOF), was selected as the representative MOF of the study. The effects of the synthetic parameters, including ligand concentration (CBTC), synthetic temperature (Ts), and synthetic time (ts) versus material properties of the Cu-MOF, were successfully characterized based on the mobility size distributions measured by ES-DMA. The results show that the mobility size of Cu-MOF was proportional to Ts, ts, and CBTC during the solvothermal growth. X-ray diffraction and Brunauer-Emmett-Teller analyses were employed complementarily to the ES-DMA, confirming that the increase in mobility size of Cu-MOF was correlated to the increase in crystallinity (i.e., larger specific surface area and crystallite size). The results of CO2 pulse adsorption show that the synthesized Cu-MOF possessed a good CO2 adsorption ability under 1 atm, 35 °C, and the cumulative amount of CO2 uptake was proportional to the measured mobility size of Cu-MOF. The work provides a proof of concept for the controlled synthesis of MOF colloids with the support of gas-phase electrophoretic analysis, and the quantitative methodology is useful for the development of MOF-based applications in CO2 capture and utilization.

Verbascoside inhibits the epithelial-mesenchymal transition of prostate cancer cells through high-mobility group box 1/receptor for advanced glycation end-products/TGF-ß pathway.

INTRODUCTION: Prostate cancer has significant mortality and metastasis rate in the male. Unfortunately, effective treatment for patients with advanced prostate cancer is still lacking. Verbascoside, a phenylethanoid glycoside, displays various pharmacological properties, such as the anti-cancer activities. The present study aimed to evaluate the effects of purified verbascoside on human prostate cancer and the associated molecular mechanisms. MATERIALS AND METHODS: The human prostate cancer cell lines, Du-145 and PC-3, were treated with various concentrations of verbascoside (0.1, 1, 10 µM) for 24 h followed by the examination of cell viability using MTT and trypan blue exclusion assays. Cell migration and invasion capacities were assessed by wound healing assay and transwell system. Western blot and immunofluorescence staining were used to detect the expression of epithelial-mesenchymal transition (EMT)-associated factors, components of transforming growth factor (TGF-ß)/Smad signaling, and high-mobility group box (HMGB)/receptor for advanced glycation end-products (RAGE) axis. RESULTS: Verbascoside treatment significantly inhibited cell proliferation, migration, and invasion abilities of Du-145 and PC-3 cells. We showed that verbascoside decreased the expression of EMT promotors, Snail and Slug, and increased the expression of E-cadherin. Moreover, the expression level of alpha-smooth muscle actin was downregulated by verbascoside as well. Besides, we found that the TGF-ß pathway was suppressed, which was demonstrated by the diminished expression of type I and II TGF-ß receptors and phosphorylated Smad2/3 along with the upregulated Smad7. Our data suggested that this downregulation of TGF-ß signaling was mediated by repression of HMGB 1 (HMGB1)/RAGE axis. CONCLUSION: Verbascoside mitigated the cell proliferation and aggressiveness of prostate cancer via downregulation of TGF-ß-associated EMT progression through HMGB1/RAGE suppression. Collectively, our findings revealed that verbascoside may be a beneficial dietary supplement for prostate cancer patients.

Long Non-Coding RNA MEG3 in Cellular Stemness.

Long non-coding RNAs (lncRNAs) regulate a diverse array of cellular processes at the transcriptional, post-transcriptional, translational, and post-translational levels. Accumulating evidence suggests that lncRNA MEG3 exerts a large repertoire of regulatory functions in cellular stemness. This review focuses on the molecular mechanisms by which lncRNA MEG3 functions as a signal, scaffold, guide, and decoy for multi-lineage differentiation and even cancer progression. The role of MEG3 in various types of stem cells and cancer stem cells is discussed. Here, we provide an overview of the functional versatility of lncRNA MEG3 in modulating pluripotency, differentiation, and cancer stemness.

Klebsiella pneumoniae Type VI Secretion System Contributes to Bacterial Competition, Cell Invasion, Type-1 Fimbriae Expression, and In Vivo Colonization.

Background: We previously isolated a Klebsiella pneumoniae strain, NTUH-K2044, from a community-acquired pyogenic liver abscess (PLA) patient. Analysis of the NTUH-K2044 genome revealed that this strain harbors 2 putative type VI secretion system (T6SS)-encoding gene clusters. Methods: The distribution of T6SS genes in the PLA and intestinal-colonizing K pneumoniae clinical isolates was examined. icmF1-, icmF2-, icmF1/icmF2-, and hcp-deficient K pneumoniae strains were constructed using an unmarked deletion method. The roles of T6SSs in antibacterial activity, type-1 fimbriae expression, cell adhesion, and invasion and intestinal colonization were determined. Results: The prevalence of T6SSs is higher in the PLA strains than in the intestinal-colonizing strains (37 of 42 vs 54 of 130). Deletion of icmF1/icmF2 and hcp genes significantly reduced interbacterial and intrabacterial killing. Strain deleted for icmF1 and icmF2 exhibited decreased transcriptional expression of type-1 fimbriae and reduced adherence to and invasion of human colorectal epithelial cells and was attenuated for in vivo competition to enable colonization of the host gut. Finally, Hcp expression in K pneumoniae was silenced by the histone-like nucleoid structuring protein via direct binding. Conclusions: These results provide new insights into T6SS-mediated bacterial competition and attachment in K pneumoniae and could facilitate the prevention of K pneumoniae infection.

A Novel Role for the Klebsiella pneumoniae Sap (Sensitivity to Antimicrobial Peptides) Transporter in Intestinal Cell Interactions, Innate Immune Responses, Liver Abscess, and Virulence.

Klebsiella pneumoniae is an important human pathogen causing hospital-acquired and community-acquired infections. Systemic K. pneumoniae infections may be preceded by gastrointestinal colonization, but the basis of this bacterium's interaction with the intestinal epithelium remains unclear. Here, we report that the K. pneumoniae Sap (sensitivity to antimicrobial peptides) transporter contributes to bacterial-host cell interactions and in vivo virulence. Gene deletion showed that sapA is required for the adherence of a K. pneumoniae blood isolate to intestinal epithelial, lung epithelial, urinary bladder epithelial, and liver cells. The ΔsapA mutant was deficient for translocation across intestinal epithelial monolayers, macrophage interactions, and induction of proinflammatory cytokines. In a mouse gastrointestinal infection model, ΔsapA yielded significantly decreased bacterial loads in liver, spleen and intestine, reduced liver abscess generation, and decreased mortality. These findings offer new insights into the pathogenic interaction of K. pneumoniae with the host gastrointestinal tract to cause systemic infection.

Antifibrotic role of PGC-1α-siRNA against TGF-ß1-induced renal interstitial fibrosis.

Peroxisome proliferator-activated receptor coactivator-1 alpha (PGC-1α) is a transcriptional coactivator that regulates energy metabolism and mitochondrial biogenesis. Recently, mitochondrial dysfunction has been indicated as an established risk factor for the development of renal fibrosis. However, whether PGC-1α is involved in the pathogenesis of renal fibrosis is unknown. In this study, we treated NRK-49F (normal rat kidney fibroblast) cells with transforming growth factor-beta 1 (TGF-ß1) for 24 h to establish an in vitro fibrosis model. TGF-ß1 induced the upregulation of type I collagen, fibronectin, TGF-ß receptor I (TGFß-RI), TGFß-RII, Smad4, and pSmad2/3, as well as PGC-1α. NRK-49F cells transfected with pcDNA-PGC-1α showed significantly increased expression of fibronectin and type I collagen, as revealed by western blot assay. Interestingly, transfection with PGC-1α-siRNA caused a stark reversal of TGF-ß1-induced cellular fibrosis, with concomitant suppression of fibronectin and type I collagen, as revealed by western blot and immunofluorescence assays. Moreover, SB431542 (TGFß-RI), LY294002 (PI3K/Akt), and SB203580 (p38 MAPK), inhibitors of TGF-ß-associated pathways, markedly suppressed TGF-ß1-induced PGC-1α upregulation. These results implicate a role of PGC-1α in renal interstitial fibrosis mediated via the TGFß-RI, PI3K/Akt, and p38 MAPK pathways. Our findings that PGC-1α-siRNA downregulates fibronectin and type I collagen suggest that it can be used as a novel molecular treatment for renal fibrosis.

Klebsiella Phage ΦK64-1 Encodes Multiple Depolymerases for Multiple Host Capsular Types.

The genome of the multihost bacteriophage ΦK64-1, capable of infecting Klebsiella capsular types K1, K11, K21, K25, K30, K35, K64, and K69, as well as new capsular types KN4 and KN5, was analyzed and revealed that 11 genes (S1-1, S1-2, S1-3, S2-1, S2-2, S2-3, S2-4, S2-5, S2-6, S2-7, and S2-8) encode proteins with amino acid sequence similarity to tail fibers/spikes or lyases. S2-5 previously was shown to encode a K64 capsule depolymerase (K64dep). Specific capsule-degrading activities of an additional eight putative capsule depolymerases (S2-4 against K1, S1-1 against K11, S1-3 against K21, S2-2 against K25, S2-6 against K30/K69, S2-3 against K35, S1-2 against KN4, and S2-1 against KN5) was demonstrated by expression and purification of the recombinant proteins. Consistent with the capsular type-specific depolymerization activity of these gene products, phage mutants of S1-2, S2-2, S2-3, or S2-6 lost infectivity for KN4, K25, K35, or K30/K69, respectively, indicating that capsule depolymerase is crucial for infecting specific hosts. In conclusion, we identified nine functional capsule depolymerase-encoding genes in a bacteriophage and correlated activities of the gene products to all ten hosts of this phage, providing an example of type-specific host infection mechanisms in a multihost bacteriophage.IMPORTANCE We currently identified eight novel capsule depolymerases in a multihost Klebsiella bacteriophage and correlated the activities of the gene products to all hosts of this phage, providing an example of carriage of multiple depolymerases in a phage with a wide capsular type host spectrum. Moreover, we also established a recombineering system for modification of Klebsiella bacteriophage genomes and demonstrated the importance of capsule depolymerase for infecting specific hosts. Based on the powerful tool for modification of phage genome, further studies can be conducted to improve the understanding of mechanistic details of Klebsiella phage infection. Furthermore, the newly identified capsule depolymerases will be of great value for applications in capsular typing.

Elucidation of the therapeutic role of mitochondrial biogenesis transducers NRF-1 in the regulation of renal fibrosis.

BACKGROUND: Mitochondrial dysfunction is a newly established risk factor for the development of renal fibrosis. Cell survival and injury repair is facilitated by mitochondrial biogenesis. Nuclear respiratory factor 1 (NRF-1) is a transcriptional regulation factor that plays a central role in the regulation of mitochondrial biogenesis. However, the transcription factor of this process in renal fibrosis is unknown. Thus, we hereby discussed the correlations of NRF-1 and renal interstitial fibrosis. MATERIALS AND METHODS: In vitro fibrosis model was established by treatment with transforming growth factor-ß1 (TGF-ß1) in NRK-49F (Normal Rat kidney fibroblast). We investigated the ROS production, mitochondrial biogenesis and fibrogenic marker (e.q. fibronectin) during the progression of renal fibrosis by kit and Western blotting assay. Here, we used that two distinct mechanisms regulate NRF-1 activation and degradation of NRF-1. NRF-1 was transfect by pcDNA-NRF-1 overexpression gene to evaluate the NRF-1 activity of the therapeutic effect in renal fibrosis. In addition, NRF-1 was silenced by shRNA-NRF-1 to evaluate the significance of NRF-1. ELISA was used to evaluate the secreted fibronectin. Immunofluorescence staining was used to assay the in situ expression of proteins (e.g. fibronectin, NRF-1). RESULTS: Under renal fibrosis conditions, TGF-ß1 (5ng/ml) increased ROS. Simultaneously, TGF-ß1-induced extracellular fibronectin by ELISA assay. In addition, TGF-ß1 decreased expression of mitochondrial biogenesis. This is the first time to demonstrate that expression of NRF-1 is significantly decreased in renal fibrosis. However, NRK49F was a transfection with pcDNA-NRF-1 (2µg/ml) expression vector dramatically reverse TGF-ß1-induced cellular fibrosis concomitantly with the suppression of fibronectin (both intracellular and extracellular fibronectin). More importantly, transfection with shRNA-NRF-1 (2µg/ml) significantly increased the expression of fibronectin of both intercellular and extracellular origins in NRK-49F cells. DISCUSSION: These finding suggest that NRF-1 plays a pivotal role on renal cellular fibrosis. Moreover, NRF-1 might act as a novel renal fibrosis antagonist by down-regulating fibrosis signaling in renal fibroblast cells.

Treatment with cytokine thymic stromal lymphopoietin short hairpin RNA substantially reduces TGF-ß1-induced interstitial cellular fibrosis.

Thymic stromal lymphopoietin (TSLP) has previously been linked to allergic inflammatory diseases, and tissue fibrosis and organ dysfunction may also arise from such inflammation. It remains unclear, however, whether TSLP plays any role in the occurrence of renal fibrosis, so this study investigated that possibility. An in vitro fibrosis model was established by treating normal rat kidney fibroblast (NRK-49F) cells with transforming growth factor-ß1 (TGF-ß1), after which the levels of various fibrogenic markers (e.g., fibronectin) and downstream fibrogenic signal proteins (e.g., smad 7) were investigated. Also, TSLP shRNA was used to silence the effects of TSLP, while an ELISA was conducted to evaluate the fibronectin secretions. The level of fibronectin in the NRK-49F cells was dose- and time-dependently increased by the administration of exogenous TSLP (P<0.05). TSLP also significantly increased the level of fibrosis signaling, in addition to inducing a marked decrease in the down-regulation of Smad7. Interestingly, the application of TSLP shRNA caused a stark reversal of the TGF-ß1-induced cellular fibrosis while simultaneously leading to the suppression of fibronectin and fibrogenic signal proteins. Taken together, these observations provide insights into how extracellular matrices develop and could thus lead to potential therapeutic interventions for the suppression of renal fibrosis.

Identification of capsular types in carbapenem-resistant Klebsiella pneumoniae strains by wzc sequencing and implications for capsule depolymerase treatment.

Klebsiella pneumoniae is an important human pathogen associated with a variety of diseases, and the prevalence of multidrug-resistant K. pneumoniae (MDRKP) is rapidly increasing. Here we determined the capsular types of 85 carbapenem-resistant K. pneumoniae (CRKP) strains by wzc sequencing and investigated the presence of carbapenemases and integrons among CRKP strains. Ten CRKP strains (12%) were positive for carbapenemase (imipenemase, 6/85 strains; K. pneumoniae carbapenemase, 3/85 strains; Verona integron-encoded metallo-ß-lactamase, 1/85 strains). Capsular type K64 accounted for 32 CRKP strains (38%), followed by K62 (13%), K24 (8%), KN2 (7%), and K28 (6%). Sequence types (STs) were determined by multilocus sequence typing (MLST), and the results indicated that ST11, which accounted for 47% of these CRKP strains (40/85 strains), was the major ST. We further isolated a K64-specific capsule depolymerase (K64dep), which could enhance serum and neutrophil killing in vitro and increase survival rates for K64 K. pneumoniae-inoculated mice. The toxicity study demonstrated that mice treated with K64dep showed normal biochemical parameters and no significant histopathological changes of liver, kidney, and spleen, indicating that enzyme treatment did not cause toxicity in mice. Therefore, the findings of capsular type clustering among CRKP strains and effective treatment with capsule depolymerase for MDRKP infections are important for capsule-based vaccine development and therapy.

The effects of diosgenin in the regulation of renal proximal tubular fibrosis.

Fibrosis is the important pathway for end-stage renal failure. Glucose has been demonstrated to be the most important fibrogenesis-inducing agent according to previous studies. Despite diosgenin has been demonstrated to be anti-inflammatory, the possible role in fibrosis regulation of diosgenin remain to be investigated. In this study, renal proximal tubular epithelial cells (designated as HK-2) were treated with high concentration of glucose (HG, 27.5mM) to determine whether diosgenin (0.1, 1 and 10 µM) has the effects to regulate renal cellular fibrosis. We found that 10 µM of diosgenin exert optimal inhibitory effects on high glucose-induced fibronectin expression in HK-2 cells. In addition, diosgenin markedly inhibited HG-induced increase in α-smooth muscle actin (α-SMA) and HG-induced decrease in E-cadherin. In addition, diosgenin antagonizes high glucose-induced epithelial-to-mesenchymal transition (EMT) signals partly by enhancing the catabolism of Snail in renal cells. Collectively, these data suggest that diosgenin has the potential to inhibit high glucose-induced renal tubular fibrosis possibly through EMT pathway.

Isolation of a bacteriophage and its depolymerase specific for K1 capsule of Klebsiella pneumoniae: implication in typing and treatment.

BACKGROUND: Klebsiella pneumoniae causing community-acquired pyogenic liver abscess complicated with metastatic meningitis and endophthalmitis has emerged recently, most frequently associated with the K1 capsular type. METHODS: A bacteriophage (NTUH-K2044-K1-1) that infects K. pneumoniae NTUH-K2044 (capsular type K1) was isolated and characterized. RESULTS: The phage infected all K1 strains, and none of the strains with other capsular types. Capsule deletion mutants were not lysed by this phage, suggesting that the capsule was essential for phage infection. Complete genome sequencing revealed the phage was a novel phiKMV-like virus. The gene-encoding capsule depolymerase was identified. The recombinant enzyme demonstrated specific lysis of the K1 capsule. Treatment with the phage or the recombinant enzyme provided significantly increased survival in mice infected with NTUH-K2044 strain, including one treated after the detection of a neck abscess by imaging. No obvious disease was observed after administration of this phage in mice. Phage was retained at detectable levels in liver, spleen, brain, and blood 24 hours after administration in mice. CONCLUSIONS: These results demonstrate this phage and its capsule depolymerase exhibit specificity for capsular type K1 and can be used for the diagnosis and treatment of K1 K. pneumoniae infections.

Identification of an immuno-dominant protein from Klebsiella pneumoniae strains causing pyogenic liver abscess: implication in serodiagnosis.

BACKGROUND: Klebsiella pneumoniae has emerged worldwide as a cause of pyogenic liver abscess (PLA) often complicated by meningitis and endophthalmitis. Early detection of this infectious disease will improve its clinical outcome. Therefore, we tried to isolate immunodominant proteins secreted by K. pneumoniae strains causing PLA. RESULTS: The secreted proteins of the NTUH-K2044 strain were separated by two-dimensional electrophoresis and then immunoblotted using convalescent sera from patients with K. pneumoniae PLA. A ~30-kDa immunodominant protein was then identified. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed an open reading frame (KP1_p307) located on the pK2044 plasmid and bioinformatic analysis identified this protein as a signal peptide of unknown function. The KP1_p307 gene was more prevalent in PLA strains and capsular type K1/K2 strains, but disruption of this gene in NTUH-K2044 strain did not decrease virulence in mice. Ten of fourteen (71%) sera from patients with K. pneumoniae PLA were immunoreactive with the recombinant KP1_p307 protein. Seroconversion demonstrated by a rise in serum titer in serial serum samples confirmed that antibodies against the KP1_p307 protein were elicited after infection. CONCLUSIONS: The KP1_p307 protein could be used as an antigen for early serodiagnosis of K. pneumoniae PLA, particularly in K1/K2 PLA strains.

Klebsiella pneumoniae peptidoglycan-associated lipoprotein and murein lipoprotein contribute to serum resistance, antiphagocytosis, and proinflammatory cytokine stimulation.

BACKGROUND: Peptidoglycan-associated lipoprotein (Pal), murein lipoprotein (LppA), and outer membrane protein A (OmpA) are dominant outer membrane proteins (OMPs) that are released by gram-negative bacteria during sepsis. OMPs are implicated in the maintenance of cell envelope integrity. Here, we characterize the roles of these OMPs in pathogenesis during bacteremia caused by Klebsiella pneumoniae. METHODS: pal-, lppA-, and ompA-deficient K. pneumoniae strains were constructed using an unmarked deletion method. Serum sensitivity, antiphagocytosis activity, outer membrane permeability, and sensitivity to anionic detergents and antimicrobial polypeptides were determined for these OMP gene deletion mutants. The ability of these OMP gene deletion mutants to induce immune responses was compared with that of the wild-type strain in a bacteremic mouse model. RESULTS: Klebsiella pneumoniae strains deleted for pal or lppA exhibited reduced protection from serum killing and phagocytosis; perturbation to the outer membrane permeability barrier and hypersensitivity to bile salts and sodium dodecyl sulfate. The strain mutated for lppA had reduced ability to activate Toll-like receptor 4. Immunization of mice with the pal or lppA mutant provided protection against infection by the wild-type strain. CONCLUSIONS: Our findings indicate that K. pneumoniae Pal and LppA proteins are important in the maintenance of cell integrity, contribute to virulence, and could be used as attenuated vaccines.

Taiwanese long-term care facility residents' experiences of caring: a qualitative study.

BACKGROUND: Until now, caring research has focused on constructs, processes and outcomes of caring traits exhibited mainly by nurses or nursing students in hospitals. Few studies have considered the perspectives of elderly residents of long-term care facilities (LTCFs). AIMS: The aim of this study was to elucidate the nature of caring by describing the experience of elderly residents of Taiwan LTCFs. METHODS: Data collected by semi-structured interviews with 12 elderly residents of LTCFs were analysed by content analysis. RESULTS: The caring perceived by the sample was thematically categorized as calming the body, respectful communication and enriching life. CONCLUSION: The findings of this study confirm the need for managers of LTCFs to address the physical and psychosocial needs of their residents. The findings can be used to develop education programmes and clinical protocols for nurses in the caring field and to develop instruments for measuring perceived caring in LTCFs.

The Regulatory Role of Nuclear Respiratory Factor 1 in Bladder Cancer Cells.

BACKGROUND/AIM: Nuclear respiratory factor 1 (NRF1) is a key mediator of genes involved in mitochondrial biogenesis and the respiratory chain; however, its role in bladder cancer remains unknown. Transitional cell carcinoma, also known as urothelial cell carcinoma, is the most common type of bladder cancer resistant to chemotherapy. An established high-grade and invasive transitional cell carcinoma line from patients with urinary bladder cancer, known as T24, has been extensively used in cancer research. In this study, we aimed to investigate the mechanisms through which NRF1 regulates proliferation and cell migration of bladder cancer cells using the T24 cell line. MATERIALS AND METHODS: Cells were transfected with plasmid cloning DNA for NRF1 to evaluate the effect of NRF1 overexpression on bladder cancer cells. Western blot was used to examine epithelial and mesenchymal markers (E-cadherin and α-smooth muscle actin), transcriptional regulators for epithelial-mesenchymal transition (snail family transcriptional repressors), components of transforming growth factor-ß1/SMADs signaling, high-mobility group box 1 (HMGB1), and receptor for advanced glycation end-products (RAGE). The in situ expression of E-cadherin, α-smooth muscle actin and SMAD7 was determined using immunofluorescence staining. Cell migration capacity was assessed by wound-healing assay. RESULTS: Transfection with NRF1 expression vector repressed the migration capacity of bladder cancer cells, diminishing HMGB1/RAGE expression and reducing transforming growth factor ß-associated epithelial-mesenchymal transition in T24 cells. CONCLUSION: Therapeutic avenues that increase NRF1 expression may serve as an adjunct to conventional treatments for bladder cancer.

Structure and immunological characterization of the capsular polysaccharide of a pyrogenic liver abscess caused by Klebsiella pneumoniae: activation of macrophages through Toll-like receptor 4.

The active components of a primary pyrogenic liver abscess (PLA) Klebsiella pneumoniae in stimulating cytokine expression in macrophages are still unclear. The capsular polysaccharide (CPS) of PLA K. pneumoniae is important in determining clinical manifestations, and we have shown that it consists of repeating units of the trisaccharide (→3)-ß-D-Glc-(1→4)-[2,3-(S)-pyruvate]-ß-D-GlcA-(1→4)-α-L-Fuc-(1→) and has the unusual feature of extensive pyruvation of glucuronic acid and acetylation of C(2)-OH or C(3)-OH of fucose. We demonstrated that PLA K. pneumoniae CPS induces secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by macrophages through Toll-like receptor 4 (TLR4) and that this effect was lost when pyruvation and O-acetylation were chemically destroyed. Furthermore, expression of TNF-α and IL-6 in PLA K. pneumoniae CPS-stimulated macrophages was shown to be regulated by the TLR4/ROS/PKC-δ/NF-κB, TLR4/PI3-kinase/AKT/NF-κB, and TLR4/MAPK signaling pathways.

Cellobiose-specific phosphotransferase system of Klebsiella pneumoniae and its importance in biofilm formation and virulence.

Klebsiella pneumoniae is a Gram-negative bacillus belonging to the family Enterobacteriaceae. In the past 20 years, K. pneumoniae has become the predominant pathogen causing community-acquired pyogenic liver abscess (PLA). The formation of biofilm facilitates bacterial colonization and has been implicated in reduced susceptibility to the host immune response. To investigate genes related to biofilm formation in a PLA-associated K. pneumoniae strain, a transposon mutant library was screened by microtiter plate assay to identify isolates impaired for biofilm formation. One of the mutants was disrupted in celB, encoding the putative cellobiose-specific subunit IIC of enzyme II (EIIC) of a carbohydrate phosphotransferase system (PTS). This transmembrane protein is responsible for recognizing and binding specific sugars and transporting them across the cell membrane into the cytoplasm. Deletion and chromosomal complementation of celB confirmed, by microtiter plate and slide culture assays, that celB was indeed responsible for biofilm formation. Cellobiose-specific PTS activities of deletion mutants grown in LB broth and 0.005% cellobiose minimal medium were markedly lower than that of the wild-type strain grown under the same conditions, thereby confirming the involvement of celB in cellobiose transport. In 0.005% cellobiose minimal medium, the celB mutant showed a delay in growth compared to the wild-type strain. In a mouse model of intragastric infection, deletion of the celB gene increased the survival rate from 12.5% to 87.5%, which suggests that the celB deletion mutant also exhibited reduced virulence. Thus, the celB locus of K. pneumoniae may contribute to biofilm formation and virulence through the metabolism of cellobiose.

Small interfering RNA targeting N-cadherin regulates cell proliferation and migration in enzalutamide-resistant prostate cancer.

Enzalutamide is one of the options for treating patients with castration-resistant or metastatic prostate cancer. However, a substantial proportion of patients become resistant to enzalutamide after a period of treatment. Cells in these tumors typically exhibit increased proliferative and migratory capabilities, in which N-cadherin (CDH2) appear to serve an important role. In the present study, by up- and downregulating the expression of CDH2, the possible effects of CDH2 on the prostate cancer cell line LNCaP were investigated. Male sex hormone-sensitive LNCaP cells treated with 10 µM enzalutamide were named LNCaP enzalutamide-resistant (EnzaR) cells. Reverse transcription-PCR, western blotting and immunofluorescence staining were used to measure CDH2, E-cadherin, α-SMA, Snail and Slug expression. Transfection with the pCMV-CDH2 plasmid was performed for CDH2 upregulation, whilst transfection with small interfering RNA (siRNA)-CDH2 was performed for CDH2 downregulation. MTT and Cell Counting Kit-4 assays were used to evaluate the proportion of viable cancer cells. Subsequently, gap closure assay was performed to evaluate the migratory capability of both LNCaP and LNCaP EnzaR cell lines. CDH2 expression was found to be increased in LNCaP EnzaR cells compared with that in LNCaP cells. CDH2 overexpression increased cell viability and migration in both LNCaP and LNCaP EnzaR cell lines. By contrast, the opposite trend was observed after CDH2 expression was knocked down. CDH2 expression also showed a high association with that of four epithelial-mesenchymal transition markers, which was confirmed by western blotting. Based on these results, it was concluded that knocking down CDH2 expression using siRNA transfection mediated significant influence on LNCaP EnzaR cell physiology, which may be a potential therapeutic option for prostate cancer treatment.

Decolonization of carbapenem-resistant Klebsiella pneumoniae from the intestinal microbiota of model mice by phages targeting two surface structures.

Background: Klebsiella pneumoniae is a normal component of the human gastrointestinal tract microbiota. However, in some cases, it can cause disease. Over the past 20 years, the prevalence of antibiotic-resistant bacteria, such as carbapenem-resistant K. pneumoniae (CRKP), has been increasing. Materials and methods: We attempted to specifically eliminate CRKP from a mouse model with the human intestinal microbiota. To establish humanized microbiota-colonized mice, we administered K64 CRKP-containing human microbiota to germ-free mice by fecal microbiota transplantation. Then, we used two phages, one targeting the capsule (φK64-1) and one targeting O1 lipopolysaccharide (φKO1-1) of K64 K. pneumoniae, to eliminate CRKP. Results: In untreated control and φKO1-1-treated K64-colonized mice, no change in CRKP was observed, while in mice treated with φK64-1, a transient reduction was observed. In half of the mice treated with both φKO1-1 and φK64-1, CRKP was undetectable in feces by PCR and culture for 60 days. However, in the other 50% of the mice, K. pneumoniae was transiently reduced but recovered 35 days after treatment. Conclusion: Combination treatment with φK64-1 and φKO1-1 achieved long-term decolonization in 52.3% of mice carrying CRKP. Importantly, the composition of the intestinal microbiota was not altered after phage treatment. Therefore, this strategy may be useful not only for eradicating drug-resistant bacterial species from the intestinal microbiota but also for the treatment of other dysbiosis-associated diseases.