Co-introduction of plasmids harbouring the carbapenemase genes, blaNDM-1 and blaOXA-232, increases fitness and virulence of bacterial host.

BACKGROUND: Bacterial isolates with multiple plasmids harbouring different carbapenemase genes have emerged and been identified repeatedly, despite a general notion that plasmids confer fitness cost in bacterial host. In this study, we investigated the effects of plasmids with carbapenemase genes on the fitness and virulence of bacteria. METHODS: Different plasmids harbouring the carbapenemase genes, blaNDM-1 and blaOXA-232, were isolated from a carbapenem-resistant K. pneumoniae strain. Each plasmid was conjugated into the Escherichia coli strain DH5α, and a transconjugant with both plasmids was also obtained by transformation. Their in vitro competitive ability, biofilm formation, serum resistance, survival ability within macrophage and fruit fly, and fly killing ability were evaluated. RESULTS: The transconjugants with a single plasmid showed identical phenotypes to the plasmid-free strain, except that they decreased fly survival after infection. However, significantly increased fitness, virulence and biofilm production were observed consistently for the transconjugant with both plasmids, harbouring blaNDM-1 and blaOXA-232. CONCLUSIONS: Our data indicate that bacteria carrying multiple plasmids encoding different carbapenemases may have increased fitness and virulence, emphasizing the need for diverse strategies to combat antimicrobial resistance.

Affecting Factors of Secondhand Smoke Exposure in Korea: Focused on Different Exposure Locations.

Exposure to secondhand smoke (SHS) not only can cause serious illness, but is also an economic and social burden. Contextual and individual factors of non-smoker exposure to SHS depend on location. However, studies focusing on this subject are lacking. In this study, we described and compared the factors related to SHS exposure according to location in Korea. Regarding individual factors related to SHS exposure, a common individual variable model and location-specific variable model was used to evaluate SHS exposure at home/work/public locations based on sex. In common individual variables, such as age, and smoking status showed different relationships with SHS exposure in different locations. Among home-related variables, housing type and family with a single father and unmarried children showed the strongest positive relationships with SHS exposure in both males and females. In the workplace, service and sales workers, blue-collar workers, and manual laborers showed the strongest positive association with SHS exposure in males and females. For multilevel analysis in public places, only SHS exposure in females was positively related with cancer screening rate. Exposure to SHS in public places showed a positive relationship with drinking rate and single-parent family in males and females. The problem of SHS embodies social policies and interactions between individuals and social contextual factors. Policy makers should consider the contextual factors of specific locations and regional and individual context, along with differences between males and females, to develop effective strategies for reducing SHS exposure.

Natural killer (NK) cells inhibit systemic metastasis of glioblastoma cells and have therapeutic effects against glioblastomas in the brain.

BACKGROUND: Glioblastoma multiforme (GBM) is characterized by extensive local invasion, which is in contrast with extremely rare systemic metastasis of GBM. Molecular mechanisms inhibiting systemic metastasis of GBM would be a novel therapeutic candidate for GBM in the brain. METHODS: Patient-derived GBM cells were primarily cultured from surgical samples of GBM patients and were inoculated into the brains of immune deficient BALB/c-nude or NOD-SCID IL2Rgamma(null) (NSG) mice. Human NK cells were isolated from peripheral blood mononucleated cells and expanded in vitro. RESULTS: Patient-derived GBM cells in the brains of NSG mice unexpectedly induced spontaneous lung metastasis although no metastasis was detected in BALB/c-nude mice. Based on the difference of the innate immunity between two mouse strains, NK cell activities of orthotopic GBM xenograft models based on BALB/c-nude mice were inhibited. NK cell inactivation induced spontaneous lung metastasis of GBM cells, which indicated that NK cells inhibit the systemic metastasis. In vitro cytotoxic activities of human NK cells against GBM cells indicated that cytotoxic activity of NK cells against GBM cells prevents systemic metastasis of GBM and that NK cells could be effective cell therapeutics against GBM. Accordingly, NK cells transplanted into orthotopic GBM xenograft models intravenously or intratumorally induced apoptosis of GBM cells in the brain and showed significant therapeutic effects. CONCLUSIONS: Our results suggest that innate NK immunity is responsible for rare systemic metastasis of GBM and that sufficient supplementation of NK cells could be a promising immunotherapeutic strategy for GBM in the brain.

Requirement of the N-terminal residues of human cytomegalovirus UL112-113 proteins for viral growth and oriLyt-dependent DNA replication.

The UL112-113 region of the human cytomegalovirus (HCMV) genome encodes four phosphoproteins of 34, 43, 50, and 84 kDa that promote viral DNA replication. Co-transfection assays have demonstrated that self-interaction of these proteins via the shared N-termini is necessary for their intranuclear distribution as foci and for the efficient relocation of a viral DNA polymerase processivity factor (UL44) to the viral replication sites. However, the requirement of UL112-113 N-terminal residues for viral growth and DNA replication has not been fully elucidated. Here, we investigated the effect of deletion of the N-terminal regions of UL112-113 proteins on viral growth and oriLyt-dependent DNA replication. A deletion of the entire UL112 region or the region encoding the 25 N-terminal amino-acid residues from the HCMV (Towne strain) bacmid impaired viral growth in bacmid-transfected human fibroblast cells, indicating their requirement for viral growth. In co-immunoprecipitation assays using the genomic gene expressing the four UL112-113 proteins together, the 25 N-terminal amino-acid residues were found to be necessary for stable expression of UL112-113 proteins and their self-interaction. These residues were also required for efficient binding to and relocation of UL44, but not for interaction with IE2, an origin-binding transcription factor. In co-transfection/replication assays, replication of the oriLyt-containing plasmid was promoted by expression of intact UL112-113 proteins, but not by the expression of 25-amino-acid residue-deleted proteins. Our results demonstrate that the 25 N-terminal amino-acid residues of UL112-113 proteins that mediate self-interaction contribute to viral growth by promoting their binding to UL44 and the initiation of oriLyt-dependent DNA replication.

Substitution of a single residue, Asp575, renders the NCKX2 K+-dependent Na+/Ca2+ exchanger independent of K+.

The Na(+)/Ca(2+)-K(+) exchanger (NCKX) is a polytopic membrane protein that uses both the inward Na(+) gradient and the outward K(+) gradient to drive Ca(2+) extrusion across the plasma membrane. NCKX1 is found in retinal rod photoreceptors, while NCKX2 is found in retinal cone photoreceptors and is also widely expressed in the brain. Here, we have identified a single residue (out of >100 tested) for which substitution removed the K(+) dependence of NCKX-mediated Ca(2+) transport. Charge-removing replacement of Asp(575) by either asparagine or cysteine rendered the mutant NCKX2 proteins independent of K(+), whereas the charge-conservative substitution of Asp(575) to glutamate resulted in a nonfunctional mutant NCKX2 protein, accentuating the critical nature of this residue. Asp(575) is conserved in the NCKX1-5 genes, while an asparagine is found in this position in the three NCX genes, coding for the K(+)-independent Na(+)/Ca(2+) exchanger.

Residues contributing to the Ca2+ and K+ binding pocket of the NCKX2 Na+/Ca2+-K+ exchanger.

The Na(+)/Ca(2+)-K(+) exchanger (NCKX) extrudes Ca(2+) from cells utilizing both the inward Na(+) gradient and the outward K(+) gradient. NCKX is thought to operate by a consecutive mechanism in which a cation binding pocket accommodates both Ca(2+) and K(+) and alternates between inward and outward facing conformations. Here we developed a simple fluorometric method to analyze changes in K(+) and Ca(2+) dependences of mutant NCKX2 proteins in which candidate residues within membrane-spanning domains were substituted. The largest shifts in both K(+) and Ca(2+) dependences compared with wild-type NCKX2 were observed for the charge-conservative substitutions of Glu(188) and Asp(548), whereas the size-conservative substitutions resulted in nonfunctional proteins. Substitution of several other residues including two proline residues (Pro(187) and Pro(547)), three additional acidic residues (Asp(258), Glu(265), Glu(533)), and two hydroxyl-containing residues (Ser(185) and Ser(545)) showed smaller shifts, but shifts in Ca(2+) dependence were invariably accompanied by shifts in K(+) dependence. We conclude that Glu(188) and Asp(548) are the central residues of a single cation binding pocket that can accommodate both K(+) and Ca(2+). Furthermore, a single set of residues lines a transport pathway for both K(+) and Ca(2+).