Patient Cranial Angle and Intrafractional Stability in CyberKnife Robotic Radiosurgery: A Retrospective Analysis.

Purpose: The aim of this study was to investigate whether variations in cranial angles and treatment accuracy during CyberKnife robotic radiosurgery necessitate adjustment of the margins of the planning target volume. Patients and Methods: Data from 66 patients receiving CyberKnife treatment for brain tumors were retrospectively analyzed. Patients were immobilized using a thermoplastic mask and headrest. The cranial angle was measured on planning CT and patients were divided into 2 groups: ≤10° (Group A) and >10° (Group B). Intrafractional motion was recorded using the CyberKnife tracking system over 50 min. Translational and rotational errors were compared between groups, and planning target volume margins were calculated. Results: In Group A, significant translational error differences were found along with the X-axis over time (P < .02). In Group B, significant differences occurred along with the Z-axis (P < .03). No significant rotational or 3-dimensional vector differences were found in either group. Group A had significantly lower Y-axis (P < .045) and roll axis (P < .005) errors compared to Group B. Estimated planning target volume margins in Group A were 0.56 mm (X), 0.46 mm (Y), and 0.47 mm (Z). In Group B, margins were 0.62 mm (X), 0.48 mm (Y), and 0.46 mm (Z). Margins covering 95% of intrafraction motion were 0.49 to 0.50 mm (X, Y, Z) and 0.69 mm (3-dimensional vector) for Group A, and 0.48 to 0.60 mm and 0.79 mm for Group B. With a 1-mm margin, complete coverage was achieved in Group A while 2.1% of vectors in Group B exceeded 1 mm. Conclusion: Adjusting cranial angle to ≤10° during thermoplastic mask molding provided better or similar intrafractional stability compared to >10°.

Evaluation of the Position Error of Wearing Surgical Masks During Radiotherapy in Head and Neck Cancer Patients.

Purpose: Wearing a mask during the coronavirus disease 2019 epidemic (COVID-19) is a preventive way to reduce droplet and aerosol transmission. The purpose of this study was to evaluate the position error of wearing a surgical mask during radiotherapy in head and neck cancer patients. Patients and Methods: We collected and analyzed 2351 kV X-ray image records of 81 patients with head and neck cancer who underwent image-guided radiotherapy (IGRT). Patients with/without a surgical mask were divided into the head-neck (HN) mask group and head-neck-shoulder (HNS) mask group. The position error in the X (left-right), Y (superior-inferior), Z (anterior-posterior), 3D (three dimensional) vectors, as well as the pitch and yaw axes were compared between the four groups. Results: We found that patients wearing surgical masks in the HN mask group showed no significant differences in the mean position error of the different types of headrest (p>0.05). In the HNS mask group, only the type C headrest group showed significant differences (P < 0.05). The X axis values were -0.05±0.07 and -0.11± 0.01 cm (P = 0.04), and the pitch axis values were 0.34±0.29° and 0.83±0.08° (P = 0.01). Conclusion: The mean position error of most patients wearing surgical masks was not greater than patients without a surgical mask. Patients wearing while receiving treatment is a low-cost and easy-to-implement prevention method.

Hepatocyte Nuclear Factor 4-Alpha Is Essential for the Active Epigenetic State at Enhancers in Mouse Liver.

Cell-fate determination is influenced by interactions between master transcription factors (TFs) and cis-regulatory elements. Hepatocyte nuclear factor 4 alpha (HNF4A), a liver-enriched TF, acts as a master controller in specification of hepatic progenitor cells by regulating a network of TFs to control onset of hepatocyte cell fate. Using analysis of genome-wide histone modifications, DNA methylation, and hydroxymethylation in mouse hepatocytes, we show that HNF4A occupies active enhancers in hepatocytes and is essential for active histone and DNA signatures, especially acetylation of lysine 27 of histone 3 (H3K27ac) and 5-hydroxymethylcytosine (5hmC). In mice lacking HNF4A protein in hepatocytes, we observed a decrease in both H3K27ac and hydroxymethylation at regions bound by HNF4A. Mechanistically, HNF4A-associated hydroxymethylation (5hmC) requires its interaction with ten-eleven translocation methylcytosine dioxygenase 3 (TET3), a protein responsible for oxidation from 5mC to 5hmC. Furthermore, HNF4A regulates TET3 expression in liver by directly binding to an enhancer region. Conclusion: In conclusion, we identified that HNF4A is required for the active epigenetic state at enhancers that amplifies transcription of genes in hepatocytes.

YAP transcriptionally regulates ErbB2 to promote liver cell proliferation.

The Hippo signaling pathway is implicated in regulation of liver size and dysregulation of this pathway contributes to tumorigenesis. The transcriptional targets and downstream pathways of the Hippo pathway effector YAP that contribute to liver growth have yet to be well-characterized. We examined the liver transcriptome in response to YAP overexpression and identify the ErbB signaling pathway as a mediator of cell growth downstream of YAP. ErbB2 is transcriptionally regulated by YAP in both the mouse liver and in HepG2 human hepatoma cells. Knockdown of YAP or pharmacological inhibition with verteporfin reduced ERBB2 levels in HepG2 cells. Analysis of ChIP-seq data revealed enrichment of the transcription factor TEAD4 at the ERBB2 promoter. Using luciferase reporter and chromatin immunoprecipitation assays, we show that YAP and TEAD4 directly bind to and activate a regulatory element in the ErbB2 promoter in both the mouse liver and HepG2 cells. Functionally, knockdown of YAP reduced EGF-induced ERBB2-mediated HepG2 cell proliferation and PI3K/AKT activation. Our findings highlight a mechanism by which YAP exerts its effects on liver cell proliferation through the ErbB signaling pathway by directly regulating the transcription of ErbB2.

S1P Stimulates Proliferation by Upregulating CTGF Expression through S1PR2-Mediated YAP Activation.

Dysregulation of the Hippo pathway in the liver results in overgrowth and eventually tumorigenesis. To date, several upstream mechanisms have been identified that affect the Hippo pathway, which ultimately regulate YAP, the major downstream effector of the pathway. However, upstream regulators of the Hippo pathway in the liver remain poorly defined. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that has been shown to stimulate hepatocellular carcinoma (HCC) cell proliferation, but whether the Hippo pathway is involved in S1P-stimulated HCC cell proliferation remains to be determined. Here it is demonstrated that S1P activates YAP and that the S1P receptor 2 (S1PR2/S1P2) mediates S1P-induced YAP activation in both human and mouse HCC cells. S1P promotes YAP-mediated upregulation of cysteine-rich protein 61 and connective tissue growth factor (CTGF), and stimulates HCC cell proliferation. By using siRNA-mediated knockdown approaches, only CTGF was required for S1P-stimulated cell proliferation. Of note, S1P activates YAP in a MST1/2-independent manner suggesting that the canonical Hippo kinase is not required for S1P-mediated proliferation in liver. The upregulation of CTGF and S1P2 were also observed in liver-specific YAP overexpression transgenic mouse hepatocytes. Moreover, YAP regulated liver differentiation-dependent gene expression by influencing the chromatin binding of HNF4α based on ChIP-seq analysis. Finally, results using gain- and loss-of-function approaches demonstrate that HNF4α negatively regulated S1P-induced CTGF expression.Implications: These findings reveal a role for S1P in stimulating HCC cell proliferation by upregulating CTGF expression through S1P2-mediated YAP activation. Mol Cancer Res; 16(10); 1543-55. ©2018 AACR.

APELA promotes tumour growth and cell migration in ovarian cancer in a p53-dependent manner.

OBJECTIVE: APELA is a small, secreted peptide that can function as a ligand for the G-protein coupled receptor, Apelin Receptor (APLNR, APJ). APELA plays an essential role in endoderm differentiation and cardiac development during embryogenesis. We investigated whether APELA exerts any functions in cancer progression. METHODS: The Cancer Genome Atlas (TCGA) RNA sequencing datasets, microarray from an OCCC mouse model, and RNA isolated from fresh frozen and FFPE patient tissue were used to assess APELA expression. APELA knockout ovarian clear cell carcinoma (OCCC) cell lines were generated using CRISPR/Cas9. RESULTS: APELA was expressed in various ovarian cancer histotypes and was especially elevated in OCCC. Disruption of APELA expression in OCCC cell lines suppressed cell growth and migration, and altered cell-cycle progression. Moreover, addition of human recombinant APELA peptide to the OCCC cell line OVISE promoted cell growth and migration. Interestingly, OVISE cells do not express APLNR, suggesting that APELA can function through an APLNR-independent pathway. Furthermore, APELA affected cell growth and cell cycle progression in a p53-dependent manner. In addition, APELA knockdown induced p53 expression in cancer cell lines. CONCLUSIONS: Our findings uncover a potential oncogenic role for APELA in promoting ovarian tumour progression and provide a possible therapeutic strategy in ovarian cancer by targeting APELA.

Genome-wide linkage analysis and regional fine mapping identified variants in the RYR3 gene as a novel quantitative trait locus for circulating adiponectin in Chinese population.

Adiponectin is adipocyte-secreted cytokine with potent insulin-sensitizing action in peripheral tissues. The heritability of plasma adiponectin is high in Han Chinese population.To identify genetic loci influencing plasma adiponectin levels in Chinese population, we performed a genome-wide linkage scan in 1949 Chinese participants of the Stanford Asia-Pacific Program for Hypertension and Insulin Resistance family study and mapped a quantitative trail locus located on chromosome 15 at 31 cM (logarithm of odds = 3.04) with 1-logarithm of odds support interval at 24 to 34 cM. Within this mapped region, we further genotyped a total of 68 single-nucleotide polymorphisms in 12 genes. Association analysis revealed that haplotypes composed of single-nucleotide polymorphisms in the ryanodine receptor 3 (RYR3) gene had strongest association with plasma adiponectin. RYR3 haplotypes were also associated with systolic (P = 0.001) and diastolic (P = 7.1 × 10) blood pressure and high-density lipoprotein cholesterol (P = 1.4 × 10). Furthermore, an inverse relationship between expression of RYR3 and adiponectin was observed in human abdominal adipose tissue. In conclusion, a genome-wide linkage scan and regional association fine-mapping identified variants in the RYR3 gene as a quantitative trail locus for plasma adiponectin levels in Chinese population.

Inhibition of Prostaglandin Reductase 2, a Putative Oncogene Overexpressed in Human Pancreatic Adenocarcinoma, Induces Oxidative Stress-Mediated Cell Death Involving xCT and CTH Gene Expressions through 15-Keto-PGE2.

Prostaglandin reductase 2 (PTGR2) is the enzyme that catalyzes 15-keto-PGE2, an endogenous PPARγ ligand, into 13,14-dihydro-15-keto-PGE2. Previously, we have reported a novel oncogenic role of PTGR2 in gastric cancer, where PTGR2 was discovered to modulate ROS-mediated cell death and tumor transformation. In the present study, we demonstrated the oncogenic potency of PTGR2 in pancreatic cancer. First, we observed that the majority of the human pancreatic ductal adenocarcinoma tissues was stained positive for PTGR2 expression but not in the adjacent normal parts. In vitro analyses showed that silencing of PTGR2 expression enhanced ROS production, suppressed pancreatic cell proliferation, and promoted cell death through increasing 15-keto-PGE2. Mechanistically, silencing of PTGR2 or addition of 15-keto-PGE2 suppressed the expressions of solute carrier family 7 member 11 (xCT) and cystathionine gamma-lyase (CTH), two important providers of intracellular cysteine for the generation of glutathione (GSH), which is widely accepted as the first-line antioxidative defense. The oxidative stress-mediated cell death after silencing of PTGR2 or addition of 15-keto-PGE2 was further abolished after restoring intracellular GSH concentrations and cysteine supply by N-acetyl-L-cysteine and 2-Mercaptomethanol. Our data highlight the therapeutic potential of targeting PTGR2/15-keto-PGE2 for pancreatic cancer.

Etoposide induces cell death via mitochondrial-dependent actions of p53.

BACKGROUND: Etoposide has been used clinically in cancer treatment, as well as in numerous research studies, for many years. However, there is incomplete information about its exact mechanism of action in induction of cell death. METHODS: Etoposide was compared at various concentrations to characterize the mechanisms by which it induces cell death. We investigated its effects on mouse embryonic fibroblasts (MEFs) and focused on both transcriptional and non-transcriptional responses of p53. RESULTS: Here we demonstrate that treatment of MEFs with higher concentrations of etoposide induce apoptosis and activate the transcription-dependent functions of p53. Interestingly, lower concentrations of etoposide also induced apoptosis, but without any evidence of p53-dependent transcription up-regulation. Treatment of MEFs with an inhibitor of p53, Pifithrin-α, blocked p53-dependent transcription but failed to rescue the cells from etoposide-induced apoptosis. Treatment with PES, which inhibits the mitochondrial arm of the p53 pathway inhibited etoposide-induced cell death at all concentrations tested. CONCLUSIONS: We have demonstrated that transcriptional functions of p53 are dispensable for etoposide-induced cell death. The more recently characterized effects of p53 at the mitochondria, likely involving its interactions with BCL-2 family members, are thus more important for etoposide's actions.

Allocation of Klebsiella pneumoniae Bloodstream Isolates into Four Distinct Groups by ompK36 Typing in a Taiwanese University Hospital.

The OmpK36 porin plays a role in carbapenem resistance and may contribute to bacterial virulence in Klebsiella pneumoniae. This study aimed to investigate the characteristics of different groups of K. pneumoniae separated by ompK36 typing. Among 226 nonduplicate K. pneumoniae bloodstream isolates collected at a Taiwanese hospital in 2011, four ompK36 types, designated types A, B, C, and D, were identified by PCR in 61, 28, 100, and 36 isolates, respectively; 1 isolate was untypeable. Statistical analysis showed significantly higher rates of antimicrobial resistance (all tested antibiotics except meropenem), extended-spectrum ß-lactamases or DHA-1 (47.5% together), Qnr-type quinolone resistance determinants (50.8%), and IncFIIA-type plasmids (49.2%) in group A than in others. Seventeen isolates were identified as belonging to 3 international high-risk clones (4 sequence type 11 [ST11], 10 ST15, and 3 ST147 isolates); all isolates but 1 ST15 isolate were classified in group A. The significant characteristics of group C were hypermucoviscosity (62.0%) and a higher virulence gene content. This group included all serotype K1 (n = 30), K2 (n = 25), and K5 (n = 3) isolates, 6 of 7 K57 isolates, all isolates of major clones associated with pyogenic liver abscesses (29 ST23, 11 ST65, 5 ST86, 7 ST373, and 1 ST375 isolates), and 16 (94.1%) of 17 isolates causing bacteremic liver abscesses. Twelve (42.9%) of the group B isolates were responsible for bacteremic biliary tract infections. Group D was predominant (83.3%) among 12 K20 isolates. This study suggests that most clinical K. pneumoniae isolates can be allocated into four groups with distinct characteristics based on ompK36 types.

Mechanism of mast cell adhesion to human tenocytes in vitro.

Mast cells and fibroblasts are two key players involved in many fibrotic and degenerative disorders. In the present study we examined the nature of binding interactions between human mast cells and tendon fibroblasts (tenocytes). In the mast cell-fibroblast co-culture model, mast cells were shown to spontaneously bind to tenocytes, in a process that was partially mediated by α5ß1 integrin receptors. The same receptors on mast cells significantly mediated binding of these cells to tissue culture plates in the presence of tenocyte-conditioned media; the tenocyte-derived fibronectin in the media was shown to also play a major role in these binding activities. Upon binding to tenocytes or tissue culture plates, mast cells acquired an elongated phenotype, which was dependent on α5ß1 integrin and tenocyte fibronectin. Additionally, tenocyte-derived fibronectin significantly enhanced mRNA expression of the adhesion molecule, THY1, by mast cells. Our data suggests that α5ß1 integrin mediates binding of mast cells to human tenocyte and to tenocyte-derived ECM proteins, in particular fibronectin.

Impairing eukaryotic elongation factor 2 kinase activity decreases atherosclerotic plaque formation.

We tested whether loss of eukaryotic elongation factor 2 kinase (eEF2K) activity in macrophages suppresses development of atherosclerosis by transplanting bone marrow from mice with mutant eEF2K into ldlr(-/-) mice. Sixteen weeks after high-fat diet feeding, mutant eEF2K hematopoietic chimeras had a dramatically reduced level of atherosclerotic plaque formation. M1-skewed macrophages from eEF2K knock-in mice have less tumour necrosis factor-α release and a lesser ability to induce expression of endothelial cell markers, providing a potential explanation for the role of eEF2K. Because eEF2K activity in cells of the hematopoietic compartment contributes to atherosclerosis development, drugs inhibiting eEF2K might have a beneficial effect in treatment of atherosclerosis.

Knockdown of RyR3 enhances adiponectin expression through an atf3-dependent pathway.

Adiponectin is an important adipose-specific protein, which possesses insulin (INS)-sensitizing, antiinflammatory, and antiatherosclerotic functions. However, its regulation remains largely unknown. In this study, we identified that ryanodine receptor (RyR)3 plays an important role in the regulation of adiponectin expression. RyR3 was expressed in 3T3-L1 preadipocytes, and its level was decreased upon adipogenesis. Silencing of RyR3 expression in 3T3-L1 preadipocytes resulted in up-regulated adiponectin promoter activity, enhanced adiponectin mRNA expression, and more adiponectin protein secreted into the medium. An inverse relation between RyR3 and adiponectin mRNA levels was also observed in adipose tissues of db/db mice. In addition, knockdown of RyR3 with small interfering RNA (siRNA) in db/db mice and high-fat diet-fed obese mice increased serum adiponectin level, improved INS sensitivity, and lowered fasting glucose levels. These effects were in parallel with decreased mitochondrial Ca(2+), increased mitochondrial mass, and reduced activating transcription factor 3 (atf3) expression. Overexpression of atf3 in 3T3-L1 preadipocytes blocked the effect of RyR3 silencing on adiponectin expression, indicating that an atf3-dependent pathway mediates the effect downstream of RyR3 silencing. Our data suggest that RyR3 may be a new therapeutic target for improving INS sensitivity and related metabolic disorders.

Cytoskeletal protein vimentin interacts with and regulates peroxisome proliferator-activated receptor gamma via a proteasomal degradation process.

Peroxisome proliferators-activated receptor gamma (PPARγ) receptor is a transcription factor that is located in and functions primarily in the nucleus. PPARγ is exported from the nucleus upon mitogen and ligand stimulation under certain circumstances. However, a cytoplasmic PPARγ interacting protein and its function have not been previously identified. Here, we report for the first time that cytosolic PPARγ interacts directly with cytoskeletal vimentin. We performed PPARγ immunoprecipitation followed by mass spectrometry to identify the vimentin-PPARγ complex. This interaction was confirmed by reciprocal vimentin and PPARγ immunoprecipitation and co-immunofluorescence examination. We demonstrated that PPARγ colocalized with vimentin in certain organelles that is golgi, mitochondria, and endoplasmic reticulum. In cells depleted of vimentin, PPARγ was ubiquitinated and targeted to a proteasomal degradation pathway. Together, these findings indicate a direct interaction of PPARγ with vimentin in the cytosolic compartment, in which vimentin appears to play a role in regulating the turnover rate of PPARγ, which may further regulate genomic or non-genomic activities through the regulation of PPARγ protein degradation.

Prostaglandin reductase 2 modulates ROS-mediated cell death and tumor transformation of gastric cancer cells and is associated with higher mortality in gastric cancer patients.

Various prostanoids and peroxisome proliferator-activated receptor γ (PPARγ) ligands play an important role in gastric cancer. Previously, we demonstrated that prostaglandin reductase 2 (PTGR2) catalyzes the reduction of the PPARγ ligand 15-keto-PGE(2) into 13,14-dihydro-15-keto-PGE(2). Here, we present functional data and clinical relevance for the role of PTGR2 in gastric cancer. Using lentiviral technology in AGS and SNU-16 gastric cancer cell lines, we either down-regulated or overexpressed PTGR2. In vitro analysis showed that PTGR2 knockdown resulted in decreased proliferation rate and colony formation, and in vivo xenograft models showed slower growth of tumors. Mechanistically, PTGR2 knockdown induced cell death, altered mitochondrial function, and increased reactive oxygen species production, which led to activation of ERK1/2 and caspase 3, with increased Bcl-2 and suppressed Bax expression. PTGR2 overexpression showed the opposite outcomes. Clinically, immunopathological staining showed strong PTGR2 expression in the gastric tumor portion, relative to nearby nontumor portions, and its expression negatively correlated with survival of patients with intestinal-type gastric cancer. Finally, in contrast to PTGR2-overexpressing cells, PTGR2-knockdown cells were more sensitive to cisplatin and 5-fluorouracil. Taken together, our findings not only provide functional and mechanistic evidence of the involvement of PTGR2 in gastric cancer, but also provide clinical observations affirming the significance of PTGR2 in gastric cancer and suggesting that PTGR2-target based therapy is worth further evaluation.

Genetic variation in the carbonyl reductase 3 gene confers risk of type 2 diabetes and insulin resistance: a potential regulator of adipogenesis.

Prostaglandins are potent modulators of insulin sensitivity. We systemically evaluated the association of 61 tag single-nucleotide polymorphisms (SNP) in 14 genes involved in prostaglandin metabolism with type 2 diabetes. Among all genotyped SNPs, rs10483032 in the CBR3 (carbonyl reductase 3) gene, which encodes for an enzyme converting prostaglandin E(2) to prostaglandin F2(α), was associated with type 2 diabetes in 760 type 2 diabetic cases and 760 controls (stage-1 study) (P = 2.0 × 10(-4)). The association was validated in 1,615 cases and 1,162 controls (stage-2 study) (P = 0.009). The A allele at rs10483032 was associated with increased risk of type 2 diabetes (odds ratio = 1.29; 95% confidence interval = 1.14-1.47; combined P < 0.0001). The association was externally validated in the Finland-United States Investigation of NIDDM Genetics (FUSION) study (P = 3.7 × 10(-4)). The risk A allele was associated with higher homeostasis model assessment of insulin resistance (HOMA-IR) in 1,012 non-diabetic controls and 1,138 non-diabetic subjects from the Stanford Asia-Pacific Program for Hypertension and Insulin Resistance (SAPPHIRe) family study. CBR3 gene expression in human abdominal adipose tissue was negatively associated with fasting insulin and HOMA-IR. CBR3 gene expression increased during differentiation of 3T3-L1 preadipocytes into adipocytes. Knockdown of CBR3 in 3T3-L1 preadipocytes enhanced adipogenesis and peroxisome proliferator-activator receptor-γ response element reporter activity. Our results indicated that genetic polymorphism in the CBR3 gene conferred risk of type 2 diabetes and insulin resistance in Chinese. The association was probably mediated through modulation of adipogenesis.

The role of nocturnin in early adipogenesis and modulation of systemic insulin resistance in human.

The deadenylase nocturnin (Noc, Ccrn4l) has been recently found to regulate lipid metabolism and to control preadipocyte differentiation. Here, we showed that among the five deadenylases tested, Noc and Pan2 exhibited a biphasic expression which is out of phase to each other during adipocyte differentiation of 3T3-L1 cells. The expression levels of other deadenylases, including Parn, Ccr4, and Caf1, were relatively unchanged or reduced. The immediate early expressed Noc during 3T3-L1 adipogenesis was involved in regulating mitotic clonal expansion (MCE) and cyclin D1 expression, as demonstrated in Noc-silenced 3T3-L1 cells and Noc(-/-) primary mouse embryonic fibroblasts (MEFs). Transcriptional profiling of Noc-depleted 3T3-L1 adipocytes revealed that most of the differentially expressed genes were related to cell growth and proliferation. In human adipose tissue, NOC mRNA level negatively associated with both fasting serum insulin and homeostasis model assessment of insulin resistance, and positively associated with both adiponectin mRNA levels and circulating adiponectin levels. Taken together, these results suggest the role of Noc in the modulation of early adipogenesis as well as systemic insulin sensitivity.

Characterization of carbapenem-non-susceptible Escherichia coli isolates from a university hospital in Taiwan.

OBJECTIVES: To investigate characteristics of nine carbapenem-non-susceptible (CP-NS) Escherichia coli isolates collected between 1999 and 2005 at a Taiwanese university hospital. METHODS: Genetic relatedness was analysed by PFGE. beta-Lactamases were characterized by PCR and isoelectric focusing. Outer membrane proteins and transcripts were investigated by SDS-PAGE and northern blotting. Cloning experiments were performed to investigate the role of membrane permeability in carbapenem non-susceptibility. RESULTS: The nine CP-NS isolates were found to produce the CMY-2 AmpC enzyme (n = 8), the CTX-M-14-type extended-spectrum beta-lactamase (ESBL) (n = 1), the SHV-12 ESBL (n = 1) and the IMP-8-type metallo-beta-lactamase (n = 1) alone or in combination. All CP-NS isolates revealed a decrease in the transcription and protein expression of ompC, and susceptibility to carbapenems was restored in one isolate by introducing the cloned ompC gene. PFGE revealed genetic diversity among the nine isolates. All patients with the CP-NS isolates had been treated with carbapenems (six patients) and/or extended-spectrum cephalosporins (five patients) before isolation. CONCLUSIONS: Our study suggests that the decreased susceptibility to carbapenems in E. coli in the hospital might arise by the stepwise accumulations of multiple drug-resistance determinants in different clones.

Prevalence of extended-spectrum beta-lactamases in Proteus mirabilis in a Taiwanese university hospital, 1999 to 2005: identification of a novel CTX-M enzyme (CTX-M-66).

A total of 1574 nonduplicate Proteus mirabilis isolates collected at a Taiwanese hospital during 1999 to 2005 were analyzed for production of extended-spectrum beta-lactamases (ESBLs). Forty-four ESBL-producing isolates including 22 CTX-M-14, 18 CTX-M-3, 2 CTX-M-24, and 2 CTX-M-66 producers were detected, and the proportion of ESBL producers increased from 0.7% in 1999 to approximately 6% after 2002. CTX-M-66 is a novel variant of CTX-M ESBLs that differs from CTX-M-3 by a Ser to Asn change at amino acid position 23. Coresistances to aminoglycosides and ciprofloxacin were very common in the CTX-M-3 producers. The presence of ArmA-type or RmtB-type 16S rRNA methylase that confers high-level aminoglycoside resistance was detected in 12 CTX-M-3 producers and 4 CTX-M-14 producers. Twenty-four clones including an endemic CTX-M-14-producing clone were observed among the 44 ESBL producers by pulsed-field gel electrophoresis, suggesting that both horizontal transfer and clonal spread contributed to the increased prevalence of bla(CTX-M) in P. mirabilis.

Binding of a venom Lys-49 phospholipase A(2) to LPS and suppression of its effects on mouse macrophages.

The Lys49-phospholipases A(2) (K49-PLAs) are abundant in many pit vipers' venom. They are highly basic myotoxins and capable of binding membranes but lack hydrolytic activity. Considerable attention has been directed to its antibacterial activity but the exact mechanisms remain unclear. We now evaluate the roles of a K49-PLA from Trimeresurus stejnegeri venom in antagonizing the effects of lipopolysaccharide (LPS) on mouse macrophages (RAW264.7 cells). The K49-PLA markedly reduced LPS-stimulated production of NO, MCP-1, RANTES, and iNOS. RT-PCR analysis also confirmed its suppression of LPS-induced transcription of these cellular proteins. Moreover, LPS-induced activation of NFkappaB was dramatically abolished, while phosphorylation and degradation of IkappaB were also inhibited. Other types of venom phospholipases tested did not show the same effects as K49-PLA. Finally, strong binding between K49-PLA and LPS with a dissociation constant at the order of 10nM was shown by microcalorimetry titration. These findings provide unprecedented evidence that a low dose of K49-PLA possesses potent anti-inflammatory and antibacterial properties, which raises the prospect of a new therapeutic approach against sepsis.