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Zeynep Ay Şenyiğit,1 Sinem Yaprak Karavana,1 Derya İlem-Özdemir,2 Çağrı Çalışkan,2 Claudia Waldner,3 Sait Şen,4 Andreas Bernkop-Schnürch,5 Esra Baloğlu1 1Faculty of Pharmacy, Department of Pharmaceutical Technology, Ege University, Bornova, İzmir, Turkey; 2Faculty of Pharmacy, Department of Radiopharmacy, Ege University, Bornova, İzmir, Turkey; 3ThioMatrix, Forschungs-Beratungs GmbH, Innsbruck, Austria; 4Faculty of Medicine, Department of Pathology, Ege University, Bornova, İzmir, Turkey; 5Institute of Pharmacy, Department of Pharmaceutical Technology, University of Innsbruck, Innsbruck, AustriaAbstract: This study aimed to develop an intravesical delivery system of gemcitabine HCl for superficial bladder cancer in order to provide a controlled release profile, to prolong the residence time, and to avoid drug elimination via urination. For this aim, bioadhesive nanoparticles were prepared with thiolated chitosan (chitosan–thioglycolic acid conjugate) and were dispersed in bioadhesive chitosan gel or in an in situ gelling poloxamer formulation in order to improve intravesical residence time. In addition, nanoparticle-loaded gels were diluted with artificial urine to mimic in vivo conditions in the bladder and were characterized regarding changes in gel structure. The obtained results showed that chitosan-thioglycolic acid nanoparticles with a mean diameter of 174.5±3.762 nm and zeta potential of 32.100±0.575 mV were successfully developed via ionotropic gelation and that the encapsulation efficiency of gemcitabine HCl was nearly 20%. In vitro/ex vivo characterization studies demonstrated that both nanoparticles and nanoparticle-loaded chitosan and poloxamer gels might be alternative carriers for intravesical administration of gemcitabine HCl, prolonging its residence time in the bladder and hence improving treatment efficacy. However, when the gel formulations were diluted with artificial urine, poloxamer gels lost their in situ gelling properties at body temperature, which is in conflict with the aimed formulation property. Therefore, 2% chitosan gel formulation was found to be a more promising carrier system for intravesical administration of nanoparticles. Keywords: chitosan–TGA, nanoparticle, gemcitabine HCl, intravesical administration, chitosan, poloxamer, superficial bladder cancer

A study is made to find whether the effect mediated by prostaglandin E2(PGE2) acting throyugh its EP2 receptor in cultured rat mesangial cells. It is seen in glomerulonephritis, cyclooxygenase 2 (COX-2) appears to suppress the expression of alpha integrins by the increased production of prostaglandin E2 activating its EP3 receptor.

Background Circulating l-lactate concentration is commonly measured in hospitalized horses by sampling from indwelling intravenous ( IV) catheters. However, there are no published evidence-based recommendations to prevent contamination by lactated Ringer's solution ( LRS). Hypothesis Withdrawing 10 mL of blood from the LRS-containing extension set connected to the IV catheter before obtaining the sample for analysis should be adequate to obtain accurate measurement of blood lactate concentration ( BLC). Animals Thirty-three adult hospitalized horses receiving constant rate infusion of LRS. Methods Immediately after disconnecting the LRS, 5 sequential 5 mL blood samples were obtained by aspiration from an extension set connected to an indwelling IV catheter, followed by 3 samples collected by direct venipuncture of the contralateral jugular vein. Samples were analyzed with 1 portable blood lactate analyzer. A linear mixed model was used to examine differences in lactate concentrations among samples collected from the catheter and by direct venipuncture. Results After considering differences in age, breed, sex, and reason for hospitalization, BLCs were higher ( P < .001) in the first and second 5 mL samples collected through the extension set/catheter than in all other extension set/catheter samples or the direct venipuncture samples. The largest difference observed between the third and subsequent catheter or venipuncture samples was 0.34 mmol/L with an upper 95% CI of 1.12 mmol/L. Conclusions and Clinical Importance Withdrawing 15 mL of blood from a LRS-containing extension set connected to an IV catheter (5.9 mL total volume capacity) before obtaining the sample for blood lactate analysis is suggested to optimize accuracy of BLC measurements. [ABSTRACT FROM AUTHOR]

Circulating immunoglobulin (Ig)G antibodies against M muscarinic acetylcholine receptors (M mAChR) have been implicated in Chagas' disease (ChD) pathophysiology. These antibodies bind to and activate their target receptor, displaying agonist-like activity through an unclear mechanism. This study tested the ability of serum anti-M mAChR antibodies from chronic ChD patients to modulate M muscarinic receptor-receptor interaction by bioluminescence resonance energy transfer (BRET). Human embryonic kidney (HEK) 293 cells co-expressing fusion proteins M mAChR-Renilla luciferase (RLuc) and M mAChR-yellow fluorescent protein (YFP) were exposed to the serum IgG fraction from ChD patients, and BRET between RLuc and YFP was assessed by luminometry. Unlike serum IgG from healthy subjects and conventional muscarinic ligands, ChD IgG promoted a time- and concentration-dependent increase in the BRET signal. This effect neither required cellular integrity nor occurred as a consequence of receptor activation. Enhancement of M receptor-receptor interaction by ChD IgG was receptor subtype-specific and mediated by the recognition of the second extracellular loop of the M mAChR. The monovalent Fab fragment derived from ChD IgG was unable to reproduce the effect of the native immunoglobulin. However, addition of ChD Fab in the presence of anti-human Fab IgG restored BRET-enhancing activity. These data suggest that the modulatory effect of ChD IgG on M receptor-receptor interaction results from receptor cross-linking by bivalent antibodies. [ABSTRACT FROM AUTHOR]

Background: In experimental glomerulonephritis, inhibition of cyclooxygenase 2 (COX-2) enhances the renocorticol expression of pathogenic αv integrins. Aims: To study whether this effect is mediated by prostoglandin E2 (PGE2) acting through its EP3 receptor in cultured rat mesangial cells (MCs). Methods: MCs were incubated with lipopolysaccharide (LPS), celecoxib, PGE2, or the selective EP3 agonist, MB28767. The expression of COX-2, EP3, and αv integrin mRNA was measured by reverse transcriptase polymerase chain reaction. Results: IPS upregulated COX-2 expression 2.8-fold and αv integrin expression twofold. The COX-2 inhibitor celecoxib increased αv integrin mRNA expression twofold. Both exogenous PGE2 and the specific EP3 receptor agonist, MB28767, reduced constitutive αv integrin mRNA expression to half normal values. COX-2 dependent PGE2 suppressed the expression of αv integrin mRNA mediated by the EP3 receptor in MCs. Conclusions: These results suggest that COX-2 suppresses the expression of αv integrins by an increased production of PGE2 activating its EP3 receptor in glomerulonephritis. [ABSTRACT FROM AUTHOR]

Introduction: Chronic pain is an important health problem adversely affecting functionality and quality of life. Though self-rated health (SRH) is a major predictor of mortality, its relationship with pain is not well understood. Early detection and treatment of pain can enhance well-being and SRH in seniors. Objective: In this analysis, we explore 1) how pain and age interact to influence SRH; and 2) provincial variations in SRH across Canada. Methods: We analyzed cross-sectional data from Statistics Canada's Canadian Community Health Survey-Healthy Aging (n = 30 685), which targeted those 45 years and older and was conducted from 2008-12-01 to 2009-11-30. The response rate was 74.4%. The topics covered included sociodemographics, well-being and chronic diseases. We performed bivariate analyses between each predictor and SRH; unadjusted odds ratios and 95% confidence intervals are reported. In the second phase of analysis, multivariate model building was used to examine the interaction between predictor variables. We used a two-level logistic regression mixed model consisting of provincial differences (first level) and individual differences (second level). An interclass correlation coefficient was computed to determine the degree of variability regarding seniors' SRH at the provincial level. All statistical analyses were completed in Stata 13 (StataCorp LP, College Station, TX, US) at a 5% level of significance. Results: Slightly more than half of respondents (52.3%) were female. In the bivariate analysis, those experiencing daily chronic pain were 4.34 times more likely to rate their health as poor (p < .001). In the multivariate analysis, being female was protective (p < .001). Those who reported being depressed, lonely, less educated and/or having a lower income were more likely to rate their health as poor (p < .001). Respondents in Atlantic Canada were more likely to report poorer health. Analgesic users were also 1.56 times more likely to report poorer health (p < .001). The association between pain and SRH was stronger among younger age groups (45-54 years) compared to older age groups (= 85 years, with an odds ratio of 4.33 [p < .001] versus 2.47 [p < .001]). Conclusion: Chronic pain, among other determinants, is associated with SRH. Individuals in rating their health may consider a variety of factors, some of which may not be apparent to health providers. We found the influence of pain on SRH was more pronounced among males and those who reported being depressed, lonely, less educated, and/or having a lower income. Also significant was the impact of pain on SRH among middle-aged Canadians in comparison to other age groups. A traditional east to west gradient in SRH in Canada was also observed in this study. [ABSTRACT FROM AUTHOR]

Abstract Background FLP recombinase mediated integration into a pre-integrated FRT site is routinely used to generate highly reproducible stable transgenic cell lines. In this study, we broaden the system of site specific integration by introducing ΦC31 integrase mediated integration into attP sites. Results We generated a HEK293 host cell line with a single copy FRT as well as an attP site allowing site specific integration of two distinct transgenes. To achieve conditional control, we used the tetracycline and Shld1 inducible systems. By introducing fluorescent reporters we show that integration and induction of two transgenes are completely independent. We applied this new technique to investigate the effect of HNF4α on proliferation of HEK293 cells by introducing HNF4α into each integration site. We obtained in two independent cell lines highly reproducible results that prove the usefulness of this novel HEK-attP/FRT cell line. Conclusions In this study we have established and applied a HEK-attP/FRT cell line that allows site specific integration of two conditional transgenes using the FLP recombinase as well as the ΦC31 integrase.

Abstract Background To analyze gene function in mammalian cells tetracycline inducible expression of a gene-of-interest at a specific genomic location (Flp-In T-REx™) is most attractive. However, leakiness of basal transgene expression and artificially high expression level upon tetracycline addition may be disadvantageous. Findings To solve these problems, we developed two different approaches to improve our pancreatic β-cell line INS-1 Flp-In T-REx™ expressing the tissue restricted transcription factor HNF4α under control of tetracycline. On the one hand we replaced the strong full length CMV promoter (CMV-Wt) with a weaker 5'-deleted CMV promoter fragment of 138 nucleotides in length (CMV-138). On the other hand we extended our INS-1 Flp-In T-REx™ cell lines with a Shield-1 dependent conditional control system of protein stability. Therefore, we fused HNF4α to the destabilization domain (DD) deduced from human FKBP12 protein. As a result in both approaches basal transgene expression level was markedly reduced, but HNF4α induction could still be maintained. Additionally, we could show that a low increase in HNF4α induces caspase activity indicating an apoptotic effect of HNF4α in these cells. Conclusion In the present study we considerably improved our INS-1 Flp-In T-REx™ cell lines conditionally expressing HNF4α to reduce leakiness and to optimize exogenous HNF4α protein expression to a physiological level. As an important result we could extend our previous results that HNF4α induces apoptosis in the pancreatic β-cell line INS-1 with the new aspect that an expression level of the HNF4α transgene marginally exceeding the endogenous level is sufficient to trigger apoptosis.

Colistin resistance in Escherichia coli is of public health significance for its use to treat multidrug-resistant Gram-negative infections. Amino acid variations in PmrB have been implicated in colistin resistance in E. coli . In this cross-sectional study, 288 generic E. coli isolates from surveillance of broiler chicken and feedlot cattle feces, retail meat, wastewater, and well water were whole-genome sequenced. Phylogroup designation and screening for two amino acid substitutions in PmrB putatively linked to colistin resistance (Y358N, E123D) were performed in silico . Three additional data sets of publicly available E. coli assemblies were similarly scrutinized: (i) E. coli isolates from studies identifying the Y358N or E123D substitutions, (ii) colistin-susceptible E. coli isolates reported in the literature, and (iii) a random sampling of 14,700 E. coli assemblies available in the National Center for Biotechnology Information public database. Within all data sets, ≥95% of phylogroup B1 and C isolates have the PmrB Y358N variation. The PmrB E123D amino acid substitution was only identified in phylogroup B2 isolates, of which 94%-100% demonstrate the substitution. Both PmrB amino acid variations were infrequent in other phylogroups. Among published colistin susceptible isolates, colistin minimum inhibitory concentrations (MICs) were not higher in isolates bearing the E123D and Y358N amino acid variations than in isolates without these PmrB substitutions. The E123D and Y358N PmrB amino acid substitutions in E. coli appear strongly associated with phylogroup. The previously observed associations between Y358N and E123D amino acid substitutions in PmrB and colistin resistance in E. coli may be spurious., Importance: Colistin is a critical last-resort treatment for extensively drug-resistant Gram-negative infections in humans. Therefore, accurate identification of the genetic mechanisms of resistance to this antimicrobial is crucial to effectively monitor and mitigate the spread of resistance. Examining over 16,000 whole-genome sequenced Escherichia coli isolates, this study identifies that PmrB E123D and Y358N amino acid substitutions previously associated with colistin resistance in E. coli are strongly associated with phylogroup and are alone not sufficient to confer a colistin-resistant phenotype. This is a critical clarification, as both substitutions are identified as putative mechanisms of colistin resistance in many publications and a common bioinformatic tool. Given the potential spurious nature of initial associations of these substitutions with colistin resistance, this study's findings emphasize the importance of appropriate experimental design and consideration of relevant biological factors such as phylogroup when ascribing causal mechanisms of resistance to chromosomal variations., Competing Interests: The authors declare no conflict of interest.