An Infection-Responsive Approach to Reduce Bacterial Adhesion in Urinary Biomaterials

Infection is an inevitable consequence of chronic urinary catheterisation, with associated problems of recurrent catheter encrustation and blockage experienced by approximately 50% of all long-term catheterised patients. In this work we have exploited, for the first time, the reported pathogen-induced elevation of urine pH as a trigger for ‘intelligent’ antimicrobial release from novel hydrogel drug delivery systems of 2-hydroxyethyl methacrylate and vinyl-functionalised nalidixic acid derivatives, developed as candidate infection-resistant urinary catheter coatings. Demonstrating up to 20-fold faster rates of drug release at pH 10, representing infected urine pH, than at pH 7, and achieving reductions of up to 96.5% in in vitro bacterial adherence, our paradigm of pH-responsive drug delivery, which requires no external manipulation, therefore represents a promising development towards the prevention of catheter-associated urinary tract infections (CAUTIs) in vivo.

Treatment of lean and diet-induced obesity (DIO) mice with a novel stable obestatin analogue alters plasma metabolite levels as detected by untargeted LC–MS metabolomics

Introduction: Obestatin is a controversial gastrointestinal peptide purported to have metabolic actions.

Objectives: This study investigated whether treatment with a stable obestatin analogue (PEG-OB(Cys10, Cys13)) changed plasma metabolite levels firstly in lean and subsequently in diet-induced obesity (DIO) C57BL6/J mice.

Methods: Untargeted LC-HRMS metabolomics experiments were carried out in ESI + mode with plasma extracts from both groups of animals. Data were normalised, multivariate and univariate statistical analysis performed and metabolites of interest putatively identified.

Results: In lean mice, 39 metabolites were significantly changed by obestatin treatment and the majority of these were increased, including various C16 and C18 moieties of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and monoacylglycerol, along with vitamin A, vitamin D3, tyrosine, acetylcarnitine and 2α-(hydroxymethyl)-5α-androstane-3β,17β-diol. Decreased concentrations of glycolithocholic acid, 3-dehydroteasterone and various phospholipids were observed. In DIO mice, 25 metabolites were significantly affected and strikingly, the magnitudes of changes here were generally much greater in DIO mice than in lean mice, and in contrast, the majority of metabolite changes were decreases. Four metabolites affected in both groups included glycolithocholic acid, and three different long-chain (C18) phospholipid molecules (phosphatidylethanolamine, platelet activating factor (PAF), and monoacylglycerol). Metabolites exclusively affected in DIO mice included various phosphatidylcholines, lysophosphatidylcholines and fatty acyls, as well as creatine and oxidised glutathione.

Conclusion: This investigation demonstrates that obestatin treatment affects phospholipid turnover and influences lipid homeostasis, whilst providing convincing evidence that obestatin may be acting to ameliorate diet-induced impairments in lipid metabolism, and it may influence steroid, bile acid, PAF and glutathione metabolism.

Bacteriophage Can Prevent Encrustation and Blockage of Urinary Catheters by Proteus mirabilis.

Proteus mirabilis forms dense crystalline biofilms on catheter surfaces that occlude urine flow, leading to serious clinical complications in long-term catheterized patients, but there are presently no truly effective approaches to control catheter blockage by this organism. This study evaluated the potential for bacteriophage therapy to control P. mirabilis infection and prevent catheter blockage. Representative in vitro models of the catheterized urinary tract, simulating a complete closed drainage system as used in clinical practice, were employed to evaluate the performance of phage therapy in preventing blockage. Models mimicking either an established infection or early colonization of the catheterized urinary tract were treated with a single dose of a 3-phage cocktail, and the impact on time taken for catheters to block, as well as levels of crystalline biofilm formation, was measured. In models of established infection, phage treatment significantly increased time taken for catheters to block (∼ 3-fold) compared to untreated controls. However, in models simulating early-stage infection, phage treatment eradicated P. mirabilis and prevented blockage entirely. Analysis of catheters from models of established infection 10 h after phage application demonstrated that phage significantly reduced crystalline biofilm formation but did not significantly reduce the level of planktonic cells in the residual bladder urine. Taken together, these results show that bacteriophage constitute a promising strategy for the prevention of catheter blockage but that methods to deliver phage in sufficient numbers and within a key therapeutic window (early infection) will also be important to the successful application of phage to this problem.

Tubular Urinary Enzymes in Acute Post-infectious Glomerulonephritis

Tubular function of 17 pediatric patients with a mild form of acute post-infectious glomerulonephritis was prospectively evaluated by assessment of the urinary activity of proximal and distal tubule enzymes. Neutral-like endopeptidase (NEP-like) and angiotensin-converting enzyme (ACE) were the proximal tubule enzymes assessed, while prolyl-endopeptidase (PE) and serine-endopeptidase H1 and H2 were the distal tubule enzymes analyzed. Urine was collected at diagnosis (T0) and after 2 (T2) and 6 (T6) months of follow-up. NEP-like enzyme activity (nmol/mg creatinine; median±quartile range) was increased at diagnosis, and this remained stable during the first 6 months (T0 18.30±83.26, T2 17.32±49.56, T6 23.38±107.18). Urinary activity of the other enzymes was as follows: ACE (mU/ml per mg creatinine) T0 0.08±0.16, T2 0.06±0.10, T6 0.18±0.29; PE (nmol/mg creatinine) T0 6.70±84.87, T2 9.55±69.00, T6 13.67±28.70; serine-endopeptidase H1 (nmol/mg creatinine) T0 7.86±26.95, T2 17.17±59.37, T6 18.19± 79.14; and serine-thiol-endopeptidase H2 (nmol/mg creatinine) T0 3.06±21.97, T2 12.06±32.42, T6 16.22± 44.06. Thirty other healthy children matched for age and gender were considered as a control group. This group was assessed once and the results were: NEP-like activity 6.05±10.54, ACE 0.11±0.22, PE 7.10±13.36, H1 5.00±17.30, and H2 6.00±20.16. In conclusion, we observed that NEP-like and H1 enzymes exhibited significant increased urinary activity 6 months after the diagnosis. This increase occurred in spite of the disappearance of clinical symptoms, which occurred 2 months after the diagnosis. We believe that the increase in urinary enzymatic activity could be a manifestation of a silent tubular dysfunction following an episode of acute post-infectious glomerulonephritis.

Liver glucose-6-phosphatase proteins in suckling and weaned grey seal pups: structural similarities to other mammals and relationship to nutrition, insulin signalling and metabolite levels

Phocid seals have been proposed as models for diabetes because they exhibit limited insulin response to glucose, high blood glucose and increasing insulin resistance when fasting. Liver glucose-6-phosphatase (G6Pase) catalyses the final step in glucose production and is central to glucose regulation in other animals. G6Pase comprises a translocase (SLC37A4) and a catalytic subunit (G6PC). G6PC and SLC37A4 expression and activity are normally regulated by nutritional state and glucostatic hormones, particularly insulin, and are elevated in diabetes. We tested the hypotheses that (1) grey seal G6PC and SLC37A4 cDNA and predicted protein sequences differ from other species’ at functional sites, (2) relative G6Pase protein abundances are lower during feeding than fasting and (3) relative G6Pase protein abundances are related to insulin, insulin receptor phosphorylation and key metabolite levels. We show that G6PC and partial SLC37A4 cDNA sequences encode proteins sharing 82–95 % identity with other mammals. Seal G6PC contained no differences in sites responsible for activity, stability or subcellular location. Several substitutions in seal SLC37A4 were predicted to be tolerated with low probability, which could affect glucose production. Suckling pups had higher relative abundance of both subunits than healthy, postweaned fasting pups. Furthermore, relative G6PC abundance was negatively related to glucose levels. These findings contrast markedly with the response of relative hepatic G6Pase abundance to feeding, fasting, insulin, insulin sensitivity and key metabolites in other animals, and highlight the need to understand the regulation of enzymes involved in glucose control in phocids if these animals are to be informative models of diabetes.

Metabolite profiling of somatic embryos of Cyclamen persicum in comparison to zygotic embryos, endosperm, and testa

Somatic embryogenesis has been shown to be an efficient in vitro plant regeneration system for many crops such as the important ornamental plant Cyclamen persicum, for which this regeneration pathway of somatic embryogenesis is of interest for the vegetative propagation of parental lines as well as elite plants. However, somatic embryogenesis is not commercially used in many crops due to several unsolved problems, such as malformations, asynchronous development, deficiencies in maturation and germination of somatic embryos. In contrast, zygotic embryos in seeds develop and germinate without abnormalities in most cases. Instead of time-consuming and labor-intensive experiments involving tests of different in vitro culture conditions and plant growth regulator supplements, we follow a more directed approach. Zygotic embryos served as a reference and were compared to somatic embryos in metabolomic analyses allowing the future optimization of the in vitro system. The aims of this study were to detect differences in the metabolite profiles of torpedo stage somatic and zygotic embryos of C. persicum. Moreover, major metabolites in endosperm and testa were identified and quantified. Two sets of extracts of two to four biological replicates each were analyzed. In total 52 metabolites were identified and quantified in the different tissues. One of the most significant differences between somatic and zygotic embryos was that the proline concentration in the zygotic embryos was about 40 times higher than that found in somatic embryos. Epicatechin, a scavenger for reactive oxygen species, was found in highest abundance in the testa. Sucrose, the most abundant metabolite was detected in significantly higher concentrations in zygotic embryos. Also, a yet unknown trisaccharide, was significantly enriched in zygotic embryos.

Firefighters' exposure biomonitoring: Impact of firefighting activities on levels of urinary monohydroxyl metabolites

The concentrations of six urinary monohydroxyl metabolites (OH-PAHs) of polycyclic aromatic hydrocarbons, namely 1-hydroxynaphthalene, 1-hydroxyacenaphthene, 2-hydroxyfluorene, 1-hydroxyphenanthrene, 1-hydroxypyrene (1OHPy), and 3-hydroxybenzo[a]pyrene, were assessed in the post-shift urine of wildland firefighters involved in fire combat activities at six Portuguese fire corporations, and compared with those of non-exposed subjects. Overall, median levels of urinary individual and total OH-PAHs (ΣOH-PAHs) suggest an increased exposure to polycyclic aromatic hydrocarbons during firefighting activities with ΣOH-PAH levels in exposed firefighters 1.7-35 times higher than in non-exposed ones. Urinary 1-hydroxynaphthalene and/or 1-hydroxyacenapthene were the predominant compounds, representing 63-98% of ΣOH-PAHs, followed by 2-hydroxyfluorene (1-17%), 1-hydroxyphenanthrene (1-13%), and 1OHPy (0.3-10%). A similar profile was observed when gender discrimination was considered. Participation in fire combat activities promoted an increase of the distribution percentage of 1-hydroxynaphthalene and 1-hydroxyacenaphthene, while contributions of 1-hydroxyphenanthrene and 1OHPy decreased. The detected urinary 1OHPy concentrations (1.73×10(-2) to 0.152μmol/mol creatinine in exposed subjects versus 1.21×10(-2) to 5.44×10(-2)μmol/mol creatinine in non-exposed individuals) were lower than the benchmark level (0.5μmol/mol creatinine) proposed by the American Conference of Governmental Industrial Hygienists. This compound, considered the biomarker of exposure to PAHs, was the less abundant one from the six analyzed biomarkers. Thus the inclusion of other metabolites, in addition to 1OHPy, in future studies is suggested to better estimate firefighters' occupational exposure to PAHs. Moreover, strong to moderate Spearman correlations were observed between individual compounds and ΣOH-PAHs corroborating the prevalence of an emission source.

Home-based urinary HPV DNA testing in women who do not attend cervical cancer screening clinics

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Urinary schistosomiasis among preschool children in Malengachanzi, Nkhotakota District, Malawi: Prevalence and risk factors

Aim: This study was designed to determine the prevalence of and risk factors for schistosomiasis among a group of preschool children in Malawi. Schistosomiasis burden among preschoolers in Malawi is not well documented in the literature. Methods: This study used field research (in the form of a snail survey), laboratory work (urinalysis and microscopy for parasite identification), and questionnaireguided interviews to determine the prevalence of and risk factors for urinary schistosomiasis among children, aged between 6 and 60 months, in Malengachanzi, Nkhotakota District, Malawi. Results: Urinary schistosomiasis prevalence among preschool children was 13%. Of the factors evaluated, only age (P = 0.027) was statistically significantly associated with urinary schistosomiasis risk. Four-year-old preschool children were five times more likely to contract urinary schistosomiasis than two-year-old children (odds ratio [OR] = 5.255; 95% confidence interval [CI] = 1.014-27.237; P = 0.048). Increased contact with infested water among older children likely explains much of their increased risk. Infestation was evidenced by the presence of infected Bulinus globosus snails in the water contact points surveyed. Multiple regression analysis showed that visiting water contact sites daily (OR = 0.898, 95% CI = 0.185-4.350, P = 0.894), bathing in these sites (OR = 9.462, 95% CI = 0.036-0.00, P = 0.430) and lack of knowledge, among caregivers, regarding the causes of urinary schistosomiasis (OR = 0.235, 95% CI = 0.005-1.102, P = 0.066) posed statistically insignificant risk increases for preschoolers contracting urinary schistosomiasis. Conclusions: Urinary schistosomiasis was prevalent among preschool children in Malengachanzi, Nkhotakota District. Contact with infested water puts these children and the general population at risk of infection and reinfection. Inclusion of preschool children in treatment programmes should be considered imperative, along with safe treatment guidelines. To prevent infection, the population in the area should be provided with health education and safe alternative water sources.

Toxicological impact of JWH-018 and its phase I metabolite N-(3-hydroxypentyl) on human cell lines

"The emergence and abuse of synthetic cannabinoids has been increasing as an alternative to cannabis, mainly among youth. As their appearance on the drug market has been recent, the pharmacological and toxicological profiles of these psychoactive substances are poorly understood. Current studies suggest that they have stronger effects compared to their natural alternatives and their metabolites retain affinity towards CB1 receptors in CNS. Since studies on its toxicological properties are scarce, the effects of the drug in human derived cell lines were investigated. The present study was designed to explore the toxicological impact of parent drug versus phase I metabolites of synthetic cannabinoids on human cells with and without CB1 receptor. The human cell line of neuroblastoma SH-SY5Y and human kidney cell line HEK-293T were exposed to JWH-018 and to its N-(3-hydroxypentyl) metabolite. Cell toxicity was evaluated using the MTT and LDH assay. Additionally, a dual staining methodology with fluorescent Annexin V-FITC and propidium iodide was performed to address the question of whether JWH-018 N-(3-hydroxypentyl) metabolite is inducing cell death through apoptosis or necrosis, in HEK293T and SH-SY5Y cell lines. The obtained results show that JWH-018 does not cause a statistically significant decrease in cell viability, in contrast to its N-(3-hydroxypentyl) metabolite, which at ≥25μM causes a significant decrease in cell viability. Both cell lines are affected by JWH-018 metabolite. Our results point to higher toxicity of JWH-018 metabolite when compared to its parent drug, suggesting a non-CB1 receptor mediated toxicological mechanism. Comparing the results from Annexin V/PI with MTT and LDH assays of SH-SY5Y and HEK293T in the presence of the synthetic cannabinoid metabolite, emerges the picture that cellular viability decreases and associated death is occurring through necrosis."