Chemical Characterization of Urinary Pheromones in Brown Antechinus, Antechinus stuartii

In the small dasyurid marsupial, Antechinus stuartii, males exhibit scent-marking in the form of cloacal marking of nesting areas during the breeding season. Females of this species show no such behavior. To characterize the potential male pheromonal scent signal, urine-derived volatiles from sexually active males were analyzed by GC-MS and compared to that of females and a castrated male. More than 10 urinary compounds were identified. A series of homologous methylketones was observed in both males and females, whereas aldehydes were present only in female urine. Urine from the castrate was virtually compound-free except for minute concentrations of a compound tentatively identified as 2,4-dithiapentane. This compound was also found in one of the sexually active males. The GC profiles of the sexually active males contained high concentrations of two pyrazine derivatives and four methylketones that were not detected in the profiles of either females or the castrate. These compounds may influence social communication in the brown antechinus, Antechinus stuartii.

Urinary tract infection of mice to model human disease: Practicalities, implications and limitations

Urinary tract infections (UTIs) are among the most common bacterial infections in humans. Murine models of human UTI are vital experimental tools that have helped to elucidate UTI pathogenesis and advance knowledge of potential treatment and infection prevention strategies. Fundamentally, several variables are inherent in different murine models, and understanding the limitations of these variables provides an opportunity to understand how models may be best applied to research aimed at mimicking human disease. In this review, we discuss variables inherent in murine UTI model studies and how these affect model usage, data analysis and data interpretation. We examine recent studies that have elucidated UTI host–pathogen interactions from the perspective of gene expression, and review new studies of biofilm and UTI preventative approaches. We also consider potential standards for variables inherent in murine UTI models and discuss how these might expand the utility of models for mimicking human disease and uncovering new aspects of pathogenesis

Clinical Escherichia coli isolates utilize alpha-hemolysin to inhibit in vitro epithelial cytokine production

Uropathogenic Escherichia coli is the primary cause of urinary tract infections, which affects over 60% of women during their lifetime. UPEC exhibits a number of virulence traits that facilitate colonization of the bladder, including inhibition of cytokine production by bladder epithelial cells. The goal of this study was to identify the mechanism of this inhibition. We observed that cytokine suppression was associated with rapid cytotoxicity toward epithelial cells. We found that cytotoxicity, cytokine suppression and alpha-hemolysin production were all tightly linked in clinical isolates. We screened a UPEC fosmid library and identified clones that gained the cytotoxicity and cytokine-suppression phenotypes. Both clones contained fosmids encoding a PAI II(J96)-like domain and expressed the alpha-hemolysin (hlyA) encoded therein. Mutation of the fosmid-encoded hly operon abolished cytotoxicity and cytokine suppression. Similarly, mutation of the chromosomal hlyCABD operon of UPEC isolate F11 also abolished these phenotypes, and they could be restored by introducing the PAI II(J96)-like domain-encoding fosmid. We also examined the role of alpha-hemolysin in cytokine production both in the murine UTI model as well as patient specimens. We conclude that E. coli utilizes alpha-hemolysin to inhibit epithelial cytokine production in vitro. Its contribution to inflammation during infection requires further study.

Current understanding of streptococcal urinary tract infection

Group B streptococcus (GBS), also known as Streptococcus agalactiae is a Gram-positive, β-hemolytic, chain-forming bacterium and a commensal within the genital tract flora in approximately 25% of healthy adult women (Campbell et al., 2000). The organism is a leading cause of serious infection in newborns, pregnant women, and older persons with chronic medical illness (Baker et al., Edwards&Baker, 2005). In neonates GBS infection most commonly causes pneumonia, meningitis, and sepsis. In addition to maternal cervicovaginal colonization and neonatal infection that can result from vertical transmission of GBS from mothers to their infants, the bacterium can also cause urinary tract infection (UTI). The spectrum of GBS UTI includes asymptomatic bacteriuria (ABU), cystitis, pyelonephritis, urethritis, and urosepsis (Bronsema et al., 1993, Edwards&Baker, 2005, Farley et al., 1993, Lefevre et al., 1991, McKenna et al., 2003, Munoz et al., 1992, Ulett et al., 2009). GBS ABU is particularly common among pregnant women, although those most at risk for cystitis due to GBS appear to be elderly individuals (Edwards&Baker, 2005, Falagas et al., 2006, Muller et al., 2006). In addition to acute and asymptomatic UTI other invasive diseases caused by GBS infection include skin infections, bacteraemia, pneumonia, arthritis, and endocarditis (Liston et al., 1979, Patil & Martin, 2010, Tissi et al., 1997, Trivalle et al., 1998). Thus, GBS is considered unique in terms of its ability to cause a spectrum of diseases in newborns and adult humans and its ability to colonize the genital tract of healthy women in a commensal-type manner...

Urinary excretion of antioxidants in healthy humans following queen garnet plum juice ingestion: a new plum variety rich in antioxidant compounds

In recent years, there has been intense interest in the potential health benefits of dietary derived plant polyphenols and antioxidants. A new variety of Prunus salicina, Queen Garnet plum (QGP), was developed as a high anthocyanin, high antioxidant plum, in a Queensland Government breeding program. Following consumption of 400 mL QGP juice (QGPJ; 1,117 mg anthocyanins) by two healthy male subjects, QGP anthocyanins (cyanidin-3-glucoside and cyanidin-3-rutinoside) were excreted mainly as methylated and glucuronidated metabolites in urine (0.5% of the ingested dose within 24 h). Furthermore, QGPJ intake resulted in a threefold increase in hippuric acid excretion (potential biomarker for total polyphenols intake and metabolite), an increased urinary antioxidant capacity and a decreased malondialdehyde excretion (biomarker for oxidative stress) within 24 h as compared with the polyphenol-/antioxidant-free control. Results from this pilot study suggest that metabolites, and not the native QGP anthocyanins/polyphenols, are most likely the bioactive compounds in vivo.

Characterization of genomic diversity in extraintestinal pathogenic Escherichia coli (ExPEC) and development of a diagnostic DNA microarray for the differentiation of ExPEC isolates causing urinary tract infections

Extraintestinal pathogenic Escherichia coli (ExPEC) represent a diverse group of strains of E. coli, which infect extraintestinal sites, such as the urinary tract, the bloodstream, the meninges, the peritoneal cavity, and the lungs. Urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC), the major subgroup of ExPEC, are among the most prevalent microbial diseases world wide and a substantial burden for public health care systems. UTIs are responsible for serious morbidity and mortality in the elderly, in young children, and in immune-compromised and hospitalized patients. ExPEC strains are different, both from genetic and clinical perspectives, from commensal E. coli strains belonging to the normal intestinal flora and from intestinal pathogenic E. coli strains causing diarrhea. ExPEC strains are characterized by a broad range of alternate virulence factors, such as adhesins, toxins, and iron accumulation systems. Unlike diarrheagenic E. coli, whose distinctive virulence determinants evoke characteristic diarrheagenic symptoms and signs, ExPEC strains are exceedingly heterogeneous and are known to possess no specific virulence factors or a set of factors, which are obligatory for the infection of a certain extraintestinal site (e. g. the urinary tract). The ExPEC genomes are highly diverse mosaic structures in permanent flux. These strains have obtained a significant amount of DNA (predictably up to 25% of the genomes) through acquisition of foreign DNA from diverse related or non-related donor species by lateral transfer of mobile genetic elements, including pathogenicity islands (PAIs), plasmids, phages, transposons, and insertion elements. The ability of ExPEC strains to cause disease is mainly derived from this horizontally acquired gene pool; the extragenous DNA facilitates rapid adaptation of the pathogen to changing conditions and hence the extent of the spectrum of sites that can be infected. However, neither the amount of unique DNA in different ExPEC strains (or UPEC strains) nor the mechanisms lying behind the observed genomic mobility are known. Due to this extreme heterogeneity of the UPEC and ExPEC populations in general, the routine surveillance of ExPEC is exceedingly difficult. In this project, we presented a novel virulence gene algorithm (VGA) for the estimation of the extraintestinal virulence potential (VP, pathogenicity risk) of clinically relevant ExPECs and fecal E. coli isolates. The VGA was based on a DNA microarray specific for the ExPEC phenotype (ExPEC pathoarray). This array contained 77 DNA probes homologous with known (e.g. adhesion factors, iron accumulation systems, and toxins) and putative (e.g. genes predictably involved in adhesion, iron uptake, or in metabolic functions) ExPEC virulence determinants. In total, 25 of DNA probes homologous with known virulence factors and 36 of DNA probes representing putative extraintestinal virulence determinants were found at significantly higher frequency in virulent ExPEC isolates than in commensal E. coli strains. We showed that the ExPEC pathoarray and the VGA could be readily used for the differentiation of highly virulent ExPECs both from less virulent ExPEC clones and from commensal E. coli strains as well. Implementing the VGA in a group of unknown ExPECs (n=53) and fecal E. coli isolates (n=37), 83% of strains were correctly identified as extraintestinal virulent or commensal E. coli. Conversely, 15% of clinical ExPECs and 19% of fecal E. coli strains failed to raster into their respective pathogenic and non-pathogenic groups. Clinical data and virulence gene profiles of these strains warranted the estimated VPs; UPEC strains with atypically low risk-ratios were largely isolated from patients with certain medical history, including diabetes mellitus or catheterization, or from elderly patients. In addition, fecal E. coli strains with VPs characteristic for ExPEC were shown to represent the diagnostically important fraction of resident strains of the gut flora with a high potential of causing extraintestinal infections. Interestingly, a large fraction of DNA probes associated with the ExPEC phenotype corresponded to novel DNA sequences without any known function in UTIs and thus represented new genetic markers for the extraintestinal virulence. These DNA probes included unknown DNA sequences originating from the genomic subtractions of four clinical ExPEC isolates as well as from five novel cosmid sequences identified in the UPEC strains HE300 and JS299. The characterized cosmid sequences (pJS332, pJS448, pJS666, pJS700, and pJS706) revealed complex modular DNA structures with known and unknown DNA fragments arranged in a puzzle-like manner and integrated into the common E. coli genomic backbone. Furthermore, cosmid pJS332 of the UPEC strain HE300, which carried a chromosomal virulence gene cluster (iroBCDEN) encoding the salmochelin siderophore system, was shown to be part of a transmissible plasmid of Salmonella enterica. Taken together, the results of this project pointed towards the assumptions that first, (i) homologous recombination, even within coding genes, contributes to the observed mosaicism of ExPEC genomes and secondly, (ii) besides en block transfer of large DNA regions (e.g. chromosomal PAIs) also rearrangements of small DNA modules provide a means of genomic plasticity. The data presented in this project supplemented previous whole genome sequencing projects of E. coli and indicated that each E. coli genome displays a unique assemblage of individual mosaic structures, which enable these strains to successfully colonize and infect different anatomical sites.

Heat-shock protein 70-2 (HSP70-2) expression in bladder urothelial carcinoma is associated with tumour progression and promotes migration and invasion

Purpose: Testis specific heat-shock protein 70-2 (HSP70-2), a member of HSP70 chaperone family, is essential for the growth of spermatocytes and cancer cells. We investigated the association of HSP70-2 expression with clinical behaviour and progression of urothelial carcinoma of bladder. Experimental design: We assessed the HSP70-2 expression by RT-PCR and HSP70-2 protein expression by immunofluorescence, flow cytometry, immunohistochemistry and Western blotting in urothelial carcinoma patient specimens and HTB-1, UMUC-3, HTB-9, HTB-2 and normal human urothelial cell lines. Further, to investigate the role of HSP70-2 in bladder tumour development, HSP70-2 was silenced in the high-grade invasive HTB-1 and UMUC-3 cells. The malignant properties of urothelial carcinoma cells were examined using colony formation, migration assay, invasion assay in vitro and tumour growth in vivo. Results: Our RT-PCR analysis and immunohistochemistry analysis revealed that HSP70-2 was expressed in both moderate to well-differentiated and high-grade invasive urothelial carcinoma cell lines studied and not in normal human urothelial cells. In consistence with these results, HSP70-2 expression was also observed in superficially invasive (70%) and muscle-invasive (90%) patient's tumours. Furthermore, HSP70-2 knockdown significantly suppressed cellular motility and invasion ability. An in vivo xenograft study showed that inhibition of HSP70-2 significantly suppressed tumour growth. Conclusions: In conclusion, our data suggest that the HSP70-2 expression is associated with early spread and progression of urothelial carcinoma of bladder cancer and that HSP70-2 can be the potential therapeutic target for bladder urothelial carcinoma. (C) 2009 Elsevier Ltd. All rights reserved.

Primary Structural Documentation of the Major Urinary Protein of the Indian Commensal Rat (Rattus rattus) Using a Proteomics Platform

Purpose: A number of proteome studies have been performed recently to identify pheromone-related protein expression and their molecular function using genetically modified rodents' urine. However, no such studies have used Indian commensal rodents; interestingly, in a previous investigation, we confirmed the presence of volatile molecules in commensal rodents urine and these molecules seem to be actively involved in pheromonal communication. Therefore, the present study aims to identify the major urinary protein [MUP] present in commensal rat urine, which will help us to understand the protein's expression pattern and intrinsic properties among the rodents globally. Experimental Design: Initially, the total urinary proteins were separated by 1-D and 2-D electrophoresis and then subsequently analyzed by Matrix Assisted Laser Desorption Ionization-Time of Flight and Mass Spectrometer (MALDI-TOF/MS). Furthermore, they were then fragmented with the aid of a Tandem Mass Spectrometer (TOF/TOF) and the identified sequences aligned and confirmed using similarity with the deduced primary structures of members of the lipocalin superfamily.Results: The SDS-PAGE protein profiles showed distinct proteins with molecular masses of 15, 22.4, 25, 28, 42, 50, 55, 68, and 91 kDa. Of these proteins, the 22.4 kDa protein was considered to be target candidate. When 2D electrophoresis was carried out, about similar to 50 spots were detected with different masses and various pI ranges. The 22.4 kDa protein was found to have a pI of about 4.9. This 22.4 kDa protein spot was digested and subjected to mass spectrometry; it was identified as rat MUP. The fragmented peptides from the rat MUP at 935, 1026, 1192, and 1303 m/z were further fragmented with the aid of MS/MS and generated de novo sequence and this confirmed this protein to be the MUP present in the urine of commensal rats.Conclusion: The present investigation confirms the presence of MUP with a molecular mass of 22.4 kDa in the urine of commensal rats. This protein may be involved in the binding of volatile molecules and opens up a discussion about how volatile and non-volatile molecules in the commensal rats' urine may contribute chemo-communication.