A human urothelial microtissue model reveals shared colonization and survival strategies between uropathogens and commensals.

Urinary tract infection is among the most common infections worldwide, typically studied in animals and cell lines with limited uropathogenic strains. Here, we assessed diverse bacterial species in a human urothelial microtissue model exhibiting full stratification, differentiation, innate epithelial responses, and urine tolerance. Several uropathogens invaded intracellularly, but also commensal Escherichia coli, suggesting that invasion is a shared survival strategy, not solely a virulence hallmark. The E. coli adhesin FimH was required for intracellular bacterial community formation, but not for invasion. Other shared lifestyles included filamentation (Gram-negatives), chaining (Gram-positives), and hijacking of exfoliating cells, while biofilm-like aggregates were formed mainly with Pseudomonas and Proteus. Urothelial cells expelled invasive bacteria in Rab-/LC3-decorated structures, while highly cytotoxic/invasive uropathogens, but not commensals, disrupted host barrier function and strongly induced exfoliation and cytokine production. Overall, this work highlights diverse species-/strain-specific infection strategies and corresponding host responses in a human urothelial microenvironment, providing insights at the microtissue, cell, and molecular level.

Inflammatory effects of Edwardsiella ictaluri lipopolysaccharide modifications in catfish gut.

Bacterial lipopolysaccharides (LPS) are structural components of the outer membranes of Gram-negative bacteria and also are potent inducers of inflammation in mammals. Higher vertebrates are extremely sensitive to LPS, but lower vertebrates, like fish, are resistant to their systemic toxic effects. However, the effects of LPS on the fish intestinal mucosa remain unknown. Edwardsiella ictaluri is a primitive member of the Enterobacteriaceae family that causes enteric septicemia in channel catfish (Ictalurus punctatus). E. ictaluri infects and colonizes deep lymphoid tissues upon oral or immersion infection. Both gut and olfactory organs are the primary sites of invasion. At the systemic level, E. ictaluri pathogenesis is relatively well characterized, but our knowledge about E. ictaluri intestinal interaction is limited. Recently, we observed that E. ictaluri oligo-polysaccharide (O-PS) LPS mutants have differential effects on the intestinal epithelia of orally inoculated catfish. Here we evaluate the effects of E. ictaluri O-PS LPS mutants by using a novel catfish intestinal loop model and compare it to the rabbit ileal loop model inoculated with Salmonella enterica serovar Typhimurium LPS. We found evident differences in rabbit ileal loop and catfish ileal loop responses to E. ictaluri and S. Typhimurium LPS. We determined that catfish respond to E. ictaluri LPS but not to S. Typhimurium LPS. We also determined that E. ictaluri inhibits cytokine production and induces disruption of the intestinal fish epithelia in an O-PS-dependent fashion. The E. ictaluri wild type and ΔwibT LPS mutant caused intestinal tissue damage and inhibited proinflammatory cytokine synthesis, in contrast to E. ictaluri Δgne and Δugd LPS mutants. We concluded that the E. ictaluri O-PS subunits play a major role during pathogenesis, since they influence the recognition of the LPS by the intestinal mucosal immune system of the catfish. The LPS structure of E. ictaluri mutants is needed to understand the mechanism of interaction.

Mechanisms of intrinsic resistance to antimicrobial peptides of Edwardsiella ictaluri and its influence on fish gut inflammation and virulence.

The genus Edwardsiella comprises a genetically distinct taxon related to other members of the family Enterobacteriaceae. It consists of bacteria differing strongly in their biochemical and physiological features, natural habitats, and pathogenic properties. Intrinsic resistance to cationic antimicrobial peptides (CAMPs) is a specific property of the genus Edwardsiella. In particular, Edwardsiella ictaluri, an important pathogen of the catfish (Ictalurus punctatus) aquaculture and the causative agent of a fatal systemic infection, is highly resistant to CAMPs. E. ictaluri mechanisms of resistance to CAMPs are unknown. We hypothesized that E. ictaluri lipopolysaccharide (LPS) plays a role in both virulence and resistance to CAMPs. The putative genes related to LPS oligo-polysaccharide (O-PS) synthesis were in-frame deleted. Individual deletions of wibT, gne and ugd eliminated synthesis of the O-PS, causing auto-agglutination, rough colonies, biofilm-like formation and motility defects. Deletion of ugd, the gene that encodes the UDP-glucose dehydrogenase enzyme responsible for synthesis of UDP-glucuronic acid, causes sensitivity to CAMPs, indicating that UDP-glucuronic acid and its derivatives are related to CAMP intrinsic resistance. E. ictaluri OP-S mutants showed different levels of attenuation, colonization of lymphoid tissues and immune protection in zebrafish (Danio rerio) and catfish. Orally inoculated catfish with O-PS mutant strains presented different degrees of gut inflammation and colonization of lymphoid tissues. Here we conclude that intrinsic resistance to CAMPs is mediated by Ugd enzyme, which has a pleiotropic effect in E. ictaluri influencing LPS synthesis, motility, agglutination, fish gut inflammation and virulence.

Safety and protective efficacy of live attenuated Salmonella Gallinarum mutants in Rhode Island Red chickens.

Salmonella enterica serovar Gallinarum is the causative agent of fowl typhoid, an important systemic disease of poultry with economic consequences in developing nations. A live attenuated orally applied S. Gallinarum vaccine could provide a low cost method for controlling this disease. We constructed S. Gallinarum strains in which the expression of the crp, rfc and rfaH genes, important for virulence of Salmonella Typhimurium in mice, were under the control of an arabinose-regulated promoter. We evaluated the virulence of these strains compared to wild-type S. Gallinarum and to mutants carrying deletions in these genes. We found that rfc mutants were fully virulent, indicating that, unlike the S. Typhimurium mouse model, the rfc gene is dispensable in S. Gallinarum for virulence in birds. In the case of rfaH, the deletion mutant was attenuated and protective, while the strain with arabinose-regulated rfaH expression retained full virulence. The strain exhibiting arabinose-regulated crp expression was attenuated. Its virulence was not affected by the inclusion of 0.2% arabinose in the drinking water. Birds immunized with this strain were protected against a lethal S. Gallinarum challenge and against colonization with the human pathogen Salmonella Enteritidis. This work shows that an arabinose-regulated crp strain provides a basis for further development of a fowl typhoid vaccine.

Comparative analysis of isocentric 3-dimensional C-arm fluoroscopy and biplanar fluoroscopy for anterior screw fixation in odontoid fractures.

STUDY DESIGN: Retrospective clinical study. OBJECTIVE: To compare long-term radiographic and clinical outcomes of patients undergoing anterior odontoid screw placement using traditional biplanar fluoroscopy or isocentric 3-dimensional C-arm (iso-C) fluoroscopy-assisted techniques. SUMMARY OF BACKGROUND DATA: Anterior screw fixation of odontoid fractures preserves motion at the C1-C2 joint, but accurate screw positioning is essential for successful outcomes. Biplanar fluoroscopy image guidance is most often used; however, iso-C imaging improves the ease and accuracy of screw placement with less radiation exposure. METHODS: Fifty-one patients underwent anterior odontoid screw fixation for type II (48 patients) and rostral type III fractures (3 patients). Procedures were guided by biplanar fluoroscopy in 25 (49%) patients, and with iso-C assistance in 26 (51%). Length of surgery, complications, and clinical outcomes based on the Smiley-Webster score were evaluated. Computed tomography confirmed adequate screw placement. Follow-up ranged from 3 to 9 months. RESULTS: At 3-month follow-up, screw position and fusion across the fracture were evident in 87% of the cases treated with biplanar fluoroscopy and in 100% treated by iso-C. The average outcome score in the iso-C group was superior to that of the biplanar group (1.08 vs. 1.33, respectively), although not statistically significant. At last follow-up, the rate of successful fusion was 88% in the biplanar group and 95% in the iso-C group. Length of surgery was significantly lower in the iso-C group compared with the biplanar group (P=0.05). The significantly longer preparation time in the iso-C group (P=0.04) accounted for no overall difference in total operating room occupancy time between the 2 groups. CONCLUSIONS: Iso-C significantly decreased surgical time. At last follow-up iso-C assistance was associated with improved rates of radiographic fusion with comparable outcome and complication profiles. This series represents the largest cohort of patients treated with intraoperative real-time navigation assistance for odontoid fractures.

Posttranscriptional mechanisms involving microRNA-27a and b contribute to fast-specific and glucocorticoid-mediated myostatin expression in skeletal muscle.

Expression of the antigrowth factor myostatin (MSTN) differs between fast and slow skeletal muscles and is increased in nearly every form of muscle atrophy, but the contribution of transcriptional vs. posttranscriptional mechanisms to its differing expression in these states has not been defined. We show here that levels of mature MSTN mRNA were sixfold greater in fast vs. slow muscle and were increased twofold in fast muscle in response to dexamethasone (Dex) injection in vivo and in C2C12 myotubes following Dex treatment in vitro, but that levels of MSTN pre-mRNA, a readout of transcription, only minimally and nonsignificantly differed in these states. Moreover, Dex treatment with or without cotransfection with a glucocorticoid receptor expression construct had only modest effects on mouse MSTN promoter activity in C2C12 myotubes. We therefore explored the potential contribution of posttranscriptional mechanisms, and the role of the microRNAs miR-27a and b in particular, on MSTN expression. The MSTN 3'-untranslated region (UTR) contains a putative recognition sequence for miR-27a and b that is conserved across a wide range of vertebrate species. Cotransfection of a MSTN 3'-UTR-luciferase construct with a miR-27b expression construct significantly attenuated by approximately half while mutation of the miR-27 recognition sequence significantly increased by approximately twofold the activity of a MSTN 3'-UTR construct and decreased mRNA degradation of a luciferase reporter construct in C2C12 myotubes. Expression of miR-27a and b was almost sixfold greater in slow-twitch than in fast-twitch muscle in vivo, and miR-27a expression was significantly decreased by nearly half by glucocorticoid treatment in vitro. Finally, the miR-27a and b promoters were activated by cotransfection with the slow-specific signaling molecules calcineurin and peroxisome proliferator-activated receptor-γ coactivator-1α. The present data represent the first demonstration that posttranscriptional mechanisms involving miR-27a and b may contribute to fast-specific and glucocorticoid-dependent myostatin expression in muscle.

Acute daily psychological stress causes increased atrophic gene expression and myostatin-dependent muscle atrophy.

Psychological stress is known to attenuate body size and lean body mass. We tested the effects of 1, 3, or 7 days of two different models of psychological stress, 1 h of daily restraint stress (RS) or daily cage-switching stress (CS), on skeletal muscle size and atrophy-associated gene expression in mice. Thymus weights decreased in both RS and CS mice compared with unstressed controls, suggesting that both models activated the hypothalamic-pituitary-adrenal axis. Body mass was significantly decreased at all time points for both models of stress but was greater for RS than CS. Mass of the tibialis anterior (TA) and soleus (SOL) muscles was significantly decreased after 3 and 7 days of RS, but CS only significantly decreased SOL mass after 7 days. TA mRNA levels of the atrophy-associated genes myostatin (MSTN), atrogin-1, and the phosphatidylinositol 3-kinase inhibitory subunit p85alpha were all significantly increased relative to unstressed mice after 1 and 3 days of RS, and expression of MSTN and p85alpha mRNA remained elevated after 7 days of RS. Expression of muscle ring finger 1 was increased after 1 day of RS but returned to baseline at 3 and 7 days of RS. MSTN, atrogin-1, and p85alpha mRNA levels also significantly increased after 1 and 3 days of CS but atrogen-1 mRNA levels had resolved back to normal levels by 3 days and p85alpha with 7 days of CS. p21CIP mRNA levels were significantly decreased by 3 days of CS or RS. Finally, body mass was minimally affected, and muscle mass was completely unaffected by 3 days of RS in mice null for the MSTN gene, and MSTN inactivation attenuated the increase in atrogin-1 mRNA levels with 4 days of RS compared with wild-type mice. Together these data suggest that acute daily psychological stress induces atrophic gene expression and loss of muscle mass that appears to be MSTN dependent.

Myostatin expression is increased by food deprivation in a muscle-specific manner and contributes to muscle atrophy during prolonged food deprivation in mice.

During food deprivation (FD), skeletal muscle protein is broken down to produce amino acids for hepatic gluconeogenesis to maintain blood glucose levels. However, it is unclear what role, if any, the secreted antigrowth factor myostatin (MSTN) plays in the muscle atrophy induced by FD. We therefore examined expression and function of MSTN in FD in mice. Two days of FD significantly decreased muscle mass and protein content and increased mRNA levels of ubiquitin ligases MuRF-1 and atrogin-1 in fast-twitch tibialis anterior (TA) muscle but not slow-twitch soleus (Sol) muscle, while 2 days of refeeding returned these to fed values in TA. MSTN mRNA levels were significantly increased approximately threefold by 2 days, but not 1 day, of FD and returned to fed levels with 2 days of refeeding in TA but were not significantly affected by FD or refeeding in Sol. TA mass decreased to a similar amount after 1 day of FD in wild-type mice and mice null for the MSTN gene but was decreased to a greater amount in wild-type than MSTN-null mice by 2 days of FD. In addition, blood glucose levels decreased and corticosterone levels increased to a greater extent in MSTN-null mice after 2 days of FD, but surprisingly muscle MuRF-1 and atrogin-1 mRNA levels were not affected by the lack of MSTN during FD. Similarly, changes in hepatic enzyme expression in response to FD were identical between wild-type and MSTN-null mice. Our data are consistent with the hypothesis that MSTN is dispensable for the initial atrophy occurring in response to FD but attenuates the decrease in fast-twitch muscle mass during prolonged FD.

Dehydrogenase based reagentless biosensor for monitoring phenylketonuria.

Phenylketonuria (PKU) is a disease characterized by an inability to metabolize the amino acid l-phenylalanine. The resulting buildup leads to brain damage and ultimately mental retardation in children if their phenylalanine intake is not carefully controlled. The National Institutes of Health recently suggested that people with PKU monitor their phenylalanine levels throughout their life and be put on a low phenylalanine diet. As an alternative approach to analysis using blood, this paper describes the first reagentless dehydrogenase based sensor for the determination of phenylalanine in human urine. The clinical range of phenylalanine in human urine is 20-60mM for people with PKU. Although most clinical analysis is performed using blood, urine was chosen due to its high concentrations of phenylalanine in phenylketonurics, as well as its simple, safe, and painless collection. The sensor is comprised of a carbon paste electrode with nicotinamide adenine dinucleotide (NAD(+)), phenylalanine dehydrogenase (PDH), uricase, and an electron mediator, 3,4-dihydroxybenzaldehyde (3,4-DHB), all mixed into the paste. The electron mediator reacts with the electrode surface to produce two redox species, which catalytically oxidize NADH. The behavior of the electron mediator mixed into a carbon paste electrode has not been previously investigated. Cyclic voltammetry was used to characterize the sensor's response to NADH, and with the addition of PDH and NAD(+) to the paste, its response to phenylalanine in human urine. The limit of detection for phenylalanine is 0.5mM (S/N=3).