Glycosylation of bacterial antigens changes epitope patterns.

Introduction: Unlike glycosylation of proteins expressed in mammalian systems, bacterial glycosylation is often neglected in the development of recombinant vaccines. Methods: Here, we compared the effects of glycosylation of YghJ, an Escherichia coli protein important for mucus attachment of bacteria causing in urinary tract infections (UTIs). A novel method based on statistical evaluation of phage display for the identification and comparison of epitopes and mimotopes of anti-YghJ antibodies in the sera was used. This is the first time that the effect of glycosylation of a recombinant bacterial antigen has been studied at the peptide epitope level. Results: The study identifies differences in the immune response for (non)-glycosylated antigens in rabbits and pigs and compares them to a large group of patients with UTI, which have been diagnosed as positive for various bacterial pathogens. We identified glycosylation-specific peptide epitopes, a large immunological similarity between different UTI pathogens, and a broad peptide epitope pattern in patients and animals, which could result in a variable response in patients upon vaccination. Discussion: This epitope analysis indicates that the vaccination of rabbits and pigs raises antibodies that translate well into the human immune system. This study underlines the importance of glycosylation in bacterial vaccines and provides detailed immune diagnostic methods to understand individual immune responses to vaccines.

Kidney stone disease.

Kidney stone disease is rapidly increasing with a strong relationship to metabolic syndrome. This review gives a brief overview of the current state and current treatment modalities. Increasing use of CT and ultrasound scans leads to increased diagnosis of asymptomatic kidney stones, which rarely require treatment. The trend in stone treatment goes towards endoscopic lithotripsy which together with ESWL enables a personalised approach. Obstructive stones with infection require urgent intervention to reduce mortality. Increased fluid intake, dietary changes as well as potassium citrate supplements are the most important elements in stone prevention in the common idiopathic stone disease.

Bladder pain syndrome.

Bladder pain syndrome is rare and leads to increased morbidity and decreased quality of life. The patients are a heterogenous group with different clinical presentations, and little is known of the different aspects of the syndrome. A thorough patient history and specialised diagnostics are required to offer these patients the best possible treatment. This review suggests an algorithm to manage these patients at all levels of the Danish health-care system. It is recommended that final diagnosis and multidisciplinary treatment should be centered at large regional hospitals.

Implementation of sacral neuromodulation for urinary indications. A Danish prospective study during the initial 15 months of a new service in a tertiary referral hospital.

OBJECTIVE: Sacral neuromodulation (SNM) is a well-established treatment modality for idiopathic overactive bladder and urgency incontinence, idiopathic fecal incontinence and non-obstructive urinary retention. This study describes the start-up phase of establishing the SNM service. Primary objective: To investigate the patient-reported outcome measures of SNM on lower urinary tract dysfunction symptoms. Secondary objectives: To investigate bowel function, sexual satisfaction and to monitor SNM safety. MATERIALS AND METHODS: Twenty-two patients with refractory idiopathic and neurogenic lower urinary tract dysfunction were offered a two-stage test-phase procedure and SNM device implantation. On completing the study, the patients rated their satisfaction with the treatment using a five-point Likert scale and a bother score of urinary, bowel and sexual symptoms on a scale of 1-10 (the worst). Their complications were assessed. RESULTS: Nineteen patients (86%) were responders during the test phase and had the pulse generator implanted. Seventeen patients were very satisfied/satisfied. A statistically significant change in urinary symptoms bother score was observed in the idiopathic and neurogenic patients, a reduction from 10 to 4 (p = .0057) and 10 to 3 (p = .014), respectively. Eleven patients (58%) had symptoms from two or three pelvic compartments. Nine patients (47%) had complications. All but one event was resolved. CONCLUSIONS: SNM is safe in this heterogeneous group of patients with refractory lower urinary tract dysfunction of various etiologies. A substantial improvement was observed in the pelvic organ dysfunction, demanding a multidisciplinary approach. More studies are required to standardize the evaluation of the subjective and objective outcomes of SNM.

[Simple, recurrent, and complicated urinary tract infections].

Urinary tract infection is a colonisation of the urinary tract with uropathogenic bacteria, leading to an inflammatory response. The increasing antimicrobial resistance presents major challenges for both the individual and our society. The goal is to reduce antimicrobial resistance and pave the way for a new era in the management of recurrent urinary tract infections. This review describes the definitions of urinary tract infection (simple, recurrent, and complicated) as well as examination and treatment recommendations. The future and most common non-antibiotic alternatives are discussed.

Resection and reconstruction of idiopathic scrotal elephantiasis: surgical approach and quality of life assessment.

Scrotal elephantiasis (SE) is a condition considered rare in western industrialised countries but common in filaria prone regions. If no apparent causes are found for SE, it is called idiopathic SE. Medical and conservative therapies are ineffective against idiopathic SE, and surgical intervention is mandatory to treat this disabling condition. Nevertheless, it remains unclear whether surgical intervention improves quality of life among patients with idiopathic SE. Herein, we report a case of a 41-year-old man who underwent acute scrotal resection and reconstruction, secondary to haemorrhage from his idiopathic SE. The aim of this study was to describe the operative approach and assess patient satisfaction after surgical treatment. The patient had no recurrence of SE after surgical treatment at 6 months follow-up and had considerable improvements assessed by general and disease-specific quality of life questionnaires.

Danish version of the National Institutes of Health-Chronic Prostatitis Symptom Index (NIH-CPSI) questionnaire: a linguistic translation, cross-cultural adaptation and test-re-test reliability study.

Objective: To translate and cross-culturally adapt the National Institutes of Health-Chronic Prostatitis Symptom Index (NIH-CPSI) into Danish and assess the reliability of the translated version. Methods: The NIH-CPSI was translated into Danish by a formalized translation procedure. Study participants suffering from Chronic Prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS) were recruited from a CP/CPPS newsletter email-list. The translated questionnaire was tested for face validity by interviewing men (n = 7) suffering from CP/CPPS. Relative reliability (interclass correlations coefficient, ICC) and absolute reliability (minimal detectable change, MDC; and standard error of measurement, SEM) were assessed on an electronic version of the Danish NIH-CPSI, including a general response assessment of symptom stability at the second assessment. Results: One hundred and twenty-nine men volunteered for the test-re-test study, 43 did not fit the eligibility criteria or had incomplete tests and 27 were excluded due to symptom changes between test and re-test, leaving 59 participants for the reliability study. The relative reliability for the total NIH-CPSI score was found to be an excellent ICC of 0.93 (95% CI = 0.91-0.96). The absolute reliability for the total NIH-CPSI score revealed an MDC of 5.0 and a SEM of 1.8, corresponding to 12% and 4%, respectively, of the maximal obtainable NIH-CPSI score. Conclusion: The NIH-CPSI questionnaire was successfully translated and cross-culturally adapted into a Danish version. An electronic version of the Danish NIH-CPSI showed excellent reliability. The questionnaire is suitable for use as an outcome measure in research studies and may also be a useful tool in the clinical setting.

[Congenital diaphragmatic hernia in Western Denmark]. / Kongenit diafragmahernie i Vestdanmark.

INTRODUCTION: The aim was to clarify the mortality rate and results of surgical treatment in children with congenital diaphragmatic hernia (CDH) born in Western Denmark. MATERIAL AND METHODS: A retrospective cohort study of all live-born infants with CDH born in Western Denmark between 1995 and 2005. The cohort enrolled 82 children, which was divided into two groups, before and after 1998, when the multidisciplinary treatment centre at Odense University Hospital (OUH) was established. RESULTS: The overall mortality between 1998 and 2005 was 23%, which was significantly lower than between 1995 and 1998, when it was 67%. The frequency of vaginal delivery, treatment with high-frequency oscillatory ventilation (HFO) and inotropic medications was higher in the second period, but the difference was not statistically significant. There was no access to extracorporal membranous oxygenation (ECMO). Postoperative complications were registered in half of the children. Three children were re-operated due to recurrence of the hernia. CONCLUSION: The overall mortality rate in live-born children with CDH was 23% in the setting of a multidisciplinary treatment centre, which was similar to that reported by other centres. In cases with prenatally diagnosed CDH, delivery in a multidisciplinary treatment centre is recommended.

Maternal Argonaute 2 is essential for early mouse development at the maternal-zygotic transition.

Activation of zygotic gene expression in the two-cell mouse embryo is associated with destruction of maternally inherited transcripts, an important process for embryogenesis about which little is understood. We asked whether the Argonaute (Ago)/RNA-induced silencing complex, providing the mRNA "slicer" activity in gene silencing, might contribute to this process. Here we show that Ago2, 3, and 4 transcripts are contributed to the embryo maternally. By systematic knockdown of maternal Ago2, 3, and 4, individually and in combination, we find that only Ago2 is required for development beyond the two-cell stage. Knockdown of Ago2 stabilizes one set of maternal mRNAs and reduces zygotic transcripts of another set of genes. Ago2 is localized in mRNA-degradation P-bodies analogous to those that function in RNAi-like mechanisms in other systems. Profiling the expression of microRNAs throughout preimplantation development identified several candidates that could potentially work with Ago2 to mediate degradation of specific mRNAs. However, their low abundance raises the possibility that other endogenous siRNAs may also participate. Together, our results demonstrate that maternal expression of Ago2 is essential for the earliest stages of mouse embryogenesis and are compatible with the notion that degradation of a proportion of maternal messages involves the RNAi-machinery.

On the suitability of carbon nanotube forests as non-stick surfaces for nanomanipulation.

A carbon nanotube forest provides a unique non-stick surface for nanomanipulation, as the nanostructuring of the surface allows micro- and nanoscale objects to be easily removed after first being deposited via a liquid dispersion. A common problem for smooth surfaces is the strong initial stiction caused by adhesion forces after deposition onto the surface. In this work, carbon nanotube forests fabricated by plasma-enhanced chemical vapour deposition are compared to structures with a similar morphology, silicon nanograss, defined by anisotropic reactive ion-etching. While manipulation experiments with latex microbeads on structured as well as smooth surfaces (gold, silicon, silicon dioxide, Teflon, diamond-like carbon) showed a very low initial stiction for both carbon nanotube forests and silicon nanograss, a homogeneous distribution of particles was significantly easier to achieve on the carbon nanotube forests. Contact-angle measurements during gradual evaporation revealed that the silicon nanograss was superhydrophic with no contact-line pinning, while carbon nanotube forests in contrast showed strong contact-line pinning, as confirmed by environmental scanning electron microscopy of microdroplets. As a consequence, latex microbeads dispersed on the surface from an aqueous solution distributed evenly on carbon nanotube forests, but formed large agglomerates after evaporation on silicon nanograss. Lateral manipulation of latex microbeads with a microcantilever was found to be easier on carbon nanotube forests and silicon nanograss compared to smooth diamond-like carbon, due to a substantially lower initial stiction force on surfaces with nanoscale roughness. Nanomanipulation of bismuth nanowires, carbon nanotubes and organic nanofibres was demonstrated on carbon nanotube forests using a sharp tungsten tip. We find that the reason for the remarkable suitability of carbon nanotube forests as a non-stick surface for nanomanipulation is indeed the strong contact-line pinning in combination with the nanostructured surface, which allows homogeneous dispersion and easy manipulation of individual particles.

Dishevelled proteins regulate cell adhesion in mouse blastocyst and serve to monitor changes in Wnt signaling.

Wnt signaling is essential for the regulation of cell polarity and cell fate in the early embryogenesis of many animal species. Multiple Wnt genes and its pathway members are expressed in the mouse early embryo, raising the question whether they play any roles in preimplantation development. Dishevelled is an important transducer of divergent Wnt pathways. Here we show that three of the mouse Dishevelled proteins are not only expressed in oocytes and during preimplantation development, but also display distinct spatio-temporal localization. Interestingly, as embryos reach blastocyst stage, Dishevelled 2 becomes increasingly associated with cell membrane in trophectoderm cells, while at E4.5, Dishevelled 3 is highly enriched in the cytoplasm of ICM cells. These changes are coincident with an increase in the active form of beta-catenin, p120catenin transcription and decrease of Kaiso expression, indicating an upregulation of Wnt signaling activity before implantation. When Dishevelled-GFP fusion proteins are overexpressed in single blastomeres of the 4-cell stage embryo, the progeny of this cell show reduction in cell adhesiveness and a rounded shape at the blastocyst stage. This suggests that perturbing Dvl function interferes with cell-cell adhesion through the non-canonical Wnt pathway in blastocysts.

Regulation of gene expression in mouse embryos and its embryonic cells through RNAi.

The ability to direct cell differentiation of embryo-derived cells should have considerable practical applications. A prerequisite for success is the availability of robust tools that allows efficient knockdown of specific genes to elucidate gene function and direct differentiation. Loss-of-function studies in the mouse have traditionally been carried out using gene knockout animals to obtain mutant embryonic cells. Despite improvements in this technology, it still remains a laborious method. In addition, the use of knockout for studying preimplantation embryonic cells is limited due to maternal contributions. RNA interference (RNAi) is a powerful genetic approach for specific gene silencing of target genes in which 21-23 nt small interfering RNAs work as sequence-specific RNAi mediators. Using RNAi approaches, gene function has been eliminated in mouse embryonic stem cells and carcinoma cells to direct cell differentiation. In mouse oocytes and embryos, RNAi eliminated both maternal and zygotic transcripts and disturbed normal development.

Strained silicon as a new electro-optic material.

For decades, silicon has been the material of choice for mass fabrication of electronics. This is in contrast to photonics, where passive optical components in silicon have only recently been realized. The slow progress within silicon optoelectronics, where electronic and optical functionalities can be integrated into monolithic components based on the versatile silicon platform, is due to the limited active optical properties of silicon. Recently, however, a continuous-wave Raman silicon laser was demonstrated; if an effective modulator could also be realized in silicon, data processing and transmission could potentially be performed by all-silicon electronic and optical components. Here we have discovered that a significant linear electro-optic effect is induced in silicon by breaking the crystal symmetry. The symmetry is broken by depositing a straining layer on top of a silicon waveguide, and the induced nonlinear coefficient, chi(2) approximately 15 pm V(-1), makes it possible to realize a silicon electro-optic modulator. The strain-induced linear electro-optic effect may be used to remove a bottleneck in modern computers by replacing the electronic bus with a much faster optical alternative.

Growth of heat-treated enterotoxin-positive Clostridium perfringens and the implications for safe cooling rates.

Clostridium perfringens 790-94 and 44071.C05 carrying a chromosomal and a plasmid cpe gene, respectively, were used to determine differences in heat resistance and growth characteristics between the genotypes. Heat inactivation experiments were conducted using an immersed coil apparatus. Spore germination, outgrowth, and lag phase, together named GOL time, as well as generation times were determined during constant temperatures in fluid thioglycollate (FTG) medium as well as in vacuum-packed, heat-treated minced turkey. GOL time and growth were also monitored during cooling scenarios from 65 to 10 degrees C for 3, 4, 5, 6, and 7 h in vacuum-packed, heat-treated minced turkey. Spores of strain 790-94 were approximately 10-fold more heat resistant at 85 degrees C than those of strain 44071.C05, and strain 790-94 also had a higher temperature growth range in FTG. The higher growth range for a chromosomal enterotoxin-producing CPE+ strain was confirmed using two other strains carrying a chromosomal (NCTC8239) and plasmid (945P) cpe gene. Moreover, strain 790-94 had shorter GOL times at 50 degrees C in turkey and approximately half the generation time compared with strain 44071.C05 at temperatures > or = 45 degrees C in both FTG and turkey. Strain 790-94 increased with 0.3, 1.0, 1.7, and 2.0 logs, respectively, during cooling from 65 to 10 degrees C in 4, 5, 6, and 7 h, which was significantly higher than for strain 44071.C05. A maximum acceptable cooling time of 5 h between 65 and 10 degrees C is suggested.

Gene structure and embryonic expression of mouse COP9 signalosome subunit 8 (Csn8).

Csn8 is the smallest subunit of the constitutively photomorphogenic 9 (COP9) signalosome (CSN), which consists of eight distinct components. CSN is homologous to the lid subcomplex of the 26S proteasome and is a regulator of the ubiquitin-proteasome pathway. Murine Csn8 is an ortholog of Arabidopsis COP9, which was originally identified as a key regulator of photomorphogenic development. CSN is essential for the viability of plants and flies, but the role of this conserved protein complex in development of vertebrate animals remains obscure. We report the genomic structure of murine Csn8 gene and the expression pattern of Csn8 during early embryo development. Mouse Csn8 protein contains 209 amino acid residues and is encoded by a single gene located on chromosome 1. Csn8 is expressed in embryonic stem (ES) cells and throughout early embryo development from zygote, preimplantation embryos, to post-implantation embryos. Immunostaining studies revealed that Csn8 is predominantly present in the inner cell mass (ICM) of E3.5 blastocyst and is widely expressed in E9.5-day embryos.

Disruption of the COP9 signalosome Csn2 subunit in mice causes deficient cell proliferation, accumulation of p53 and cyclin E, and early embryonic death.

Csn2 (Trip15/Cops2/Alien) encodes the second subunit of the COP9 signalosome (CSN), an eight-subunit heteromeric complex homologous to the lid subcomplex of the 26S proteasome. CSN is a regulator of SCF (Skp1-cullin-F-box protein)ubiquitin ligases, mostly through the enzymatic activity that deconjugates the ubiquitin-like protein Nedd8 from the SCF Cul1 component. In addition, CSN associates with protein kinase activities targeting p53, c-Jun, and IkappaB for phosphorylation. Csn2 also interacts with and regulates a subset of nuclear hormone receptors and is considered a novel corepressor. We report that targeted disruption of Csn2 in mice caused arrest of embryo development at the peri-implantation stage. Csn2(-/-) blastocysts failed to outgrow in culture and exhibited a cell proliferation defect in inner cell mass, accompanied by a slight decrease in Oct4. In addition, lack of Csn2 disrupted the CSN complex and resulted in a drastic increase in cyclin E, supporting a role for CSN in cooperating with the SCF-ubiquitin-proteasome system to regulate protein turnover. Furthermore, Csn2(-/-) embryos contained elevated levels of p53 and p21, which may contribute to premature cell cycle arrest of the mutant.

Growth of whaleworm (Anisakis simplex, Nematodes, Ascaridoidea, Anisakidae) third-stage larvae in paratenic fish hosts.

The growth of Anisakis simplex L3 larvae in the three paratenic hosts saithe ( Pollachius virens), cod (Gadus morhua) and redfish (Sebastes marinus) was studied based on material collected off the island of Vega on the west coast of Norway over a period of 1 year. In all three fish species, a positive correlation between the length of larvae and the age of the fish was shown. The number of large larvae, here defined as L3>28 mm, increases with increasing age of the host. These findings clearly indicate an accumulation of large L3 larvae and larval growth in the fish host. The larvae were additionally grouped according to the tissue--muscle, liver or other viscera--they were located in. When the average lengths of the three groups of larvae were compared, the gadoids saithe and cod had somewhat higher values for L3 larvae found in the liver. Redfish, on the other hand, which store fat to a relatively higher degree in the viscera, had the highest average length of larvae in this tissue. The lowest average values in all three species were found in the musculature. These trends suggest that the growth of A. simplex L3 larvae is positively correlated with the fat content of the tissue in which it is encapsulated. Of the three host species included in this study, saithe seemed to be the best suited for A. simplex L3, as the growth rate and average length of the parasite was greatest in this species. The growth and average length of L3 was least pronounced in redfish while cod held an intermediate position.

Population dynamics of sealworm, Pseudoterranova decipienssensu lato, in sculpins, Myoxocephalus scorpius, from two areas in Norway between 1990 and 1996.

The abundance of sealworms (Pseudoterranova decipienss.l.) in sculpins (Myoxocephalus scorpius) was studied at Torbjørnskjaer, southeast Norway, and Vega, northern Norway, in samples from 1990 to 1996. The harbour seal ( Phoca vitulina) population at Torbjørnskjaer declined by 70% during the phocine distemper virus epizootic in 1988, and has increased during the 1990s. The abundance of sealworms in sculpins was relatively stable from 1992 to 1996 and any effect of the reduction in the number of harbour seals on the abundance of sealworms has been short-term. When correcting for size differences, young sculpins (2-4 years) from Torbjørnskjaer had higher abundances than those from Vega, while in old sculpins (5-7 years) the abundances seemed similar and stabilised. This happened at a younger age among the more heavily infected sculpins at Torbjørnskjaer than at Vega, and we interpret this as density dependence in the fish host.

CAND1 binds to unneddylated CUL1 and regulates the formation of SCF ubiquitin E3 ligase complex.

The SCF ubiquitin E3 ligase regulates ubiquitin-dependent proteolysis of many regulatory proteins such as p27(Kip1), IkappaB, and beta-catenin. We report the isolation of a CUL1 binding protein, p120(CAND1). We found the majority of CUL1 is in a complex with CAND1 and ROC1 independent of SKP1 and F box protein SKP2. Both in vivo and in vitro, CAND1 prevents the binding of SKP1 and SKP2 to CUL1 while dissociation of CAND1 from CUL1 promotes the reverse reaction. Neddylation of CUL1 or the presence of SKP1 and ATP causes CAND1 dissociation. Our data suggest that CAND1 regulates the formation of the SCF complex, and its dissociation from CUL1 is coupled with the incorporation of F box proteins into the SCF complex, causing their destabilization.

The COP9 signalosome inhibits p27(kip1) degradation and impedes G1-S phase progression via deneddylation of SCF Cul1.

The COP9 signalosome (CSN) is a conserved protein complex with homologies to the lid subcomplex of the 26S proteasome. It promotes cleavage of the Nedd8 conjugate (deneddylation) from the cullin component of SCF ubiquitin ligases. We provide evidence that cullin neddylation and deneddylation is highly dynamic, that its equilibrium can be effectively modulated by CSN, and that neddylation allows Cul1 to form larger protein complexes. CSN2 integrates into the CSN complex via its C-terminal region and its N-terminal half region is necessary for direct interaction with Cul1. The polyclonal antibodies against CSN2 but not other CSN subunits cause accumulation of neddylated Cul1/Cul2 in HeLa cell extract, indicating that CSN2 is essential in cullin deneddylation. Further, CSN inhibits ubiquitination and degradation of the cyclin-dependent kinase inhibitor p27(kip1) in vitro. Microinjection of the CSN complex impeded the G1 cells from entering the S phase. Moreover, anti-CSN2 antibodies negate the CSN-dependent p27 stabilization and the G1/S blockage, suggesting that these functions require the deneddylation activity. We conclude that CSN inhibits SCF ubiquitin ligase activity in targeting p27 proteolysis and negatively regulates cell cycle at the G1 phase by promoting deneddylation of Cul1.