Exploration of New Contrasts, Targets, and MR Imaging and Spectroscopy Techniques for Neuromuscular Disease - A Workshop Report of Working Group 3 of the Biomedicine and Molecular Biosciences COST Action BM1304 MYO-MRI.
Neuromuscular diseases are characterized by progressive muscle degeneration and muscle weakness resulting in functional disabilities. While each of these diseases is individually rare, they are common as a group, and a large majority lacks effective treatment with fully market approved drugs. Magnetic resonance imaging and spectroscopy techniques (MRI and MRS) are showing increasing promise as an outcome measure in clinical trials for these diseases. In 2013, the European Union funded the COST (co-operation in science and technology) action BM1304 called MYO-MRI (www.myo-mri.eu), with the overall aim to advance novel MRI and MRS techniques for both diagnosis and quantitative monitoring of neuromuscular diseases through sharing of expertise and data, joint development of protocols, opportunities for young researchers and creation of an online atlas of muscle MRI and MRS. In this report, the topics that were discussed in the framework of working group 3, which had the objective to: Explore new contrasts, new targets and new imaging techniques for NMD are described. The report is written by the scientists who attended the meetings and presented their data. An overview is given on the different contrasts that MRI can generate and their application, clinical needs and desired readouts, and emerging methods.


A remarkable pattern of a tibial plateau fracture - use of a safe technique with practical advantages in the surgical field.
Bicondylar plateau fractures are complex injuries often requiring a challenging treatment. We report a case of a 68 year-old-male patient with a complicated tibial plateau bicondylar fracture. The fracture of the tibial plateau involved all columns (lateral, medial, posterior). The fracture pattern of the proximal tibia managed by triple plating through dual posteromedial and anterolateral incisions. Posterior-medial and the medial plating result in increased stability. The posteromedial approach to the knee that we used in our case, offers various advantages. We recommend the option of the posteromedial access, as an approach that allows excellent control of the posterior involvement of this pattern of tibial plateau fracture.


H3K23me2 is a new heterochromatic mark in Caenorhabditis elegans.
Genome-wide analyses in Caenorhabditis elegans show that post-translational modifications (PTMs) of histones are evolutionary conserved and distributed along functionally distinct genomic domains. However, a global profile of PTMs and their co-occurrence on the same histone tail has not been described in this organism. We used mass spectrometry based middle-down proteomics to analyze histone H3 N-terminal tails from C. elegans embryos for the presence, the relative abundance and the potential cross-talk of co-existing PTMs. This analysis highlighted that the lysine 23 of histone H3 (H3K23) is extensively modified by methylation and that tri-methylated H3K9 (H3K9me3) is exclusively detected on histone tails with di-methylated H3K23 (H3K23me2). Chromatin immunoprecipitation approaches revealed a positive correlation between H3K23me2 and repressive marks. By immunofluorescence analyses, H3K23me2 appears differentially regulated in germ and somatic cells, in part by the action of the histone demethylase JMJD-1.2. H3K23me2 is enriched in heterochromatic regions, localizing in H3K9me3 and heterochromatin protein like-1 (HPL-1)-positive foci. Biochemical analyses indicated that HPL-1 binds to H3K23me2 and interacts with a conserved CoREST repressive complex. Thus, our study suggests that H3K23me2 defines repressive domains and contributes to organizing the genome in distinct heterochromatic regions during embryogenesis.


CLL intraclonal fractions exhibit established and recently acquired patterns of DNA methylation.
Intraclonal subpopulations of circulating chronic lymphocytic leukemia (CLL) cells with different proliferative histories and reciprocal surface expression of CXCR4 and CD5 have been observed in the peripheral blood of CLL patients and named proliferative (PF), intermediate (IF), and resting (RF) cellular fractions. Here, we found that these intraclonal circulating fractions share persistent DNA methylation signatures largely associated with the mutation status of the immunoglobulin heavy chain locus (IGHV) and their origins from distinct stages of differentiation of antigen-experienced B cells. Increased leukemic birth rate, however, showed a very limited impact on DNA methylation of circulating CLL fractions independent of IGHV mutation status. Additionally, DNA methylation heterogeneity increased as leukemic cells advanced from PF to RF in the peripheral blood. This frequently co-occurred with heterochromatin hypomethylation and hypermethylation of Polycomb-repressed regions in the PF, suggesting accumulation of longevity-associated epigenetic features in recently born cells. On the other hand, transcriptional differences between paired intraclonal fractions confirmed their proliferative experience and further supported a linear advancement from PF to RF in the peripheral blood. Several of these differentially expressed genes showed unique associations with clinical outcome not evident in the bulk clone, supporting the pathological and therapeutic relevance of studying intraclonal CLL fractions. We conclude that independent methylation and transcriptional landscapes reflect both preexisting cell-of-origin fingerprints and more recently acquired hallmarks associated with the life cycle of circulating CLL cells.


Antioxidant active packaging systems to extend the shelf life of sliced cooked ham.
The effectiveness of active packaging systems with green tea extract and oregano essential oil was checked for their use in sliced cooked ham. Three packaging systems were evaluated: i) control group without active film, ii) ATGT packed with active film of green tea extract (1%) and iii) ATRX with a mixture of green tea extract and oregano essential oil (1%). The evolution of microbiological, physicochemical (pH, aw, colour and lipid oxidation) and sensory attributes were analysed after 0, 7, 14 and 21 days of refrigerated storage. Microbial populations were below the limits established by the European Regulations (106 UFC/g). The samples packed with ATGT showed the better antimicrobial activity against total viable counts (TVC) and lactic acid bacteria (BAL), while lower counts of Brochothrix thermosphacta was observed in ATRX film (1.48 vs. 1.78 and 2.59 UFC/g for ATRX vs. ATGT and CON, respectively). Regarding colour, low differences were found between the samples packaged with active and control films. Unlike L*, a* and b* parameters showed a progressive diminution throughout the storage in all batches, being the films that contained green tea (ATGT) were the ones that showed the less discolouration at the end of storage (8.86 vs. 8.63 and 7.50 for ATGT vs. CON and ATRX, respectively). The low fat content of this type of product and the use of anaerobic atmosphere for the packaging of cooked ham did not allow to show an antioxidant effect on lipid oxidation (values below 0.15 mg MDA/kg). Finally, the use of ATGT and ATRX did not suppose a modification of the sensorial attributes of the product, being acceptance scores under the acceptance limit during the whole display.


EvoPipes.net: Bioinformatic Tools for Ecological and Evolutionary Genomics.
Recent increases in the production of genomic data are yielding new opportunities and challenges for biologists. Among the chief problems posed by next-generation sequencing are assembly and analyses of these large data sets. Here we present an online server, http://EvoPipes.net, that provides access to a wide range of tools for bioinformatic analyses of genomic data oriented for ecological and evolutionary biologists. The EvoPipes.net server includes a basic tool kit for analyses of genomic data including a next-generation sequence cleaning pipeline (SnoWhite), scaffolded assembly software (SCARF), a reciprocal best-blast hit ortholog pipeline (RBH Orthologs), a pipeline for reference protein-based translation and identification of reading frame in transcriptome and genomic DNA (TransPipe), a pipeline to identify gene families and summarize the history of gene duplications (DupPipe), and a tool for developing SSRs or microsatellites from a transcriptome or genomic coding sequence collection (findSSR). EvoPipes.net also provides links to other software developed for evolutionary and ecological genomics, including chromEvol and NU-IN, as well as a forum for discussions of issues relating to genomic analyses and interpretation of results. Overall, these applications provide a basic bioinformatic tool kit that will enable ecologists and evolutionary biologists with relatively little experience and computational resources to take advantage of the opportunities provided by next-generation sequencing in their systems.


Hydrogen treatment reduces tendon adhesion and inflammatory response.
A rat model of tendon repair was established to investigate the effects of hydrogen water on tendon adhesion reduction. Thirty-six Sprague Dawley rats were randomly divided into a normal saline (NS) group and a hydrogen water (HS) group according to the processing reagents after a tendon repairing operation. Pre- and postoperative superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) in subjects' serum were observed. Skin fibroblasts were grouped into an NS group, H2 O2 group, H2 group, and H2 O2 H2 group. Expressions of Nrf2, CATA, and γ-GCS were also tested by Western blot analysis. 8-OHdG, GSH, MDA, and SOD of the cells were analyzed by the enzyme-linked immunosorbent assay method. The postoperative SOD activity and GSH contents were significantly reduced (P < 0.05), whereas the postoperative MDA level was significantly increased (P < 0.05). Similarly, the postoperative HS group showed significantly higher SOD activity and GSH contents (P < 0.05) but lower MDA (P < 0.05) compared with the postoperative NS group. MDA and 8-OHdG were significantly decreased in hydrogen-rich medium, while SOD and GSH were increased. The expression of Nrf2, CATA, and γ-GCS in antioxidant system were reduced after H2 O2 processing, which were restored after the application of hydrogen-rich medium. Hydrogen water can reduce tendon adhesion after tendon repairing and prohibit excessive inflammatory response, which could be associated with the activation of the Nrf2 pathway.


Influenza H1 Mosaic Hemagglutinin Vaccine Induces Broad Immunity and Protection in Mice.
Annually, influenza A virus (IAV) infects ~5-10% of adults and 20-30% of children worldwide. The primary resource to protect against infection is by vaccination. However, vaccination only induces strain-specific and transient immunity. Vaccine strategies that induce cross-protective immunity against the broad diversity of IAV are needed. Here we developed and tested a novel mosaic H1 HA immunogen. The mosaic immunogen was optimized in silico to include the most potential B and T cell epitopes (PBTE) across a diverse population of human H1 IAV. Phylogenetic analysis showed that the mosaic HA localizes towards the non-pandemic 2009 strains which encompasses the broadest diversity in the H1 IAV population. We compared the mosaic H1 immunogen to wild-type HA immunogens and the commercial inactivated influenza vaccine, Fluzone. When analyzed by ELISA, the mosaic immunogen induced stronger antibody responses against all four diverse H1 HA proteins. When analyzing T cell responses, again the mosaic immunogen induced stronger cellular immunity against all 4 diverse HA strains. Not only was the magnitude of T cell responses strongest in mosaic immunized mice, the number of epitopes recognized was also greater. The mosaic vaccinated mice showed strong cross-protection against challenges with three divergent IAV strains. These data show that the mosaic immunogen induces strong cross-protective immunity and should be investigated further as a universal influenza vaccine.


Targeted ultrasound contrast agent for molecular imaging of inflammation in high-shear flow.
Targeted ultrasound contrast materials (gas-filled microbubbles carrying ligands to endothelial selectins or integrins) have been investigated as potential molecular imaging agents. Such microbubbles normally exhibit good targeting capability at the slower flow conditions. However, in the conditions of vigorous flow, binding may be limited. Here, we describe a microbubble capable of efficient binding to targets both in slow and fast flow (exceeding 4 dyne/cm(2) wall shear stress) using a clustered polymeric form of the fast-binding selectin ligand sialyl Lewis(X). Microbubbles were prepared from decafluorobutane gas and stabilized with a monolayer of phosphatidylcholine, PEG stearate and biotin-PEG-lipid. Biotinylated PSLe(x) (sialyl Lewis(X) polyacrylamide) or biotinylated anti-P-selectin antibody (RB40.34) was attached to microbubbles via a streptavidin bridge. In a parallel plate flow chamber targeted adhesion model, PSLe(x) bubbles demonstrated specific adhesion, retention and slow rolling on P-selectin-coated plates. Efficiency of firm targeted adhesion to a P-selectin surface (140 molecules/microm(2)) was comparable for antibody-carrying bubbles and PSLe(x)-targeted bubbles at 0.68 dyne/cm(2) shear stress. At fast flow (4.45 dyne/cm(2)), PSLe(x)-targeted bubbles maintained their ability to bind, while antibody-mediated targeting dropped more than 20-fold. At lower surface density of P-selectin (7 molecules/microm(2)), targeting via PSLe(x) was more efficient than via antibody under all the flow conditions tested. Negative control casein-coated plates did not retain bubbles in the range of flow conditions studied. To confirm echogenicity, targeted PSLe(x)-bubbles were visualized on P-selectin-coated polystyrene plates by ultrasound imaging with a clinical scanner operated in pulse inversion mode; control plates lacking targeted bubbles did not show significant acoustic backscatter. In vivo, in a murine model of inflammation in the femoral vein setting, targeting efficacy of intravenously administered PSLe(x)-microbubbles was comparable with targeting mediated by anti-P-selectin antibody, and significantly exceeded the accumulation of non-targeted control bubbles. In the inflamed femoral artery setting, PSLe(x)-mediated microbubble targeting was superior to antibody-mediated targeting.


An evidence-based approach in school prevention means an everyday fight: a case study of the Czech Republic's experience with national quality standards and a national certification system.
The Czech Republic has reached the end of a 15-year-long period of the development of a nationwide preventive system in schools. Reflection on this development can offer an interesting case study that demonstrates the general difficulties involved in creating a national prevention policy and implementing the principles of an evidence-based approach. Through its historical context the up-to-date outputs of the latest projects are presented as "key documents" (quality standards, textbook, explanatory dictionary, examples of good practice etc.) and a national system of assessment of quality called a certification procedure, which has a practical impact on the grant system of the Ministry of Education of the Czech Republic. This context is also used to show how certain European networks (EUSPR, IREFREA, etc.) can be very helpful in generalizing this idea across Europe. All the examples of activities on the national or international level seem to be promising and supportive of the increasingly noticeable trend of using research evidence in real practice and making the whole field more attractive for students and young researchers.


Crystal structure of the Helicobacter pylori vacuolating toxin p55 domain.
Helicobacter pylori VacA, a pore-forming toxin secreted by an autotransporter pathway, causes multiple alterations in human cells, contributes to the pathogenesis of peptic ulcer disease and gastric cancer, and is a candidate antigen for inclusion in an H. pylori vaccine. Here, we present a 2.4-A crystal structure of the VacA p55 domain, which has an important role in mediating VacA binding to host cells. The structure is predominantly a right-handed parallel beta-helix, a feature that is characteristic of autotransporter passenger domains but unique among known bacterial protein toxins. Notable features of VacA p55 include disruptions in beta-sheet contacts that result in five beta-helix subdomains and a C-terminal domain that contains a disulfide bond. Analysis of VacA protein sequences from unrelated H. pylori strains, including m1 and m2 forms of VacA, allows us to identify structural features of the VacA surface that may be important for interactions with host receptors. Docking of the p55 structure into a 19-A cryo-EM map of a VacA dodecamer allows us to propose a model for how VacA monomers assemble into oligomeric structures capable of membrane channel formation.


A microfluidic platform using molecular beacon-based temperature calibration for thermal dehybridization of surface-bound DNA.
This work presents a simple microfluidic device with an integrated thin-film heater for studies of DNA hybridization kinetics and double-stranded DNA melting temperature measurements. The heating characteristics of the device were evaluated with a novel, noninvasive indirect technique using molecular beacons as temperature probes inside reaction chambers. This is the first microfluidic device in which thermal dehybridization of surface-bound oligonucleotides was performed for measurement of double-stranded DNA melting temperatures with +/- 1 degrees C precision. Surface modification and oligonucleotide immobilization were performed by continuously flowing reagents through the microchannels. The resulting reproducibility of oligonucleotide surface densities, at 9% RSD, was better than for the same modification chemistries on glass slides in unstirred reagent solutions (RSD=20%). Moreover, the surface density of immobilized DNA probe molecules could be varied controllably by changing the concentration of the reagent solution used for immobilization. Thus, excellent control of surface characteristics was made possible, something which is often difficult to achieve with larger devices. Solid-phase hybridization reactions, a fundamental aspect of microarray technologies often taking several hours in conventional systems, were reduced to minutes in this device. It was also possible to determine forward rate constants for hybridization, k. These varied from 820,000 to 72,000 M(-1) s(-1), decreasing as surface densities increased. Surface densities could therefore be optimized to obtain rapid hybridization using such an approach. Taken together, this combined microfluidic/small-volume heating approach represents a powerful tool for surface-based DNA analysis.


Complex biogeographic history of a Holarctic passerine.
Our analysis of the ND2 sequences revealed six clades within winter wrens (Troglodytes troglodytes). These clades corresponded to six geographical regions: western Nearctic, eastern Nearctic, eastern Asia, Nepal, Caucasus and Europe, and differed by 3-8.8% of sequence divergence. Differences among regions explained 96% of the sequence variation in winter wren. Differences among individuals within localities explained 3% of the sequence variation, and differences among localities within regions explained 1%. Grouping sequences into subspecies instead of localities did not change these proportions. Proliferation of the six clades coincided with Early and Middle Pleistocene glaciations. The distribution of winter wren clades can be explained by a series of five consecutive vicariant events. Western Nearctic wrens diverged from the Holarctic ancestor 1.6 Myr before the present time (MYBP). Eastern Nearctic and Palaearctic wrens diverged 1 MYBP. Eastern and western Palaearctic birds diverged 0.83 MYBP. Nepalese and east Asian wrens diverged 0.67 MYBP, and Caucasian birds diverged from European wrens 0.54 MYBP. The winter wren has a much greater degree of inter- and intracontinental differentiation than the three other Holarctic birds studied to date--dunlin (Calidris alpina), common raven (Corvus corax) and three-toed woodpecker (Picoides trydactylus)--and represents an example of cryptic speciation that has been overlooked.


Towards better measures of research utilization: a collaborative study in Canada and Sweden.
This paper is a report of a study examining research utilization in nursing. The specific aims were to (1) clarify the construct of research utilization, and (2) identify observable indicators of research utilization. Robust measures of research utilization do not exist despite steadily increasing numbers of studies in the field. One reason for this is theoretical confusion surrounding the central concepts in the field. A qualitative (focus group) design was used to explore the construct of research utilization in two countries: Canada and Sweden. A systematic and sequential (three phases) approach to expert sampling framed the study. Phase 1 consisted of initial construct clarification by the research team (2005). In Phase 2, a face-to-face meeting with a panel of international research utilization nursing experts was held (2005). Phase 3 consisted of a series of focus groups with nursing care (direct and non-direct) providers (2005-2007). Data were analysed using content analysis. The nursing care providers did not commonly use the term 'research utilization'. Several examples of research utilization were provided; a majority of these examples related to instrumental research utilization and became increasingly concrete as one moved from non-direct to direct care participants. Participants identified several indicators of research utilization (instrumental and conceptual). From these indicators, a measurement schematic was derived. The construct of research utilization is multi-faceted. Several indicators of research utilization were identified, which can be used to augment existing or develop a new and improved measure that taps both instrumental and conceptual use.


Primary Sjögren's syndrome with antibodies to HTLV-I: clinical and laboratory features.
The prevalence of antibodies to human T lymphotropic virus type I (HTLV-I) was studied in patients with primary Sjögren's syndrome. Thirteen of 36 serum samples were positive by enzyme linked immunosorbent assay (ELISA) and particle agglutination assay for antibodies to HTLV-I and were confirmed by western blotting. The presence of antibodies to HTLV-I may signify an HTLV-I carrier state. These patients had a high occurrence of extraglandular manifestations such as uveitis, myopathy, and recurrent high fever compared with patients who did not have antibodies to HTLV-I. Patients with antibodies to HTLV-I had an increased spontaneous proliferation of peripheral blood mononuclear cells compared with those without the antibodies. The proportions of activated and memory T cells (HLA-DR+ CD3+, CD25+ CD3+, and CD29+ CD4+ cells) were higher in HTLV-I carriers than in non-carriers. The presence of antibodies to HTLV-I in some patients with primary Sjögren's syndrome suggests that HTLV-I may cause primary Sjögren's syndrome or its extraglandular manifestations, or both.


Level of inflammatory cytokines in rheumatoid arthritis patients: Correlation with 25-hydroxy vitamin D and reactive oxygen species.
Rheumatoid arthritis (RA) is an autoimmune inflammatory disorder. Reactive oxygen species (ROS) and pro-inflammatory cytokines have been believed to be involved in the etiopathogenesis of the disease. The aim of the study was to determine the correlation of inflammatory cytokines with 25-hydroxy vitamin D and ROS. 100 RA patients and 50 healthy age and sex matched individuals were included in the study. Patients were further divided on the basis of presence or absence of rheumatoid factor and disease severity. Serum 25-hydroxy vitamin D levels were monitored by chemiluminescent immunoassay. 10% hematocrit was used to detect the level of ROS by spectro fluorometer. The levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-10 and IL-17) were determined in plasma by ELISA. The level of 25-hydroxy vitamin D was found to be decreased in RA patients in comparison to the control group. However the level of ROS and inflammatory cytokines were found to be elevated in RA patients in comparison with the healthy controls, with the increase being more pronounced in seropositive and RA patients having high disease severity. Inflammatory cytokines showed negative correlation with 25-hydroxy vitamin D and positive correlation with ROS. This study for the first time shows the association of inflammatory cytokines with 25-hydroxy vitamin D and ROS in RA patients. The results suggest that 25-hydroxy vitamin D being an immune modulator is decreased in the serum of RA patients. Further ROS and cytokines play an important role in the pathogenesis of RA and are responsible for increasing the severity of disease.


Estimation by simulation of the efficiency of the French marker-assisted selection program in dairy cattle.
The efficiency of the French marker-assisted selection (MAS) was estimated by a simulation study. The data files of two different time periods were used: April 2004 and 2006. The simulation method used the structure of the existing French MAS: same pedigree, same marker genotypes and same animals with records. The program simulated breeding values and new records based on this existing structure and knowledge on the QTL used in MAS (variance and frequency). Reliabilities of genetic values of young animals (less than one year old) obtained with and without marker information were compared to assess the efficiency of MAS for evaluation of milk, fat and protein yields and fat and protein contents. Mean gains of reliability ranged from 0.015 to 0.094 and from 0.038 to 0.114 in 2004 and 2006, respectively. The larger number of animals genotyped and the use of a new set of genetic markers can explain the improvement of MAS reliability from 2004 to 2006. This improvement was also observed by analysis of information content for young candidates. The gain of MAS reliability with respect to classical selection was larger for sons of sires with genotyped progeny daughters with records. Finally, it was shown that when superiority of MAS over classical selection was estimated with daughter yield deviations obtained after progeny test instead of true breeding values, the gain was underestimated.


Poly(ADP-ribose) polymerase inhibitors combined with external beam and radioimmunotherapy to treat aggressive lymphoma.
To assess the possible radiosensitizing capabilities of two different poly(ADP-ribose) polymerase (PARP) inhibitors in combination with external beam and I-tositumomab in a non-Hodgkin's lymphoma cell line. Epstein-Barr virus-infected human Raji lymphoma cells with lentivirally transfected green fluorescent protein and luciferase in log-phase growth were incubated with various doses of AZD-2281 and ABT-888 24 h before external beam radiation exposure. A 500 nmol/l concentration of AZD-2281 and ABT-888 was used to assess the growth curve of Raji lymphoma cells over 5 days. The number of double-stranded breaks was visually assessed using a H2AX antibody and confocal microscopy. Intracellular PARP activity was measured 2 h after incubation with AZD-2281 (500 nmol/l) and ABT-888 using a colorimetric PARP assay kit. The radiosensitizing effect of AZD-2281 (500 nmol/l) with various doses of I-tositumomab was assessed after 24 h. A volume of 500 nmol/l of AZD-2281 and 500 nmol/l of ABT-888, in combination with 0, 4, 8, and 12 Gy external beam radiation, showed a 5.2, 7.1, 10.1, and 33.1% radiosensitization. A measure of 500 nmol/l AZD-2281 and ABT-888 significantly reduced the percentage of viable cells on days 3-5 compared with controls. The maximal relative reduction in viable cells was 78.5%, and this occurred with AZD-2281 (500 nmol/l) on day 5. AZD-2281 revealed a higher number of double-stranded breaks with confocal microscopy than did ABT-888. Two hours after incubation of Raji cells with 500 nmol/l of AZD-2281 or ABT-888, the colorimetric PARP activity assay showed a reduction of 30.36% with ABT-888 and of 47.8% with AZD-2281. Combining AZD-2281 (500 nmol/l) with 0, 5 μCi (0.185 MBq), 10 μCi (0.37 MBq) and 20 μCi (0.74 MBq) ¹³¹I-tositumomab revealed a significant reduction in cell viability after 24 h with 5 μCi (0.185 MBq) (P<0.01) and 10 μCi (0.37 MBq) (P<0.01) radiation dose. PARP inhibitors AZD-2281 and ABT-888 are highly radiosensitizing agents when used before external beam radiation and ¹³¹I-tositumomab.


The comparative economic burden of mild, moderate, and severe fibromyalgia: results from a retrospective chart review and cross-sectional survey of working-age U.S. adults.
Patients with fibromyalgia report persistent widespread pain, fatigue, and substantial functional limitations, which may lead to high health resource use (HRU) and lost productivity. Previous analyses of the U.S. population have not examined the direct and indirect costs of fibromyalgia by severity level. To assess (a) HRU, direct and indirect costs associated with fibromyalgia in routine clinical practice in the United States using a patient-centric approach, and (b) the relationship of fibromyalgia severity level to HRU and costs. This study recruited a nonprobability convenience sample of 203 subjects aged 18 through 65 years between August 2008 and February 2009 from 20 U.S. community-based physician offices. Subjects had a prior diagnosis of fibromyalgia by a rheumatologist, neurologist, or pain specialist; received treatment at the enrolling physician's practice for at least 3 months; experienced widespread pain for at least 3 months; and experienced pain in the previous 24 hours. Subjects completed a 106-item patient questionnaire that included 5 validated health-related quality-of-life instruments and study-specific questions about demographics; clinical history; overall health; treatment satisfaction; and impact of fibromyalgia on cognitive function, daily activities, and employment status. Subjects also self-reported hours of unpaid informal caregiver time because of inability to perform daily activities (e.g., housework, child care), out-of-pocket expenses for medical and nonmedical services, and lost productivity related to fibromyalgia for the previous 4 weeks. The 20-item Fibromyalgia Impact Questionnaire total score was used to stratify subjects into fibromyalgia severity groups (0 to less than 39 = mild, 39 to less than 59 = moderate, 59 to 100 = severe). Staff at each site recorded clinical characteristics, HRU, and medication use attributable to fibromyalgia on a paper clinical case report form (CRF) based on a 3-month retrospective medical chart review. Unit costs for 2009 were assigned to the 3-month HRU data reported on the CRF and 4-week subject-reported lost productivity. Costs were then annualized and reported in the following categories: direct medical, direct nonmedical, and indirect. Differences across severity levels were evaluated using the Kruskal-Wallis test (continuous measures) and Pearson chi-square or Fisher's exact tests (categorical measures) at the 0.05 alpha level. Of the 203 subjects, 21 (10.3%) had mild, 49 (24.1%) had moderate, and 133 (65.5%) had severe fibromyalgia. For subjects with mild, moderate, and severe fibromyalgia, respectively, the number of fibromyalgia-related medications (3-month means: 1.8, 2.3, and 2.8, P = 0.011) and office visits to health care providers (3-month means: 2.7, 5.2, and 6.9, P < 0.001) significantly differed across severity levels. Across severity levels, total medical and nonmedical out-of-pocket costs also differed (P = 0.025). Mean [median] 3-month total direct costs (including payer costs for HRU and out-of-pocket costs for medical and nonmedical services) were $1,213 [$1,150], $1,415 [$1,215], and $2,329 [$1,760] for subjects with mild, moderate, and severe fibromyalgia, respectively (P = 0.002); and mean [median] 3-month indirect costs (including subject-reported absenteeism, unemployment, disability, and the estimated value of unpaid informal care) were $1,341 [$0], $5,139 [$1,680], and $8,285 [$7,030] (P < 0.001). Mean total indirect costs accounted for 52.5%, 78.4%, and 78.1% of mean total costs for subjects with mild, moderate, and severe fibromyalgia, respectively. Direct and indirect costs related to fibromyalgia are higher among subjects with worse fibromyalgia severity. Indirect costs account for a majority of fibromyalgia-related costs at all fibromyalgia severity levels.


Finite element analysis of idealised unit cell cancellous structure based on morphological indices of cancellous bone.
Human bones can be categorised into one of two types--the compact cortical and the porous cancellous. Whilst the cortical is a solid structure macroscopically, the structure of cancellous bone is highly complex with plate-like and strut-like structures of various sizes and shapes depending on the anatomical site. Reconstructing the actual structure of cancellous bone for defect filling is highly unfeasible. However, the complex structure can be simplified into an idealised structure with similar properties. In this study, two idealised architectures were developed based on morphological indices of cancellous bone: the tetrakaidecahedral and the prismatic. The two architectures were further subdivided into two types of microstructure, the first consists of struts only and the second consists of a combination of plates and struts. The microstructures were transformed into finite element models and displacement boundary condition was applied to all four idealised cancellous models with periodic boundary conditions. Eight unit cells extracted from the actual cancellous bone obtained from micro-computed tomography were also analysed with the same boundary conditions. Young's modulus values were calculated and comparison was made between the idealised and real cancellous structures. Results showed that all models with a combination of plates and struts have higher rigidity compared to the one with struts only. Values of Young's modulus from eight unit cells of cancellous bone varied from 42 to 479 MPa with an average of 234 MPa. The prismatic architecture with plates and rods closely resemble the average stiffness of a unit cell of cancellous bone.


Forensic investigation approaches of searching relatives in DNA databases.
There are several indirect database searching approaches to identify the potential source of a forensic biological sample. These DNA-based approaches are familial searching, Y-STR database searching, and investigative genetic genealogy (IGG). The first two strategies use forensic DNA databases managed by the government, and the latter uses databases managed by private citizens or companies. Each of these search strategies relies on DNA testing to identify relatives of the donor of the crime scene sample, provided such profiles reside in the DNA database(s). All three approaches have been successfully used to identify the donor of biological evidence, which assisted in solving criminal cases or identifying unknown human remains. This paper describes and compares these approaches in terms of genotyping technologies, searching methods, database structures, searching efficiency, data quality, data security, and costs, and raises some potential privacy and legal considerations for further discussion by stakeholders and scientists. Y-STR database searching and IGG are advantageous since they are able to assist in more cases than familial searching readily identifying distant relatives. In contrast, familial searching can be performed more readily with existing laboratory systems. Every country or state may have its own unique economic, technical, cultural, and legal considerations and should decide the best approach(es) to fit those circumstances. Regardless of the approach, the ultimate goal should be the same: generate investigative leads and solve active and cold criminal cases to public safety, under stringent policies and security practices designed to protect the privacy of its citizenry.


A clinical, microbiological, and pathological study of intestinal perforation associated with typhoid fever.
One of the most serious complications of typhoid fever is intestinal perforation. Of 27 patients admitted to a provincial hospital in the Mekong Delta region of Vietnam who had gastrointestinal perforation secondary to suspected typhoid fever, 67% were male, with a median age of 23 years and a median duration of illness of 10 days. Salmonella enterica subspecies enterica serotype Typhi (S. Typhi) was isolated from 11 (41%) of 27 patients; of 27 patients, only 4 (15%) had positive cultures from gut biopsies. S. Typhi DNA was detected by polymerase chain reaction for all perforation biopsy samples. Detailed histological examination of the gastrointestinal mucosa at the site of perforation in all cases showed a combination of discrete acute and chronic inflammation. Acute inflammation at the serosal surface indicated additional tissue damage after perforation. Immunohistochemical results showed that the predominant infiltrating cell types at the site of perforation were CD68+ leukocytes (macrophages) or CD3+ leukocytes (T lymphocytes).


Oscillation and polarity of E-cadherin asymmetries control actomyosin flow patterns during morphogenesis.
Actomyosin flows are involved in a variety of cellular processes, including cytokinesis, cell migration, polarization, and morphogenesis. In epithelia, flow polarization orients cell deformations. It is unclear, however, how flows are polarized and how global patterns of junction remodeling emerge from flow polarization locally. We address this question during intercalation-driving extension of the Drosophila germband. Intercalation is associated with polarized junction remodeling, whereby actomyosin pulses flow anisotropically toward dorsal-ventral junctions and shrink them. Here, we show that planar polarization of flows emerges from polarized fluctuations in the levels of E-cadherin clusters that produce transient and oscillating asymmetries of coupling. These fluctuations are triggered by polarized E-cadherin endocytosis and are amplified by flow itself. This work suggests that fluctuations and mechanical instability are not the consequences of limited control over the systems key parameters, but rather that they define the axis of symmetry breaking.


Randomized, controlled trial of division of tongue-tie in infants with feeding problems.
To determine whether, in infants with a tongue-tie and a feeding problem, the current medical treatment (referral to a lactation consultant) or immediate division works best and enables the infants to feed normally. Between March and July 2002, all the babies in the district of Southampton with tongue-ties were followed in order to see if they had any feeding problems. If they developed problems, the mothers gave written consent and were enrolled in an ethics committee approved, randomized, controlled trial, comparing 48 h of intensive lactation consultant support (control) with immediate division. A total of 201 babies had tongue-tie, of whom 88 had breast-feeding or bottle-feeding problems. Thirty-one were not enrolled, so 57 were randomized. Of the 29 controls, one improved (3%) and breast-fed for 8 months, but 28 did not. At 48 h, these 28 were offered division, which all accepted, and 27 improved (96%) and fed normally. Of the 28 babies who had immediate division, 27 improved and fed normally but one remained on a nipple shield (P < 0.001). Twenty-four mothers breast-fed for 4 months (24/40, 60%). Overall, division of the tongue-tie babies resulted in improved feeding in 54/57 (95%) babies. This randomized, controlled trial has clearly shown that tongue-ties can affect feeding and that division is safe, successful and improved feeding for mother and baby significantly better than the intensive skilled support of a lactation consultant.


Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen.
Studies of nuclear function in many organisms, especially those with tough cell walls, are limited by lack of availability of simple, economical methods for large-scale preparation of clean, undamaged nuclei. Here we present a useful method for nuclear isolation from the important model organism, the fission yeast, Schizosaccharomyces pombe. To preserve in vivo molecular configurations, we flash-froze the yeast cells in liquid nitrogen. Then we broke their tough cell walls, without damaging their nuclei, by grinding in a precision-controlled motorized mortar-and-pestle apparatus. The cryo-ground cells were resuspended and thawed in a buffer designed to preserve nuclear morphology, and the nuclei were enriched by differential centrifugation. The washed nuclei were free from contaminating nucleases and have proven well-suited as starting material for genome-wide chromatin analysis and for preparation of fragile DNA replication intermediates. We have developed a simple, reproducible, economical procedure for large-scale preparation of endogenous-nuclease-free, morphologically intact nuclei from fission yeast. With appropriate modifications, this procedure may well prove useful for isolation of nuclei from other organisms with, or without, tough cell walls.


Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes.
In recent years, there has been an exponential increase in the number of studies aiming to understand the biology of exosomes, as well as other extracellular vesicles. However, classification of membrane vesicles and the appropriate protocols for their isolation are still under intense discussion and investigation. When isolating vesicles, it is crucial to use systems that are able to separate them, to avoid cross-contamination. EVS RELEASED FROM THREE DIFFERENT KINDS OF CELL LINES: HMC-1, TF-1 and BV-2 were isolated using two centrifugation-based protocols. In protocol 1, apoptotic bodies were collected at 2,000×g, followed by filtering the supernatant through 0.8 µm pores and pelleting of microvesicles at 12,200×g. In protocol 2, apoptotic bodies and microvesicles were collected together at 16,500×g, followed by filtering of the supernatant through 0.2 µm pores and pelleting of exosomes at 120,000×g. Extracellular vesicles were analyzed by transmission electron microscopy, flow cytometry and the RNA profiles were investigated using a Bioanalyzer(®). RNA profiles showed that ribosomal RNA was primary detectable in apoptotic bodies and smaller RNAs without prominent ribosomal RNA peaks in exosomes. In contrast, microvesicles contained little or no RNA except for microvesicles collected from TF-1 cell cultures. The different vesicle pellets showed highly different distribution of size, shape and electron density with typical apoptotic body, microvesicle and exosome characteristics when analyzed by transmission electron microscopy. Flow cytometry revealed the presence of CD63 and CD81 in all vesicles investigated, as well as CD9 except in the TF-1-derived vesicles, as these cells do not express CD9. Our results demonstrate that centrifugation-based protocols are simple and fast systems to distinguish subpopulations of extracellular vesicles. Different vesicles show different RNA profiles and morphological characteristics, but they are indistinguishable using CD63-coated beads for flow cytometry analysis.


Topological Two-Dimensional Floquet Lattice on a Single Superconducting Qubit.
Current noisy intermediate-scale quantum (NISQ) devices constitute powerful platforms for analog quantum simulation. The exquisite level of control offered by state-of-the-art quantum computers make them especially promising to implement time-dependent Hamiltonians. We implement quasiperiodic driving of a single qubit in the IBM Quantum Experience and thus experimentally realize a temporal version of the half-Bernevig-Hughes-Zhang Chern insulator. Using simple error mitigation, we achieve consistently high fidelities of around 97%. From our data we can infer the presence of a topological transition, thus realizing an earlier proposal of topological frequency conversion by Martin, Refael, and Halperin. Motivated by these results, we theoretically study the many-qubit case, and show that one can implement a wide class of Floquet Hamiltonians, or time-dependent Hamiltonians in general. Our study highlights promises and limitations when studying many-body systems through multifrequency driving of quantum computers.


Identifying alternative models of healthcare service delivery to inform health system improvement: scoping review of systematic reviews.
To describe available evidence from systematic reviews of alternative healthcare delivery arrangements relevant to high-income countries to inform decisions about healthcare system improvement. Scoping review of systematic reviews. Systematic reviews of interventions indexed in Pretty Darn Quick-Evidence. All English language systematic reviews evaluating the effects of alternative delivery arrangements relevant to high-income countries, published between 1 January 2012 and 20 September 2017. Eligible reviews had to summarise evidence on at least one of the following outcomes: patient outcomes, quality of care, access and/or use of healthcare services, resource use, impacts on equity and/or social outcomes, healthcare provider outcomes or adverse effects. Journal, publication year, number and design of primary studies, populations/health conditions represented and types of outcomes were extracted. Of 829 retrieved records, 531 reviews fulfilled our inclusion criteria. Almost all (93%) reviews reported on patient outcomes, while only about one-third included resource use as an outcome of interest. Just over a third (n=189, 36%) of reviews focused on alternative information and communications technology interventions (including 162 reviews on telehealth). About one-quarter (n=122, 23%) of reviews focused on alternative care coordination interventions. 15% (n=80) of reviews examined interventions involving changes to who provides care and how the healthcare workforce is managed. Few reviews investigated the effects of interventions involving changes to how and when care is delivered (n=47, 9%) or interventions addressing a goal-focused question (n=38, 7%). A substantial body of evidence about the effects of a wide range of delivery arrangements is available to inform health system improvements. The lack of economic evaluations in the majority of systematic reviews of delivery arrangements means that the value of many of these models is unknown. This scoping review identifies evidence gaps that would be usefully addressed by future research.


Mid-life microbiota crises: middle age is associated with pervasive neuroimmune alterations that are reversed by targeting the gut microbiome.
Male middle age is a transitional period where many physiological and psychological changes occur leading to cognitive and behavioural alterations, and a deterioration of brain function. However, the mechanisms underpinning such changes are unclear. The gut microbiome has been implicated as a key mediator in the communication between the gut and the brain, and in the regulation of brain homeostasis, including brain immune cell function. Thus, we tested whether targeting the gut microbiome by prebiotic supplementation may alter microglia activation and brain function in ageing. Male young adult (8 weeks) and middle-aged (10 months) C57BL/6 mice received diet enriched with a prebiotic (10% oligofructose-enriched inulin) or control chow for 14 weeks. Prebiotic supplementation differentially altered the gut microbiota profile in young and middle-aged mice with changes correlating with faecal metabolites. Functionally, this translated into a reversal of stress-induced immune priming in middle-aged mice. In addition, a reduction in ageing-induced infiltration of Ly-6Chi monocytes into the brain coupled with a reversal in ageing-related increases in a subset of activated microglia (Ly-6C+) was observed. Taken together, these data highlight a potential pathway by which targeting the gut microbiome with prebiotics can modulate the peripheral immune response and alter neuroinflammation in middle age. Our data highlight a novel strategy for the amelioration of age-related neuroinflammatory pathologies and brain function.


Comparison of hepatotoxicity and mechanisms induced by triclosan (TCS) and methyl-triclosan (MTCS) in human liver hepatocellular HepG2 cells.
Triclosan (TCS) is used as an antimicrobial agent and has been widely dispersed and detected in the environment and organisms including human samples. Methyl-triclosan (MTCS) is the predominant bacterial TCS metabolite. At present, the toxicological effects and mechanism of TCS and MTCS are still not fully understood. In this study, the cytotoxic effects of TCS and MTCS in HepG2 cells were investigated in terms of cell proliferation, comet assay, cell cycle, and apoptosis. In addition, the expressions of related proteins were detected with western blotting analysis. The results showed that TCS could significantly inhibit cell proliferation, while MTCS had no obvious effect on cell growth. Both TCS and MTCS caused oxidative injury associated with HO-1 induction and increased DNA strand breaks, which consequently initiated the damage repair process via up-regulation of DNA-PKcs. In addition, TCS blocked the HepG2 cells in S and G2/M phases of cell cycle through down-regulation of cyclin A2 and CDK; while MTCS induced cell cycle arrest at the S phase through up-regulation of cyclin A2 and CDK. Furthermore, TCS activated p53 mediated apoptosis in HepG2 cells in a caspase-independent manner, while MTCS induced apoptosis was dependent on caspase. Moreover, TCS exposure exhibited more severe toxicity in HepG2 cells as compared with MTCS exposure, indicating that the replacement of the ionizable proton in TCS by the methyl group in MTCS is correlated with the cellular toxicity and the molecular mechanism.


Temporal trends in deep ocean Redfield ratios.
The Redfield ratio [carbon:nitrogen:phosphorus (C:N:P)] of particle flux to the deep ocean is a key factor in marine biogeochemical cycling. Changes in oceanic carbon sequestration have been linked to variations in the Redfield ratio on geological time scales, but this ratio generally is assumed to be constant with time in the modern ocean. However, deep-water Redfield ratios in the northern hemisphere show evidence for temporal trends over the past five decades. The North Atlantic Ocean exhibits a rising N:P ratio, which may be related to increased deposition of atmospheric nitrous oxides from anthropogenic N emissions. In the North Pacific Ocean, increasing C:N and C:P ratios are accompanied by rising remineralization rates, which suggests intensified export production. Stronger export of carbon in this region may be due to enhanced bioavailability of aeolian iron. These findings imply that the biological part of the marine carbon cycle currently is not in steady state.


Angiotensin II inhibits luteinizing hormone-stimulated cholesterol side chain cleavage expression and stimulates basic fibroblast growth factor expression in bovine luteal cells in primary culture.
Angiotensin II has been identified immunohistochemically in the ovaries of both rats and humans. Here we present evidence that angiotensin II (an extremely vasoactive agent in a wide range of tissues) may be involved in the regulation of the major steroidogenic enzyme in the ovary, cholesterol side chain cleavage cytochrome P-450 (P-450scc), as well as of basic fibroblast growth factor (bFGF), which has been implicated as an angiogenic factor in the bovine corpus luteum. We have used primary cultures of bovine luteal cells to examine the effect of angiotensin II and its receptor antagonist, saralasin, on expression of mRNA encoding bFGF as well as on progesterone production and the expression of mRNA encoding cholesterol side chain cleavage cytochrome P-450 (P-450scc). Neither angiotensin II nor saralasin when added alone to the culture medium had any effect on basal progesterone production. Luteinizing hormone (LH) caused a 15-fold increase in progesterone accumulation after 24 h of exposure which was reduced to 5-fold in the presence of angiotensin II. This appeared to be receptor-mediated in that although saralasin alone had no effect on LH-stimulated progesterone accumulation, it significantly reversed the inhibition by angiotensin II. This pattern was mirrored by the levels of mRNA encoding P-450scc, i.c., LH induced the highest levels of expression of this message, these levels were reduced by angiotensin II, and saralasin partially overcame this reduction. Levels of mRNA encoding bFGF were elevated by both LH and angiotensin II. Treatment with saralasin, however, resulted in complete inhibition of bFGF mRNA expression in the presence of both LH and angiotensin II. These results suggest a role for angiotensin II to mediate the action of LH as a regulator of bFGF expression and hence, potentially, angiogenesis. Local production of angiotensin II might also contribute to the refractoriness of luteal progesterone secretion to LH at the time of luteal regression.


A new genus of African monkey, Rungwecebus: morphology, ecology, and molecular phylogenetics.
A new species of African monkey, Lophocebus kipunji, was described in 2005 based on observations from two sites in Tanzania. We have since obtained a specimen killed by a farmer on Mount Rungwe, the type locality. Detailed molecular phylogenetic analyses of this specimen demonstrate that the genus Lophocebus is diphyletic. We provide a description of a new genus of African monkey and of the only preserved specimen of this primate. We also present information on the animal's ecology and conservation.


Frequency of asymptomatic and symptomatic respiratory virus detection in pediatric hematopoietic cell transplant patients.
Respiratory viral infections are common and can cause significant morbidity and mortality in pediatric patients undergoing hematopoietic cell transplantation (HCT). The prevalence of disease has been primarily identified from retrospective studies using standard-of-care specimens. The incidence of both asymptomatic respiratory viral detection and symptomatic respiratory viral detection in this high-risk population is not well described. We performed longitudinal, active, prospective surveillance in pediatric HCT patients. Subjects underwent weekly midturbinate swabs (MTSs) for the detection of 18 respiratory viruses and subtypes peri-HCT and 100 days post-HCT. Clinical data were obtained from the medical record. From September 2015 to February 2017, 24 children underwent 29 HCT, and 284 MTSs were collected. Forty-two (15%) specimens were virus-positive from 10 (42%) subjects. Specimens from children undergoing allogeneic HCT were more likely to have a virus detected (17% vs 8%, P = .04) compared with specimens from children undergoing autologous HCT. Sixteen (38%) detections were not associated with symptoms. Almost half (8/17) of the unique viral infections occurred during the HCT hospitalization after a negative specimen, suggesting nosocomial acquisition, and preceded detection from a clinical specimen. Rhinovirus, the most common virus detected, was the only virus detected in 33 (81%) virus-positive specimens; only 11 (33%) rhinovirus detections were asymptomatic. Asymptomatic detection of coronavirus and bocavirus occurred. Asymptomatic respiratory virus detection occurred in more than one-third of the children undergoing HCT. The acquisition of respiratory viruses during HCT hospitalization suggests nosocomial acquisition. Early detection of respiratory viruses during asymptomatic periods could have infection prevention and treatment implications.


The regulation of yeast mating-type chromatin structure by SIR: an action at a distance affecting both transcription and transposition.
The two yeast mating-type alleles MATa and MAT alpha each produce two mRNAs that are transcribed in opposite and diverging directions from central promoters. Silent copies of MATa (HMRa) and MAT alpha (HML alpha) contain identical DNA sequences throughout the transcribed region, yet are not transcribed, except in sir- strains. Since SIR represses not only transcription from a silent copy but also its ability to act as a recipient in a mating-type interconversion, we have investigated whether it might act by regulating the entire chromatin structure of a silent locus. We have therefore compared the profile of DNAase I and micrococcal nuclease cleavage at HML alpha with MAT alpha and HMRa with MATa in sir- and SIR+ strains. We find that SIR is necessary for the maintenance of a different chromatin structure at HM loci from their active counterparts at MAT. One particularly striking change that SIR induces provides a simple explanation for one of its biological properties: control of directionality of switching. SIR causes the disappearance of a DNAase I-hypersensitive site at Y-Z boundary (found at MAT or HM sir-) that is coincident with a double-strand cleavage possibly created by HO in the initiation of a mating-type switch.


Uterine rejection after allogeneic uterus transplantation in the rat is effectively suppressed by tacrolimus.
To evaluate the effects of the immunosuppressant tacrolimus on rejection of a transplanted uterus and on uterine expression of markers of inflammation and implantation. Experimental study. University laboratory. Female rats. Uteri from brown Norway rats were transplanted to Lewis rats, receiving either tacrolimus or no treatment. Sham groups underwent either hemihysterectomy or tacrolimus treatment. Gross morphology, histology, density of T-lymphocytes by immunohistochemistry, and mRNA levels of interleukin (IL)-1α, leukemia inhibitory factor (LIF), galectin-1, CD200, IL-15, interferon-inducible protein-10 (IP-10), and nuclear factor-κB (NF-κB) at 14 days' post-transplantation. Nontreated uterine grafts showed rejection with necrosis. Sham groups and the tacrolimus-treated transplanted group exhibited normal uterine morphology with low numbers of T-lymphocytes in all uteri except in two out of seven uteri of the tacrolimus-treated transplant group. Uteri of the nontreated transplanted group showed elevated mRNA expression of IL-1α and IP-10 and reduced galectin-1, compared with the tacrolimus-treated transplanted group. There was no difference between any groups concerning uterine expression of LIF, NF-κB, IL-15, and CD200. Tacrolimus monotherapy suppresses rejection of an allotransplanted uterus and normalizes the expression of IL-1α and IP-10 and prevents T-lymphocyte infiltration.


The Avon patellofemoral joint replacement: Five-year results from an independent centre.
Between May 1998 and May 2007 we carried out 50 Avon patellofemoral joint replacements in 32 patients with isolated patellofemoral osteoarthritis. There were no revisions in the first five years, giving a cumulative survival of 100% for those with a minimum follow-up of five years. The mean follow-up was 5.3 years (2.1 to 10.2). The median Oxford knee score was 30.5 (interquartile range 22.25 to 42.25). In patients with bilateral replacements the median Euroqol General health score was 50 which was significantly lower than that of 75 in those with a unilateral replacement (p = 0.047). The main complication was progression of disease, which was identified radiologically in 11 knees (22%). This highlights the need for accurate selection of patients. Our findings suggest that the Avon prosthesis survives well and gives a satisfactory functional outcome in the medium term.


Posterior parietal cortex represents sensory history and mediates its effects on behaviour.
Many models of cognition and of neural computations posit the use and estimation of prior stimulus statistics: it has long been known that working memory and perception are strongly impacted by previous sensory experience, even when that sensory history is not relevant to the current task at hand. Nevertheless, the neural mechanisms and regions of the brain that are necessary for computing and using such prior experience are unknown. Here we report that the posterior parietal cortex (PPC) is a critical locus for the representation and use of prior stimulus information. We trained rats in an auditory parametric working memory task, and found that they displayed substantial and readily quantifiable behavioural effects of sensory-stimulus history, similar to those observed in humans and monkeys. Earlier proposals that the PPC supports working memory predict that optogenetic silencing of this region would impair behaviour in our working memory task. Contrary to this prediction, we found that silencing the PPC significantly improved performance. Quantitative analyses of behaviour revealed that this improvement was due to the selective reduction of the effects of prior sensory stimuli. Electrophysiological recordings showed that PPC neurons carried far more information about the sensory stimuli of previous trials than about the stimuli of the current trial. Furthermore, for a given rat, the more information about previous trial sensory history in the neural firing rates of the PPC, the greater the behavioural effect of sensory history, suggesting a tight link between behaviour and PPC representations of stimulus history. Our results indicate that the PPC is a central component in the processing of sensory-stimulus history, and could enable further neurobiological investigation of long-standing questions regarding how perception and working memory are affected by prior sensory information.


Independent Multiple Correlates of Post-Exercise Systolic Blood Pressure Recovery in Healthy Adults.
The aim of the present study was to evaluate the independent relationships of systolic blood pressure recovery (SBPR) with age, sex, body mass index (BMI), waist circumference (WC), resting heart rate (HR), physical activity, and cigarette smoking in healthy adults. Subjects performed cycle ergometer exercise at progressive incremental workloads until subjects reached 80% of their age-predicted maximum HR. Blood pressure (BP) was measured before exercise (after 10 and 15 minutes of rest), during exercise (at 2-minute intervals), immediately after exercise (within the first minute) and subsequently at 2-minute intervals until recovery to baseline. The ratio of third-minute SBP relative to first-minute post-exercise SBP was used as the SBPR variable. Our results indicated independent correlations (p<0.05) between SBPR and age, resting HR, physical activity and cigarette smoking (r =0.473; 0.192; -0.262; 0.102 respectively in males and r =0.113; 0.315; -0.637; 0.104 respectively in females). BMI associated positively (r =0.106; p<0.01) with SBPR in males but not in females (r =0.092), while WC was predictive of SBPR in females (r =0.212; p<0.01) but not in males (r =0.005). Age in men and physical activity in females were the strongest predictors of SBPR. The present findings in which SBPR is associated with risk factors of cardiovascular abnormalities strengthen the previously reported significance of SBPR after exercise test as a prognostic tool for the evaluation of cardiovascular abnormalities. Additionally, it may help clinicians to define and interpret the mechanisms behind changes in postexercise SBP responses in adults in future investigations.


Inhibition of receptor tyrosine kinase signalling by small molecule agonist of T-cell protein tyrosine phosphatase.
T-cell protein tyrosine phosphatase (TCPTP/TC45) is a ubiquitously expressed intra-cellular non-receptor protein tyrosine phosphatase involved in the negative regulation of several cancer relevant cellular signalling pathways. We have previously shown that interaction between the alpha-cytoplasmic tail of alpha1beta1 integrin and TCPTP activates TCPTP by disrupting an inhibitory intra-molecular bond in TCPTP. Thus, inhibition of the regulatory interaction in TCPTP is a desirable strategy for TCPTP activation and attenuation of oncogenic RTK signalling. However, this is challenging with low molecular weight compounds. We developed a high-throughput compatible assay to analyse activity of recombinant TCPTP in vitro. Using this assay we have screened 64280 small molecules to identify novel agonists for TCPTP. Dose-dependent response to TCPTP agonist was performed using the in vitro assay. Inhibition effects and specificity of TCPTP agonists were evaluated using TCPTP expressing and null mouse embryonic fibroblasts. Western blot analysis was used to evaluate attenuation of PDGFRbeta and EGFR phosphorylation. Inhibition of VEGF signalling was analysed with VEGF-induced endothelial cell sprouting assays. From the screen we identified six TCPTP agonists. Two compounds competed with alpha1-cytoplasmic domain for binding to TCPTP, suggesting that they activate TCPTP similar to alpha1-cyt by disrupting the intra-molecular bond in TCPTP. Importantly, one of the compounds (spermidine) displayed specificity towards TCPTP in cells, since TCPTP -/- cells were 43-fold more resistant to the compound than TCPTP expressing cells. This compound attenuates PDGFRbeta and VEGFR2 signalling in cells in a TCPTP-dependent manner and functions as a negative regulator of EGFR phosphorylation in cancer cells. In this study we showed that small molecules mimicking TCPTP-alpha1 interaction can be used as TCPTP agonists. These data provide the first proof-of-concept description of the use of high-throughput screening to identify small molecule PTP activators that could function as RTK antagonists in cells.


[The effect of processing on the Vitamin A content of human milk].
To analyze the effect of processing on the Vitamin A levels of milk that is donated to the Human Milk Bank at the University Maternity Hospital, and to evaluate whether this milk supplies the infants' Vitamin A requirement. Sixty milk samples from the Human Milk Bank were divided into two equal portions. One milk portion was extracted prior to processing, while another fraction was subjected to pasteurization and later extracted. The samples were analyzed using high performance liquid chromatography. Analysis with Student's t test indicated that the difference between mean retinol levels was statistically significant at the p < 0.05 level. The retinol content found in the milk prior to processing was 55.4+/-34.0 microg/100 ml, whereas for the processed milk this level was 36.6+/-26.1 microg/100 ml (p < 0.001). It was found that retinol loss occurs during milk processing and that the milk from the Human Milk Bank does not meet infants' Vitamin A requirement.


Subtraction-free and bisulfite-free specific sequencing of 5-methylcytosine and its oxidized derivatives at base resolution.
Although various methods have been developed for sequencing cytosine modifications, it is still challenging for specific and quantitative sequencing of individual modification at base-resolution. For example, to obtain both true 5-methylcytosine (5mC) and true 5-hydroxymethylcytosine (5hmC) information, the two major epigenetic modifications, it usually requires subtraction of two methods, which increases noise and requires high sequencing depth. Recently, we developed TET-assisted pyridine borane sequencing (TAPS) for bisulfite-free direct sequencing of 5mC and 5hmC. Here we demonstrate that two sister methods, TAPSβ and chemical-assisted pyridine borane sequencing (CAPS), can be effectively used for subtraction-free and specific whole-genome sequencing of 5mC and 5hmC, respectively. We also demonstrate pyridine borane sequencing (PS) for whole-genome profiling of 5-formylcytosine and 5-carboxylcytosine, the further oxidized derivatives of 5mC and 5hmC. This work completes the set of versatile borane reduction chemistry-based methods as a comprehensive toolkit for direct and quantitative sequencing of all four cytosine epigenetic modifications.


A chromosome-wide QTL study on BTA29 affecting temperament traits in German Angus beef cattle and mapping of DRD4.
The behaviour of beef cattle is important for the safety and welfare of stockmen and animals. Ten microsatellites spanning BTA29 and, in addition, the candidate gene, dopamine receptor D4 gene, were analysed in 545 German Angus calves of six sires and included in a quantitative trait locus (QTL) study on the basis of three different behaviour tests. A putative QTL for the score while entering the scale (ScE) was detected at BMS764. The DRD4 fragment was mapped in the distal region of BTA29 15.3 cM distal of ILSTS081. The results clearly indicate that BTA29 with a putative QTL in the proximal part and the candidate gene, DRD4, in the distal part plays an important role in the regulation of temperament. During the study one of the sires was detected to be a blood chimera.


Vaccines for preventing influenza in healthy children.
In children and adults the consequences of influenza are mainly absences from school and work, however the risk of complications is greatest in children and people over 65 years old. To appraise all comparative studies evaluating the effects of influenza vaccines in healthy children; assess vaccine efficacy (prevention of confirmed influenza) and effectiveness (prevention of influenza-like illness) and document adverse events associated with receiving influenza vaccines. We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library Issue 1, 2005); OLD MEDLINE (1966 to 1969); MEDLINE (1969 to December 2004); EMBASE (1974 to December 2004); Biological Abstracts (1969 to December 2004); and Science Citation Index (1974 to December 2004). We wrote to vaccine manufacturers and a number of corresponding authors of studies in the review. Any randomised controlled trials (RCTs), cohort and case-control studies of any influenza vaccine in healthy children under 16 years old. Two authors independently assessed trial quality and extracted data. Fifty-one studies involving 263,987 children were included. Seventeen papers were translated from Russian. Fourteen RCTs and 11 cohort studies were included in the analysis of vaccine efficacy and effectiveness. From RCTs, live vaccines showed an efficacy of 79% (95% confidence interval (CI) 48% to 92%) and an effectiveness of 33% (95% CI 28% to 38%) in children older than two years compared with placebo or no intervention. Inactivated vaccines had a lower efficacy of 59% (95% CI 41% to 71%) than live vaccines but similar effectiveness: 36% (95% CI 24% to 46%). In children under two, the efficacy of inactivated vaccine was similar to placebo. Thirty-four reports containing safety outcomes were included, 22 including live vaccines, 8 inactivated vaccines and 4 both types. The most commonly presented short-term outcomes were temperature and local reactions. The variability in design of studies and presentation of data was such that meta-analysis of safety outcome data was not feasible. Influenza vaccines are efficacious in children older than two years but little evidence is available for children under two. There was a marked difference between vaccine efficacy and effectiveness. That no safety comparisons could be carried out emphasizes the need for standardisation of methods and presentation of vaccine safety data in future studies. It was surprising to find only one study of inactivated vaccine in children under two years, given recent recommendations to vaccinate healthy children from six months old in the USA and Canada. If immunisation in children is to be recommended as public-health policy, large-scale studies assessing important outcomes and directly comparing vaccine types are urgently required.


Formation of chimeric genes with essential functions at the origin of eukaryotes.
Eukaryotes evolved from the symbiotic association of at least two prokaryotic partners, and a good deal is known about the timings, mechanisms, and dynamics of these evolutionary steps. Recently, it was shown that a new class of nuclear genes, symbiogenetic genes (S-genes), was formed concomitant with endosymbiosis and the subsequent evolution of eukaryotic photosynthetic lineages. Understanding their origins and contributions to eukaryogenesis would provide insights into the ways in which cellular complexity has evolved. Here, we show that chimeric nuclear genes (S-genes), built from prokaryotic domains, are critical for explaining the leap forward in cellular complexity achieved during eukaryogenesis. A total of 282 S-gene families contributed solutions to many of the challenges faced by early eukaryotes, including enhancing the informational machinery, processing spliceosomal introns, tackling genotoxicity within the cell, and ensuring functional protein interactions in a larger, more compartmentalized cell. For hundreds of S-genes, we confirmed the origins of their components (bacterial, archaeal, or generally prokaryotic) by maximum likelihood phylogenies. Remarkably, Bacteria contributed nine-fold more S-genes than Archaea, including a two-fold greater contribution to informational functions. Therefore, there is an additional, large bacterial contribution to the evolution of eukaryotes, implying that fundamental eukaryotic properties do not strictly follow the traditional informational/operational divide for archaeal/bacterial contributions to eukaryogenesis. This study demonstrates the extent and process through which prokaryotic fragments from bacterial and archaeal genes inherited during eukaryogenesis underly the creation of novel chimeric genes with important functions.


Computer simulation study of the structural stability and materials properties of DNA-intercalated layered double hydroxides.
The intercalation of DNA into layered double hydroxides (LDHs) has various applications, including drug delivery for gene therapy and origins of life studies. The nanoscale dimensions of the interlayer region make the exact conformation of the intercalated DNA difficult to elucidate experimentally. We use molecular dynamics techniques, performed on high performance supercomputing grids, to carry out large-scale simulations of double stranded, linear and plasmid DNA up to 480 base pairs in length intercalated within a magnesium-aluminum LDH. Currently only limited experimental data have been reported for these systems. Our models are found to be in agreement with experimental observations, according to which hydration is a crucial factor in determining the structural stability of DNA. Phosphate backbone groups are found to align with aluminum lattice positions. At elevated temperatures and pressures, relevant to origins of life studies which maintain that the earliest life forms originated around deep ocean hydrothermal vents, the structural stability of LDH-intercalated DNA is substantially enhanced as compared to DNA in bulk water. We also discuss how the materials properties of the LDH are modified due to DNA intercalation.


An Online Survey on Consumer Knowledge and Understanding of Added Sugars.
Evidence of an association between added sugars (AS) and the risk of obesity has triggered public health bodies to develop strategies enabling consumers to manage their AS intake. The World Health Organisation (WHO) has strongly recommended a reduction of free sugars to 10% of total dietary energy (TE) and conditionally recommended a reduction to 5% TE to achieve health benefits. Despite food labelling being a policy tool of choice in many countries, there is no consensus on the mandatory addition of AS to the nutrition panel of food labels. An online survey was conducted to explore consumer ability to identify AS on food labels and to investigate consumer awareness of the WHO guidelines in relation to sugar intakes. The questionnaire was tested for participant comprehension using face-to-face interviews prior to conducting the online study. The online survey was conducted in Northern Ireland during May 2015 and was completed by a convenient sample of 445 subjects. Results showed that just 4% of respondents correctly classified 10 or more ingredients from a presented list of 13 items, while 65% of participants were unaware of the WHO guidelines for sugar intake. It may be timely to reopen dialogue on inclusion of AS on food product nutrition panels.


α-Synuclein aggregation in the olfactory bulb of middle-aged common marmoset.
The synaptic protein α-synuclein has been identified as a major component of Lewy bodies, a pathological hallmark of Parkinson's disease (PD). Prior to the formation of Lewy bodies, mislocalization and aggregation of the α-synuclein in brain tissue is frequently observed in various neurodegenerative diseases. Aberrant accumulation and localization of α-synuclein are also observed in the aging human brain, for which reason aging is regarded as a risk factor for neurodegenerative disease. To investigate changes in α-synuclein properties in the aging brain, we compared α-synuclein immunoreactivity in brain tissue of young (2-years-old) and middle-aged (6-years-old) common marmoset (Callithrix jacchus). Our analyses revealed marked changes in α-synuclein immunoreactivity in the olfactory bulb of common marmosets of these age cohorts. Perikaryal α-synuclein aggregations were formed in the olfactory bulb in middle-aged animals. We also observed signals of α-synuclein accumulation in hippocampus in this cohort; however, unlike in the olfactory bulb, hippocampal α-synuclein signals were localized in the synaptic terminals. We did not observe either of these features in younger marmosets, which suggest that aging may play a role in these phenomena. Our results using common marmoset brain corresponded with the observation that the α-synuclein aggregations were first occurred from olfactory bulb in human normal aged and PD brain. Therefore, common marmoset is expected as useful model for α-synuclein pathology.


Paired involvement of human-specific Olduvai domains and NOTCH2NL genes in human brain evolution.
Sequences encoding Olduvai (DUF1220) protein domains show the largest human-specific increase in copy number of any coding region in the genome and have been linked to human brain evolution. Most human-specific copies of Olduvai (119/165) are encoded by three NBPF genes that are adjacent to three human-specific NOTCH2NL genes that have been shown to promote cortical neurogenesis. Here, employing genomic, phylogenetic, and transcriptomic evidence, we show that these NOTCH2NL/NBPF gene pairs evolved jointly, as two-gene units, very recently in human evolution, and are likely co-regulated. Remarkably, while three NOTCH2NL paralogs were added, adjacent Olduvai sequences hyper-amplified, adding 119 human-specific copies. The data suggest that human-specific Olduvai domains and adjacent NOTCH2NL genes may function in a coordinated, complementary fashion to promote neurogenesis and human brain expansion in a dosage-related manner.


Remote Learning Barriers and Opportunities for Graduate Student and Postdoctoral Learners in Career and Professional Skill Development: A Case Study.
Career and professional development competencies are critical for biomedical PhD and postdoctoral training. In the current educational landscape, programs that meet these competencies are offered and attended in an ad hoc manner. During the COVID-19 pandemic and the accompanying switch to virtual learning, our team observed a surge in interest for our weekly nonsequential programs. In this study, we surveyed our learners to better understand motivators for attending these programs during the pandemic and to identify barriers for participating in such events before and during Work-from-Home. Our data indicate that conflict with research responsibilities, time spent to get to the event location, and planning time to attend are all significant barriers to engagement. Notably, feelings of being overwhelmed, which increased slightly during the pandemic, stood out as an identified barrier. Per our results, the virtual format was an attraction. While 58% of respondents would prefer to access professional development programs virtually in the future, almost 42% indicated a preference for in-person events when normalcy resumes, as the physical presence of an instructor and of peers result in a deeper engagement. Our collective analysis here suggests that learners will benefit from a hybrid or combination of synchronous and asynchronous career and professional development programming in the future, even postpandemic, to reduce identified barriers. Alongside hybrid learning engagements, we strongly recommend structured time for learners to enhance their professional competencies, enabled by a commitment from departments and faculty mentors to bring equity in professional skill building and foster a life-long growth mindset.


First report of Trypanosoma vivax outbreak in dairy cattle in São Paulo state, Brazil.
This is the first description of a Trypanosoma vivax outbreak in the state of São Paulo (municipality of Lins). Fever, jaundice, decreased milk production, weight loss, profuse diarrhea, abortion, anemia, leukocytosis and hyperfibrinogenemia were observed in the affected animals. Thirty-one cows and calves died out of a total of 1080 in the herd. Three cows showed neurological symptoms like dysmetria, ataxia, muscle weakness, ptyalism, lymph node enlargement and submandibular edema. Flagellated hemoparasites were observed in blood smears. The species was diagnosed as T. vivax by means of PCR. This T.vivax strain showed resistance to diaminazene aceturate and the infection spread quickly at the herd. From the ELISA test, 599 serum samples (98.36%) were positive for anti-T.vivax IgG antibodies. This outbreak occurred during a very dry period, which indicates that other factors were involved in the outbreak, such as absence of tabanids and large populations of Haematobia irritans and Stomoxys calcitrans. The increases in these populations may have been due to the use of biosolid waste from sugar and ethanol plants in the sugarcane plantations surrounding the dairy farm.


Tracking in low frame rate video: a cascade particle filter with discriminative observers of different life spans.
Tracking objects in low frame rate (LFR) video or with abrupt motion poses two main difficulties which most conventional tracking methods can hardly handle: 1) poor motion continuity and increased search space; 2) fast appearance variation of target and more background clutter due to increased search space. In this paper, we address the problem from a view which integrates conventional tracking and detection, and present a temporal probabilistic combination of discriminative observers of different lifespans. Each observer is learned from different ranges of samples, with different subsets of features, to achieve varying levels of discriminative power at varying cost. An efficient fusion and temporal inference is then done by a cascade particle filter which consists of multiple stages of importance sampling. Experiments show significantly improved accuracy of the proposed approach in comparison with existing tracking methods, under the condition of LFR data and abrupt motion of both target and camera.


Prion diseases are efficiently transmitted by blood transfusion in sheep.
The emergence of variant Creutzfeld-Jakob disease, following on from the bovine spongiform encephalopathy (BSE) epidemic, led to concerns about the potential risk of iatrogenic transmission of disease by blood transfusion and the introduction of costly control measures to protect blood supplies. We previously reported preliminary data demonstrating the transmission of BSE and natural scrapie by blood transfusion in sheep. The final results of this experiment, reported here, give unexpectedly high transmission rates by transfusion of 36% for BSE and 43% for scrapie. A proportion of BSE-infected transfusion recipients (3 of 8) survived for up to 7 years without showing clinical signs of disease. The majority of transmissions resulted from blood collected from donors at more than 50% of the estimated incubation period. The high transmission rates and relatively short and consistent incubation periods in clinically positive recipients suggest that infectivity titers in blood were substantial and/or that blood transfusion is an efficient method of transmission. This experiment has established the value of using sheep as a model for studying transmission of variant Creutzfeld-Jakob disease by blood products in humans.


Ebola virus disease in West Africa--the first 9 months of the epidemic and forward projections.
On March 23, 2014, the World Health Organization (WHO) was notified of an outbreak of Ebola virus disease (EVD) in Guinea. On August 8, the WHO declared the epidemic to be a "public health emergency of international concern." By September 14, 2014, a total of 4507 probable and confirmed cases, including 2296 deaths from EVD (Zaire species) had been reported from five countries in West Africa--Guinea, Liberia, Nigeria, Senegal, and Sierra Leone. We analyzed a detailed subset of data on 3343 confirmed and 667 probable Ebola cases collected in Guinea, Liberia, Nigeria, and Sierra Leone as of September 14. The majority of patients are 15 to 44 years of age (49.9% male), and we estimate that the case fatality rate is 70.8% (95% confidence interval [CI], 69 to 73) among persons with known clinical outcome of infection. The course of infection, including signs and symptoms, incubation period (11.4 days), and serial interval (15.3 days), is similar to that reported in previous outbreaks of EVD. On the basis of the initial periods of exponential growth, the estimated basic reproduction numbers (R0 ) are 1.71 (95% CI, 1.44 to 2.01) for Guinea, 1.83 (95% CI, 1.72 to 1.94) for Liberia, and 2.02 (95% CI, 1.79 to 2.26) for Sierra Leone. The estimated current reproduction numbers (R) are 1.81 (95% CI, 1.60 to 2.03) for Guinea, 1.51 (95% CI, 1.41 to 1.60) for Liberia, and 1.38 (95% CI, 1.27 to 1.51) for Sierra Leone; the corresponding doubling times are 15.7 days (95% CI, 12.9 to 20.3) for Guinea, 23.6 days (95% CI, 20.2 to 28.2) for Liberia, and 30.2 days (95% CI, 23.6 to 42.3) for Sierra Leone. Assuming no change in the control measures for this epidemic, by November 2, 2014, the cumulative reported numbers of confirmed and probable cases are predicted to be 5740 in Guinea, 9890 in Liberia, and 5000 in Sierra Leone, exceeding 20,000 in total. These data indicate that without drastic improvements in control measures, the numbers of cases of and deaths from EVD are expected to continue increasing from hundreds to thousands per week in the coming months.


Antitumor effects of a refined polysaccharide peptide fraction isolated from Coriolus versicolor: in vitro and in vivo studies.
RPSP, a refined polysaccharide peptide fraction isolated by fast performance liquid chromatography (FPLC) from the crude powder of total peptide-bound polysaccharides of cultivated Coriolus versicolor Cov-1 dose-dependently inhibited the proliferation of a human hepatoma cell line (HEPG2). The effective dose causing 50% inhibition following 3-day exposure to RPSP was 243 +/- 36 micrograms/ml for HEPG2. However, little or no inhibitory effects were detected in normal human foetal hepatocytes. On the other hand, in the pretreatment group, in which RPSP was administered i.p. for two weeks before sarcoma 180 inoculation in nude mice, the incidence of tumor growth was less (2 out of 5 mice) than that of the control group (all 5 mice). The tumor size of the control group was about 3-5 times bigger than that of the pretreatment group. In tumor-bearing nude mice, 5 days after sarcoma 180 inoculation, i.v. administration of RPSP significantly suppressed the growth of tumor mass. The inhibition rate was 93.6% on day 13. Furthermore, administration of RPSP did not cause any pathological lesions in vital organs of rabbits such as heart, liver, spleen, lung and kidney. In conclusion, these results indicate that RPSP acts by directly suppressing tumor cell growth in vitro and the prevention of in vivo growth of tumor mass is probably mediated also via its immunomodulating effects.


The use of bi-planar tissue expanders to augment abdominal domain in a pediatric intestinal transplant recipient.
Intestinal transplantation is a well-accepted treatment for SBS. However, patients with SBS often have decreased abdominal capacity, which makes size-matching of donor organs more difficult, thus decreasing organ availability. Reported approaches for addressing this problem include surgically reducing the graft size, leaving an open abdomen for a prolonged period, and cotransplanting rectus fascia as a non-vascularized allograft. Each approach has significant disadvantages. There has been one previous report of tissue expanders used intra-abdominally and two reports of subcutaneous use to increase intra-abdominal capacity prior to transplantation. We report the first use of bi-planar expander placement for this purpose. In our case, a two-yr-old male child with SBS due to malrotation was treated with tissue expanders 10 months prior to intestinal transplantation, thus allowing transplantation of a larger graft with the ability to close the abdomen safely. There were no complications, and the patient is now doing well and tolerating diet off PN. The use of tissue expanders prior to intestinal transplantation is a promising approach for such patients and avoids the morbidity associated with other approaches. This approach requires a multidisciplinary effort by gastroenterology, transplant surgery, and plastic surgery teams.


Relationship of the physical and enzymatic properties of Escherichia coli recA protein to its strand exchange activity.
We have shown that performing the recA protein catalyzed strand exchange reaction in the presence of acetate anions, rather than chloride which is commonly used, greatly increases the rate of the reaction. The initial rate of the reaction in an acetate-based buffer is approximately 3-4 times higher in the presence of Escherichia coli single-stranded DNA binding protein (SSB protein) and 2 times higher in its absence than the initial rate in chloride. To determine the enzymatic basis for this stimulatory effect of acetate buffer, we investigated the relationship between a number of physical and enzymatic properties of recA protein and the strand exchange reaction. We have found that although the acetate anion has some effect on the aggregation properties and the single-stranded DNA-dependent ATPase activity of recA protein, these effects cannot explain the enhanced strand exchange activity in an acetate-based buffer. We do find, however, that two aspects of recA protein activity closely parallel the ability of this protein to catalyze strand exchange. The first is the ability of recA protein to displace SSB protein from single-stranded DNA, an event critical to presynaptic complex formation. RecA protein is able to resist displacement by SSB protein at a lower magnesium concentration in acetate than in chloride buffer. The magnesium ion concentration dependence of strand exchange coincides exactly with this behavior. The second activity correlated to strand exchange is the duplex DNA-dependent ATPase activity of recA protein. We find that over a wide variety of sodium chloride and sodium acetate concentrations, this duplex DNA-dependent ATPase activity is linearly related to the amount of product formed in the strand exchange reaction. We postulate that this duplex DNA-dependent ATPase activity is important in the denaturation of the duplex DNA during the branch migration step of strand exchange and have also determined that this reaction is quite efficient, with the number of ATP molecules hydrolyzed per base pair exchanged being 0.75 +/- 0.25. In addition, recA protein catalyzed strand exchange between circular single-strand and linear duplex DNA molecules is shown to be irreversible, and a possible explanation for this irreversibility is presented.


Subinhibitory antibiotic therapy alters recurrent urinary tract infection pathogenesis through modulation of bacterial virulence and host immunity.
The capacity of subinhibitory levels of antibiotics to modulate bacterial virulence in vitro has recently been brought to light, raising concerns over the appropriateness of low-dose therapies, including antibiotic prophylaxis for recurrent urinary tract infection management. However, the mechanisms involved and their relevance in influencing pathogenesis have not been investigated. We characterized the ability of antibiotics to modulate virulence in the uropathogens Staphylococcus saprophyticus and Escherichia coli. Several antibiotics were able to induce the expression of adhesins critical to urothelial colonization, resulting in increased biofilm formation, colonization of murine bladders and kidneys, and promotion of intracellular niche formation. Mice receiving subinhibitory ciprofloxacin treatment were also more susceptible to severe infections and frequent recurrences. A ciprofloxacin prophylaxis model revealed this strategy to be ineffective in reducing recurrences and worsened infection by creating larger intracellular reservoirs at higher frequencies. Our study indicates that certain agents used for antibiotic prophylaxis have the potential to complicate infections. Antibiotics are the mainstay treatment for bacterial infections; however, evidence is emerging that argues these agents may have off-target effects if sublethal concentrations are present. Most studies have focused on changes occurring in vitro, leaving questions regarding the clinical relevance in vivo. We utilized a murine urinary tract infection model to explore the potential impact of low-dose antibiotics on pathogenesis. Using this model, we showed that subinhibitory antibiotics prime uropathogens for adherence and invasion of murine urothelial tissues. These changes in initial colonization promoted the establishment of chronic infection. Furthermore, treatment of chronically infected mice with subtherapeutic ciprofloxacin served to exacerbate infection. A part of these changes was thought to be due to suppression of mucosal immunity, as demonstrated through reductions in cytokine secretion and migration of leukocytes into the urinary tract. This work identifies novel risk factors associated with antibiotic therapy when dosing strategies fall below subtherapeutic levels.


Myeloid-derived suppressor cells in the tumor microenvironment: expect the unexpected.
Our understanding of the role of myeloid-derived suppressor cells (MDSCs) in cancer is becoming increasingly complex. In addition to their eponymous role in suppressing immune responses, they directly support tumor growth, differentiation, and metastasis in a number of ways that are only now beginning to be appreciated. It is because of this increasingly complex role that these cells may become an important factor in the treatment of human cancer. In this Review, we discuss the most pertinent and controversial issues of MDSC biology and their role in promoting cancer progression and highlight how these cells may be used in the clinic, both as prognostic factors and as therapeutic targets.


Providing effective and preferred care closer to home: a realist review of intermediate care.
Intermediate care is one of the number of service delivery models intended to integrate care and provide enhanced health and social care services closer to home, especially to reduce reliance on acute care hospital beds. In order for health and social care practitioners, service managers and commissioners to make informed decisions, it is vital to understand how to implement the admission avoidance and early supported discharge components of intermediate care within the context of local care systems. This paper reports the findings of a theory-driven (realist) review conducted in 2011-2012. A broad range of evidence contained in 193 sources was used to construct a conceptual framework for intermediate care. This framework forms the basis for exploring factors at service user, professional and organisational levels that should be considered when designing and delivering intermediate care services within a particular local context. Our synthesis found that involving service users and their carers in collaborative decision-making about the objectives of care and the place of care is central to achieving the aims of intermediate care. This pivotal involvement of the service user relies on practitioners, service managers and commissioners being aware of the impact that organisational structures at the local level can have on enabling or inhibiting collaborative decision-making and care co-ordination. Through all interactions with service users and their care networks, health and social care professionals should establish the meaning which alternative care environments have for different service users. Doing so means decisions about the best place of care will be better informed and gives service users choice. This in turn is likely to support psychological and social stability, and the attainment of functional goals. At an organisational level, integrated working can facilitate the delivery of intermediate care, but there is not a straightforward relationship between integrated organisational processes and integrated professional practice.