Molecular Biology

Background: Alteration in epigenetic methylation can affect gene expression and other processes. In Prokaryota, DNA methyltransferase genes frequently move between genomes and present a potential threat. A methyl-specific deoxyribonuclease, McrBC, of Escherichia coli cuts invading methylated DNAs. Here we examined whether McrBC competes with genome methylation systems through host killing by chromosome cleavage. Results: McrBC inhibited the establishment of a plasmid carrying a PvuII methyltransferase gene but lacking its recognition sites, likely through the lethal cleavage of chromosomes that become methylated. Indeed, its phage-mediated transfer caused McrBC-dependent chromosome cleavage. Its induction led to cell death accompanied by chromosome methylation, cleavage and degradation. RecA/RecBCD functions affect the chromosome processing and, together with SOS response, reduce the lethality. Our evolutionary/genomic analyses of mcrBC homologs revealed (i) wide distribution in Prokaryota, (ii) frequent distant horizontal transfer and linkage with mobility-related genes, (iii) diversification in the DNA binding domain. In these features, McrBCs resemble Type II restriction-modification systems, which behave as selfish mobile elements maintaining their frequency by host killing. McrBCs are frequently found linked with a methyltransferase homolog, which suggests a functional association. Conclusions: Our experiments indicate McrBC can respond to genome methylation systems by host killing. Combined with our evolutionary/genomic analyses, they support our hypothesis McrBCs have evolved as mobile elements competing with specific genome methylation systems through host killing. To our knowledge, this represents the first report of a defense system against epigenetic systems through cell death.

Hedgehog proteins, which are important in developmental signaling, have been joined by other newly discovered proteins from throughout the eukaryotes in the Hint-domain-containing group of proteins.

Background: Alternative splicing of messenger RNA permits the formation of a wide range of mature RNA transcripts and has the potential to generate a diverse spectrum of functional proteins. Although there is extensive evidence for large scale alternative splicing at the transcript level there have been no comparable studies demonstrating the existence of alternatively spliced protein isoforms. Results: Recent advances in proteomics technology have allowed us to carry out a comprehensive identification of protein isoforms in Drosophila. The analysis of this proteomic data confirmed the presence of multiple alternative gene products for over a hundred Drosophila genes. Conclusions: We demonstrate that proteomics techniques can detect the expression of stable alternative splice isoforms on a genome-wide scale. Many of these alternative isoforms are likely to have regions that are disordered in solution, and specific proteomics methodologies may be required to identify these peptides.

Background: Decompensated heart failure may present with severe hypertension in patients with preserved (PreEF) or reduced left ventricular (LV) ejection fraction (RedEF) and is clinically indistinguishable. Previously, we demonstrated that arterial pressure elevation increases LV filling pressures in a canine model of chronic LV dysfunction with PreEF or RedEF. It is not clear whether any differences in hemodynamics, LV volume or performance, or diastolic function can be demonstrated between canines with PreEF or RedEF in response to arterial pressure elevation. We hypothesized that the LV systolic, diastolic, and hemodynamic response to pressure loading would be similar in RedEF or PreEF. Methods: We studied 25 dogs with chronic LV dysfunction due to coronary microsphere embolization with RedEF (35+4%) and 20 dogs with PreEF (50+3%). Arterial pressure was increased with methoxamine infusion and hemodynamics and echo-Doppler parameters of LV size, function, transaortic and transmitral pulsed Doppler prior to and with methoxamine infusion was obtained. Results: Though LV filling pressures were similar at baseline, LV size was larger (p<0.01) and ejection fraction lower in dogs with RedEF (p<0.001). With methoxamine, there were similar increases in LV size, LV pressures, and index of myocardial performance with the ejection fraction reduced similarly. Diastolic parameters demonstrated similar tau increases, E/A reduction, and diastolic filling shortening in RedEF and PreEF dogs. A similar extent of isovolumic contraction and relaxation times and index of myocardial performance prolongation occurred with pressure loading. Conclusion: Pressure loading in a canine model of LV dysfunction with PreEF and RedEF resulted in similar degrees of LV dilatation, increased filling pressures, and increased index of myocardial performance.

Background: Poly(amidoamine)s (PAAs) are synthetic polymers endowed with many biologically interesting properties, being highly biocompatible, non toxic and biodegradable. Hydrogels based on PAAs can be easily modified during the synthesis by the introduction of functional co-monomers. Aim of this work is the development and testing of novel amphoteric nanosized poly(amidoamine) hydrogel film incorporating 4-aminobutylguanidine (agmatine) moieties to create RGD-mimicking repeating units for promoting cell adhesion. Results: A systematic comparative study of the response of an epithelial cell line was performed on hydrogels with agmatine and on non-functionalized amphoteric poly(amidoamine) hydrogels and tissue culture plastic substrates. The cell adhesion on the agmatine containing substrates was comparable to that on plastic substrates and significantly enhanced with respect to the non-functionalized controls. Interestingly, spreading and proliferation on the functionalized supports are slower than on plastic exhibiting the possibility of an easier control of the cell growth kinetics. In order to favor the handling of the samples, a procedure for the production of bi-layered constructs was also developed by means the deposition via spin coating of a thin layer of hydrogel on a pre-treated cover slip. Conclusions: The obtained results reveal that PAAs hydrogels can be profitably functionalized and, in general, undergo physical and chemical modifications to meet specific requirements. In particular the incorporation of agmatine warrants good potential in the field of cell culturing and the development of supported functionalized hydrogels on cover glass are very promising substrates for applications in cell screening devices.

Background: The magnetic resonance technique of arterial spin labeling (ASL) allows myocardial perfusion to be quantified without the use of a contrast agent. This study aimed to use a modified ASL technique and T1 regression algorithm, previously validated in canine models, to calculate myocardial blood flow (MBF) in normal human subjects and to compare the accuracy and repeatability of this calculation at 1.5 T and 3.0 T. A computer simulation was performed and compared with experimental findings. Results: Eight subjects were imaged, with scans at 3.0 T showing significantly higher T1 values (P < 0.001) and signal-to-noise ratios (SNR) (P < 0.002) than scans at 1.5 T. The average MBF was found to be 0.990 +/- 0.302 mL/g/min at 1.5 T and 1.058 +/- 0.187 mL/g/min at 3.0 T. The repeatability at 3.0 T was improved 43% over that at 1.5 T, although no statistically significant difference was found between the two field strengths. In the simulation, the accuracy and the repeatability of the MBF calculations were 61% and 38% higher, respectively, at 3.0 T than at 1.5 T, but no statistically significant differences were observed. There were no significant differences between the myocardial perfusion data sets obtained from the two independent observers. Additionally, there was a trend toward less variation in the perfusion data from the two observers at 3.0 T as compared to 1.5 T. Conclusions: This suggests that this ASL technique can be used, preferably at 3.0 T, to quantify myocardial perfusion in humans and with further development could be useful in the clinical setting as an alternative method of perfusion analysis.

Iatrogenic fornix rupture caused during retrograde manipulation of the ureter is a rather rare or rarely diagnosed phenomenon. A 22 year-old female patient presented with a fornix rupture following endoscopic ureteral stone extraction under uretero-renoscopy, the rupture having become symptomatic two days later.

Background: Gram-positive bacteria of the genus Anoxybacillus have been found in diverse thermophilic habitats such as geothermal hot springs, manure, and in processed foods such as gelatin and milk powder. Anoxybacillus flavithermus is a facultatively anaerobic bacterium found in super-saturated silica solutions and in opaline silica sinter. The ability of A. flavithermus to grow in super-saturated silica solutions makes it an ideal subject to study the processes of sinter formation, which might be similar to the biomineralization processes that occurred at the dawn of life. Results: We report here the complete genome sequence of Anoxybacillus flavithermus strain WK1, isolated from the waste water drain at the Wairakei geothermal power station in New Zealand. It consists of a single chromosome of 2,846,746 base pairs and is predicted to encode 2,863 proteins. In silico genome analysis identified several enzymes that could be involved in silica adaptation and biofilm formation, and their predicted functions were experimentally validated in vitro. Proteomic analysis confirmed the regulation of biofilm-related proteins and crucial enzymes for the synthesis of long-chain polyamines as constituents of silica nanospheres. Conclusions: Microbial fossils preserved in silica and silica sinters are excellent objects for studying ancient life, a new paleobiological frontier. An integrated analysis of the A. flavithermus genome and proteome provides the first glimpse of metabolic adaptation during silicification and sinter formation. Comparative genome analysis suggests an extensive gene loss in the Anoxybacillus/Geobacillus branch after its divergence from other bacilli.

Background: Neck problems are often recurring or chronic. After pain, unsteadiness and balance problems are among the most frequent symptoms reported by chronic neck pain (CNP) patients. Altered sensorimotor control of the cervical spine and sensorimotor integration problems affecting postural control have been observed in CNP patients. Very few data are available regarding the post-intervention effects of rehabilitation programs on postural control in CNP.Case presentationThis is a case study of a traumatic CNP patient (a 45-year old female) with postural unsteadiness who participated in an 8-week rehabilitation program combining therapeutic exercises with spinal manipulative therapy. Pre-intervention data revealed that the postural control system was challenged when postural control sensory inputs were altered, particularly during the head-extended-backward condition. Post-intervention centre of pressure measurements indicated a drastic reduction in postural sway during trials with changes in neck orientation. Conclusion: This case report indicates that an 8-week rehabilitation program combining therapeutic exercises with spinal manipulative therapy may have had an effect on improvement of postural control in a trauma CNP patient with unsteadiness. These results warrant further studies to investigate the relationships between pain amelioration, sensorimotor control of the cervical spine, muscle fitness and postural steadiness.

Background: Throughout the period of one year, approximately 50% of recreational runners will sustain an injury that disrupts their training regimen. Foot orthoses have been shown to be clinically effective in the prevention and treatment of several common running injuries, yet the physical effect of this intervention during running remains poorly understood. The aim of this literature review was to evaluate the biomechanical trials in which the effect of foot orthoses on lower extremity forces and pressure (kinetics) was investigated. Methods: A systematic search of electronic databases including Medline (1966-present), CINAHL, SportDiscus, and The Cochrane Library occurred on 7 May 2008. Eligible articles were selected according to pre-determined criteria. Methodological quality was evaluated by use of the Quality Index as described by Downs & Black, followed by critical analysis according to outcome variables. Results: The most widely reported kinetic outcomes were loading rate and impact force, however the effect of foot orthoses on these variables remains unclear due to inconsistent findings. In contrast, current evidence suggests that a reduction in the rearfoot inversion moment is the most consistent kinetic effect of foot orthoses during running. Conclusions: The findings of this review demonstrate systematic effects that can be used to inform the direction of future research in this field, as further evidence is required to adequately define the mechanism of action of foot orthoses during running. Continuation of research in this field will enable targeting of design parameters towards biomechanical variables that are supported by evidence, and may lead to advancements in clinical efficacy.

Background: Regionalization of adult critical care services may improve outcomes for critically ill patients. We sought to develop a framework for understanding clinician attitudes toward regionalization and potential barriers to developing a tiered, regionalized system of care in the United States. Methods: We performed a qualitative study using semi-structured interviews of critical care stakeholders in the United States, including physicians, nurses and hospital administrators. Stakeholders were identified from a stratified-random sample of United States general medical and surgical hospitals. Key barriers and potential solutions were identified by performing content analysis of the interview transcriptions. Results: We interviewed 30 stakeholders from 24 different hospitals, representing a broad range of hospital locations and sizes. Key barriers to regionalization included personal and economic strain on families, loss of autonomy on the part of referring physicians and hospitals, loss of revenue on the part of referring physicians and hospitals, the potential to worsen outcomes at small hospitals by limiting services, and the potential to overwhelm large hospitals. Improving communication between destination and source hospitals, provider education, instituting voluntary objective criteria to become a designated referral center, and mechanisms to feed back patients and revenue to source hospitals were identified as potential solutions to some of these barriers. Conclusions: Regionalization efforts will be met with significant conceptual and structural barriers. These data provide a foundation for future research and can be used to inform policy decisions regarding the design and implementation of a regionalized system of critical care.

A simpler way for manipulating droplets on a flat surface was demonstrated, eliminating the complications in the existing methods of open-surface digital microfluidics. Programmed and motorized movements of 10 uL droplets were demonstrated using stepper motors and microcontrollers, including merging, complicated movement along the programmed path, and rapid mixing. Latex immunoagglutination assays for mouse immunoglobulin G, bovine viral diarrhea virus and Escherichia coli were demonstrated by merging two droplets on a superhydrophobic surface (contact angle = 155 degrees) and using subsequent back light scattering detection, with detection limits of 50 pg/mL, 2.5 TCID50/mL and 85 CFU/mL, respectively, all significantly lower than the other immunoassay demonstrations in conventional microfluidics (~1 ng/mL for proteins, ~100 TCID50/mL for viruses and ~100 CFU/mL for bacteria). Advantages of this system over conventional microfluidics or microwell plate assays include: (1) minimized biofouling and repeated use (>100 times) of a platform; (2) possibility of nanoliter droplet manipulation; (3) reprogrammability with a computer or a game pad interface.

Background: Pain is often a dominant clinical feature of chronic pancreatitis but the frequency and severity is highly variable between subjects. We hypothesized that genetic polymorphisms contribute to variations in clinical pain patterns. Since genetic variations in the GTP cyclohydrolase (GCH1) gene have been reported to protect some patients from pain, we investigated the effect of the "pain protective haplotype" in well characterized patients with chronic pancreatitis (CP) or recurrent acute pancreatitis (RAP) from the North American Pancreatitis Study 2 (NAPS2). Results: Subjects in the NAPS2 study were asked to rank their pain in one of 5 categories reflecting different levels of pain frequency and severity. All subjects were genotyped at rs8007267 and rs3783641 to determine the frequency of the GCH1 pain-protective haplotype. In Caucasian subjects the frequency of the pain-protective GCH1 haplotype was no different in the control group (n=236), CP patients (n=265), RAP patients (N=131), or in CP patients subclassified by pain category compared to previously reported haplotype frequencies in the general Caucasian population. Conclusions: The GCH1 pain-protective haplotype does not have a significant effect on pain patterns or severity in RAP or CP. These results are important for helping to define the regulators of visceral pain, and to distinguish different mechanisms of pain.

One of the least-appreciated advances in pediatric rheumatology over the past 25 years has been the delineation of the many ways in which children with rheumatic disease differ from adults with the same illnesses. Furthermore, we are now learning that paradigms that are useful in evaluating adults with musculoskeletal complaints have limited utility in children. Nowhere is that more true than in the use of commonly used laboratory tests, particularly antinuclear antibody (ANA) and rheumatoid factor (RF) assays. This short review will provide the practitioner with the evidence base that supports a more limited use of ANA and RF testing in children.

Background: Employing genomic DNA clones to characterise gene attributes has several advantages over the use of cDNA clones, including the presence of native transcription and translation regulatory sequences as well as a representation of the complete repertoire of potential splice variants encoded by the gene. However, working with genomic DNA clones has traditionally been tedious due to their large size relative to cDNA clones and the presence, absence or position of particular restriction enzyme sites that may complicate conventional in vitro cloning procedures. Results: To enable efficient cloning and manipulation of genomic DNA fragments for the purposes of gene expression and reporter-gene studies we have combined aspects of the Gateway system and a bacteriophage-based homologous recombination (i.e. recombineering) system. To apply the method for characterising plant genes we developed novel Gateway and plant transformation vectors that are of small size and incorporate selectable markers which enable efficient identification of recombinant clones. We demonstrate that the genomic coding region of a gene can be directly cloned into a Gateway Entry vector by recombineering enabling its subsequent transfer to Gateway Expression vectors. We also demonstrate how the coding and regulatory regions of a gene can be directly cloned into a plant transformation vector by recombineering. This construct was then rapidly converted into a novel Gateway Expression vector incorporating cognate 5' and 3' regulatory regions by using recombineering to replace the intervening coding region with the Gateway Destination cassette. Such expression vectors can be applied to characterise gene regulatory regions through development of reporter-gene fusions, using the Gateway Entry clones of GUS and GFP described here, or for ectopic expression of a coding region cloned into a Gateway Entry vector. We exemplify the utility of this approach with the Arabidopsis PAP85 gene and demonstrate that the expression profile of a PAP85::GUS transgene highly corresponds with native PAP85 expression. Conclusions: We describe a novel combination of the favourable attributes of the Gateway and recombineering systems to enable efficient cloning and manipulation of genomic DNA clones for more effective characterisation of gene function. Although the system and plasmid vectors described here were developed for applications in plants, the general approach is broadly applicable to gene characterisation studies in many biological systems.

Background: Self-directed and other violence as well as subsequent coercive interventions occur in a substantial proportion of patients with personality disorders during in-patient treatment. Different strategies may be required to reduce coercive interventions for patients of different diagnostic groups. Methods: We specialised one of our acute admission wards in the treatment of personality disorders and adjustment disorders (ICD-10 F4 and F6). Patients are not transferred to other acute wards in case of suicidal or violent behaviour. Violent behaviour and coercive interventions such as seclusion or restraint were recorded in the same way as in the rest of the hospital. We recorded the percentage of subjects affected by diagnostic group and average length of an intervention in the year before and after the change in organisational structure. Results: The total number of coercive interventions decreased by 85 % both among patients with an F4 and those with an F6 primary diagnosis. Violent behaviours decreased by about 50 %, the proportion of involuntary committed patients decreased by 70 %. Conclusion: The organisational change turned out to be highly effective without any additional cost of personnel or other resources.

We report a case of a 67-year-old Caucasian man with right upper quadrant abdominal pain. He underwent radiologic investigations that revealed a solid, focal mass, at the V hepatic segment. Because a definitive diagnosis, based on imaging appearance of the lesion, was impossible in our case, we performed a hystopathological investigation but the biopsies were inconclusive. So, the definitive diagnosis of intraductal papillary mucinous tumor of bile ducts was made on surgical resected material. Intraductal papillary neoplasm of the liver (IPNL) is a recently recognized entity which closely resembles an intraductal papillary mucinous tumor (IPMT) of the pancreas.

In 2004, a team comprised of researchers and service providers launched the Safer Crack Use, Outreach, Research and Education (SCORE) project in the Downtown Eastside of Vancouver, British Columbia, Canada. The project was aimed at developing a better understanding of the harms associated with crack cocaine smoking and determining the feasibility of introducing specific harm reduction strategies. Specifically, in partnership with the community, we constructed and distributed kits that contained harm reduction materials. We were particularly interested in understanding what people thought of these kits and how the kits contents were used. To obtain this information, we conducted 27 interviews with women and men who used crack cocaine and received safer crack kits. Four broad themes were generated from the data: 1) the context of crack use practices; 2) learning /transmission of harm reduction education; 3) changing practice; 4) barriers to change. This project suggests that harm reduction education is most successful when it is informed by current practices with crack use. In addition it is most effectively delivered through informal interactions with people who use crack and includes repeated demonstrations of harm reduction equipment by peers and outreach workers. This paper also suggests that barriers to harm reduction are systemic: lack of safe housing and private space shape crack use practices.

Background: Most patients with advanced Ewing's sarcoma (EWS) respond poorly to conventional chemotherapy, indicating the need for new treatment approaches. Epigenetic events, such as promoter hypermethylation and chromatin histone deacetylation, silence the expression of tumor suppressor genes (TSGs) and play an important role in tumorigenesis. These epigenetic changes can be reversed by using 5-aza-2'-deoxycytidine (5AZA-CdR), a potent inhibitor of DNA methylation, in combination with an inhibitor of histone deacetylase (HDAC). Results: Here, we used a clonogenic assay to evaluate the in vitro antineoplastic activity of 5AZA-CdR in combination with different HDAC inhibitors on EWS cells. We observed that the HDAC inhibitors, MS-275, trichostatin-A, phenylbutyrate, LAQ824 and depsipeptide, enhanced the antineoplastic action of 5AZA-CdR on EWS cells. The combination of 5AZA-CdR and MS-275 showed marked synergy, and was correlated with significant reactivation of the expression of two TSGs, E-cadherin and tumor suppressor lung cancer-1 (TSLC1), in a EWS cell line. Conclusion: These results suggest the value of future clinical studies investigating the combination of 5AZA-CdR and MS-275 in patients with advanced EWS.

Background: Under the current healthcare system in China, there is no government-sponsored health insurance program for children. Children from families who move from rural and interior regions to large urban centres without a valid residency permit might be at higher risk of being uninsured due to their low socioeconomic status. We conducted a survey in Shanghai to describe children's health insurance coverage according to their migration status. MethodBetween 2005 and 2006, we conducted an in-person health survey of the adult care-givers of children aged 7 and under, residing in five districts of Shanghai. We compared uninsurance rates between temporary and permanent child residents, and investigated factors associated with child health uninsurance. Results: Even though cooperative insurance eligibility has been extended to temporary residents of Shanghai, the uninsurance rate was significantly higher among temporary (65.6%) than permanent child residents (21.1%, adjusted odds ratio (OR): 5.85, 95% confidence interval (95% CI): 4.62-7.41). For both groups, family income was associated with having child health insurance; children in lower income families were more likely to be uninsured (OR: 1.96, 95% CI: 1.40-2.96). Conclusions: Children must rely on their parents to make the insurance purchase decision, which is constrained by their income and the perceived benefits of the insurance program. Children from migrant families are at even higher risk for uninsurance due to their lower socioeconomic status. Government initiatives specifically targeting temporary residents and providing health insurance benefits for their children are urgently needed.