Medical grade honey for the treatment of paediatric abdominal wounds: a case series.
Children are at high risk of injuries and wounds. The application of medical grade honey is a promising approach to improving the healing of wounds of various origin and severity. However, the use of medical grade honey in young paediatric patients remains limited. The aim of this study is to show the safety, efficacy and usefulness of medical grade honey in abdominal wounds, of different causes, in paediatric patients. This was a prospective, observational case series evaluating five young infants with abdominal wounds at the General Hospital in Thessaloniki. All wounds were treated in the same manner with daily medical grade honey applied to the wound area and closely monitored. All treated wounds rapidly presented granulation tissue formation and underwent re-epithelialisation. Peripheral oedema and inflammation decreased upon initial application. Necrotic tissue was effectively debrided when present. Slough was removed and no signs of infection were detected, irrespective of initial wound presentations. Scar formation was minimal and the full range of motion was preserved in all cases. Based on this case study, medical grade honey is safe and effective in treating different abdominal wounds, including infected or dehisced wounds as well as burns. The easy application and broad applicability make medical grade honey recommendable as a first-line treatment in paediatric patients.


Chemical mutagenesis: selective post-expression interconversion of protein amino acid residues.
The ability to alter protein structure by site-directed mutagenesis has revolutionized biochemical research. Controlled mutations at the DNA level, before protein translation, are now routine. These techniques allow specific, high fidelity interconversion largely between 20 natural, proteinogenic amino acids. Nonetheless, there is a need to incorporate other amino acids, both natural and unnatural, that are not accessible using standard site-directed mutagenesis and expression systems. Post-translational chemistry offers access to these side chains. Nearly half a century ago, the idea of a 'chemical mutation' was proposed and the interconversion between amino acid side chains was demonstrated on select proteins. In these isolated examples, a powerful proof-of-concept was demonstrated. Here, we revive the idea of chemical mutagenesis and discuss the prospect of its general application in protein science. In particular, we consider amino acids that are chemical precursors to a functional set of other side chains. Among these, dehydroalanine has much potential. There are multiple methods available for dehydroalanine incorporation into proteins and this residue is an acceptor for a variety of nucleophiles. When used in conjunction with standard genetic techniques, chemical mutagenesis may allow access to natural, modified, and unnatural amino residues on translated, folded proteins.


Novel inhibitor for prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis and details of substrate-recognition mechanism.
A new inhibitor, H-Ala-Ile-pyrrolidin-2-yl boronic acid, was developed as an inhibitor against prolyl tripeptidyl aminopeptidase with a K(i) value of 88.1 nM. The structure of the prolyl tripeptidyl aminopeptidase complexed with the inhibitor (enzyme-inhibitor complex) was determined at 2.2 A resolution. The inhibitor was bound to the active site through a covalent bond between Ser603 and the boron atom of the inhibitor. This structure should closely mimic the structure of the reaction intermediate between the enzyme and substrate. We previously proposed that two glutamate residues, Glu205 and Glu636, are involved in the recognition of substrates. In order to clarify the function of these glutamate residues in substrate recognition, three mutant enzymes, E205A, E205Q, and E636A were generated by site-directed mutagenesis. The E205A mutant was expressed as an inclusion body. The E205Q mutant was expressed in soluble form, but no activity was detected. Here, the structures of the E636A mutant and its complex with the inhibitor were determined. The inhibitor was located at almost the same position as in the wild-type enzyme-inhibitor complex. The amino group of the inhibitor interacted with Glu205 and the main-chain carbonyl group of Gln203. In addition, a water molecule in the place of Glu636 of the wild-type enzyme interacted with the amino group of the inhibitor. This water molecule was located near the position of Glu636 in the wild-type and formed a hydrogen bond with Gln203. The k(cat)/K(M) values of the E636A mutant toward the two substrates used were smaller than those of the wild-type by two orders of magnitude. The K(i) value of our inhibitor for the E636A mutant was 48.8 microM, which was 554-fold higher than that against the wild-type enzyme. Consequently, it was concluded that Glu205 and Glu636 are significant residues for the N-terminal recognition of a substrate.


How to Teach Health IT Evaluation: Recommendations for Health IT Evaluation Courses.
Systematic health IT evaluation studies are needed to ensure system quality and safety and to provide the basis for evidence-based health informatics. Well-trained health informatics specialists are required to guarantee that health IT evaluation studies are conducted in accordance with robust standards. Also, policy makers and managers need to appreciate how good evidence is obtained by scientific process and used as an essential justification for policy decisions. In a consensus-based approach with over 80 experts in health IT evaluation, recommendations for the structure, scope and content of health IT evaluation courses on the master or postgraduate level have been developed, supported by a structured analysis of available courses and of available literature. The recommendations comprise 15 mandatory topics and 15 optional topics for a health IT evaluation course.


Rapid assessment of postfire plant invasions in coniferous forests of the western United States.
Fire is a natural part of most forest ecosystems in the western United States, but its effects on nonnative plant invasion have only recently been studied. Also, forest managers are engaging in fuel reduction projects to lessen fire severity, often without considering potential negative ecological consequences such as nonnative plant species introductions. Increased availability of light, nutrients, and bare ground have all been associated with high-severity fires and fuel treatments and are known to aid in the establishment of nonnative plant species. We use vegetation and environmental data collected after wildfires at seven sites in coniferous forests in the western United States to study responses of nonnative plants to wildfire. We compared burned vs. unburned plots and plots treated with mechanical thinning and/or prescribed burning vs. untreated plots for nonnative plant species richness and cover and used correlation analyses to infer the effect of abiotic site conditions on invasibility. Wildfire was responsible for significant increases in nonnative species richness and cover, and a significant decrease in native cover. Mechanical thinning and prescribed fire fuel treatments were associated with significant changes in plant species composition at some sites. Treatment effects across sites were minimal and inconclusive due to significant site and site x treatment interaction effects caused by variation between sites including differences in treatment and fire severities and initial conditions (e.g., nonnative species sources). We used canonical correspondence analysis (CCA) to determine what combinations of environmental variables best explained patterns of nonnative plant species richness and cover. Variables related to fire severity, soil nutrients, and elevation explained most of the variation in species composition. Nonnative species were generally associated with sites with higher fire severity, elevation, percentage of bare ground, and lower soil nutrient levels and lower canopy cover. Early assessments of postfire stand conditions can guide rapid responses to nonnative plant invasions.


Dopamine assisted PMOXA/PAA brushes for their switchable protein adsorption/desorption.
Protein adsorption on interfaces immersed in biological fluids is a prevalent and mostly irreversible phenomenon of great importance and is often uncontrolled. Poly(2-methyl-2-oxazoline) (PMOXA) is shown to possess excellent protein-resistant properties, whereas proteins can be adsorbed deeply inside the swollen brushes of poly(acrylic acid) (PAA). In this work, mixed brushes of PMOXA/PAA were prepared by sequentially grafting amine-terminated PMOXA (PMOXA-NH2) and thiol-terminated PAA (PAA-SH) onto poly(dopamine) (PDA)-coated substrates. The constituents of the mixed brushes can be adjusted by changing the grafting time of PMOXA-NH2 or PAA-SH to the PDA-coated surfaces. PAA chains with different molecular weights in the mixed brushes of PMOXA/PAA were used to investigate the effect of the length of the PAA chains on the surface wetting and protein adsorption on the prepared interfaces. The prepared brushes were rigorously characterized in terms of the surface chemical composition, thickness and hydrophilicity using X-ray photoelectron spectroscopy (XPS), ellipsometry and the static water contact angle (WCA) tests. Protein adsorption/desorption on the brushes were monitored using fluorescence microscopy as well as surface plasmon resonance (SPR) for in situ and real-time measurements. The results demonstrated that the WCA on mixed brushes could be reversibly tuned with the variation of pH and I, the amount of bovine serum albumin (BSA) adsorbed on mixed brushes could be well regulated by the grafting time and the molecular weight of PAA-SH, and the maximal amount (87%) of adsorbed BSA could then be desorbed upon changing pH and I. The highly switchable protein adsorption/desorption properties are therefore a nice combination of the protein-resistance of PMOXA and the salt and pH-sensitivity of PAA.


The Drosophila MI-2 chromatin-remodeling factor regulates higher-order chromatin structure and cohesin dynamics in vivo.
dMi-2 is a highly conserved ATP-dependent chromatin-remodeling factor that regulates transcription and cell fates by altering the structure or positioning of nucleosomes. Here we report an unanticipated role for dMi-2 in the regulation of higher-order chromatin structure in Drosophila. Loss of dMi-2 function causes salivary gland polytene chromosomes to lose their characteristic banding pattern and appear more condensed than normal. Conversely, increased expression of dMi-2 triggers decondensation of polytene chromosomes accompanied by a significant increase in nuclear volume; this effect is relatively rapid and is dependent on the ATPase activity of dMi-2. Live analysis revealed that dMi-2 disrupts interactions between the aligned chromatids of salivary gland polytene chromosomes. dMi-2 and the cohesin complex are enriched at sites of active transcription; fluorescence-recovery after photobleaching (FRAP) assays showed that dMi-2 decreases stable association of cohesin with polytene chromosomes. These findings demonstrate that dMi-2 is an important regulator of both chromosome condensation and cohesin binding in interphase cells.


Complications associated with NRTI therapy: update on clinical features and possible pathogenic mechanisms.
The availability of durable, effective antiretroviral therapy for HIV-infected patients has fundamentally altered the prognosis of this disease and has also increased awareness that long-term drug toxicities have the potential to cause significant morbidity and even mortality in this patient population. The long-term use of nucleoside analogue reverse transcriptase inhibitor (NRTI) drugs has been associated with a number of clinically relevant toxicities including hyperlactataemia and lactic acidosis, neuropathy, pancreatitis and, more recently, a syndrome of pathological loss of subcutaneous fat tissue (lipoatrophy). Importantly, the toxicity profile of each NRTI drug within this class is unique in terms of the overall risk of long-term complications, as well as the tissue specificity of its toxic effects. In this review, the clinical manifestations, risk factors and pathological basis for NRTI-associated toxicity syndromes are explored, with an emphasis on clinical assessment and management.


Improving electrokinetic microdevice stability by controlling electrolysis bubbles.
The voltage-operating window for many electrokinetic microdevices is limited by electrolysis gas bubbles that destabilize microfluidic system causing noise and irreproducible responses above ∼3 V DC and less than ∼1 kHz AC at 3 Vpp. Surfactant additives, SDS and Triton X-100, and an integrated semipermeable SnakeSkin® membrane were employed to control and assess electrolysis bubbles from platinum electrodes in a 180 by 70 μm, 10 mm long microchannel. Stabilized current responses at 100 V DC were observed with surfactant additives or SnakeSkin® barriers. Electrolysis bubble behaviors, visualized via video microscopy at the electrode surface and in the microchannels, were found to be influenced by surfactant function and SnakeSkin® barriers. Both SDS and Triton X-100 surfactants promoted smaller bubble diameters and faster bubble detachment from electrode surfaces via increasing gas solubility. In contrast, SnakeSkin® membranes enhanced natural convection and blocked bubbles from entering the microchannels and thus reduced current disturbances in the electric field. This data illustrated that electrode surface behaviors had substantially greater impacts on current stability than microbubbles within microchannels. Thus, physically blocking bubbles from microchannels is less effective than electrode functionalization approaches to stabilize electrokinetic microfluidic systems.


An improved hybrid of SVM and SCAD for pathway analysis.
Pathway analysis has lead to a new era in genomic research by providing further biological process information compared to traditional single gene analysis. Beside the advantage, pathway analysis provides some challenges to the researchers, one of which is the quality of pathway data itself. The pathway data usually defined from biological context free, when it comes to a specific biological context (e.g. lung cancer disease), typically only several genes within pathways are responsible for the corresponding cellular process. It also can be that some pathways may be included with uninformative genes or perhaps informative genes were excluded. Moreover, many algorithms in pathway analysis neglect these limitations by treating all the genes within pathways as significant. In previous study, a hybrid of support vector machines and smoothly clipped absolute deviation with groups-specific tuning parameters (gSVM-SCAD) was proposed in order to identify and select the informative genes before the pathway evaluation process. However, gSVM-SCAD had showed a limitation in terms of the performance of classification accuracy. In order to deal with this limitation, we made an enhancement to the tuning parameter method for gSVM-SCAD by applying the B-Type generalized approximate cross validation (BGACV). Experimental analyses using one simulated data and two gene expression data have shown that the proposed method obtains significant results in identifying biologically significant genes and pathways, and in classification accuracy.


Effect of immobilization on the activity of rat hepatic microsomal cytochrome P450 enzymes.
Cytochrome P450 enzyme preparations were obtained from phenobarbital-treated rats. On immobilization in Romicon PM 10 polysulfone anisotropic hollow fibers, 20-25% of the original activity remained. The immobilization procedure did not significantly alter the Km of the preparations when 7-ethoxycoumarin was the substrate. Coimmobilization of the preparations with glucose-6-phosphate dehydrogenase allowed cofactor recycling to take place but resulted in an apparent drop in the value for the Km. The Vmax of immobilized preparations with or without cofactor recycling was about 25% of that observed with the nonimmobilized preparations. The flow rate through the reactors greatly affected the time at which product was detected in the effluent from the reactor and the amount of 7-hydroxycoumarin produced. It is suggested that this reflects alternative fates of the NADPH cofactor when residence times in the reactor are lengthened. Increasing the amounts of protein immobilized increased the Km and decreased the Vmax values in comparison to the free enzyme. Immobilization increased the stability of preparations with respect to ionic strength and storage. Little effect was noted with regard to changes in temperature stability or pH optima of immobilized preparations.


Total Resection of Cervical Ventral Intramedullary Cavernous Hemangiomas with an Anterior Corpectomy.
Intramedullary lesions and tumors are generally accessed by a posterior approach. However, if the lesion is located on the ventral side of the spinal cord, a posterior resection with myelotomy poses technical difficulties. We report two cases of complete resection of a cervical ventral intramedullary cavernous hemangioma using an anterior approach. Two cases of intramedullary cavernous hemangioma located on the ventral side of the spinal cord were successfully treated by total resection with anterior cervical corpectomy followed by anterior spinal fusion with an autologous bone strut from the iliac crest. In both cases, the postoperative course was uneventful, and there was no neurological deficit. Bony fusion was achieved, and there was no recurrence or complication during a follow-up period of at least two years. Here, we describe an anterior approach for total resection of cavernous hemangiomas on the ventral side of the cervical spinal cord. Outcomes were stable two years after the operations. Although the method should be assessed with more patients and a longer follow-up time, this anterior approach may be useful for the radical resection of a vascular malformation or tumor.


The RNA Ontology Consortium: an open invitation to the RNA community.
The aim of the RNA Ontology Consortium (ROC) is to create an integrated conceptual framework-an RNA Ontology (RO)-with a common, dynamic, controlled, and structured vocabulary to describe and characterize RNA sequences, secondary structures, three-dimensional structures, and dynamics pertaining to RNA function. The RO should produce tools for clear communication about RNA structure and function for multiple uses, including the integration of RNA electronic resources into the Semantic Web. These tools should allow the accurate description in computer-interpretable form of the coupling between RNA architecture, function, and evolution. The purposes for creating the RO are, therefore, (1) to integrate sequence and structural databases; (2) to allow different computational tools to interoperate; (3) to create powerful software tools that bring advanced computational methods to the bench scientist; and (4) to facilitate precise searches for all relevant information pertaining to RNA. For example, one initial objective of the ROC is to define, identify, and classify RNA structural motifs described in the literature or appearing in databases and to agree on a computer-interpretable definition for each of these motifs. To achieve these aims, the ROC will foster communication and promote collaboration among RNA scientists by coordinating frequent face-to-face workshops to discuss, debate, and resolve difficult conceptual issues. These meeting opportunities will create new directions at various levels of RNA research. The ROC will work closely with the PDB/NDB structural databases and the Gene, Sequence, and Open Biomedical Ontology Consortia to integrate the RO with existing biological ontologies to extend existing content while maintaining interoperability.


Inhibition of thrombin by antithrombin III and heparin cofactor II in vivo.
The critical role of thrombin in the pathogenesis of venous and arterial thrombosis, and the effectiveness of glycosaminoglycans as antithrombotic drugs are well known. Antithrombin III is a major inhibitor of thrombin and augmentation of its inhibitory actions by heparin is the basis for the clinical uses of heparin. Recent clinical and experimental studies have demonstrated that another glycosaminoglycan, dermatan sulfate, is an effective antithrombotic drug. Dermatan sulfate catalyses the inhibition of thrombin by heparin cofactor II. The concentrations of heparin cofactor II are higher in the plasmas of individuals with congenital antithrombin III deficiency and pregnant women than controls. The role of heparin cofactor II as a physiologic thrombin inhibitor is unknown. Enzyme-linked immunosorbent assays were used to quantify thrombin-heparin cofactor II and thrombin-antithrombin III endogenous to the plasmas of adult antithrombin III-Hamilton deficient subjects, their siblings with normal antithrombin III levels, pregnant women at term and 3 to 5 days after delivery. Both thrombin-antithrombin III and thrombin-heparin cofactor II complexed with vitronectin were detected in all the plasmas. Significantly, the concentrations of thrombin-heparin cofactor II-vitronectin were higher in the plasmas of congenital antithrombin III deficient subjects and in pre- and post-delivery plasmas than those of normal subjects. In addition, the concentrations of thrombin-heparin cofactor II decreased 3 to 5 days after delivery, reflecting the disappearance of the catalytically active dermatan sulfate elaborated by the placenta. Thus, heparin cofactor II normally inactivates thrombin in vivo, with its role increasing in conditions associated with high levels of heparin cofactor II and/or dermatan sulfate.


Parental monitoring in late adolescence: relations to ADHD symptoms and longitudinal predictors.
In this study, we aimed to replicate Stattin and Kerr's (2000) study on parental monitoring and adolescents' deviant behavior, to extend their findings to ADHD symptoms, and to examine the longitudinal predictors (8-18 years) of parental knowledge and child disclosure. Results showed that conduct problems were primarily associated with parental knowledge and child disclosure, but not with parental solicitation and control. A similar pattern was observed for ADHD symptoms. However, while the relations for conduct problems were generally independent of ADHD symptoms, the relations for ADHD symptoms were primarily non-significant after controlling for conduct problems. Moreover, early behavior problems, but not insecure/disorganized attachment, were associated with parental knowledge and child disclosure in adolescence. In conclusion, child disclosure is primarily associated with deviant behavior rather than ADHD, and early child problem behavior is a more important predictor of child disclosure (implicating reciprocal relations between these two constructs) than is insecure/disorganized attachment.


Detritus-dependent development of the microbial community in an experimental system: qualitative analysis by denaturing gradient gel electrophoresis.
Correlations between the biomass of phytoplankton and the biomass of bacteria and between the biomass of bacteria and the biomass of protozoans suggest that there is coupling between these compartments of the "microbial loop." To investigate this coupling on the species level, bacteria and protozoans from untreated lake water inocula were allowed to grow on detritus of the green alga Ankistrodesmus falcatus or the cyanobacterium Oscillatoria limnetica in continuous-flow systems for 1 month. Denaturing gradient gel electrophoresis (DGGE) of the 16S and 18S rRNA genes was used to monitor the development of the bacterial community structure and the eukaryotic community structure, respectively. Nonmetric multidimensional scaling of the DGGE profiles revealed the changes in the microbial community structure. This analysis showed that significantly different bacterial communities developed on the green algal detritus and on the cyanobacterial detritus. Although similar results were obtained for the eukaryotic communities, the differences were not significant. Hence, our findings indicate that the origin of detritus can affect the structure of at least the bacterial community. A phylogenetic analysis of 20 18S ribosomal DNA clones that were isolated from the continuous cultures revealed that many sequences were related to the sequences of bacterivorous protozoans (members of the Ciliophora, Rhizopoda, Amoeba, and Kinetoplastida). One clone grouped in a recently established clade whose previously described members are all parasites. The affiliations of about 20% of the clones could not be determined.


Identification of novel downstream molecules of tissue factor activation by comparative proteomic analysis.
Tissue factor (TF) is both an initiator of blood coagulation and a signaling receptor. Using a proteomic approach, we investigated the role of TF in cell signaling when stimulated by its ligand, activated factor VII (FVIIa). From a 2-D difference gel electrophoresis (DIGE) study we found forty one spots that were differentially regulated over time in FVIIa stimulated cells or in comparison to nonstimulated cells. Mass spectrometry identifies 23 out of these as 13 different proteins. One of them, elongation factor 2 (EF-2), was investigated in greater detail by Western blot, a protein synthesis assay and cell cycle analysis. When tissue factor was stimulated by FVIIa, the phosphorylation of EF-2 increased which inactivates this protein. Analyzing the effect using site inactivated FVIIa (FVIIai), as well as the protease activated receptor 2 (PAR-2) agonist SLIGKV, indicated that the inactivation was not PAR-2 dependent. A panel of tissue factor mutants was analyzed further to try to pinpoint what part of the cytoplasmic domain that is needed for this effect. Performing a protein synthesis assay in two different cell lines we could confirm that protein synthesis decreased upon stimulation by FVIIa. Cell cycle analysis showed that FVIIa also promotes a higher degree of cell proliferation.


Colorectal distension enforce acute urinary bladder distension-induced hepatic vasoconstriction in the rat.
Acute urinary bladder distension (AUBD) and colorectal distension (CRD) activated mechanical afferents from the urinary bladder and colon and the activated afferent signaling affected visceral organ behavior. AUBD and CRD have been reported to affect the arterial blood pressure (ABP) response in the rat. We hypothesized that AUBD and CRD may influence the cardiovascular response in the liver by neurokinin receptor-mediated afferent transmission and sympathetic nerve-mediated vesicovascular reflex. We simultaneously evaluated the bladder and colorectal pressure, hepatic microcirculation and arterial blood pressure (ABP) in response to AUBD (0-60mmHg) and/or CRD stimulation (0-80mmHg) in the urethane anesthetized rat. Hepatic sympathetic denervation and intravenous CP96,345 (neurokinin 1 receptor antagonist) and SR48968 (neurokinin 2 receptor antagonist) were adopted to determine the possible neural pathways in response to AUBD and/or CRD stimulation. Our results showed that AUBD or CRD evoked a pressor response in ABP and a hepatic vasoconstriction in a pressure-dependent manner. The pressor response was demonstrated in an order AUBD>AUBD+CRD>CRD. The hepatic vasoconstrictor response was displayed in an order AUBD+CRD>AUBD>CRD. Hepatic sympathetic denervation and CP96,345 (neurokinin 1 receptor antagonist), not SR48968 (neurokinin 2 receptor antagonist), significantly inhibited AUBD, CRD and AUBD+CRD induced hepatic vasoconstriction. CRD significantly inhibited AUBD evoked spontaneous bladder contractions and ABP elevation. In conclusion, our results indicated that AUBD and CRD via neurokinin 1 receptors activate hepatic sympathetic nerve-mediated vesicovascular reflex and consequently lead to hepatic vasoconstriction. CRD exerts different cross-talk effects to influence AUBD-evoked bladder contractions and vesicovascular reflex.


Structure of the recombinant Paramecium tetraurelia calmodulin at 1.68 A resolution.
The crystal structure of the recombinant calmodulin from Paramecium tetraurelia (rPCaM, M(r) = 16 700, 148 residues) has been determined at 1.68 A resolution. X-ray intensity data were collected at 263 K using a Siemens-Nicolet area detector and Cu Kalpha radiation from a rotating-anode source. A total of 35 936 observations were processed with XENGEN1.3 and scaled to yield 16 255 unique reflections with R(symm)(I) of 4.1%. The crystals are triclinic, with unit-cell dimensions a = 29.89, b = 53.42, c = 25.35 A, alpha = 93.67, beta = 96.88, gamma = 89.24 degrees, space group P1, with one molecule in the unit cell. The atomic coordinates of the wild-type Paramecium calmodulin (PCaM) studied in our laboratory provided the starting model. Refinement of the structure by X-PLOR and refitting it into omit maps yielded an R value of 0.194 for 15 965 reflections greater than 3sigma(F) in the 6.0-1.68 A resolution range. The final model contained 1165 protein atoms for all of the 148 residues, four Ca(2+) ions, and 172 water molecules. The dumbbell structure has seven alpha-helices including a long 7.8 turn central helix connecting the two terminal domains each containing two EF-hand (helix-loop-helix motif) calcium-binding sites. The loops within each pair of EF-hand motifs in the N- and C-terminal domains are brought into juxtaposition to form a pair of hydrogen-bonded antiparallel beta-sheets which are extended at either ends by water bridges. The four calcium-binding EF-hands are superposable with r.m.s. deviations of 0.31-0.79 A. The best agreement is between site 1 and site 3 and the worst agreement is between site 1 and 4. The largest differences are in the ninth and tenth residues of the calcium-binding loops probably because of their involvement in the mini beta-sheets. The calcium coordination distances vary between 2.04 and 2.69 A, average 2.34 A. The rPCaM and wild-type PCaM have an r.m.s. deviation of 0.36 A for equivalent C(alpha) atoms. The side chains of Lys13 and Lys115 are more extended in rPCaM compared to the wild type where the post-translational modified di- and tri-methylated lysine residues are more folded. The sequence of PCaM differs from those of mammalian (MCaM) and Drosophila calmodulin (DCaM), but the overall structures are very similar, with r.m.s,. deviations of 0.44 and 1.68 A for equivalent C(alpha) atoms, respectively. However, in rPCaM, the first four N-terminal residues stretch out and make intermolecular crystal contacts, in contrast to those in recombinant Drosophila calmodulin (rDCaM), they stretch out in the opposite direction and towards the second calcium-binding site (see note below), while in MCaM and wild-type PCaM, the N-terminal residues are not visible. The central helix in rPCaM has all its backbone hydrogen bonds intact with no unusually long separation between the carbonyl and amide groups as found in MCaM and rDCaM.


A randomized trial comparing colloid preload to coload during spinal anesthesia for elective cesarean delivery.
Hypotension after spinal anesthesia for cesarean delivery is common. Previous studies have demonstrated that a crystalloid fluid "coload" (rapid administration of a fluid bolus starting at the time of intrathecal injection) is superior to the conventional crystalloid preload (fluid administered before the intrathecal injection) for preventing hypotension. Colloid preload provides a sustained increase in central blood volume. We hypothesized that, in contrast to crystalloid, a colloid preload may be more effective than colloid coload for reducing the incidence of spinal anesthesia-induced hypotension. In this double-blind study, 178 patients were randomly assigned to receive a preload of 500 mL of hydroxyethyl starch over a period of 15-20 min before initiation of spinal anesthesia (n = 90) or an identical fluid bolus of hydroxyethyl starch starting at the time of identification of cerebrospinal fluid (n = 88). Vasopressors (ephedrine or phenylephrine) were administered if systolic arterial blood pressure decreased less than 80% of the baseline pressure and <100 mm Hg, or with smaller decreases in blood pressure if accompanied by nausea, vomiting, or dizziness. The primary outcome was the incidence of hypotension (defined as the administration of at least one dose of vasopressor). There was no significant difference between the groups in the incidence of hypotension (68% in preload group and 75% in coload group, 95% confidence interval of difference -6%-20%; P = 0.28), doses of ephedrine and phenylephrine, and number of vasopressor unit doses. The incidence of severe hypotension (systolic blood pressure <80 mm Hg) was 16% in the preload group and 22% in the coload group (P = 0.30). There were no differences in the incidence of nausea and/or vomiting, or neonatal outcome between the groups. There was no difference in the incidence of hypotension in women who received colloid administration before the initiation of spinal anesthesia compared with at the time of initiation of anesthesia. Both modalities are inefficient as single interventions to prevent hypotension.


Development of a nude mouse model to study human sebaceous gland physiology and pathophysiology.
Study of human sebaceous gland physiology and pathophysiology is limited by lack of an adequate animal model. This study was designed to develop an animal model using human face skin grafted onto the nude mouse to study human sebaceous glands. Full-thickness human face skin was grafted onto 60 adult male nude mice. 4 wk after grafting, androgens, which are known to stimulate sebaceous glands, were administered to test the system. Androgens were administered to 21 animals by implanted catheters that were filled with testosterone (T) or dihydrotestosterone (DHT). Empty catheters were implanted in 15 control animals. Graft biopsies and blood for androgen levels were obtained at time 1 (pre-catheter) and time 2 (26 d after catheter implantation). Three assessments were made on each biopsy: sebaceous gland volume, using an image analyzing computer; sebaceous cell size; and sebaceous gland labeling index. 29 mice completed the study through time 2. In the androgen-treated group, T levels (nanogram per milliliter) five times increased to 4.92 +/- 0.35, and DHT levels (nanogram per milliliter) increased 50 times to 16.70. In the androgen-treated group, sebaceous gland volume (micron 3 X 10(-3) increased from 896 +/- 194 to 3,233 +/- 754 (P less than 0.001), sebaceous cell area (micron 2) increased from 167 +/- 12 to 243 +/- 19 (P less than 0.001), and labeling index (percentage) increased from 2.7 +/- 0.7 to 6.4 +/- 0.9 (P less than 0.01). In the control group, sebaceous gland volume fell from 1,070 +/- 393 to 417 +/- 99 (NS), sebaceous cell size remained the same, and the labeling index fell from 5.1 +/- 1.9 to 3.2 +/- 1.1. After androgen administration, Halowax N-34, a known comedogen, or its vehicle, was applied to 15 grafts for 2-6 wk. Twice as many microcomedones were seen in the Halowax-treated grafts, compared with vehicle-treated grafts at the end of this time period. No visible comedones were produced. This study demonstrated that: (a) human sebaceous glands can be successfully transplanted and studied on the nude mouse; (b) after androgen stimulation, sebaceous gland volume, cell size, and labeling index increase; (c) microcomedones can be produced in the human skin grafts by the application of a comedogenic substance. Thus, this model demonstrates significant potential for the future study of human sebaceous gland physiology and pathology.


Using patient-derived iPSCs to develop humanized mouse models for chronic myelomonocytic leukemia and therapeutic drug identification, including liposomal clodronate.
Chronic myelomonocytic leukemia (CMML) is an entity of myelodysplastic syndrome/myeloproliferative neoplasm. Although CMML can be cured with allogeneic stem cell transplantation, its prognosis is generally very poor due to the limited efficacy of chemotherapy and to the patient's age, which is usually not eligible for transplantation. Comprehensive analysis of CMML pathophysiology and the development of therapeutic agents have been limited partly due to the lack of cell lines in CMML and the limited developments of mouse models. After successfully establishing patient's derived disease-specific induced pluripotent stem cells (iPSCs) derived from a patient with CMML, we utilized these CMML-iPSCs to achieve hematopoietic re-differentiation in vitro, created a humanized CMML mouse model via teratomas, and developed a drug-testing system. The clinical characteristics of CMML were recapitulated following hematopoietic re-differentiation in vitro and a humanized CMML mouse model in vivo. The drug-testing system using CMML-iPSCs identified a MEK inhibitor, a Ras inhibitor, and liposomal clodronate as potential drugs for treating CMML. Clodronate is a drug commonly used as a bisphosphonate for osteoporosis. In this study, the liposomalization of clodronate enhanced its effectiveness in these assays, suggesting that this variation of clodronate may be adopted as a repositioned drug for CMML therapy.


A Partial Response to Reintroduced Chemotherapy in a Resistant Small Cell Lung Cancer Patient After Priming with RRx-001.
As an exceedingly recalcitrant and highly aggressive tumor type without Food and Drug Administration-approved treatment or a known cure, the prognosis of recurrent extensive stage platinum-resistant/refractory small cell lung cancer (SCLC) is worse than other types of lung cancer, and many other tumor types, given a response rate of less than 10% and an overall survival of less than six months. It was broadly classified into three groups based on the initial response to cisplatin/etoposide therapy, platinum-refractory, platinum-resistant, and platinum-sensitive, extensive stage SCLC inevitably relapses, at which point the only standard options are to rechallenge with the first-line chemotherapeutic regimen in the case of sensitive disease or to start the topoisomerase I inhibitor, topotecan. Sensitive disease is defined by a response to the first-line therapy and a treatment-free interval of at least 90 days, while the definitions of refractory and resistant disease, respectively, are nonresponse to the first-line treatment or relapse within 90 days. As an important predictor of response to the second-line treatment, the clinical cutoff of three months (or two months in some cases) for resistant and sensitive disease, which along with performance status prognostically separates patients into high- and low-risk categories, dictates subsequent management. This case report presents a resistant SCLC patient enrolled on a Phase II clinical trial called QUADRUPLE THREAT (formerly TRIPLE THREAT; NCT02489903) who responded to reintroduced platinum doublets after sequential priming with the resistance-reversing epi-immunotherapeutic agent, RRx-001. In the QUADRUPLE THREAT clinical trial, both during priming with RRx-001 and during sequential treatment with platinum doublets, the patient maintained a good quality of life and performance status.


[Own experience endovascular prosthetic repair of abdominal aortic aneurysms].
Endovascular prosthetic repair has increasingly been used over the last several decades. This type of intervention is indicated for patients running a high surgical and anaesthesiological risk of an open surgical procedure. The experience gained in endovascular prosthetic repair of abdominal aortic aneurysms makes it possible to extend the indications for its practical application. The authors describe herein their experience gained in a total of 20 procedures of endovascular prosthetic repair of abdominal aortic aneurysms using the Gore Excluder endograft device in patients running a high risk of an open surgical intervention. There were neither short -nor long-term lethal outcomes. Three patients after endovascular prosthetic repair were found to have type 1 endoleak and two appeared to have type 2 endoleak. Type 1 endoleaks were coped with intraoperationally. Dynamic follow-up (at 1, 6, 12 months after surgery) of patients including computed tomography showed no complications whatsoever (i. e., no endoleaks, endograft migration, nor increase in the aneurysm's diameter). After endografting of abdominal aortic aneurysms there were neither cardiac nor respiratory complications in the immediate postoperative period. Hence, endografting of an abdominal aortic aneurysm is a method of choice for high-risk patients.


Effect of pH, polymer concentration and molecular weight on the physical state properties of tolfenamic acid.
Tolfenamic acid (TA) has been transformed from crystalline to amorphous state through freeze-drying by using varying ratios of polyacrylic acid (PA) at various pH values. The characterization of the films has been carried out using X-ray diffraction, differential scanning calorimetry, Fourier transform infrared (FTIR) spectrometry and scanning electron microscopy. The results showed a gradual change in the solid state properties of TA and a complete transformation into its amorphous form in 1:8, 1:4, 1:2 and 1:1 ratios at pH 3, 4, 5 and 6, respectively. FTIR spectrometry reveals the formation of a yellow polymorphic form of TA. Polymer molecular weight has also been observed to affect the drug transformation and interaction as the low molecular weight PA (Mw ∼ 1800) was found to be most effective followed by its medium (Mv ∼ 450 000) and high molecular weight (Mv ∼ 3 000 000) forms. No signs of recrystallization in the TA-PA films were noted during the 12-week storage period. PA of low molecular weight has also been found more effective in inhibiting the recrystallization of the melt upon cooling thus proving a valuable polymer in producing stable amorphous solid dispersions of TA.


Sarcoidosis: correlation of pulmonary parenchymal pattern at CT with results of pulmonary function tests.
The appearances of the lungs on radiographs and computed tomographic (CT) scans were correlated with degree of uptake on gallium scans and results of pulmonary function tests (PFTs) in 27 patients with sarcoidosis. CT scans were evaluated both qualitatively and quantitatively. Patients were divided into five categories on the basis of the pattern of abnormality at CT: 1 = normal (n = 4); 2 = segmental air-space disease (n = 4); 3 = spherical (alveolar) masslike opacities (n = 4); 4 = multiple, discrete, small nodules (n = 6); and 5 = distortion of parenchymal structures (fibrotic end-stage sarcoidosis) (n = 9). The percentage of the volume judged to be abnormal (CT grade) was correlated with PFT results for each CT and radiographic category. CT grades were also correlated with gallium scanning results and percentage of lymphocytes recovered from bronchoalveolar lavage (BAL). Patients in CT categories 1 and 2 had normal lung function, those in category 3 had mild functional impairment, and those in categories 4 and 5 showed moderate to severe dysfunction. The overall CT grade correlated well with PFT results expressed as a percentage of the predicted value. In five patients, CT scans showed extensive parenchymal disease not seen on radiographs. CT grades did not correlate with the results of gallium scanning or BAL lymphocytes. The authors conclude that patterns of parenchymal sarcoidosis seen at CT correlate with the PFT results and can be used to indicate respiratory impairment.


Model to Select On-Therapy vs Off-Therapy Tests for Patients With Refractory Esophageal or Extraesophageal Symptoms.
It is not clear whether we should test for reflux in patients with refractory heartburn or extraesophageal reflux (EER) symptoms, such as cough, hoarseness, or asthma. Guidelines recommend testing patients by pH monitoring when they are on or off acid-suppressive therapies based on pretest probability of reflux, determined by expert consensus. However, it is not clear what constitutes a low or high pretest probability of reflux in these patients. We aimed to develop a model that clinicians can use at bedside to estimate pretest probability of abnormal reflux. We performed a prospective study of 471 adult patients with refractory heartburn (n = 214) or suspected EER symptoms (n = 257) who underwent endoscopy with wireless pH monitoring while they were off acid-suppressive treatment and assigned them to groups based on symptoms at presentation (discovery cohort). Using data from the discovery cohort, we performed proportional odds ordinal logistic regression to select factors (easy to obtain demographic criteria and clinical symptoms such as heartburn, regurgitation, asthma, cough, and hoarseness) associated with esophageal exposure to acid. We validated our findings in a cohort of 118 patients with the same features from 2 separate tertiary care centers (62% women; median age 59 years; 62% with cough as presenting symptom). Abnormal pH (>5.5% of time spent at pH <4) was found in 56% of patients with heartburn and 63% of patients with EER (P = .15). Within EER groups, abnormal pH was detected in a significantly larger proportion (80%) of patients with asthma compared with patients with cough (60%) or hoarseness (51%; P < .01). Factors significantly associated with abnormal pH in patients with heartburn were presence of hiatal hernia and body mass index >25 kg/m2. In patients with EER, the risk of reflux was independently associated with the presence of concomitant heartburn (odds ratio [OR] 2.0; 95% confidence interval [CI] 1.3-3.1), body mass index >25 kg/m2 (OR 2.1; 95% CI 1.5-3.1), asthma (OR 2.0; 95% CI 1.2-3.5), and presence of hiatal hernia (OR 1.9; 95% CI 1.2-3.1). When we used these factors to create a scoring system, we found that a score of ≤2 excluded patients with moderate to severe reflux, with a negative predictive value of 80% in the discovery cohort and a negative predictive value of 85% in the validation cohort. We developed a clinical model to estimate pretest probability of abnormal pH in patients who were failed by proton pump inhibitor therapy. This system can help guide clinicians at bedside in determining the most appropriate diagnostic test in this challenging group of patients.


Loss of murine Paneth cell function alters the immature intestinal microbiome and mimics changes seen in neonatal necrotizing enterocolitis.
Necrotizing enterocolitis (NEC) remains the leading cause of gastrointestinal morbidity and mortality in premature infants. Human and animal studies suggest a role for Paneth cells in NEC pathogenesis. Paneth cells play critical roles in host-microbial interactions and epithelial homeostasis. The ramifications of eliminating Paneth cell function on the immature host-microbial axis remains incomplete. Paneth cell function was depleted in the immature murine intestine using chemical and genetic models, which resulted in intestinal injury consistent with NEC. Paneth cell depletion was confirmed using histology, electron microscopy, flow cytometry, and real time RT-PCR. Cecal samples were analyzed at various time points to determine the effects of Paneth cell depletion with and without Klebsiella gavage on the microbiome. Deficient Paneth cell function induced significant compositional changes in the cecal microbiome with a significant increase in Enterobacteriacae species. Further, the bloom of Enterobacteriaceae species that occurs is phenotypically similar to what is seen in human NEC. This further strengthens our understanding of the importance of Paneth cells to intestinal homeostasis in the immature intestine.


How different are baby-led weaning and conventional complementary feeding? A cross-sectional study of infants aged 6-8 months.
To compare the food, nutrient and 'family meal' intakes of infants following baby-led weaning (BLW) with those of infants following a more traditional spoon-feeding (TSF) approach to complementary feeding. Cross-sectional study of dietary intake and feeding behaviours in 51 age-matched and sex-matched infants (n=25 BLW, 26 TSF) 6-8 months of age. Parents completed a questionnaire, and weighed diet records (WDRs) on 1-3 non-consecutive days, to investigate food and nutrient intakes, the extent to which infants were self-fed or parent-fed, and infant involvement in 'family meals'. BLW infants were more likely than TSF infants to have fed themselves all or most of their food when starting complementary feeding (67% vs 8%, p<0.001). Although there was no statistically significant difference in the large number of infants consuming foods thought to pose a choking risk during the WDR (78% vs 58%, p=0.172), the CI was wide, so we cannot rule out increased odds with BLW (OR, 95% CI: 2.57, 0.63 to 10.44). No difference was observed in energy intake, but BLW infants appeared to consume more total (48% vs 42% energy, p<0.001) and saturated (22% vs 18% energy, p<0.001) fat, and less iron (1.6 vs 3.6 mg, p<0.001), zinc (3.0 vs 3.7 mg, p=0.001) and vitamin B12 (0.2 vs 0.5 μg, p<0.001) than TSF infants. BLW infants were more likely to eat with their family at lunch and at the evening meal (both p≤0.020). Infants following BLW had similar energy intakes to those following TSF and were eating family meals more regularly, but appeared to have higher intakes of fat and saturated fat, and lower intakes of iron, zinc and vitamin B12. A high proportion of both groups were offered foods thought to pose a choking risk.


R2* Relaxation Affects Pharmacokinetic Analysis of Dynamic Contrast-Enhanced MRI in Cancer and Underestimates Treatment Response at 7 T.
Effective transverse relaxivity of gadolinium-based contrast agents is often neglected in dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Here, we assess time and tissue dependence of R2* enhancement and its impact on pharmacokinetic parameter quantification and treatment monitoring. Multiecho DCE-MRI was performed at 7 T on mice bearing subcutaneous TOV-21G human ovarian cancer xenografts (n = 8) and on the transgenic adenocarcinoma of the mouse prostate (TRAMP) model (n = 7). Subsequently, the TOV-21G tumor-bearing mice were treated with bevacizumab and rescanned 2 days later. Pharmacokinetic analysis (extended Tofts model) was performed using either the first echo signal only (standard single-echo DCE-MRI) or the estimated signal at TE = 0 derived from exponential fitting of R2* relaxation (R2*-corrected). Neglecting R2* enhancement causes underestimation of Gd-DOTA concentration (peak enhancement underestimated by 9.4%-16% in TOV-21G tumors and 13%-20% in TRAMP prostates). Median Ktrans and ve were underestimated in every mouse (TOV-21G Ktrans: 11%-19%, TOV-21G ve: 5.3%-8.9%; TRAMP Ktrans: 8.6%-19%, TRAMP ve: 12%-21%). Bevacizumab treatment reduced Ktrans in all TOV-21G tumors after 48 hours. Treatment effect was significantly greater in all tumors after R2* correction (median change of -0.050 min-1 in R2*-corrected Ktrans vs. -0.037 min-1 in uncorrected Ktrans). R2* enhancement in DCE-MRI is both time- and tissue-dependent and may not be negligible at 7 T in tissue with high Ktrans. This has consequences for the use of Ktrans and other DCE-MRI parameters as biomarkers, because treatment effect size can be underestimated when R2* enhancement is neglected.


Development of 5006 full-length CDNAs in barley: a tool for accessing cereal genomics resources.
A collection of 5006 full-length (FL) cDNA sequences was developed in barley. Fifteen mRNA samples from various organs and treatments were pooled to develop a cDNA library using the CAP trapper method. More than 60% of the clones were confirmed to have complete coding sequences, based on comparison with rice amino acid and UniProt sequences. Blastn homologies (E<1E-5) to rice genes and Arabidopsis genes were 89 and 47%, respectively. Of the 5028 possible amino acid sequences derived from the 5006 FLcDNAs, 4032 (80.2%) were classified into 1678 GreenPhyl multigenic families. There were 555 cDNAs showing low homology to both rice and Arabidopsis. Gene ontology annotation by InterProScan indicated that many of these cDNAs (71%) have no known molecular functions and may be unique to barley. The cDNAs showed high homology to Barley 1 GeneChip oligo probes (81%) and the wheat gene index (84%). The high homology between FLcDNAs (27%) and mapped barley expressed sequence tag enabled assigning linkage map positions to 151-233 FLcDNAs on each of the seven barley chromosomes. These comprehensive barley FLcDNAs provide strong platform to connect pre-existing genomic and genetic resources and accelerate gene identification and genome analysis in barley and related species. Sequence data from this article have been deposited with the DDBJ/EMBL/GenBank Data Libraries under accession nos AK248134-AK253139. The online database with annotation is available at http://www.shigen.nig.ac.jp/barley/.


Computational examination of the mechanism of alkene epoxidation catalyzed by gallium(III) complexes with N-donor ligands.
The ability of gallium(III) complexes to catalyze the epoxidation of alkenes by peracetic acid has been examined with density functional theory calculations. According to the calculations, the chloride anions of the precatalyst [Ga(phen)2Cl2](+) (phen = 1,10-phenanthroline) can be displaced by either acetic or peracetic acid through dissociative ligand exchange pathways; both acetic and peracetic acid deprotonate upon binding to the formally tricationic metal center. Because of the high basicity of peracetate relative to that of chloride, only the acetate for chloride exchange occurs spontaneously, providing a rationale for the preponderance of gallium acetate adducts observed in the reaction mixtures. With respect to the mechanism of olefin epoxidation, the computational results suggest that the peracetic acid is most efficiently activated for redox activity when it binds to the metal center in a κ(2) fashion, with the carbonyl oxygen atom serving as the second point of attachment. The phen ligands' coordination to the gallium is essential for the catalysis, and the lowest energy pathways for alkene oxidation proceed through hexacoordinate Ga(III) species with four Ga-N bonds. A natural bond order analysis confirms the electrophilic nature of the metal-containing oxidant.


Fontan conversion with concomitant arrhythmia surgery for the failing atriopulmonary connections: mid-term results from a single centre.
Classical Atriopulmonary Fontan connections tend to fail in the long term due to progressive anastomotic site obstruction, right atrial enlargement, and refractory atrial arrhythmias. Conversion to total cavopulmonary connection with concomitant arrhythmia surgery is a promising treatment but optimal timing of the procedure remains controversial. Between the years 2002 and 2009, 15 patients with a median age of 26.2 (12-43) years underwent Fontan conversion operation with concomitant arrhythmia surgery. All were symptomatic and 14 out of the 15 patients had refractory arrhythmias. The duration of pre-operative arrhythmia and the outcome of surgery were correlated to study the impact of delay in surgical intervention on post-operative survival and arrhythmia control. There were two patients who died in the early post-operative period (13.3%). At the mid-term follow-up, 53 (20-86) months, late atrial arrhythmias had recurred in two of the 13 surviving patients (15.30%) and one patient developed late sinus node dysfunction. The need for anti-arrhythmic drugs decreased considerably from 93.5% to 15.3% on mid-term follow-up. There was no late death or need for cardiac transplantation. The duration of arrhythmia before surgery was prolonged for more than 10 years in patients who died as well as in those who had complications like late recurrence of arrhythmias, dependence on anti-arrhythmic medications, and worsening of ventricular dysfunction. Fontan conversion is a well-established treatment option for salvaging the failing atriopulmonary connections. Concomitant arrhythmia surgery effectively resolves the refractory atrial arrhythmias and improves survival, but we need to optimise the timing of Fontan conversion to improve the long-term outcome.


Bone-protecting effect of Rubus coreanus by dual regulation of osteoblasts and osteoclasts.
Bone loss occurs with increasing age and/or as a secondary occurrence to chronic metabolic disease. Certain nutritional and pharmacological, as well as nonpharmacologic interventions such as weight-bearing exercise and muscle strengthening help prevent bone loss. We examined the effect of the methanol extract from the fruit of Rubus coreanus (RCM) on postmenopausal osteoporosis. Ovariectomized rats were assigned to sham (negative control), vehicle control, positive control, safflower seed 200 mg/kg, RCM 100 mg/kg (RCM 100), RCM 200 mg/kg (RCM 200), and RCM 400 mg/kg (RCM 400) groups for 10 weeks after the operation. Serum biochemistry, histochemistry, immunohistochemistry, and other related biomarkers of bone metabolism were investigated. We observed that RCM could prevent bone loss by increasing the femur trabecular bone area in a dose-dependent manner in ovariectomized rats. The mineral composition of RCM contains many more valuable elements, especially potassium, magnesium, and vitamins D and B2, than safflower seed. The effect of RCM increased not only osteoblast differentiation but also osteoclast apoptosis. In addition, the extract of RCM contained in quercetin suggests that the extract of RCM resulted in improved aging-related bone loss through an antioxidant effect. The present data provide the first direct in vivo evidence that RCM has a bone-protecting effect caused by estrogen deficiency, without undesirable side effects on the uterus and other solid organs. The beneficial effect of RCM may be mediated, at least in part, by dual regulation of the enhancement of osteoblast function and induction of osteoclast apoptosis.


Control of protein immobilization: coupling immobilization and site-directed mutagenesis to improve biocatalyst or biosensor performance.
Mutagenesis and immobilization are usually considered to be unrelated techniques with potential applications to improve protein properties. However, there are several reports showing that the use of site-directed mutagenesis to improve enzyme properties directly, but also how enzymes are immobilized on a support, can be a powerful tool to improve the properties of immobilized biomolecules for use as biosensors or biocatalysts. Standard immobilizations are not fully random processes, but the protein orientation may be difficult to alter. Initially, most efforts using this idea were addressed towards controlling the orientation of the enzyme on the immobilization support, in many cases to facilitate electron transfer from the support to the enzyme in redox biosensors. Usually, Cys residues are used to directly immobilize the protein on a support that contains disulfide groups or that is made from gold. There are also some examples using His in the target areas of the protein and using supports modified with immobilized metal chelates and other tags (e.g., using immobilized antibodies). Furthermore, site-directed mutagenesis to control immobilization is useful for improving the activity, the stability and even the selectivity of the immobilized protein, for example, via site-directed rigidification of selected areas of the protein. Initially, only Cys and disulfide supports were employed, but other supports with higher potential to give multipoint covalent attachment are being employed (e.g., glyoxyl or epoxy-disulfide supports). The advances in support design and the deeper knowledge of the mechanisms of enzyme-support interactions have permitted exploration of the possibilities of the coupled use of site-directed mutagenesis and immobilization in a new way. This paper intends to review some of the advances and possibilities that these coupled strategies permit.


Enzyme immunoassay of Cryptosporidium-specific immunoglobulin G antibodies to assess longitudinal infection trends in six communities in British Columbia, Canada.
A newly developed enzyme-linked immunosorbent assay (ELISA) that detects immunoglobulin G antibodies to the 27-kDa Cryptosporidium parvum sporozoite surface antigen was used to test 4,097 sera collected from pregnant women in 6 communities in British Columbia, Canada, between January 1996, and December 1997. Waterborne outbreaks of cryptosporidiosis occurred in two of the study communities during the period of follow-up, and ELISA seropositivity was high in all six communities during the study period (77% positive to 92% positive). In the community with the largest outbreak, levels of antibody to the 27-kDa antigen increased rapidly and then decayed to background levels within 3-4 months of the peak of the epidemic curve. Trends in serologic reactivity were complex in all communities, and increased antibody levels not related temporally to known waterborne outbreaks were also observed. Serological assays may provide more accurate information regarding community levels of Cryptosporidium infection.


High-resolution mapping and functional analysis of se2.1: a major stigma exsertion quantitative trait locus associated with the evolution from allogamy to autogamy in the genus Lycopersicon.
The degree to which stigmas are exserted above the stamen in flowers is a key determinant of cross-pollination (and hence allogamy) in many plant species. Most species in the genus Lycopersicon are obligate or facultative outcrossers and bear flowers with highly exserted stigmas. In contrast, the cultivated tomato (Lycopersicon esculentum) bears flowers with flush or inserted stigmas promoting self-fertilization. It has been observed that a major QTL, se2.1, on chromosome 2 is responsible for a large portion of phenotypic variation for this trait and that mutation(s) at this locus were likely involved in the evolution from allogamy to autogamy in this genus. To understand the genetic and molecular basis of stigma exsertion, we have conducted a high-resolution mapping at the chromosome region harboring the se2.1 QTL. The results indicate that this is a compound locus, comprising at least five tightly linked genes, one controlling style length, three controlling stamen length, and the other affecting anther dehiscence, a taxonomic character used to distinguish Lycopersicon species from other solanaceous species. This cluster of genes may represent the vestiges of an ancient coadapted gene complex in controlling mating behavior.


A Computerized Test Battery to Study Pharmacodynamic Effects on the Central Nervous System of Cholinergic Drugs in Early Phase Drug Development.
Investigating potential pharmacodynamic effects in an early phase of central nervous system (CNS) drug research can provide valuable information for further development of new compounds. A computerized and thoroughly validated battery of neuropsychological and neurophysiological tests has been shown to be sensitive to detect drug-induced effects of multiple new and existing compounds. The test battery covers the main CNS domains, which have been shown to respond to drug effects and can be repeatedly administered following drug administration to characterize the concentration-effect profile of a drug. The standard tests in the battery are saccadic eye movement, smooth pursuit eye movement, the Bowdle visual analog scale (VAS), the Bond and Lader VAS, body sway, adaptive tracking, visual verbal learning, and quantitative electroencephalography (qEEG). However, the test battery is adaptive in nature, meaning that it can be composed and adjusted with tests fit to investigate specific drug classes, or even specific receptors. Showing effects of new cholinergic drugs designed to have a pro-cognitive outcome has been difficult. The pharmacological challenge model is a tool for early proof-of-pharmacology. Here, a marketed drug is used to induce temporary and reversible disease-like symptoms in healthy subjects, via a pharmacological mechanism related to the disease that is targeted as indication for the new compound. The test battery was implemented to investigate the potential of the nicotinic receptor antagonist mecamylamine to be used as a challenge model for cholinergic dysfunction, as seen in neurodegenerative disorders. A worsening of scores in a dose dependent manner on the visual verbal learning test (VVLT; a test for learning and memory abilities) and the adaptive tracking test (a measure of visuomotor control and arousal), in particular, showed that the test battery is sensitive to showing acute pharmacodynamic effect after administration of anti-cholinergic drugs.


Preventing Fusarium head blight of wheat and cob rot of maize by inhibition of fungal deoxyhypusine synthase.
Upon posttranslational activation, the eukaryotic initiation factor-5A (eIF-5A) transports a subset of mRNAs out of the nucleus to the ribosomes for translation. Activation of the protein is an evolutionary highly conserved process that is unique to eIF-5A, the conversion of a lysine to a hypusine. Instrumental for the synthesis of hypusine is the first of two enzymatic reactions mediated by deoxyhypusine synthase (DHS). We show that DHS of wheat and the pathogenic fungus Fusarium graminearum, which causes one of the most destructive crop diseases worldwide, are transcriptionally upregulated during their pathogenic interaction. Although DHS of wheat, fungus, and human can be equally inhibited by the inhibitor CNI-1493 in vitro, application during infection of wheat and maize flowers results in strong inhibition of the pathogen without interference with kernel development. Our studies provide a novel strategy to selectively inhibit fungal growth without affecting plant growth. We identified fungal DHS as a target for the development of new inhibitors, for which CNI-1493 may serve as a lead substance.


Metabolic response of prostate cancer to nicotinamide phophoribosyltransferase inhibition in a hyperpolarized MR/PET compatible bioreactor.
Metabolic shifts in disease are of great interest for the development of novel therapeutics. In cancer treatment, these therapies exploit the metabolic phenotype associated with oncogenesis and cancer progression. One recent strategy involves the depletion of the cofactors needed to maintain the high rate of glycolysis seen with the Warburg effect. Specifically, blocking nicotinamide adenine dinucleotide (NAD) biosynthesis via nicotinamide phosphoribosyltransferase (NAMPT) inhibition depletes cancer cells of the NAD needed for glycolysis. To characterize this metabolic phenotype in vivo and describe changes in flux with treatment, non-invasive biomarkers are necessary. One such biomarker is hyperpolarized (HP) [1-(13) C] pyruvate, a clinically translatable probe that allows real-time assessment of metabolism. We therefore developed a cell perfusion system compatible with HP magnetic resonance (MR) and positron emission tomography (PET) to develop translatable biomarkers of response to NAMPT inhibition in reduced volume cell cultures. Using this platform, we observed a reduction in pyruvate flux through lactate dehydrogenase with NAMPT inhibition in prostate cancer cells, and showed that both HP lactate and 2-[(18) F] fluoro-2-deoxy-D-glucose (FDG) can be used as biomarkers for treatment response of such targeted agents. Moreover, we observed dynamic flux changes whereby HP pyruvate was re-routed to alanine, providing both positive and negative indicators of treatment response. This study demonstrated the feasibility of a MR/PET compatible bioreactor approach to efficiently explore cell and tissue metabolism, the understanding of which is critical for developing clinically translatable biomarkers of disease states and responses to therapeutics.


Traditional alcohol production and use in three provinces in Vietnam: an ethnographic exploration of health benefits and risks.
Gaps exist in knowledge about the production and use of traditional alcohols, particularly in Asia. This study adds new information about the nature, production and sale of traditional distilled spirit alcohol in Vietnam. This was an ethnographic study of traditional distilled spirit alcohol production in rural areas of three provinces in Vietnam. Researchers interviewed more than 300 individuals and recorded responses to general open-ended questions about local alcohol production. Interviews were recorded, transcribed, and studied to discern what information about traditional alcohol was important to the speakers. Methods of production followed long-held traditions. Participants listed both personal and community benefits (economic, health, and social) from traditional alcohol making. Older people favoured traditional alcohol, while younger people favoured brand-name beer. Typically people consumed 2-4 drinks daily, mainly at meal times. People consumed more alcohol at special events and festivals. Distribution patterns ranged from low-risk distribution to family and neighbours to high-risk distribution by an agent who might combine alcohol from several producers, which increases the opportunity for dilution and adulteration. The most commonly listed health risks associated with locally-made alcohol were local air pollution and water pollution; participants also mentioned traffic crashes and bad public behaviour. Depending on the location, community leaders reported that production may be relatively stable or it may be declining. Traditional alcohol manufacture, sale, and use in Vietnam is a long-standing practice and low- to moderate-risk to health. There do not appear to be instances of accidental or intentional contamination. Urbanization seems to be affecting the market share of traditional alcohol as urbanized youth turn to branded products, mainly beer, making traditional alcohol making and consumption an activity mainly linked to older people in rural areas. In the rural areas surveyed, significant economic and social benefits are derived from traditional alcohol manufacture, sale, and use. Policy makers designing ways to reduce alcohol-related risks and harms need to give thoughtful consideration to the role traditional alcohol plays in the local society and to suggest changes that do not create unintended problems.


Atovaquone is active against AML by upregulating the integrated stress pathway and suppressing oxidative phosphorylation.
Atovaquone, a US Food and Drug Administration-approved antiparasitic drug previously shown to reduce interleukin-6/STAT3 signaling in myeloma cells, is well tolerated, and plasma concentrations of 40 to 80 µM have been achieved with pediatric and adult dosing. We conducted preclinical testing of atovaquone with acute myeloid leukemia (AML) cell lines and pediatric patient samples. Atovaquone induced apoptosis with an EC50 <30 µM for most AML lines and primary pediatric AML specimens. In NSG mice xenografted with luciferase-expressing THP-1 cells and in those receiving a patient-derived xenograft, atovaquone-treated mice demonstrated decreased disease burden and prolonged survival. To gain a better understanding of the mechanism of atovaquone, we performed an integrated analysis of gene expression changes occurring in cancer cell lines after atovaquone exposure. Atovaquone promoted phosphorylation of eIF2α, a key component of the integrated stress response and master regulator of protein translation. Increased levels of phosphorylated eIF2α led to greater abundance of the transcription factor ATF4 and its target genes, including proapoptotic CHOP and CHAC1. Furthermore, atovaquone upregulated REDD1, an ATF4 target gene and negative regulator of the mechanistic target of rapamycin (mTOR), and caused REDD1-mediated inhibition of mTOR activity with similar efficacy as rapamycin. Additionally, atovaquone suppressed the oxygen consumption rate of AML cells, which has specific implications for chemotherapy-resistant AML blasts that rely on oxidative phosphorylation for survival. Our results provide insight into the complex biological effects of atovaquone, highlighting its potential as an anticancer therapy with novel and diverse mechanisms of action, and support further clinical evaluation of atovaquone for pediatric and adult AML.


Efficacy of potential chemical control compounds for removing invasive American bullfrogs (Rana catesbeiana).
Invasive American bullfrogs [Rana catesbeiana (Lithobates catesbeianus)] are outcompeting and predating on native biota and contributing to reductions in biodiversity worldwide. Current methods for controlling American bullfrogs are incapable of stopping their expansion, thus more cost-effective and broadly applicable methods are needed. Although chemical control compounds have been identified as effective for removing other invasive amphibians, none have been tested for American bullfrogs. Our objective was to expand on previous research and test the efficacy of 10 potential chemical control compounds for removing invasive American bullfrogs. After a dermal spray-application of 4 ml, we found 3 compounds (i.e., chloroxylenol, rotenone with permethrin, and caffeine) at 5-10 % concentrations in water were 100 % lethal for adult American bullfrogs. Chloroxylenol and rotenone with permethrin were fast acting with time-to-death <2 h. This research presents a first-step toward incorporating chemical control as part of integrated pest management strategy for controlling invasive American bullfrogs. Follow-up studies on delivery systems and reducing non-target hazards should ensue with these compounds to confirm their effectiveness and safety for removing invasive American bullfrogs.


Bubbly cavitating flow generation and investigation of its erosional nature for biomedical applications.
This paper presents a study that investigates the destructive energy output resulting from hydrodynamic bubbly cavitation in microchannels and its potential use in biomedical applications. The research performed in this study includes results from bubbly cavitation experiments and findings showing the destructive effects of bubbly cavitating flow on selected solid specimens and live cells. The bubbles generated by hydrodynamic cavitation are highly destructive at the surfaces of the target medium on which they are carefully focused. The resulting destructive energy output could be effectively used for biomedical treatments, such as destroying kidney stones (renal calculi) or killing cancer cells. Motivated by this potential, the cavitation damage to cancerous cells and material removal from chalk pieces (which possess similar material properties as some kidney stones) was investigated. Our results showed that cavitation could induce damage both on chalk pieces and leukemia/lymphoma cells. We discovered that hydrodynamic cavitation exposure had early and delayed effects on cancer cell survival. Hence, the potential of hydrodynamic bubbly cavitation generated at the microscale for biomedical treatments was revealed using the microchannel configuration as a microorifice (with an inner diameter of 147 μm and a length of 1.52 cm), which acts as the source of bubbly cavitating flows.


Origin of the polycomb repressive complex 2 and gene silencing by an E(z) homolog in the unicellular alga Chlamydomonas.
Polycomb group proteins play an essential role in the maintenance of cell identity and the regulation of development in both animals and plants. The Polycomb Repressive Complex 2 (PRC2) is involved in the establishment of transcriptionally silent chromatin states, in part through its ability to methylate lysine 27 of histone H3 by the Enhancer of zeste [E(z)] subunit. The absence of PRC2 in unicellular model fungi and its function in the repression of genes vital for the development of higher eukaryotes led to the proposal that this complex may have evolved together with the emergence of multicellularity. However, we report here on the widespread presence of PRC2 core subunits in unicellular eukaryotes from the Opisthokonta, Chromalveolata and Archaeplastida supergroups. To gain insight on the role of PRC2 in single celled organisms, we characterized an E(z) homolog, EZH, in the green alga Chlamydomonas reinhardtii. RNAi-mediated suppression of EZH led to defects in the silencing of transgenes and retrotransposons as well as to a global increase in histone post-translational modifications associated with transcriptional activity, such as trimethylation of histone H3 lysine 4 and acetylation of histone H4. On the basis of the parsimony principle, our findings suggest that PRC2 appeared early in eukaryotic evolution, even perhaps in the last unicellular common ancestor of eukaryotes. One of the ancestral roles of PCR2 may have been in defense responses against intragenomic parasites such as transposable elements, prior to being co-opted for lineage specific functions like developmental regulation in multicellular eukaryotes.


Antenatal care: provision and inequality in rural north India.
The objectives of this paper are to examine factors associated with use of antenatal care in rural areas of north India, to investigate access to specific critical components of care and to study differences in the pattern of services received via health facilities versus home visits. We used the 1998-1999 Indian National Family Health Survey of ever-married women in the reproductive age group and analysed data from the states of Bihar, Madhya Pradesh, Rajasthan, and Uttar Pradesh (n = 11,369). Overall, about three-fifths of rural women did not receive any antenatal check-up during their last pregnancy. Services actually received were predominantly provision of tetanus toxoid vaccination and supply of iron and folic acid tablets. Only about 13% of pregnant women had their blood pressure checked and a blood test done at least once. Women visited by health workers received fewer services compared to women who visited a health facility. Home visits were biased towards households with a better standard of living. There was significant under-utilisation of nurse/midwives in the provision of antenatal services and doctors were often the lead providers. The average number of antenatal visits reported in this study was 2.4 and most visits were in the second trimester. Higher social and economic status was associated with increased chances of receiving an antenatal check-up, and of receiving specific components including blood pressure measurement, a blood test and urine testing but not the obstetric physical examination, which was however linked to ever-use of family planning and the education of women and their husbands. Thus, pregnant women from poor and uneducated backgrounds with at least one child were the least likely to receive antenatal check-ups and services in the four large north Indian states. Basic antenatal care components are effective means to prevent a range of pregnancy complications and reduce maternal mortality. The findings indicate substantial limitations of the health services in overcoming socio-economic and cultural barriers to access.


Automatic network fingerprinting through single-node motifs.
Complex networks have been characterised by their specific connectivity patterns (network motifs), but their building blocks can also be identified and described by node-motifs-a combination of local network features. One technique to identify single node-motifs has been presented by Costa et al. (L. D. F. Costa, F. A. Rodrigues, C. C. Hilgetag, and M. Kaiser, Europhys. Lett., 87, 1, 2009). Here, we first suggest improvements to the method including how its parameters can be determined automatically. Such automatic routines make high-throughput studies of many networks feasible. Second, the new routines are validated in different network-series. Third, we provide an example of how the method can be used to analyse network time-series. In conclusion, we provide a robust method for systematically discovering and classifying characteristic nodes of a network. In contrast to classical motif analysis, our approach can identify individual components (here: nodes) that are specific to a network. Such special nodes, as hubs before, might be found to play critical roles in real-world networks.


The epidemiology of low birthweight.
In this paper, I review the epidemiology of low birthweight (LBW). I begin by defining LBW and emphasizing the distinction between infants who are born early (preterm) and those who are born small for their gestational age (SGA). I then review data on the global burden of preterm birth and SGA and the evidence bearing on whether 'one size fits all', i.e. whether a single birthweight (or birthweight for gestational age) cutoff is appropriate for different regions and population subgroups. I summarize what is known about the etiologic determinants of preterm and SGA birth, how they differ among countries of varying socioeconomic development and prevalence of risk factors, and how they are changing over time. I conclude with a critical appraisal of LBW prevention as a public health priority and argue that reducing fetal and infant mortality is a more important and achievable target for intervention.


Are treatments for cervical precancerous lesions in less-developed countries safe enough to promote scaling-up of cervical screening programs? A systematic review.
Since the mid-1990 s, there have been growing efforts to prevent cervical cancer in less-developed countries through the development of innovative screening approaches such as visual inspection of the cervix associated with same day management of cervical lesions with cryotherapy or loop electrosurgical excision procedure (LEEP). In the past, promising cancer screening interventions have been widely promoted despite incomplete evidence, only to become the subject of intense controversies about ensuing net health benefit. Because the efficacy and effectiveness of the new protocols for global cervical cancer screening have not been well characterized yet, and as a contribution to the evaluation of the balance between the benefits and risks of these protocols, we reviewed the literature on the safety of cryotherapy and LEEP for cervical intraepithelial neoplasia (CIN) in low- and middle-income countries. We searched 12 databases (Medline, Google Scholar, Scopus, Cochrane Library, Web of Science, OCLC, PAIS International Database, WHO Global Health Library, CINAHL, Science.gov, NYAM Grey Literature Report, and POPLINE) for original research published between January 1995 and April 2009. Both peer-reviewed publications and items of "grey" literature were retrieved; no language restriction was applied. We calculated the median (minimum, maximum) reported rate for each harm considered. Because of limitations and heterogeneity in the data, no formal meta-analysis was performed. The search identified 32 articles that reported safety data from 24 cryotherapy and LEEP studies. The combined sample consisted of 6,902 women treated by cryotherapy and 4,524 women treated by LEEP. Most studies were conducted in reference or research settings in Asia and Africa. Short-term harms of cryotherapy and LEEP appeared to be similar to those described in the literature from high-income countries. Information was sparse on HIV-related harms and long-term reproductive outcomes of treatment. When performed in resource-limited settings by qualified providers, cryotherapy and LEEP are not associated with excess harm. However, available data are insufficient to propose fully evidence-based protocols for routine screening of HIV-infected women and women of reproductive age.


Modeling neural tube development by differentiation of human embryonic stem cells in a microfluidic WNT gradient.
The study of brain development in humans is limited by the lack of tissue samples and suitable in vitro models. Here, we model early human neural tube development using human embryonic stem cells cultured in a microfluidic device. The approach, named microfluidic-controlled stem cell regionalization (MiSTR), exposes pluripotent stem cells to signaling gradients that mimic developmental patterning. Using a WNT-activating gradient, we generated a neural tissue exhibiting progressive caudalization from forebrain to midbrain to hindbrain, including formation of isthmic organizer characteristics. Single-cell transcriptomics revealed that rostro-caudal organization was already established at 24 h of differentiation, and that the first markers of a neural-specific transcription program emerged in the rostral cells at 48 h. The transcriptomic hallmarks of rostro-caudal organization recapitulated gene expression patterns of the early rostro-caudal neural plate in mouse embryos. Thus, MiSTR will facilitate research on the factors and processes underlying rostro-caudal neural tube patterning.


Second to fourth digit ratio, testosterone and perceived male dominance.
Previous studies have shown that male faces with extreme features associated with testosterone are perceived as dominant and masculine. Women have been reported to prefer more masculinized male faces as they may consider testosterone markers to be an 'honest' indication of good health, and such considerations may underlie their aesthetic preferences. However, pronounced testosterone facial markers are also associated with dominance, and several negative personality traits. This suggests that female aesthetic preferences may be an adaptive compromise between positive attributes associated with higher than average testosterone, and negative attributes associated with more extreme masculinization. This current study attempts to clarify the role of hormone markers in female perceptions of dominance, masculinity and attractiveness, in male facial images. Recent evidence suggests that the relative length of the 2nd to 4th finger (2D : 4D ratio) is a pointer to prenatal testosterone levels and may thus serve as a window to the prenatal hormonal environment. We measured 2D : 4D in a sample of male college students and took salivary samples to analyse circulating levels of testosterone. Women rated facial images of these males for dominance, masculinity and attractiveness. Our results show that male 2D : 4D was significantly negatively related to perceived dominance and masculinity but not attractiveness. Circulating testosterone levels were not related to dominance, masculinity or attractiveness. These findings suggest that: (i) high prenatal levels of testosterone serve to 'organize' male facial features to subsequently reflect dominance and masculine characteristics presumably activated during puberty; and (ii) attractiveness is not directly related to testosterone levels. We conclude that facial dominance and masculinity reflect a male's perceived status rather than his physical attraction to women.


Experimental and computational determination of the hydrodynamics of mini vessel dissolution testing systems.
Mini vessel dissolution testing systems consist of a small-scale 100-mL vessel with a small paddle impeller, similar to the USP Apparatus 2, and are typically utilized when only small amounts of drug product are available during drug development. Despite their common industrial use, mini vessels have received little attention in the literature. Here, Computational Fluid Dynamics (CFD) was used to predict velocity profiles, flow patterns, and strain rate distribution in a mini vessel at different agitation speeds. These results were compared with experimental velocity measurements obtained with Particle Image Velocimetry (PIV). Substantial agreement was observed between CFD results and PIV data. The flow is strongly dominated by the tangential velocity component. Secondary flows consist of vertical upper and lower recirculation loops above and below the impeller. A low recirculation zone was observed in the lower part of the vessel. The radial and axial velocities in the region just below the impeller are very small especially in the innermost core zone below the paddle, where tablet dissolution occurs. Increasing agitation speed reduces the radius of this zone, which is always present at any speed, and only modestly increases the tangential flow intensity, with significant implication for dissolution testing in mini vessels.


Trocar types in laparoscopy.
Laparoscopic surgery has led to great clinical improvements in many fields of surgery; however, it requires the use of trocars, which may lead to complications as well as postoperative pain. The complications include intra-abdominal vascular and visceral injury, trocar site bleeding, herniation and infection. Many of these are extremely rare, such as vascular and visceral injury, but may be life-threatening; therefore, it is important to determine how these types of complications may be prevented. It is hypothesised that trocar-related complications and pain may be attributable to certain types of trocars. This systematic review was designed to improve patient safety by determining which, if any, specific trocar types are less likely to result in complications and postoperative pain. To analyse the rates of trocar-related complications and postoperative pain for different trocar types used in people undergoing laparoscopy, regardless of the condition. Two experienced librarians conducted a comprehensive search for randomised controlled trials (RCTs) in the Menstrual Disorders and Subfertility Group Specialised Register, Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, PsycINFO, CINAHL, CDSR and DARE (up to 26 May 2015). We checked trial registers and reference lists from trial and review articles, and approached content experts. RCTs that compared rates of trocar-related complications and postoperative pain for different trocar types used in people undergoing laparoscopy. The primary outcomes were major trocar-related complications, such as mortality, conversion due to any trocar-related adverse event, visceral injury, vascular injury and other injuries that required intensive care unit (ICU) management or a subsequent surgical, endoscopic or radiological intervention. Secondary outcomes were minor trocar-related complications and postoperative pain. We excluded trials that studied non-conventional laparoscopic incisions. Two review authors independently conducted the study selection, risk of bias assessment and data extraction. We used GRADE to assess the overall quality of the evidence. We performed sensitivity analyses and investigation of heterogeneity, where possible. We included seven RCTs (654 participants). One RCT studied four different trocar types, while the remaining six RCTs studied two different types. The following trocar types were examined: radially expanding versus cutting (six studies; 604 participants), conical blunt-tipped versus cutting (two studies; 72 participants), radially expanding versus conical blunt-tipped (one study; 28 participants) and single-bladed versus pyramidal-bladed (one study; 28 participants). The evidence was very low quality: limitations were insufficient power, very serious imprecision and incomplete outcome data. Primary outcomesFour of the included studies reported on visceral and vascular injury (571 participants), which are two of our primary outcomes. These RCTs examined 473 participants where radially expanding versus cutting trocars were used. We found no evidence of a difference in the incidence of visceral (Peto odds ratio (OR) 0.95, 95% confidence interval (CI) 0.06 to 15.32) and vascular injury (Peto OR 0.14, 95% CI 0.0 to 7.16), both very low quality evidence. However, the incidence of these types of injuries were extremely low (i.e. two cases of visceral and one case of vascular injury for all of the included studies). There were no cases of either visceral or vascular injury for any of the other trocar type comparisons. No studies reported on any other primary outcomes, such as mortality, conversion to laparotomy, intensive care admission or any re-intervention. Secondary outcomesFor trocar site bleeding, the use of radially expanding trocars was associated with a lower risk of trocar site bleeding compared to cutting trocars (Peto OR 0.28, 95% CI 0.14 to 0.54, five studies, 553 participants, very low quality evidence). This suggests that if the risk of trocar site bleeding with the use of cutting trocars is assumed to be 11.5%, the risk with the use of radially expanding trocars would be 3.5%. There was insufficient evidence to reach a conclusion regarding other trocar types, their related complications and postoperative pain, as no studies reported data suitable for analysis. Data were lacking on the incidence of major trocar-related complications, such as visceral or vascular injury, when comparing different trocar types with one another. However, caution is urged when interpreting these results because the incidence of serious complications following the use of a trocar was extremely low. There was very low quality evidence for minor trocar-related complications suggesting that the use of radially expanding trocars compared to cutting trocars leads to reduced incidence of trocar site bleeding. These secondary outcomes are viewed to be of less clinical importance.Large, well-conducted observational studies are necessary to answer the questions addressed in this review because serious complications, such as visceral or vascular injury, are extremely rare. However, for other outcomes, such as trocar site herniation, bleeding or infection, large observational studies may be needed as well. In order to answer these questions, it is advisable to establish an international network for recording these types of complications following laparoscopic surgery.


Measuring the outcomes of a comprehensive HIV care course: pilot test at the Infectious Diseases Institute, Kampala, Uganda.
To evaluate the effects of the Infectious Diseases Institute's 4-week course for African doctors on comprehensive management of HIV including antiretroviral therapy on four outcomes: (1) clinical skills, (2) clinical activities, (3) monitoring of HIV patients, and (4) training activities Clinical exam at beginning and end of course and at follow-up 3 to 4 months later, and a cross-section telephone survey. Forty-seven doctors attending the course (October 2004, November 2004, March 2005, and April 2005) agreed to participate. A 17-item Clinical Exam Checklist was used to assess clinical skills. A telephone survey was conducted 1 month after the course to collect data in four areas: clinical activities, monitoring of HIV patients, case studies on initiation of ART, and training activities. The course improved the clinical skills of doctors. Between the beginning and end of the course, their clinical skills improved significantly in 11 of 17 areas (n = 34). Between the end of the course and follow-up, their skills improved significantly in three areas (n = 14). The trainees were practicing HIV care and training. The telephone survey (n = 46) showed that 93% of trainees treated HIV patients, 35% provided training on HIV, and 47% monitored the weight of the last HIV patient treated (patient's weight was a clinical end point to measure health status). At follow-up, everyone provided training and trained an average of 20 people per month.


Deliberations about deliberative methods: issues in the design and evaluation of public participation processes.
A common thread weaving through the current public participation debate is the need for new approaches that emphasize two-way interaction between decision makers and the public as well as deliberation among participants. Increasingly complex decision making processes require a more informed citizenry that has weighed the evidence on the issue, discussed and debated potential decision options and arrived at a mutually agreed upon decision or at least one by which all parties can abide. We explore the recent fascination with deliberative methods for public involvement first by examining their origins within democratic theory, and then by focusing on the experiences with deliberative methods within the health sector. In doing so, we answer the following questions "What are deliberative methods and why have they become so popular? What are their potential contributions to the health sector?" We use this critical review of the literature as the basis for developing general principles that can be used to guide the design and evaluation of public involvement processes for the health-care sector in particular.