Peptides as adjuvants for ampicillin and oxacillin against methicillin-resistant Staphylococcus aureus (MRSA).
Fast emerging antibiotic resistance in pathogens requires special attention for strengthening the reservoir of antimicrobial compounds. In view of this, several peptides with known antimicrobial activities have been reported to enhance the efficacy of antibiotics against multidrug resistant (MDR) pathogens. In the present study, potential of peptides having distinct mechanism of action, if any, was evaluated to improve the efficacy of conventional antibiotics against methicillin-resistant S. aureus (MRSA). After primary screening of six peptides, two peptides namely T3 and T4 showing very high minimum inhibitory concentrations (MICs) were selected to assess their role in altering the MICs of antibiotics to which the pathogen was resistant. In the presence of the peptides, the MICs of the antibiotics were found to be reduced as per the fractional inhibitory concentration indices (FICI) and time kill assay. These observations prompted us to look for their mechanism of action. The effect of peptides on the morphology of pathogen by field emission scanning electron microscopy (FE-SEM) revealed no damage to the cells at the sub-inhibitory concentrations of the peptide which correlated well with the higher MIC of the peptide, indicating no direct impact on the pathogen. However, dielectric spectroscopy, confocal microscopy and flow cytometry confirmed the interaction and localization of peptides with the bacterial membrane. The peptides were also found to inhibit efflux of ethidium bromide which is the substrate for many proteins involved in efflux system. Therefore, it is speculated that the peptides after interacting with the membrane of the pathogen might have resulted in the inhibition of the efflux of antibiotics thereby reducing their effective concentrations. The study thus suggests that peptides with no antimicrobial activity of their own, can also enhance the efficacy of the antibiotics by interacting with the pathogen thereby, acting as adjuvants for the antibiotics.


Genome-Wide Analysis of Glioblastoma Patients with Unexpectedly Long Survival.
Glioblastoma (GBM), representing WHO grade IV astrocytoma, is a relatively common primary brain tumor in adults with an exceptionally dismal prognosis. With an incidence rate of over 10 000 cases in the United States annually, the median survival rate ranges from 10-15 months in IDH1/2-wildtype tumors and 24-31 months in IDH1/2-mutant tumors, with further variation depending on factors such as age, MGMT methylation status, and treatment regimen. We present a cohort of 4 patients, aged 37-60 at initial diagnosis, with IDH1-mutant GBMs that were associated with unusually long survival intervals after the initial diagnosis, currently ranging from 90 to 154 months (all still alive). We applied genome-wide profiling with a methylation array (Illumina EPIC Array 850k) and a next-generation sequencing panel to screen for genetic and epigenetic alterations in these tumors. All 4 tumors demonstrated methylation patterns and genomic alterations consistent with GBM. Three out of four cases showed focal amplification of the CCND2 gene or gain of the region on 12p that included CCND2, suggesting that this may be a favorable prognostic factor in GBM. As this study has a limited sample size, further evaluation of patients with similar favorable outcome is warranted to validate these findings.


A novel therapeutic combination for neuroblastoma: the vascular endothelial growth factor receptor/epidermal growth factor receptor/rearranged during transfection inhibitor vandetanib with 13-cis-retinoic acid.
High-risk cases of neuroblastoma have poor survival rates, and novel therapies are needed. Vandetanib (ZD6474, Zactima) is an inhibitor of the vascular endothelial growth factor receptor, epidermal growth factor receptor, and rearranged during transfection (RET) tyrosine kinases, which have each been implicated in neuroblastoma pathogenesis. The authors hypothesized that vandetanib combined with 13-cis-retinoic acid (CRA), a differentiating agent used in most current neuroblastoma treatment regimens, would be effective against neuroblastoma tumor models. The authors evaluated the effects of vandetanib with and without CRA on RET phosphorylation and on the proliferation and survival of human neuroblastoma cell lines in vitro. Using a subcutaneous mouse xenograft model of human neuroblastoma, they analyzed tumors treated with CRA, vandetanib, and the combination of vandetanib plus CRA for growth, gross and histologic appearance, vascularity, and apoptosis. Vandetanib treatment inhibited RET phosphorylation and resulted in induction of apoptosis in the majority of neuroblastoma cell lines in vitro, whereas CRA treatment induced morphologic differentiation and cell-cycle arrest. Treatment with vandetanib plus CRA resulted in more significant reduction in neuroblastoma cell viability than either alone. In a mouse xenograft model, the combination of vandetanib with CRA demonstrated significantly more growth inhibition than either alone, via both reduction in tumor vascularity and induction of apoptosis. Vandetanib induces neuroblastoma tumor cell death in vitro and reduces tumor growth and vascularity in vivo. The combination of vandetanib with CRA was more effective in reducing tumor growth than either treatment alone. The antitumor effects of vandetanib plus CRA suggest a novel combination for use in neuroblastoma patients.


Random phase data masks: fabrication tolerances and advantages of four phase level masks.
An analysis is presented of the intensity variations in the Fourier transform plane resulting from the use of a bilevel random phase mask for holographic data recording as suggested by Burckhardt. To maintain a fixed ratio of peak intensities between in-phase amplitude components and out-of-phase components for an imperfect phase mask, the permissible error in the phase shift varies as 1/N, where N is the number of apertures in the data mask. A factor of 2(1/2) reduction in the rms intensity fluctuations in both the Fourier transform plane where the hologram is recorded and in the retrieved image may be obtained by using four levels of phase shift. No further improvement is obtained with additional levels.


A randomized, controlled trial of platelet transfusions in thrombocytopenic premature infants.
A multicenter prospective, randomized controlled trial was conducted to determine whether early use of platelet concentrates would reduce the incidence or extension of intracranial hemorrhage or both in sick preterm infants with thrombocytopenia. The effects on bleeding as reflected by the amount of blood product support administered and a shortened bleeding time were assessed as secondary outcomes. Premature infants with a platelet count < 150 x 10(9)/L within the first 72 hours of life were randomly assigned to receive either conventional therapy or conventional therapy plus platelet concentrates (10 ml/kg). The platelet count was maintained < 150 x 10(9)/L until day 7 of life by one to three platelet transfusions. In 22 (28%) of the 78 treated infants and 19 (26%) of the 74 control infants, either a new intracranial hemorrhage developed or an already-present one became more extensive (p = 0.73). Similar numbers of infants had each grade of intracranial hemorrhage on both initial and follow-up ultrasonography. Similar numbers of infants received fresh frozen plasma and packed red blood cells, but treated infants received less of both. The bleeding time was prolonged in the treated group before the infusion of platelet concentrates but subsequently shortened (mean difference, 79.0; 95% confidence interval, 73.1 to 84.9). Subanalysis of the control group showed that infants with platelet counts < 60 x 10(9)/L (n = 21) on at least one occasion received more fresh frozen plasma and packed red blood cells than did those with platelet counts > 60 x 10(9)/L.(ABSTRACT TRUNCATED AT 250 WORDS)


c-Src-mediated phosphorylation of hnRNP K drives translational activation of specifically silenced mRNAs.
hnRNPK and hnRNP E1/E2 mediate translational silencing of cellular and viral mRNAs in a differentiation-dependent way by binding to specific regulatory sequences. The translation of 15-lipoxygenase (LOX) mRNA in erythroid precursor cells and of the L2 mRNA of human papilloma virus type 16 (HPV-16) in squamous epithelial cells is silenced when either of these cells is immature and is activated in maturing cells by unknown mechanisms. Here we address the question of how the silenced mRNA can be translationally activated. We show that hnRNP K and the c-Src kinase specifically interact with each other, leading to c-Src activation and tyrosine phosphorylation of hnRNP K in vivo and in vitro. c-Src-mediated phosphorylation reversibly inhibits the binding of hnRNP K to the differentiation control element (DICE) of the LOX mRNA 3' untranslated region in vitro and specifically derepresses the translation of DICE-bearing mRNAs in vivo. Our results establish a novel role of c-Src kinase in translational gene regulation and reveal a mechanism by which silenced mRNAs can be translationally activated.


An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy.
Scanning confocal microscopes offer improved rejection of out-of-focus noise and greater resolution than conventional imaging. In such a microscope, the imaging and condenser lenses are identical and confocal. These two lenses are replaced by a single lens when epi-illumination is used, making confocal imaging particularly applicable to incident light microscopy. We describe the results we have obtained with a confocal system in which scanning is performed by moving the light beam, rather than the stage. This system is considerably faster than the scanned stage microscope and is easy to use. We have found that confocal imaging gives greatly enhanced images of biological structures viewed with epifluorescence. The improvements are such that it is possible to optically section thick specimens with little degradation in the image quality of interior sections.


A new vision of evaluating gene expression signatures.
Gene expression profiles based on high-throughput technologies contribute to molecular classifications of different cell lines and consequently to clinical diagnostic tests for cancer types and other diseases. Statistical techniques and dimension reduction methods have been devised for identifying minimal gene subset with maximal discriminative power. For sets of in silico candidate genes, assuming a unique gene signature or performing a parsimonious signature evaluation seems to be too restrictive in the context of in vitro signature validation. This is mainly due to the high complexity of largely correlated expression measurements and the existence of various oncogenic pathways. Consequently, it might be more advantageous to identify and evaluate multiple gene signatures with a similar good predictive power, which are referred to as near-optimal signatures, to be made available for biological validation. For this purpose we propose the bead-chain-plot approach originating from swarm intelligence techniques, and a small scale computational experiment is conducted in order to convey our vision. We simulate the acquisition of candidate genes by using a small pool of differentially expressed genes derived from microarray-based CNS tumour data. The application of the bead-chain-plot provides experimental evidence for improved classifications by using near-optimal signatures in validation procedures.


Engineered cystine knot peptides that bind alphavbeta3, alphavbeta5, and alpha5beta1 integrins with low-nanomolar affinity.
There is a critical need for compounds that target cell surface integrin receptors for applications in cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium trypsin inhibitor (EETI-II), a knottin peptide from the squash family of protease inhibitors, as a new class of integrin-binding agents. We generated yeast-displayed libraries of EETI-II by substituting its 6-amino acid trypsin binding loop with 11-amino acid loops containing the Arg-Gly-Asp integrin binding motif and randomized flanking residues. These libraries were screened in a high-throughput manner by fluorescence-activated cell sorting to identify mutants that bound to alpha(v)beta(3) integrin. Select peptides were synthesized and were shown to compete for natural ligand binding to integrin receptors expressed on the surface of U87MG glioblastoma cells with half-maximal inhibitory concentration values of 10-30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both alpha(v)beta(3) and alpha(v)beta(5) integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to alpha(v)beta(3), alpha(v)beta(5), and alpha(5)beta(1) integrins. This finding has important clinical implications because all three of these receptors can be coexpressed on tumors. In addition, we showed that engineered knottin peptides inhibit tumor cell adhesion to the extracellular matrix protein vitronectin, and in some cases fibronectin, depending on their integrin binding specificity. Collectively, these data validate EETI-II as a scaffold for protein engineering, and highlight the development of unique integrin-binding peptides with potential for translational applications in cancer.


Biomimetic Tissue-Engineered Bone Substitutes for Maxillofacial and Craniofacial Repair: The Potential of Cell Sheet Technologies.
Maxillofacial defects are complex lesions stemming from various etiologies: accidental, congenital, pathological, or surgical. A bone graft may be required when the normal regenerative capacity of the bone is exceeded or insufficient. Surgeons have many options available for bone grafting including the "gold standard" autologous bone graft. However, this approach is not without drawbacks such as the morbidity associated with harvesting bone from a donor site, pain, infection, or a poor quantity and quality of bone in some patient populations. This review discusses the various bone graft substitutes used for maxillofacial and craniofacial repair: allografts, xenografts, synthetic biomaterials, and tissue-engineered substitutes. A brief overview of bone tissue engineering evolution including the use of mesenchymal stem cells is exposed, highlighting the first clinical applications of adipose-derived stem/stromal cells in craniofacial reconstruction. The importance of prevascularization strategies for bone tissue engineering is also discussed, with an emphasis on recent work describing substitutes produced using cell sheet-based technologies, including the use of thermo-responsive plates and the self-assembly approach of tissue engineering. Indeed, considering their entirely cell-based design, these natural bone-like substitutes have the potential to closely mimic the osteogenicity, osteoconductivity, osteoinduction, and osseointegration properties of autogenous bone for maxillofacial and craniofacial reconstruction.


Infrared spectroscopic characterization of phosphate binding at the goethite-water interface.
The interaction between phosphates and soil mineral surfaces, such as Fe- and Al-(oxyhydr)oxides, plays a crucial role in the immobilization of P and thus its availability for plants. The reactions of phosphates with Fe-hydroxides and especially goethite have been studied extensively. But a molecular-level picture of the phosphate binding mechanisms at the goethite-water interface is still lacking. Therefore, in the current contribution we have explored the molecular binding mechanisms for the adsorbed phosphate at the goethite-water interface by performing sorption kinetics experiments for orthophosphate and characterizing the adsorbed species by FT-IR spectroscopy. In parallel, periodic DFT calculations have been performed to explore the interaction mechanisms and to assign the IR spectra for monodentate (M) and bidentate (B) orthophosphate complexes at two different goethite surface planes (010 and 100) in the presence of water. In general, our interaction energy results give evidence that the mono-protonated B phosphate complex is favored to be formed at the goethite-water interface, although the M motif could exist as a minor fraction. Moreover, it was found that water plays an important role in controlling the phosphate adsorption process at the goethite surfaces. The interfacial water molecules form H-bonds (HBs) with the phosphate as well as with the goethite surface atoms. Furthermore, some water molecules form covalent bonds with goethite's Fe atoms while others dissociate at the surface to protons and hydroxyl groups. The present theoretical assignment of IR spectra introduces a benchmark for characterizing experimental IR data for the adsorbed KH2PO4 species at the goethite-water interface. In particular, the IR spectra of the mono-protonated (2O + 1Fe) B complex at the 010 goethite surface plane and the M complex at the 100 goethite surface plane were found to be consistent with the experimental data. In order to explore the role of different abundances of surface planes and binding motifs, IR spectra obtained from weighted averages have been analyzed. The results confirmed the conclusions drawn from interaction energy calculations.


Epidemiology and Medication Pattern Change of Psoriatic Diseases in Taiwan from 2000 to 2013: A Nationwide, Population-based Cohort Study.
To analyze the trend of prevalence and incidence rates for psoriatic arthritis (PsA) and psoriasis in Taiwan, and to determine the changes in medication patterns. Data were collected from the Taiwan National Health Insurance Research Database, which covered at least 95% of the population from 2000 to 2013. International Classification of Diseases, 9th edition (ICD-9) was used to identify PsA (ICD-9 696.0) and other psoriasis (ICD-9 696.1). Medications were identified by Anatomical Therapeutic Chemical Classification code. We calculated the annual age standardized prevalence and incidence rate of PsA and psoriasis in individuals aged ≥ 16 years from 2000 to 2013, and used the Poisson regression to test the trends by Wald chi-square statistic. The prevalence (per 100,000 population) of psoriatic diseases between 2000 and 2013 increased from 11.12 to 37.75 for PsA, and from 179.2 to 281.5 for psoriasis. The incidence (per 100,000 person-yrs) increased from 3.64 to 6.91 in PsA, while there was no significant change in psoriasis. Prevalence and incidence in PsA were more rapidly increased than in psoriasis. Sex ratio (men to women) of PsA decreased from 2.0 to 1.5 in 2000 and 2013, respectively. There was an increase in the use of disease-modifying antirheumatic drugs (DMARD), especially biologics, which is significantly different from topical therapies. The prevalence and incidence rates of psoriatic disease, especially PsA, were increasing in Taiwan. The medication pattern showed an increase in DMARD and biologics, while use of topical therapies decreased.


Integrated Transcriptional Regulatory Network of Quorum Sensing, Replication Control, and SOS Response in Dinoroseobacter shibae.
Quorum sensing (QS) coordinates population wide gene expression of bacterial species. Highly adaptive traits like gene transfer agents (GTA), morphological heterogeneity, type 4 secretion systems (T4SS), and flagella are QS controlled in Dinoroseobacter shibae, a Roseobacter model organism. Its QS regulatory network is integrated with the CtrA phosphorelay that controls cell division in alphaproteobacteria. To elucidate the network topology, we analyzed the transcriptional response of the QS-negative D. shibae strain ΔluxI1 toward externally added autoinducer (AI) over a time period of 3 h. The signaling cascade is initiated by the CtrA phosphorelay, followed by the QS genes and other target genes, including the second messenger c-di-GMP, competence, flagella and pili. Identification of transcription factor binding sites in promoters of QS induced genes revealed the integration of QS, CtrA phosphorelay and the SOS stress response mediated by LexA. The concentration of regulatory genes located close to the origin or terminus of replication suggests that gene regulation and replication are tightly coupled. Indeed, addition of AI first stimulates and then represses replication. The restart of replication comes along with increased c-di-GMP levels. We propose a model in which QS induces replication followed by differentiation into GTA producing and non-producing cells. CtrA-activity is controlled by the c-di-GMP level, allowing some of the daughter cells to replicate again. The size of the GTA producing subpopulation is tightly controlled by QS via the AI Synthase LuxI2. Finally, induction of the SOS response allows for integration of GTA DNA into the host chromosome.


Antibody-catalyzed degradation of cocaine.
Immunization with a phosphonate monoester transition-state analog of cocaine provided monoclonal antibodies capable of catalyzing the hydrolysis of the cocaine benzoyl ester group. An assay for the degradation of radiolabeled cocaine identified active enzymes. Benzoyl esterolysis yields ecgonine methyl ester and benzoic acid, fragments devoid of cocaine's stimulant activity. Passive immunization with such an artificial enzyme could provide a treatment for dependence by blunting reinforcement.


A Comparison of Cognitive and Behavioral Approaches for Reducing Cost Bias in Social Anxiety.
Cognitive-behavioral theories suggest that anxiety is maintained in part by estimates of the probability and cost of feared negative outcomes. Social phobia may be unique among the anxiety disorders in that it is characterized by overestimates of the cost of events that are objectively noncatastrophic (e.g., committing social mishaps). As such, treatment approaches that target cost bias may be particularly effective in reducing social phobia symptoms. This study examined the efficacy of 2 cost-specific techniques in a single-session intervention for social anxiety. Individuals (n = 61) with elevated social interaction anxiety were randomly assigned to an expressive writing control condition, a cognitive restructuring condition, or a behavioral experiment condition. Results demonstrated that the cognitive restructuring condition produced significantly greater improvement in indices of social anxiety than the other conditions. Reduction in cost bias fully mediated the significantly greater improvement in social interaction anxiety in the cognitive restructuring condition relative to the behavioral experiment condition. The present findings highlight the value of techniques designed to reduce cost biases in social anxiety. Clinical implications are discussed.


Identification of lymphatic vessels by using a monoclonal antibody specific for the human thoracic duct.
Light microscopic identification of lymphatic vessels of the human small intestine was studied immuno-histochemically with a monoclonal antibody specific for human thoracic duct (mAb-D) and rabbit anti-human laminin antibody (anti-L). Two types of vessels were observed with anti-L; one strongly reacted with regularly outlined vessels and the other weakly reacted with irregularly outlined vessels and thin walls. Immunoreactivity to mAb-D was strong in the weakly reacting vessels, and the strongly reacting vessels did not react to mAb-D. Immunoreactivity of lymphatic vessels to anti-L has been reported to be weaker than that of the blood vessels; only irregularly outlined vessels which were weakly immunostained with anti-L reacted to mAb-D and no cross-reaction to mAb-D was observed in the regularly outlined vessels. This indicates that the vessels strongly reacting with anti-L are blood vessels and the vessels immunostained with mAb-D must be lymphatics. The study shows that it is possible to identify lymphatic vessels light microscopically by mAb-D. This method will be useful in studying the fine distribution of lymphatic vessels in normal tissue and also in pathological tissue such as metastasis of malignant tumors.


Neural correlates of semantic competition during processing of ambiguous words.
The current study investigated the neural correlates that underlie the processing of ambiguous words and the potential effects of semantic competition on that processing. Participants performed speeded lexical decisions on semantically related and unrelated prime-target pairs presented in the auditory modality. The primes were either ambiguous words (e.g., ball) or unambiguous words (e.g., athlete), and targets were either semantically related to the dominant (i.e., most frequent) meaning of the ambiguous prime word (e.g., soccer) or to the subordinate (i.e., less frequent) meaning (e.g., dance). Results showed increased activation in the bilateral inferior frontal gyrus (IFG) for ambiguous-related compared to unambiguous-related stimulus pairs, demonstrating that prefrontal areas are activated even in an implicit task where participants are not required to explicitly analyze the semantic content of the stimuli and to make an overt selection of a particular meaning based on this analysis. Additionally, increased activation was found in the left IFG and the left cingulate gyrus for subordinate meaning compared to dominant meaning conditions, suggesting that additional resources are recruited in order to resolve increased competition demands in accessing the subordinate meaning of an ambiguous word.


Pazopanib in advanced vascular sarcomas: an EORTC Soft Tissue and Bone Sarcoma Group (STBSG) retrospective analysis.
Pazopanib is a multitargeted tyrosine kinase inhibitor approved for the treatment of patients with selective subtypes of advanced soft tissue sarcoma (STS) who have previously received standard chemotherapy including anthracyclines. Data on the efficacy in vascular sarcomas are limited. The main objective of this study was to investigate the activity of pazopanib in vascular sarcomas. A retrospective study of patients with advanced vascular sarcomas, including angiosarcoma (AS), epithelioid hemangioendothelioma (HE) and intimal sarcoma (IS) treated with pazopanib in real life practice at EORTC centers as well as patients treated within the EORTC phase II and III clinical trials (62043/62072) was performed. Patient and tumor characteristics were collected. Response was assessed according to RECIST 1.1. and survival analysis was performed. Fifty-two patients were identified, 40 (76.9%), 10 (19.2%) and two (3.8%) with AS, HE and IS, respectively. The response rate was eight (20%), two (20%) and two (100%) in the AS, HE and IS subtypes, respectively. There was no significant difference in response rate between cutaneous and non-cutaneous AS and similarly between radiation-associated and non-radiation-associated AS. Median progression-free survival (PFS) and median overall survival (OS; from commencing pazopanib) were three months (95% CI 2.1-4.4) and 9.9 months (95% CI 6.5-11.3) in AS, respectively. The activity of pazopanib in AS is comparable to its reported activity in other STS subtypes. In this study, the activity of pazopanib was similar in cutaneous/non-cutaneous and in radiation/non-radiation-associated AS. In addition, pazopanib showed promising activity in HE and IS, worthy of further evaluation.


Association of tibialis posterior tendon pathology with other radiographic findings in the foot: a case-control study.
The purpose of this study was to analyze the prevalence of spring ligament pathology and other radiographic changes related to flatfoot deformity in the presence of different degrees of tibialis posterior tendon pathology. A total of 72 patients (24 with tibialis posterior tendon abnormality and 48 sex- and age-matched controls) were evaluated for tibialis posterior tendon pathology, spring ligament pathology, and plain pedal radiographic angles, including cuboid abduction, talar declination, calcaneal inclination, and Meary's angles. The patients with tibialis posterior tendon pathology were subdivided into either minor (Type I) or severe (Type II/III), according to the Conti classification of tibialis posterior tendon pathology on MRI. All the continuous data of radiographic angles were dichotomized into either a flatfoot group or normal/cavus foot group. Associations between these nominal variables were analyzed. There was no association between Type I tibialis posterior tendon pathology and spring ligament pathologies (OR = 0.8, 95% CI = 0.15-4.65). Conversely, every patient with Type II/III tibialis posterior tendon pathology had spring ligament abnormality. Type II/III group also showed statistically significant associations with both increased talar declination angle (OR = 10.4, 95% CI = 1.62-109.22) and Meary's angle (OR = 7.5, 95% CI = 1.35-51.12), while no such associations were found with Type I tibialis posterior tendon pathology (OR = 1.0, 95% CI = 0.18-6.18 with talar declination angle; OR = 3.9, 95% CI = 0.65-27.71 with Meary's angle). In this investigation, only advanced tibialis posterior tendon pathology was statistically significantly associated with adult-acquired flatfoot deformity and spring ligament pathology.


A CaV1.1 Ca2+ channel splice variant with high conductance and voltage-sensitivity alters EC coupling in developing skeletal muscle.
The Ca(2+) channel alpha(1S) subunit (Ca(V)1.1) is the voltage sensor in skeletal muscle excitation-contraction (EC) coupling. Upon membrane depolarization, this sensor rapidly triggers Ca(2+) release from internal stores and conducts a slowly activating Ca(2+) current. However, this Ca(2+) current is not essential for skeletal muscle EC coupling. Here, we identified a Ca(V)1.1 splice variant with greatly distinct current properties. The variant of the CACNA1S gene lacking exon 29 was expressed at low levels in differentiated human and mouse muscle, and up to 80% in myotubes. To test its biophysical properties, we deleted exon 29 in a green fluorescent protein (GFP)-tagged alpha(1S) subunit and expressed it in dysgenic (alpha(1S)-null) myotubes. GFP-alpha(1S)Delta 29 was correctly targeted into triads and supported skeletal muscle EC coupling. However, the Ca(2+) currents through GFP-alpha(1S)Delta 29 showed a 30-mV left-shifted voltage dependence of activation and a substantially increased open probability, giving rise to an eightfold increased current density. This robust Ca(2+) influx contributed substantially to the depolarization-induced Ca(2+) transient that triggers contraction. Moreover, deletion of exon 29 accelerated current kinetics independent of the auxiliary alpha(2)delta-1 subunit. Thus, characterizing the Ca(V)1.1 Delta 29 splice variant revealed the structural bases underlying the specific gating properties of skeletal muscle Ca(2+) channels, and it suggests the existence of a distinct mode of EC coupling in developing muscle.


Spitz and Wingless, emanating from distinct borders, cooperate to establish cell fate across the Engrailed domain in the Drosophila epidermis.
A key step in development is the establishment of cell type diversity across a cellular field. Segmental patterning within the Drosophila embryonic epidermis is one paradigm for this process. At each parasegment boundary, cells expressing the Wnt family member Wingless confront cells expressing the homeoprotein Engrailed. The Engrailed-expressing cells normally differentiate as one of two alternative cell types. In investigating the generation of this cell type diversity among the 2-cell-wide Engrailed stripe, we previously showed that Wingless, expressed just anterior to the Engrailed cells, is essential for the specification of anterior Engrailed cell fate. In a screen for additional mutations affecting Engrailed cell fate, we identified anterior open/yan, a gene encoding an inhibitory ETS-domain transcription factor that is negatively regulated by the Rasl-MAP kinase signaling cascade. We find that Anterior Open must be inactivated for posterior Engrailed cells to adopt their correct fate. This is achieved by the EGF receptor (DER), which is required autonomously in the Engrailed cells to trigger the Ras1-MAP kinase pathway. Localized activation of DER is accomplished by restricted processing of the activating ligand, Spitz. Processing is confined to the cell row posterior to the Engrailed domain by the restricted expression of Rhomboid. These cells also express the inhibitory ligand Argos, which attenuates the activation of DER in cell rows more distant from the ligand source. Thus, distinct signals flank each border of the Engrailed domain, as Wingless is produced anteriorly and Spitz posteriorly. Since we also show that En cells have the capacity to respond to either Wingless or Spitz, these cells must choose their fate depending on the relative level of activation of the two pathways.


The specification of heart mesoderm occurs during gastrulation in Xenopus laevis.
The establishment of heart mesoderm during Xenopus development has been examined using an assay for heart differentiation in explants and explant combinations in culture. Previous studies using urodele embryos have shown that the heart mesoderm is induced by the prospective pharyngeal endoderm during neurula and postneurula stages. In this study, we find that the specification of heart mesoderm must begin well before the end of gastrulation in Xenopus embryos. Explants of prospective heart mesoderm isolated from mid- or late neurula stages were capable of heart formation in nearly 100% of cases, indicating that the specification of heart mesoderm is complete by midneurula stages. Moreover, inclusion of pharyngeal endoderm had no statistically significant effect upon either the frequency of heart formation or the timing of the initiation of heartbeat in explants of prospective heart mesoderm isolated after the end of gastrulation. When the superficial pharyngeal endoderm was removed at the beginning of gastrulation, experimental embryos formed hearts, as did explants of prospective heart mesoderm from such embryos. These results indicate that the inductive interactions responsible for the establishment of heart mesoderm occur prior to the end of gastrulation and do not require the participation of the superficial pharyngeal endoderm.


CD81 protein is expressed at high levels in normal germinal center B cells and in subtypes of human lymphomas.
CD81 is a tetraspanin cell surface protein that regulates CD19 expression in B lymphocytes and enables hepatitis C virus infection of human cells. Immunohistologic analysis in normal hematopoietic tissue showed strong staining for CD81 in normal germinal center B cells, a cell type in which its increased expression has not been previously recognized. High-dimensional flow cytometry analysis of normal hematopoietic tissue confirmed that among B- and T-cell subsets, germinal center B cells showed the highest level of CD81 expression. In more than 800 neoplastic tissue samples, its expression was also found in most non-Hodgkin lymphomas. Staining for CD81 was rarely seen in multiple myeloma, Hodgkin lymphoma, or myeloid leukemia. In hierarchical cluster analysis of diffuse large B-cell lymphoma, staining for CD81 was most similar to other germinal center B cell-associated markers, particularly LMO2. By flow cytometry, CD81 was expressed in diffuse large B-cell lymphoma cells independent of the presence or absence of CD10, another germinal center B-cell marker. The detection of CD81 in routine biopsy samples and its differential expression in lymphoma subtypes, particularly diffuse large B-cell lymphoma, warrant further study to assess CD81 expression and its role in the risk stratification of patients with diffuse large B-cell lymphoma.


Titration of histidine 62 in R67 dihydrofolate reductase is linked to a tetramer<-->two-dimer equilibrium.
R67 dihydrofolate reductase (DHFR) is an R-plasmid encoded protein that confers clinical resistance to the antibacterial drug trimethoprim. To determine whether an acidic titration in kinetic pH profiles is related to titration of histidines 62, 162, 262, and 362, the stability of tetrameric R67 DHFR has been monitored as a function of pH. For the pH range 5-8, tetrameric R67 DHFR reversibly dissociates into dimers, as monitored by ultracentrifugation and molecular sieving techniques. From the crystal structures of dimeric and tetrameric R67 DHFR [Matthews et al. (1986) Biochemistry 25, 4194-4204] (Narayana, Matthews, and Xuong, personal communication), symmetry-related histidines 62, 162, 262, and 362 occur at the two dimer-dimer interfaces and protonation of these residues could destabilize tetrameric R67 DHFR. Ionization of these histidines was confirmed by monitoring the chemical shifts of the C2 proton in NMR experiments, and best fits of an incomplete titration curve yield a pKa of 6.77. Since tryptophans 38, 138, 238, and 338 also occur at the dimer-dimer interfaces, fluorescence additionally monitors the tetramer-two dimers equilibrium. When fluorescence was monitored over the pH range 5-8, a protein concentration dependence of fluorescence was observed and global fitting of three titration curves yielded Kd = 9.72 nM and pKa = 6.84 for the linked reactions: [formula: see text] Modification of H62, H162, H262, and H362 by diethyl pyrocarbonate stabilizes dimeric R67 DHFR and causes a 200-600-fold decrease in catalytic efficiency. Decreased catalytic activity in dimeric R67 DHFR is presumably due to loss of the putative single active site pore found in tetrameric R67 DHFR.


Quantum codes give counterexamples to the unique preimage conjecture of the N-representability problem.
It is well known that the ground state energy of many-particle Hamiltonians involving only 2-body interactions can be obtained using constrained optimizations over density matrices which arise from reducing an N-particle state. While determining which 2-particle density matrices are "N-representable" is a computationally hard problem, all known extreme N-representable 2-particle reduced density matrices arise from a unique N-particle preimage, satisfying a conjecture established in 1972. We present explicit counterexamples to this conjecture through giving Hamiltonians with 2-body interactions which have degenerate ground states that cannot be distinguished by any 2-body operator. We relate the existence of such counterexamples to quantum error correction codes and topologically ordered spin systems.


Fast segmentation and high-quality three-dimensional volume mesh creation from medical images for diffuse optical tomography.
Multimodal approaches that combine near-infrared (NIR) and conventional imaging modalities have been shown to improve optical parameter estimation dramatically and thus represent a prevailing trend in NIR imaging. These approaches typically involve applying anatomical templates from magnetic resonance imaging/computed tomography/ultrasound images to guide the recovery of optical parameters. However, merging these data sets using current technology requires multiple software packages, substantial expertise, significant time-commitment, and often results in unacceptably poor mesh quality for optical image reconstruction, a reality that represents a significant roadblock for translational research of multimodal NIR imaging. This work addresses these challenges directly by introducing automated digital imaging and communications in medicine image stack segmentation and a new one-click three-dimensional mesh generator optimized for multimodal NIR imaging, and combining these capabilities into a single software package (available for free download) with a streamlined workflow. Image processing time and mesh quality benchmarks were examined for four common multimodal NIR use-cases (breast, brain, pancreas, and small animal) and were compared to a commercial image processing package. Applying these tools resulted in a fivefold decrease in image processing time and 62% improvement in minimum mesh quality, in the absence of extra mesh postprocessing. These capabilities represent a significant step toward enabling translational multimodal NIR research for both expert and nonexpert users in an open-source platform.


A diversity of localized timescales in network activity.
Neurons show diverse timescales, so that different parts of a network respond with disparate temporal dynamics. Such diversity is observed both when comparing timescales across brain areas and among cells within local populations; the underlying circuit mechanism remains unknown. We examine conditions under which spatially local connectivity can produce such diverse temporal behavior. In a linear network, timescales are segregated if the eigenvectors of the connectivity matrix are localized to different parts of the network. We develop a framework to predict the shapes of localized eigenvectors. Notably, local connectivity alone is insufficient for separate timescales. However, localization of timescales can be realized by heterogeneity in the connectivity profile, and we demonstrate two classes of network architecture that allow such localization. Our results suggest a framework to relate structural heterogeneity to functional diversity and, beyond neural dynamics, are generally applicable to the relationship between structure and dynamics in biological networks. DOI: http://dx.doi.org/10.7554/eLife.01239.001.


The effects of season and agriculture on nitrous oxide production in headwater streams.
Streams and rivers are a globally significant source of nitrous oxide (N(2)O), a potent greenhouse gas. However, there remains much uncertainty in the magnitude of N(2)O emissions from these sources, partly due to an incomplete understanding of the factors that control microbial N(2)O production in lotic sediments. During 2004-2005 we measured sediment N(2)O production in 12 headwater streams across an agricultural land use gradient. Stream water nitrate (NO(3)(-)) concentrations were positively related to the proportion of agricultural land use in the basin and frequently exceeded 20 mg N L(-1) in the stream draining the most agricultural basin. Stream sediments were nearly always a net source of N(2)O, and production rates were positively related to stream water NO(3)(-) concentrations and sediment carbon content. There were no seasonal patterns in N(2)O production rates during 2004, but stream water NO(3)(-) and N(2)O production both peaked during the winter of 2005. The spike in NO(3)(-) concentrations likely resulted from winter rain and snowmelt that flushed NO(3)(-) from the soils following a dry summer and fall. In turn, the elevated stream water NO(3)(-) concentrations stimulated in-stream N(2)O production rates. Overall, we were only able to explain 29% of the variation in N(2)O production rates on a log scale. The unexplained variation may be due to differences in the fraction of denitrified NO(3)(-) that is converted to N(2)O among the study sites, or that our measures of substrate availability in the water column were not reflective of substrate availability in the porewater used by denitrifiers.


Transport Barriers Influence the Activation of Anti-Tumor Immunity: A Systems Biology Analysis.
Effective anti-cancer immune responses require activation of one or more naïve T cells. If the correct naïve T cell encounters its cognate antigen presented by an antigen presenting cell, then the T cell can activate and proliferate. Here, mathematical modeling is used to explore the possibility that immune activation in lymph nodes is a rate-limiting step in anti-cancer immunity and can affect response rates to immune checkpoint therapy. The model provides a mechanistic framework for optimizing cancer immunotherapy and developing testable solutions to unleash anti-tumor immune responses for more patients with cancer. The results show that antigen production rate and trafficking of naïve T cells into the lymph nodes are key parameters and that treatments designed to enhance tumor antigen production can improve immune checkpoint therapies. The model underscores the potential of radiation therapy in augmenting tumor immunogenicity and neoantigen production for improved ICB therapy, while emphasizing the need for careful consideration in cases where antigen levels are already sufficient to avoid compromising the immune response.


Tractor overturn concerns in Iowa: perspectives from the Keokuk county rural health study.
Agriculture remains one of the most hazardous industries in the U.S., with tractor overturns producing the greatest number of agricultural machinery-related fatalities. Rollover protective structures (ROPS) and seatbelts effectively reduce tractor overturn deaths. However, a large proportion of tractors in use in American agriculture are older tractors without ROPS and seatbelts. This article describes the tractor-related responses from participants in a population-based study conducted in Keokuk County, Iowa. This study was designed to measure rural and agricultural adverse health and injury outcomes and their respective risk factors. Questionnaires were partially developed from well-documented national surveys. Questions about agricultural machinery use, presence of safety equipment on the machinery, work practices, and attitudes about farm safety were included. Study participants on farms who owned tractors had an average of 3.1 tractors with an average age of 27 years. Only 39% of the 665 tractors had ROPS. Tractor age was associated with the presence of ROPS; 84% of tractors manufactured after 1984 were ROPS-equipped, whereas only 3% of tractors manufactured before 1960 were ROPS-equipped. ROPS-equipped tractors were significantly more common on larger farms and households with higher income. Only 4% of the farmers reported that their tractors had seatbelts and they wore them when operating their tractors. The results of this study support the findings of other studies, which indicate that many older tractors without ROPS and seatbelts remain in use in American agriculture. Until a dramatic reduction in the number of tractors in the U.S. operated without ROPS and seatbelts is achieved, the annual incidence of 120 to 130 deaths associated with tractor overturns will persist.


Nucleotide excision repair gene variants and association with survival in osteosarcoma patients treated with neoadjuvant chemotherapy.
The aim of this study was to investigate the role of common polymorphisms in the nucleotide excision repair pathway genes in the tumorigenesis of osteosarcoma and in the response to DNA damaging therapies, such as cisplatin-based neoadjuvant therapy. Excision repair cross-complementing (ERCC) group 2 (XPD; rs13181 and rs1799793), group 5 (XPG; rs17655) and group 1 (XPA; rs3212986 and rs11615) polymorphisms were analyzed in a group of 130 homogenously treated patients with high-grade osteosarcoma, for association with event-free survival (EFS), using the Kaplan-Meier plots and log-rank test. A positive association was observed between both XPD single-nucleotide polymorphisms and an increased EFS (hazards ratio (HR) = 0.34, 95% confidence interval (CI) 0.12-0.98 and HR = 0.19, 95% CI 0.05-0.77, respectively). We had also performed a case-control study for relative risk to develop osteosarcoma. Patients carrying at least one variant allele of XPD rs1799793 had a reduced risk of developing osteosarcoma, compared with wild-type patients (odds ratio = 0.55, 95% CI 0.36-0.84). This study suggests that XPD rs1799793 could be a marker of osteosarcoma associated with features conferring either a better prognosis or a better outcome after platinum therapy, or both.


Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo.
Pore-forming toxins (PFTs) constitute the single largest class of proteinaceous bacterial virulence factors and are made by many of the most important bacterial pathogens. Host responses to these toxins are complex and poorly understood. We find that the endoplasmic reticulum unfolded protein response (UPR) is activated upon exposure to PFTs both in Caenorhabditis elegans and in mammalian cells. Activation of the UPR is protective in vivo against PFTs since animals that lack either the ire-1-xbp-1 or the atf-6 arms of the UPR are more sensitive to PFT than wild-type animals. The UPR acts directly in the cells targeted by the PFT. Loss of the UPR leads to a normal response against unrelated toxins or a pathogenic bacterium, indicating its PFT-protective role is specific. The p38 mitogen-activated protein (MAPK) kinase pathway has been previously shown to be important for cellular defenses against PFTs. We find here that the UPR is one of the key downstream targets of the p38 MAPK pathway in response to PFT since loss of a functional p38 MAPK pathway leads to a failure of PFT to properly activate the ire-1-xbp-1 arm of the UPR. The UPR-mediated activation and response to PFTs is distinct from the canonical UPR-mediated response to unfolded proteins both in terms of its activation and functional sensitivities. These data demonstrate that the UPR, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack.


Nonoverlapping T and B cell determinants on an hepatitis B surface antigen pre-S(2) region synthetic peptide.
We have examined T cell recognition of a hepatitis B surface antigen (HBsAg), pre-S(2)-region synthetic peptide, p120-145, in terms of fine specificity, H-2-linked genetic influences, comparison to antibody binding, and relevance to T cell recognition of the native protein. We showed that the immune response to the synthetic peptide is regulated by H-2-linked genes, but that the pattern of H-2 restriction differed from that observed for the native anti-pre-S(2) response. Dominant and nonoverlapping T cell and B cell recognition sites were identified on the synthetic peptide p120-145. T cell recognition is focussed on the NH2-terminal sequence, and antibody (B cell) recognition is focussed on the COOH-terminal sequence. The fine specificity of T cell recognition of p120-145 was defined by a single, subtype-dependent amino acid substitution. With respect to the immunogenicity of p120-145, the synthetic peptide containing both T and B cell determinants is highly immunogenic in responder strains, whereas separate T or B cell peptide determinants are minimally immunogenic. Furthermore, the synthetic T cell recognition site can prime T cell help for antibody production to the synthetic B cell site, which is crossreactive with the native pre-S(2) region of HBsAg/p33 particles. This system provides evidence that totally synthetic T cell and B cell recognition sites can be combined to yield a functional immunogen.


Staged Inertial Microfluidic Focusing for Complex Fluid Enrichment.
Microfluidic inertial focusing reliably and passively aligns small particles and cells through a combination of competing inertial fluid forces. The equilibrium behavior of inertially focused particles in straight channels has been extensively characterized and has been shown to be a strong function of channel size, geometry and particle size. We demonstrate that channels of varying geometry may be combined to produce a staged device capable of high throughput particle and cell concentration and efficient single pass complex fluid enrichment. Straight and asymmetrically curved microchannels were combined in series to accelerate focusing dynamics and improve concentration efficiency. We have investigated single and multiple pass concentration efficiency and results indicate that these devices are appropriate for routine cell handling operations, including buffer exchange. We demonstrate the utility of these devices by performing a ubiquitous fluorescence staining assay on-chip while sacrificing very little sample or processing time relative to centrifugation. Staged concentration is particularly desirable for point of care settings in which more conventional instrumentation is impractical or cost-prohibitive.


Evolution of calcium-carbonate skeletons in the Hydractiniidae.
Biomineralization has mostly been studied in the class Anthozoa (Phylum Cnidaria), but very little is known about the evolution of the calcified skeleton in the class Hydrozoa or about the processes leading to its formation. The evolution of the calcified skeleton is here investigated in the hydrozoan family Hydractiniidae. A phylogenetic analysis of ribosomal, mitochondrial, and nuclear-protein-coding DNA sequences supported two independent origins of the calcified skeleton within the Hydractiniidae and indicates a case of parallel evolution, as suspected in the Anthozoa. Neither of the two origins of skeleton in the Hydractiniidae has led to either speciose or numerically abundant species, in contrast with other skeletonized hydrozoan families. Finally, we show that the origin of calcified skeletons in the Hydractiniidae is significantly correlated with the distribution of species with calcium carbonate granules within a polyp's gastrodermal cells. This suggests that the presence of these granules precedes the origin of a full skeleton.


Choice in the context of informal care-giving.
Extending choice and control for social care service users is a central feature of current English policies. However, these have comparatively little to say about choice in relation to the informal carers of relatives, friends or older people who are disabled or sick. To explore the realities of choice as experienced by carers, the present paper reviews research published in English since 1985 about three situations in which carers are likely to face choices: receiving social services; the entry of an older person to long-term care; and combining paid work and care. Thirteen electronic databases were searched, covering both the health and social care fields. Databases included: ASSIA; IBSS; Social Care Online; ISI Web of Knowledge; Medline; HMIC; Sociological Abstracts; INGENTA; ZETOC; and the National Research Register. The search strategy combined terms that: (1) identified individuals with care-giving responsibilities; (2) identified people receiving help and support; and (3) described the process of interest (e.g. choice, decision-making and self-determination). The search identified comparatively few relevant studies, and so was supplemented by the findings from another recent review of empirical research on carers' choices about combining work and care. The research evidence suggests that carers' choices are shaped by two sets of factors: one relates to the nature of the care-giving relationship; and the second consists of wider organisational factors. A number of reasons may explain the invisibility of choice for carers in current policy proposals for increasing choice. In particular, it is suggested that underpinning conceptual models of the relationship between carers and formal service providers shape the extent to which carers can be offered choice and control on similar terms to service users. In particular, the exercise of choice by carers is likely to be highly problematic if it involves relinquishing some unpaid care-giving activities.


The ACF1 complex is required for DNA double-strand break repair in human cells.
DNA double-strand breaks (DSBs) are repaired via nonhomologous end-joining (NHEJ) or homologous recombination (HR), but cellular repair processes remain elusive. We show here that the ATP-dependent chromatin-remodeling factors, ACF1 and SNF2H, accumulate rapidly at DSBs and are required for DSB repair in human cells. If the expression of ACF1 or SNF2H is suppressed, cells become extremely sensitive to X-rays and chemical treatments producing DSBs, and DSBs remain unrepaired. ACF1 interacts directly with KU70 and is required for the accumulation of KU proteins at DSBs. The KU70/80 complex becomes physically more associated with the chromatin-remodeling factors of the CHRAC complex, which includes ACF1, SNF2H, CHRAC15, and CHRAC17, after treatments producing DSBs. Furthermore, the frequency of NHEJ as well as HR induced by DSBs in chromosomal DNA is significantly decreased in cells depleted of either of these factors. Thus, ACF1 and its complexes play important roles in DSBs repair.


Robust radiotherapy planning.
Motion and uncertainty in radiotherapy is traditionally handled via margins. The clinical target volume (CTV) is expanded to a larger planning target volume (PTV), which is irradiated to the prescribed dose. However, the PTV concept has several limitations, especially in proton therapy. Therefore, robust and probabilistic optimization methods have been developed that directly incorporate motion and uncertainty into treatment plan optimization for intensity modulated radiotherapy (IMRT) and intensity modulated proton therapy (IMPT). Thereby, the explicit definition of a PTV becomes obsolete and treatment plan optimization is directly based on the CTV. Initial work focused on random and systematic setup errors in IMRT. Later, inter-fraction prostate motion and intra-fraction lung motion became a research focus. Over the past ten years, IMPT has emerged as a new application for robust planning methods. In proton therapy, range or setup errors may lead to dose degradation and misalignment of dose contributions from different beams - a problem that cannot generally be addressed by margins. Therefore, IMPT has led to the first implementations of robust planning methods in commercial planning systems, making these methods available for clinical use. This paper first summarizes the limitations of the PTV concept. Subsequently, robust optimization methods are introduced and their applications in IMRT and IMPT planning are reviewed.


Prevalence of post-traumatic osteoarthritis after anterior cruciate ligament injury remains high despite advances in surgical techniques.
The aim of this study was to report the pooled prevalence of post-traumatic osteoarthritis (PTOA) and examine whether the risk of developing PTOA after anterior cruciate ligament (ACL) injury has decreased in recent decades. The PubMed and Web of Science databases were searched from 1 January 1980 to 11 May 2022. Patient series, observational studies, and clinical trials having reported the prevalence of radiologically confirmed PTOA after ACL injury, with at least a ten-year follow-up, were included. All studies were analyzed simultaneously, and separate analyses of the operative and nonoperative knees were performed. The prevalence of PTOA was calculated separately for each study, and pooled prevalence was reported with 95% confidence intervals (CIs) using either a fixed or random effects model. To examine the effect of the year of injury on the prevalence, a logit transformed meta-regression analysis was used with a maximum-likelihood estimator. Results from meta-regression analyses were reported with the unstandardized coefficient (β). The pooled prevalence of PTOA was 37.9% (95% CI 32.1 to 44) for operatively treated ACL injuries with a median follow-up of 14.6 years (interquartile range (IQR) 10.6 to 16.7). For nonoperatively treated ACL injuries, the prevalence was 40.5% (95% CI 28.9 to 53.3), with a median of follow-up of 15 years (IQR 11.7 to 20.0). The association between the year of operation and the prevalence of PTOA was weak and imprecise and not related to the choice of treatment (operative β -0.038 (95% CI -0.076 to 0.000) and nonoperative β -0.011 (95% CI -0.101 to 0.079)). The initial injury, irrespective of management, has, by the balance of probability, resulted in PTOA within 20 years. In addition, the prevalence of PTOA has only slightly decreased during past decades. Therefore, further research is warranted to develop strategies to prevent the development of PTOA after ACL injuries.


Vitamin K status in healthy volunteers.
Vitamin K's recommended dietary allowance (RDA) is based on the hepatic requirement for clotting factor synthesis, but substantial concentrations of undercarboxylated extra-hepatic Gla-proteins are found in the circulation of non-supplemented individuals. This suggests that vitamin K intake above the RDA is required for an optimal extra-hepatic vitamin K status. Circulating uncarboxylated osteocalcin (ucOC) and desphospho-uncarboxylated matrix Gla-protein (dp-ucMGP) are considered markers of the vitamin K status in bone and the vasculature, respectively. We measured these markers in 896 samples of healthy volunteers and defined target groups for vitamin K supplementation based on increased levels indicative of tissue-specific vitamin K deficiency. We studied the response to vitamin K supplements at different states of vitamin K deficiency by measuring the circulating dp-ucMGP level in samples from two short-term trials on menaquinone-7 (MK-7, vitamin K2) supplementation in 42 children and 68 adults. Children had high ucOC levels (3.4-96.9 ng ml(-1)); other age groups had values in the range of 1.5-5.0 ng ml(-1). From the age of 40 years, dp-ucMGP levels gradually increased. Children and adults with more pronounced vitamin K deficiency gave the highest responses to MK-7 supplementation. Children and adults above 40 years showed the largest tissue-specific vitamin deficiency and accordingly may benefit from MK-7 supplementation to improve their extra-hepatic vitamin K status.


In vivo influence of sodium fluoride on sperm chemotaxis in male mice.
Reproductive process covers lots of procedures, including capacitation, hyperactivation, chemotaxis and the acrosome reaction. Each plays an important role in the success of fertilization. Although multiple studies have reported the toxic effects of fluoride on the male reproduction, the effect of fluoride on sperm chemotaxis is little known. This study is to examine the effect of fluoride on the sperm chemotaxis and then to reveal the underling mechanisms of fluoride toxicity in sperm chemotaxis. 260 healthy Kunming male mice (8 weeks old) were randomly divided into four groups and exposed to 50, 100, 150 mg NaF/L in the drinking water for 8 weeks. At the end of the exposure, sperm chemotaxis was examined using a microchannel-based device. Ca(2+) concentration, adenylate cyclase (AC) content and mRNA expression of mACIII, mACVIII, Golf alpha, CatSper1, CatSper2 were measured to elucidate the possible molecular mechanisms. The results showed that the percentage of chemotactic sperm was decreased by NaF in a dose-dependent manner. In the 100 and 150 mg/L groups, Ca(2+) concentration and AC content were notably lower than the control group. Compared with the control group, mRNA expression of CatSper1 in the 100 and 150 mg/L treatment groups was decreased significantly, and other genes showed no statistical difference. These data suggested that excessive fluoride did adversely affect sperm chemotaxis. The alteration of Ca(2+) concentration, AC content and CatSper1 mRNA expression level may play a key role in the mechanism underlying the affection.


Heart failure after conventional metal-on-metal hip replacements.
Background and purpose - It is unclear whether metal particles and ions produced by mechanical wear and corrosion of hip prostheses with metal-on-metal (MoM) bearings have systemic adverse effects on health. We compared the risk of heart failure in patients with conventional MoM total hip arthroplasty (THA) and in those with metal-on-polyethylene (MoP) THA. Patients and methods - We conducted a retrospective cohort study using data from the Australian Government Department of Veterans' Affairs health claims database on patients who received conventional THA for osteoarthritis between 2004 and 2012. The MoM THAs were classified into groups: Articular Surface Replacement (ASR) XL Acetabular System, other large-head (LH) (> 32 mm) MoM, and small-head (SH) (≤ 32 mm) MoM. The primary outcome was hospitalization for heart failure after THA. Results - 4,019 patients with no history of heart failure were included (56% women). Men with an ASR XL THA had a higher rate of hospitalization for heart failure than men with MoP THA (hazard ratio (HR) = 3.2, 95% CI: 1.6-6.5). No statistically significant difference in the rate of heart failure was found with the other LH MoM or SH MoM compared to MoP in men. There was no statistically significant difference in heart failure rate between exposure groups in women. Interpretation - An association between ASR XL and hospitalization for heart failure was found in men. While causality between ASR XL and heart failure could not be established in this study, it highlights an urgent need for further studies to investigate the possibility of systemic effects associated with MoM THA.


Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site.
Ufo/Axl belongs to a new family of receptor tyrosine kinases with an extracellular structure similar to that of neural cell adhesion molecules. In order to elucidate intracellular signaling, the cytoplasmic moiety of Ufo/Axl was used to screen an expression library according to the CORT (cloning of receptor targets) method. Three putative Ufo substrates were identified: phospholipase Cgamma1 (PLCgamma), as well as p85alpha and p85beta subunits of phosphatidylinositol 3'-kinase (PI3-kinase). Subsequently, chimeric EGFR/Ufo receptors consisting of the extracellular domains of the epidermal growth factor receptor (EGFR) and the transmembrane and intracellular moiety of Ufo were engineered. Using different far-Western blot analyses and coimmunoprecipitation assays, receptor binding of PLCgamma and p85 proteins as well as GRB2, c-src and lck was examined in vitro and in vivo. Competitive inhibition of substrate binding and mutagenesis experiments with EGFR/Ufo constructs revealed C-terminal tyrosine 821 (EILpYVNMDEG) as a docking site for multiple effectors, namely PLCgamma, p85 proteins, GRB2, c-src and lck. Tyrosine 779 (DGLpYALMSRC) demonstrated an additional, but lower binding affinity for the p85 proteins in vitro. In addition, binding of PLCgamma occurred through tyrosine 866 (AGRpYVLCPST). Moreover, our in vivo data indicate that further direct or indirect binding sites for PLCgamma, GRB2, c-src and lck on the human Ufo receptor may exist.


Assessment of mindfulness by self-report.
Assessment of mindfulness is essential to understanding its relationships with psychological functioning. Currently, mindfulness is most often assessed with self-report questionnaires. Although additional work is required, mindfulness questionnaires have reasonable psychometric properties and are making important contributions to the understanding of mindfulness and its effects on health and wellbeing. For example, measurement of mindfulness as a multidimensional construct shows that present-moment awareness can be unhelpful unless accompanied by a nonjudgmental, nonreactive stance; moreover, nonjudgment and nonreactivity may be only weakly related to present-moment awareness in people with no meditation experience. Differences between psychological and Buddhist conceptions of mindfulness, though often a source of criticism of mindfulness questionnaires, are argued here to be inevitable and not necessarily problematic.


Impact of a critical care postgraduate certificate course on nurses' self-reported competence and confidence: A quasi-experimental study.
Postgraduate education is said to support the development of nurses' professional competence and confidence, essential to the delivery of safe and effective care. However, there is a shortness of empirical evidence to demonstrate an increase to nurses' self-reported confidence and competence on completion of critical care postgraduate certificate-level education. To explore the impact of a critical care postgraduate certificate course on nurses' self-reported competence and confidence. To explore the psychometric properties and performance of the Critical Care Competence and Confidence Questionnaire. A quasi-experimental pre/post-test design. A total population sample of nurses completing a critical care postgraduate certificate course at an Australian University. The Critical Care Competence and Confidence Questionnaire was developed for this study to measure nurses' self-reported competence and confidence at baseline and follow up. Descriptive and inferential statistics were used to explore sample characteristics and changes between baseline and follow-up. Reliability of the questionnaire was explored using Cronbach's Alpha and item-total correlations. There was a statistically significant increase in competence and confidence between baseline and follow-up across all questionnaire domains. Satisfactory reliability estimates were found for the questionnaire. Completion of a critical care postgraduate certificate course significantly increased nurses' perceived competence and confidence. The Critical Care Competence and Confidence Questionnaire was found to be psychometrically sound for measuring nurses' self-reported competence and confidence.


Isolation and characterisation of a novel sulphate-reducing bacterium of the Desulfovibrio genus.
A novel sulphate-reducing bacterium (Ind 1) was isolated from a biofilm removed from a severely corroded carbon steel structure in a marine environment. Light microscopy observations revealed that cells were Gram-negative, rod shaped and very motile. Partial 16S rRNA gene sequencing and analysis of the fatty acid profile demonstrated a strong similarity between the new species and members from the Desulfovibrio genus. This was confirmed by the results obtained following purification and characterisation of the key proteins involved in the sulphate-reduction pathway. Several metal-containing proteins, such as two periplasmic proteins: hydrogenase and cytochrome c3, and two cytoplasmic proteins: ferredoxin and sulphite reductase, were isolated and purified. The latter proved to be of the desulfoviridin type which is typical of the Desulfovibrio genus. The study of the remaining proteins revealed a high degree of similarity with the homologous proteins isolated from Desulfovibrio gigas. However, the position of the strain within the phylogenetic tree clearly indicates that the bacterium is closely related to Desulfovibrio gabonensis, and these three strains form a separate cluster in the delta subdivision of the Proteobacteria. On the basis of the results obtained, it is suggested that Ind 1 belongs to a new species of the genus Desulfovibrio, and the name Desulfovibrio indonensis is proposed.


Workplace social capital and adherence to antihypertensive medication: a cohort study.
While hypertension is a common and treatable health problem, adherence to antihypertensive medication remains a challenge. This study examines the hypothesis that workplace social capital may influence adherence to antihypertensive medication among hypertensive employees. We linked survey responses to nationwide pharmacy records for a cohort of 3515 hypertensive employees (mean age 53.9 years, 76% women) who required continuous antihypertensive drug therapy (the Finnish Public Sector study). A standard scale was used to measure workplace social capital from co-workers' assessments and self-reports in 2000-2004. Non-adherence to antihypertensive medication was determined based on the number of days-not-treated at the year following the survey using comprehensive prescription records. Negative binomial regression models were conducted adjusting for socio-demographic characteristics, duration of hypertension, behaviour-related risk factors, and co-morbid conditions. The overall rate of days-not-treated was 20.7 per person-year (78% had no days-not-treated). Higher age, obesity, and presence of somatic co-morbidities were all associated with better adherence, but this was not the case for co-worker-assessed or self-reported workplace social capital. The rate of days-not-treated was 19.7 per person-year in the bottom fourth of co-worker-assessed workplace social capital, compared to 20.4 in the top fourth. The corresponding rate ratio from the fully-adjusted model was 0.95 (95% confidence interval (CI) 0.58-1.56). In a subgroup of 907 new users of antihypertensive medication this rate ratio was 0.98 (95% CI 0.42-2.29). We found no consistent evidence to support the hypothesized effect of workplace social capital on adherence to drug therapy among employees with chronic hypertension.


Fully automated, sequential focused ion beam milling for cryo-electron tomography.
Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and determining structures of macromolecular complexes in their cellular context. A limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here, we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.


Which Patients Should We Follow up beyond 5 Years after Definitive Therapy for Localized Renal Cell Carcinoma?
Up to 10% of recurrences develop beyond 5 years after curative treatment of localized renal cell carcinoma (RCC). Clinicopathologic features were evaluated to determine which factors are associated with late recurrence. A total of 753 patients were diagnosed with localized RCC from January 2000 to June 2008. We enrolled 225 patients who were treated surgically and had a minimal recurrence-free survival of 60 months. Patients who had recurrence beyond 5 years after nephrectomy were defined as the late recurrence group and the remaining patients as the recurrence-free group. Multivariate logistic regression analyses and the Cox proportional hazard model were used for determination of features associated with late recurrence. In multivariate analyses, age older than 60 (p=0.030), Fuhrman grade ≥ 3 (p=0.042), and pT stage ≥ pT2 (p=0.010) showed statistical association with late recurrence. The Cox proportional hazard model showed significant differences in recurrence-free survival when we classified the patients based on pT2 (p=0.007) and on patient age ≥ 60 years (p=0.039). Patient age greater than 60 years, Fuhrman grade ≥ 3, and tumor stage ≥ pT2 are independent risk factors of recurrence more than 5 years after surgery in patients with RCC. Therefore, close lifelong follow-up is recommended for patients with these risk factors.


Re-shaping models of E. coli population dynamics in livestock faeces: increased bacterial risk to humans?
Dung-pats excreted directly on pasture from grazing animals can contribute a significant burden of faecal microbes to agricultural land. The aim of this study was to use a combined field and modelling approach to determine the importance of Escherichia coli growth in dung-pats when predicting faecal bacteria accumulation on grazed grassland. To do this an empirical model was developed to predict the dynamics of an E. coli reservoir within 1ha plots each grazed by four beef steers for six months. Published first-order die-off coefficients were used within the model to describe the expected decline of E. coli in dung-pats. Modelled estimates using first-order kinetics led to an underestimation of the observed E. coli land reservoir, when using site-specific die-off coefficients. A simultaneous experiment determined the die-off profiles of E. coli within fresh faeces of beef cattle under field relevant conditions and suggested that faecal bacteria may experience growth and re-growth in the period post defecation when exposed to a complex interaction of environmental drivers such as variable temperature, UV radiation and moisture levels. This growth phase in dung-pats is not accounted for in models based on first-order die-off coefficients. When the model was amended to incorporate the growth of E. coli, equivalent to that observed in the field study, the prediction of the E. coli reservoir was improved with respect to the observed data and produced a previously unquantified step-change improvement in model predictions of the accumulation of these faecal bacteria on grasslands. Results from this study suggest that the use of first-order kinetic equations for determining land-based reservoirs of faecal bacteria should be approached with caution and greater emphasis placed on accounting for actual survival patterns observed under field relevant conditions.


Coordinate regulation of Cyp2e1 by β-catenin- and hepatocyte nuclear factor 1α-dependent signaling.
Depending on their position within the liver lobule, hepatocytes fulfill different metabolic functions. Cytochrome P450 (CYP) 2E1 is a drug-metabolizing enzyme which is exclusively expressed in hepatocytes surrounding branches of the hepatic central vein. Previous publications have shown that signaling through the Wnt/β-catenin pathway, a major determinant of liver zonation, and the hepatocyte-enriched transcription factor HNF (hepatocyte nuclear factor) 1α participate in the regulation of the gene. This study was aimed to decipher the molecular mechanisms by which the two transcription factors, β-catenin and HNF1α, jointly regulate CYP2E1 at the gene promoter level. Chromatin immunoprecipitation identified a conserved Wnt/β-catenin-responsive site (WRE) in the murine Cyp2e1 promoter adjacent to a known HNF1α response element (HNF1-RE). In vitro analyses demonstrated that both, activated β-catenin and HNF1α, are needed for the full response of the promoter. The WRE was dispensable for β-catenin-mediated effects on the Cyp2e1 promoter, while activity of β-catenin was integrated into the promoter response via the HNF1-RE. Physical interaction of β-catenin and HNF1α was demonstrated by co-immunoprecipitation. In conclusion, present data the first time identify and characterize the interplay of HNF1α and β-catenin and elucidate molecular determinants of CYP2E1 expression in the liver.


Perioperative Arnica montana for Reduction of Ecchymosis in Rhinoplasty Surgery.
Studies of homeopathic therapies to decrease postrhinoplasty ecchymosis have previously used subjective measurements, limiting their clinical significance. Recently, Arnica montana was shown to decrease postoperative ecchymosis after rhytidectomy, using an objective measuring tool. We believe that oral A. montana, given perioperatively, can be objectively shown to reduce extent and intensity of postoperative ecchymosis in rhinoplasty surgery. Subjects scheduled for rhinoplasty surgery with nasal bone osteotomies by a single surgeon were prospectively randomized to receive either oral perioperative A. montana (Alpine Pharmaceuticals, San Rafael, Calif) or placebo in a double-blinded fashion. Ecchymosis was measured in digital "three-quarter"-view photographs at 3 postoperative time points. Each bruise was outlined with Adobe Photoshop (Adobe Systems Incorporated, San Jose, Calif), and the extent was scaled to a standardized reference card. Cyan, magenta, yellow, black, and luminosity were analyzed in the bruised and control areas to calculate change in intensity. P value of <0.1 was set as a meaningful difference with statistical significance. Compared with 13 subjects receiving placebo, 9 taking A. montana had 16.2%, 32.9%, and 20.4% less extent on postoperative days 2/3, 7, and 9/10, a statistically significant difference on day 7 (P = 0.097). Color change initially showed 13.1% increase in intensity with A. montana but 10.9% and 36.3% decreases on days 7 and 9/10, a statistically significant difference on day 9/10 (P = 0.074). One subject experienced mild itching and rash with the study drug that resolved during the study period. Arnica montana seems to accelerate postoperative healing, with quicker resolution of the extent and the intensity of ecchymosis after osteotomies in rhinoplasty surgery, which may dramatically affect patient satisfaction.


Conservation conflicts across Africa.
There is increasing evidence that areas of outstanding conservation importance may coincide with dense human settlement or impact. We tested the generality of these findings using 1 degree-resolution data for sub-Saharan Africa. We find that human population density is positively correlated with species richness of birds, mammals, snakes, and amphibians. This association holds for widespread, narrowly endemic, and threatened species and looks set to persist in the face of foreseeable population growth. Our results contradict earlier expectations of low conflict based on the idea that species richness decreases and human impact increases with primary productivity. We find that across Africa, both variables instead exhibit unimodal relationships with productivity. Modifying priority-setting to take account of human density shows that, at this scale, conflicts between conservation and development are not easily avoided, because many densely inhabited grid cells contain species found nowhere else.


Observations on pulpal response to carbon dioxide laser drilling of dentine in healthy human third molars.
Preservation of pulpal health is the primary prerequisite for successful application of laser systems in the hard tissue management of vital teeth. The purpose of this study was to investigate the short and long-term pulpal effects to cavity preparations in healthy human teeth using carbon dioxide (CO2) laser. A total of seven, healthy, third molars that were scheduled to be removed due to space problems were used. After the laser drilling, the occlusal cavities were closed temporarily, and the teeth were extracted 7 days (n=5) and 3 months (n=2) after the operation. The specimens were fixed, decalcified, subdivided and processed for light and transmission electron microscopy. Seven days postoperatively all the five teeth that had been irradiated with the CO2 laser did not reveal any pathological changes in the pulpo-dentine complex. Three months postoperatively the two teeth that were prepared with the laser showed subtle but distinct apposition of tertiary dentine that was lined with intact odontoblasts. One of the specimens at 3 months revealed the presence of a mild, but very circumscribed, pulpal infiltration of chronic inflammatory cells subjacent to the cavity preparation. The latter is unlikely to be due to a direct effect of the laser irradiation but a possible consequence of microleakage of oral antigens and/or other tissue-irritating molecules through the temporary restoration and the remaining dentine thickness (RDT). Although these preliminary histological results suggest that the CO2 laser under investigation induced only minimal response of the dentine-pulp complex when used as a hard-tissue drilling tool, with specific energy settings, pulse duration within thermal relaxation time and emitting radiations at 9.6 microm of wavelength, larger clinical trials involving various types of teeth are necessary to reach definite conclusions for large-scale clinical application of the laser device.


Isolation of Lyme disease Borrelia from puffins (Fratercula arctica) and seabird ticks (Ixodes uriae) on the Faeroe Islands.
This is the first report on the isolation of Lyme disease Borrelia from seabirds on the Faeroe Islands and the characteristics of its enzootic cycle. The major components of the Borrelia cycle include the puffin (Fratercula arctica) as the reservoir and Ixodes uriae as the vector. The importance of this cycle and its impact on the spread of human Lyme borreliosis have not yet been established. Borrelia spirochetes isolated from 2 of 102 sampled puffins were compared to the borreliae previously obtained from seabird ticks, I. uriae. The rrf-rrl intergenic spacer and the rrs and the ospC genes were sequenced and a series of phylogenetic trees were constructed. Sequence data and restriction fragment length polymorphism analysis grouped the strains together with Borrelia garinii. In a seroepidemiological survey performed with residents involved in puffin hunting on the Faeroe Islands, 3 of 81 serum samples were found to be positive by two commonly used clinical tests: a flagellin-based enzyme-linked immunosorbent assay (ELISA) and Western blotting. These three positive serum samples also had high optical density values in a whole-cell ELISA. The finding of seropositive Faeroe Islanders who are regularly exposed to I. uriae indicate that there may be a transfer of B. garinii by this tick species to humans.


A conceptual framework for the molecular pathogenesis of progressive kidney disease.
The data regarding the pathogenesis of progressive kidney disease implicate cytokine effects, physiological factors, and myriad examples of relatively nonspecific cellular dysfunction. The sheer volume of information being generated on this topic threatens to overwhelm our efforts to understand progression in chronic kidney disease or to derive rational strategies to treat it. Here, a conceptual framework is offered for organizing and considering these data. Disease is initiated by an injury that evokes a tissue-specific cellular response. Subsequent structural repair may be effective, or the new structure may be sufficiently changed that it requires an adaptive physiological response. If this adaptation is not successful, subsequent cycles of misdirected repair or maladaptation may lead to progressive nephron loss. To illustrate how this framework can be used to organize our approach to disease pathogenesis, the role of cytokines in proteinuria and progressive glomerular disease is discussed. Finally, this theoretical framework is reconsidered to examine its implications for the diagnosis and treatment of clinical conditions. Application of this schema could have significant relevance to both research inquiry and clinical practice.


Synaptic interactions between mammalian central neurons in cell culture. II. Quantal Analysis of EPSPs.
The presynaptic release mechanism involved in excitatory synaptic connections between neurons in cell cultures of fetal mouse spinal cord were studied by statistical analysis of intracellularly recorded postsynaptic responses. Quantal parameters were determined for the EPSPs evoked in spinal cord (SC) neurons by stimulation of either other SC or dorsal root ganglion (DRG) neurons. Transmitter release was manipulated by varying the Ca2+ and Mg2+ content of the culture medium. The release process was represented better by binomial than by Poisson statistics. A method was derived for obtaining the probability of release and the number of release elements. The quantal content and the number of release elements were substantially higher for the SC-SC connection than for the DRG-SC connection. This was partially compensated for by a larger quantal amplitude for the DRG-SC connection. There was some indication that the probability of release was higher for the SC-SC connection. The relationship between transmitter output and effective external Ca2+ ion concentration was approximately linear.


Prevalence of dry eye syndrome in an adult population.
To determine the prevalence of dry eye syndrome in the general 40- to 64-year-old population of Shahroud, Iran. Population-based cross-sectional study. Through cluster sampling, 6311 people were selected and 5190 participated. Assessment of dry eye was done in a random subsample of 1008 people. Subjective assessment for dry eye syndrome was performed using Ocular Surface Disease Index questionnaire. In addition, the following objective tests of dry eye syndrome were employed: Schirmer test, tear break-up time, and fluorescein and Rose Bengal staining using the Oxford grading scheme. Those with an Ocular Surface Disease Index score ≥23 were considered symptomatic, and dry eye syndrome was defined as having symptoms and at least one positive objective sign. The prevalence of dry eye syndrome was 8.7% (95% confidence interval 6.9-10.6). Assessment of signs showed an abnormal Schirmer score in 17.8% (95% confidence interval 15.5-20.0), tear break-up time in 34.2% (95% confidence interval 29.5-38.8), corneal fluorescein staining (≥1) in 11.3% (95% confidence interval 8.5-14.1) and Rose Bengal staining (≥3 for cornea and/or conjunctiva) in 4.9% (95% confidence interval 3.4-6.5). According to the Ocular Surface Disease Index scores, 18.3% (95% confidence interval 15.9-20.6) had dry eye syndrome symptoms. The prevalence of dry eye syndrome was significantly higher in women (P = 0.010) and not significantly associated with age (P = 0.291). The objective dry eye syndrome signs significantly increased with age. Based on the findings, the prevalence of dry eye syndrome in the studied population is in the mid-range. The prevalence is higher in women. Also, objective tests tend to turn abnormal at higher age. Pterygium is associated with dry eye syndrome and increased its symptoms.


Impact of length or relevance of questionnaires on attrition in online trials: randomized controlled trial.
There has been limited study of factors influencing response rates and attrition in online research. Online experiments were nested within the pilot (study 1, n = 3780) and main trial (study 2, n = 2667) phases of an evaluation of a Web-based intervention for hazardous drinkers: the Down Your Drink randomized controlled trial (DYD-RCT). The objective was to determine whether differences in the length and relevance of questionnaires can impact upon loss to follow-up in online trials. A randomized controlled trial design was used. All participants who consented to enter DYD-RCT and completed the primary outcome questionnaires were randomized to complete one of four secondary outcome questionnaires at baseline and at follow-up. These questionnaires varied in length (additional 23 or 34 versus 10 items) and relevance (alcohol problems versus mental health). The outcome measure was the proportion of participants who completed follow-up at each of two follow-up intervals: study 1 after 1 and 3 months and study 2 after 3 and 12 months. At all four follow-up intervals there were no significant effects of additional questionnaire length on follow-up. Randomization to the less relevant questionnaire resulted in significantly lower rates of follow-up in two of the four assessments made (absolute difference of 4%, 95% confidence interval [CI] 0%-8%, in both study 1 after 1 month and in study 2 after 12 months). A post hoc pooled analysis across all four follow-up intervals found this effect of marginal statistical significance (unadjusted difference, 3%, range 1%-5%, P = .01; difference adjusted for prespecified covariates, 3%, range 0%-5%, P = .05). Apparently minor differences in study design decisions may have a measurable impact on attrition in trials. Further investigation is warranted of the impact of the relevance of outcome measures on follow-up rates and, more broadly, of the consequences of what we ask participants to do when we invite them to take part in research studies. ISRCTN Register 31070347; http://www.controlled-trials.com/ISRCTN31070347/31070347 Archived by WebCite at (http://www.webcitation.org/62cpeyYaY).


Takotsubo cardiomyopathy following lightning strike.
Lightning strike is the most common environmental cause of sudden cardiac death, but may also be associated with a myriad of injuries to various organ systems. Direct myocardial injury may be manifest as electrocardiographic alterations or elevation in cardiac-specific isoenzymes; however, significant electrical cardiac trauma appears uncommon. A case is presented of severe acute cardiomyopathy in a "Takotsubo" distribution causing cardiogenic shock following lightning strike in a previously healthy 37-year-old woman. Although rarely identified in this context, Takotsubo cardiomyopathy (also known as "transient left ventricular apical ballooning syndrome") is characterised by transient cardiac dysfunction, electrocardiographic changes that may mimic acute myocardial infarction and minimal release of cardiac-specific enzymes in the absence of obstructive coronary artery disease. The condition is associated with a substantial female bias (up to 90% of cases) in reported series, and despite occasionally dramatic presentations recovery of left ventricular function is almost universal over days to weeks. In rare instances, however, the syndrome has been associated with more catastrophic complications such as papillary muscle or cardiac free wall rupture, necessitating emergency surgical intervention to preserve life. In clinical practice, non-lethal lightning strike-induced cardiac injury is frequently associated with small elevations of cardiac isoenzymes without overt clinical sequelae; however, the incidence of silent myocardial mechanical dysfunction remains unknown. Cases such as the one presented highlight the potential for serious, albeit usually transient, cardiac sequelae from lightning strike injury and remind us that our mothers' advice to remain indoors during thunderstorms is probably worth heeding.


Revision of the Competency Standards for Occupational Therapy Driver Assessors: An overview of the evidence for the inclusion of cognitive and perceptual assessments within fitness-to-drive evaluations.
Determination of fitness-to-drive after illness or injury is a complex process typically requiring a comprehensive driving assessment, including off-road and on-road assessment components. The competency standards for occupational therapy driver assessors (Victoria, Australia) define the requirements for performance of a comprehensive driving assessment, and we are currently revising these. Assessment of cognitive and perceptual skills forms an important part of the off-road assessment. The aim of this systematic review of systematic reviews (known as an overview) is to identify what evidence exists for including assessment of cognitive and perceptual skills within fitness-to-drive evaluations to inform revision of the competency standards. Five electronic databases (MEDLINE, CINAHL, PsycINFO, The Cochrane Library, OT Seeker) were systematically searched. Systematic review articles were appraised by two authors for eligibility. Methodological quality was independently assessed using the AMSTAR tool. Narrative analysis was conducted to summarise the content of eligible reviews. A total of 1228 results were retrieved. Fourteen reviews met the inclusion criteria. Reviews indicated that the components of cognition and perception most frequently identified as being predictive of fitness-to-drive were executive function (n = 13), processing speed (n = 12), visuospatial skills, attention, memory and mental flexibility (n = 11). Components less indicative were perception, concentration (n = 10), praxis (n = 9), language (n = 7) and neglect (n = 6). This overview of systematic reviews supports the inclusion of assessment of a range of cognitive and perceptual skills as key elements in a comprehensive driver assessment and therefore should be included in the revised competency standards for occupational therapy driver assessors.


Locomotor activity, core body temperature, and circadian rhythms in mice selected for high or low heat loss.
Daily locomotor activity, core body temperature, and their circadian rhythms were measured in lines of mice selected for high (MH) or low (ML) heat loss and unselected controls (MC). Lines were created by selecting for 16 generations in each of three replicates. Collection of locomotor activity and core temperature data spanned Generations 20 and 21 for a total of 352 mice. Physical activity and core body temperature data were accumulated using implanted transmitters and continuous automated collection. Measurement for each animal was for 3 d. Activity was recorded for each half hour and then averaged for the day; temperature was averaged daily; circadian rhythm was expressed in 12-h (light vs dark) or 6-h periods as well as by fitting cyclic models. Activity means were transformed to log base 2 to lessen heterogeneity of variance within lines. Heat loss for a 15-h period beginning at 1630 and feed intake for 7 d were measured on 74 additional mice in order to estimate the relationship between locomotor activity and heat loss or feed intake. Selection lines were different (P < 0.01) for both locomotor activity and core body temperature. Differences were due to selection (MH-ML, P < 0.01), and there was no evidence of asymmetry of response (P > 0.38). Retransformed from log base 2 to the scale of measurement, mean activity counts were 308, 210, and 150 for MH, MC, and ML, respectively. Mean core temperatures were 37.2, 36.9, and 36.7 degrees C for MH, MC, and ML (P < 0.01), respectively. Females had greater physical activity (P < 0.01) and body temperature (P < 0.01) than males. There was no evidence of a sex x selection criterion interaction for either activity or temperature (P > 0.20). Overall phenotypic correlation between body temperature and log base 2 activity was 0.43 (P < 0.01). Periods during the day were different for both 12- and 6-h analyses (P < 0.01), but there were no period x selection criterion interactions (P > 0.1) for physical activity or body temperature. More sensitive cyclic models revealed significant (P < 0.01) 24-, 12-, 8-, and 6-h cycles that differed (P < 0.01) among lines. Estimated differences between MH and ML mice in feed intake and heat loss due to locomotor activity were 36 and 11.5%, respectively. Variation in activity thus contributed to variation in feed intake.