Airway clearance devices for cystic fibrosis: an evidence-based analysis.
The purpose of this evidence-based analysis is to examine the safety and efficacy of airway clearance devices (ACDs) for cystic fibrosis and attempt to differentiate between devices, where possible, on grounds of clinical efficacy, quality of life, safety and/or patient preference. Cystic fibrosis (CF) is a common, inherited, life-limiting disease that affects multiple systems of the human body. Respiratory dysfunction is the primary complication and leading cause of death due to CF. CF causes abnormal mucus secretion in the airways, leading to airway obstruction and mucus plugging, which in turn can lead to bacterial infection and further mucous production. Over time, this almost cyclical process contributes to severe airway damage and loss of respiratory function. Removal of airway secretions, termed airway clearance, is thus an integral component of the management of CF. A variety of methods are available for airway clearance, some requiring mechanical devices, others physical manipulation of the body (e.g. physiotherapy). Conventional chest physiotherapy (CCPT), through the assistance of a caregiver, is the current standard of care for achieving airway clearance, particularly in young patients up to the ages of six or seven. CF patients are, however, living much longer now than in decades past. The median age of survival in Canada has risen to 37.0 years for the period of 1998-2002 (5-year window), up from 22.8 years for the 5-year window ending in 1977. The prevalence has also risen accordingly, last recorded as 3,453 in Canada in 2002, up from 1,630 in 1977. With individuals living longer, there is a greater need for independent methods of airway clearance. AIRWAY CLEARANCE DEVICES: THERE ARE AT LEAST THREE CLASSES OF AIRWAY CLEARANCE DEVICES: positive expiratory pressure devices (PEP), airway oscillating devices (AOD; either handheld or stationary) and high frequency chest compression (HFCC)/mechanical percussion (MP) devices. Within these classes are numerous different brands of devices from various manufacturers, each with subtle iterations. At least 10 devices are licensed by Health Canada (ranging from Class 1 to Class 3 devices). EVIDENCE-BASED ANALYSIS OF EFFECTIVENESS: Does long-term use of ACDs improve outcomes of interest in comparison to CCPT in patients with CF?Does long-term use of one class of ACD improve outcomes of interest in comparison to another class of ACD in CF patients? A comprehensive literature search was performed on March 7, 2009 using OVID MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations, EMBASE, the Cumulative Index to Nursing & Allied Health Literature (CINAHL), the Cochrane Library, and the International Agency for Health Technology Assessment (INAHTA) for studies published from January 1, 1950 to March 7, 2009. All randomized controlled trials including those of parallel and crossover design,Systematic reviews and/or meta-analyses. Randomized controlled trials (RCTs), systematic reviews and meta-analyses Abstracts were generally excluded because their methods could not be examined; however, abstract data was included in several Cochrane meta-analyses presented in this paper;Studies of less than seven days duration (including single treatment studies);Studies that did not report primary outcomes;Studies in which less than 10 patients completed the study. Primary outcomes under review were percent-predicted forced expiratory volume (FEV-1), forced vital capacity (FVC), and forced expiratory flow between 25%-75% (FEF25-75). Secondary outcomes included number of hospitalizations, adherence, patient preference, quality of life and adverse events. All outcomes were decided a priori. Literature searching and back-searching identified 13 RCTs meeting the inclusion criteria, along with three Cochrane systematic reviews. The Cochrane reviews were identified in preliminary searching and used as the basis for formulating this review. Results were subgrouped by comparison and according to the available literature. For example, results from Cochrane meta-analyses included abstract data and therefore, additional meta-analyses were also performed on trials reported as full publications only (MAS generally excludes abstracted data when full publications are available as the methodological quality of trials reported in abstract cannot be properly assessed). Executive Summary Table 1 summarizes the results across all comparisons and subgroupings for primary outcomes of pulmonary function. Only two comparisons yielded evidence of moderate or high quality according to GRADE criteria-the comparisons of CCPT vs. PEP and handheld AOD vs. PEP-but only the comparison of CCPT vs. PEP noted a significant difference between treatment groups. In comparison to CCPT, there was a significant difference in favour of PEP for % predicted FEV-1 and FVC according to one long-term, parallel RCT. This trial was accepted as the best available evidence for the comparison. The body of evidence for the remaining comparisons was low to very low, according to GRADE criteria, being downgraded most often because of poor methodological quality and low generalizability. Specifically, trials were likely not adequately powered (low sample sizes), did not conduct intention-to-treat analyses, were conducted primarily in children and young adolescents, and outdated (conducted more than 10 years ago). Secondary outcomes were poorly or inconsistently reported, and were generally not of value to decision-making. Of note, there were a significantly higher number of hospitalizations among participants undergoing AOD therapy in comparison to PEP therapy. ES Table 1:Summarization of results for primary outcomes by comparison and subgroupingsOutcome or SubgroupNo. of StudiesEstimate of Effectiveness (95% CI)P-valueHeterogeneity (I(2))GRADECCPT vs. PEP Cochrane FEV-1 FVC FEF(25-75%)6640.08 (-1.45 to 1.62)0.38 (-1.56 to 2.23)-0.44 (-3.38 to 2.50)0.910.700.7746%63%36 N/A Full publications only FEV-1 FVC FEF(25-75%)332-0.50 (-3.93 to 2.92)-0.86 (-4.66 to 2.95)-0.12 (-6.22 to 5.98)0.770.660.9777%74%0% N/A Long-term, parallel RCTs only FEV-1 FVC FEF(25-75%)111-8.25 (-15.77 to -0.75)-8.74 (-16.03 to -1.45)-3.56 (-13.30 to 6.18)0.030.020.47N/AN/AN/A 1 TrialMODERATECCPT vs. HFCC/MP Cochrane FEV-1 FVC FEF(25-75%)332-1.76 (-4.67 to 1.16)-1.42 (-5.17 to 2.33)0.49 (-2.54 to 3.52)0.240.460.750%70%0% N/A Full publications only FEV-1 FVC FEF(25-75%)332-2.10 (-5.49 to 1.29)-3.86 (-8.05 to 0.33)0.49 (-2.54 to 3.52)0.230.070.750%0%0% 3 TrialsLOWCCPT vs. AOD 2 of 3 RCTs/Cochrane FEV-1 FVC FEF(25-75%)2220.80 (-5.79 to 7.39)6.06 (-2.42 to 14.55)1.26 (-7.56 to 10.09)0.810.160.780%12%0% 3 TrialsLOWAOD vs. PEP Long-term, parallel RCTs only/Cochrane FEV-1 FVC FEF(25-75%)2220.29 (-4.17 to 4.75)-0.55 (-4.60 to 3.50)0.10 (-4.86 to 5.06)0.900.790.9773%77%0% 2 TrialsMODERATEAOD vs. HFCC/MP Long-term, parallel RCTs only/Cochrane FEV-1 FVC FEF(25-75%)111-1.6 (-3.44 to 0.24)-1.80 (-4.32 to 0.72)-1.40 (-3.07 to 0.27)0.090.080.16N/AN/AN/A 1 TrialVERY LOWBolding indicates significant differencePositive summary statistics favour the former intervention AOD, airway oscillating device; CCPT, conventional chest physiotherapy; CI, confidence interval; HFCC, high frequency chest compression; MP, mechanical percussion; N/A: not applicable; PEP, positive expiratory pressure Devices ranged in cost from around $60 for PEP and handheld AODs to upwards of $18,000 for a HFCC vest device. Although the majority of device costs are paid out-of-pocket by the patients themselves, their parents, or covered by third-party medical insurance, Ontario did provide funding assistance through the Assistive Devices Program (ADP) for postural drainage boards and MP devices. These technologies, however, are either obsolete or their clinical efficacy is not supported by evidence. ADP provided roughly $16,000 in funding for the 2008/09 fiscal year. Using device costs and prevalent and incident cases of CF in Ontario, budget impact projections were generated for Ontario. Prevalence of CF in Ontario for patients from ages 6 to 71 was cited as 1,047 cases in 2002 while incidence was estimated at 46 new cases of CF diagnosed per year in 2002. Budget impact projections indicated that PEP and handheld AODs were highly economically feasible costing around $90,000 for the entire prevalent population and less than $3,000 per year to cover new incident cases. HFCC vest devices were by far the most expensive, costing in excess of $19 million to cover the prevalent population alone. There is currently a lack of sufficiently powered, long-term, parallel randomized controlled trials investigating the use of ACDs in comparison to other airway clearance techniques. While much of the current evidence suggests no significant difference between various ACDs and alternative therapies/technologies, at least according to outcomes of pulmonary function, there is a strong possibility that past trials were not sufficiently powered to identify a difference. Unfortunately, it is unlikely that there will be any future trials comparing ACDs to CCPT as withholding therapy using an ACD may be seen as unethical at present. Conclusions of clinical effectiveness are as follows: Moderate quality evidence suggests that PEP is at least as effective as or more effective than CCPT, according to primary outcomes of pulmonary function. (ABSTRACT TRUNCATED)


First Report of Cankers and Dieback Caused by Neofusicoccum mediterraneum and Diplodia corticola on Grapevine in Spain.
In November 2010, four grapevine plants of cv. Crimson from a vineyard located in Sevilla (south Spain) revealed trunk cankers. Several pathogens were isolated, including Cylindrocarpon liriodendri (2), Phaeoacremonium aleophilum (2), Pleurostomophora richardsiae, Neofusicoccum parvum, and Botryosphaeria dothidea (2). Among Botryosphaeriaceae fungi isolated on potato dextrose agar (PDA) were two types that did not fit the above mentioned species. Isolates of type 1 produced an abundant, gray mycelium with a diurnal zonation that gradually became dark olivaceous. Mycelium growth occurred from 5 to 37°C with an optimum at 28°C. Conidia were hyaline, fusiform, aseptate, thin walled, but gradually became obscured and septate with age, and measured (18.4-) 21.4 (-24.3) × (4.2-) 5.5 (-7.2) μm with a length/width (L/W) ratio of 4.0 ± 0.5 (n = 100). Isolates of type 1 were identified as N. mediterraneum (3). Single-spore cultures of type 2 developed a whitish, dense, aerial mycelium and remained white up to 10 days on PDA and darkened to gray thereafter. Mycelium growth occurred from 3 to 37°C with an optimum at 29 to 30°C. Conidia were hyaline, aseptate, thick walled, oblong to cylindrical, sometimes becoming light brown and one or two septate after discharge, and measured (24.6-) 30.2 (-42.8) × (10.9-) 14.3 (-18.6) μm with a L/W ratio of 2.1 ± 0.2 (n =100). Isolates of type 2 were identified as Diplodia corticola (1). Nucleotide sequences of the ribosomal internal transcribed spacer (ITS) region and the -tubulin genes were used to confirm the identifications through BLAST searches in GenBank. Comparison of the sequences of types 1 and 2 showed 99 to 100% homology with N. mediterraneum (HM443604 (4) and GU251836) and D. corticola (AY268421 (1) and EU673117), respectively. Representative sequences of N. mediterraneum (JF949757 and JF949756) and D. corticola (JF949758 and JF949759) were deposited in GenBank. The pathogenicity of one representative isolate of each of N. mediterraneum and D. corticola was confirmed by inoculating 10 detached grapevine canes (averaging 12 mm in diameter and 30 cm long) per isolate. A shallow wound was made with a scalpel on the internodes. A colonized 6-mm agar plug, from the margin of an actively growing colony, was inserted in every wound and sealed with Parafilm. Ten grapevine canes controls received only sterile PDA agar plugs. Canes were maintained at 25°C and 70% humidity. After 5 weeks, all inoculated canes developed cankers and pycnidia around the inoculation site. Vascular necroses that developed on the inoculated canes were an average of 28.6 mm for N. mediterraneum and 27.7 mm for D. corticola. One-way analysis of variance and Tukey's test confirmed significant differences in the extent of vascular necroses. The average necroses length in the inoculated canes was significantly greater (P < 0.05) than the average length of discoloration induced by the simulated inoculation process in the control. Both pathogens were reisolated from all inoculated plants but not from controls. To our knowledge, this is the first report of N. mediterraneum and D. corticola as pathogens on grapevine in Spain. References: (1) A. Alves et al. Mycologia 96:603, 2004. (2) A. Aroca and D. Gramaje et al. Eur. J. Plant. Pathol. 126:165, 2010. (3) P. W. Crous et al. Fungal Planet. No. 19, 2007. (4) F. P. Trouillas et al. Plant. Dis. 94:1267, 2010.


Increased mitochondrial oxidative phosphorylation in the liver is associated with obesity and insulin resistance.
Obesity is the result of excess energy intake relative to expenditure, however little is known about why some individuals are more prone to weight gain than others. Inbred strains of mice also vary in their susceptibility to obesity and therefore represent a valuable model to study the genetics and physiology of weight gain and its co-morbidities such as type 2 diabetes. C57BL/6J mice are susceptible to obesity and insulin resistance when fed an obesogenic diet, whereas A/J mice are resistant despite increased caloric intake. Analysis of B6- and A/J-derived chromosome substitution strains and congenic strains revealed a complex genetic and physiological basis for this phenotype. To improve our understanding of the molecular mechanisms underlying susceptibility to metabolic disease we analyzed global gene expression patterns in 6C1 and 6C2 congenic strains. 6C1 is susceptible whereas 6C2 is resistant to diet-induced obesity. In addition, we demonstrate that 6C1 is glucose intolerant and insulin resistant relative to 6C2. Pathway analysis of global gene expression patterns in muscle, adipose, and liver identified expression level differences between 6C1 and 6C2 in pathways related to basal transcription factors, endocytosis, and mitochondrial oxidative phosphorylation (OxPhos). The OxPhos expression differences were subtle but evident in each complex of the electron transport chain and were associated with a marked increase in mitochondrial oxidative capacity in the livers of the obese strain 6C1 relative to the obesity-resistant strain 6C2. These data suggests the importance of hepatic mitochondrial function in the development of obesity and insulin resistance.


Establishment of tandem mass spectrometric fingerprint of novel antineoplastic curcumin analogues using electrospray ionization.
Curcumin analogues are antineoplastic agents, designed based on the structure of the spice turmeric with structural modifications aiming at enhancing potency. The goal is to identify the common tandem mass spectrometric (MS/MS) behavior of 13 novel curcumin analogues. Such knowledge is critical for their biological assessment, including metabolite identification and pharmacokinetic evaluation. Both detection of the protonated molecules [M + H](+) of the synthesized compounds and determination of their exact molecular masses were achieved with hybrid quadrupole orthogonal time-of-flight mass spectrometry (QqTOF-MS). Low-energy collision-induced dissociation (CID)-MS/MS analysis was performed using triple quadrupole linear ion trap mass spectrometry (QqLIT-MS). Both instruments were equipped with an electrospray ionization (ESI) source. MS(3) and neutral loss experiments were performed using QqLIT-MS to confirm the genesis of the observed product ions. Abundant [M + H](+) molecules were formed using the QqTOF-MS hybrid instrument with mass accuracies below 6 ppm. CID-MS/MS dissociation studies were centered on the piperidone ring of curcumin analogues; twelve common product ions have been identified from the fission of the various bonds within the piperidone moiety. There was a tendency for the formation of highly conjugated product ions, stabilized via resonance. The variety of the side-chain substituents at the nitrogen atom resulted in side-chain-specific product ions. The ESI-CID-MS/MS analysis of curcumin analogues revealed a common fragmentation behavior of all tested compounds, which gave diagnostic product ions identified for each molecule. The established MS/MS behavior will be applied to determine metabolic by-products of curcumin analogues as well as to develop targeted identification/quantification methods within biological extracts.


Single crystal metals encapsulated in carbon nanoparticles.
Single-domain microcrystals of LaC(2) encapsulated within nanoscale polyhedral carbon particles have been synthesized in a carbon arc. Typical particle sizes are on the order of 20 to 40 nanometers. The stoichiometry and phase of the La-containing crystals have been assigned from characteristic lattice spacings observed by high-resolution transmission electron microscopy and energy dispersive spectroscopy (EDS). EDS spectra show that La and C are the only elements present. Characteristic interatomic distances of 3.39 and 2.78 angstroms identify the compound inside the nanoparticle cavities as alpha-LaC(2), the phase of LaC(2) that is stable at room temperature. Bulk alpha-LaC(2) is metallic and hydrolytic. Observation of crystals of pure encapsulated alpha-LaC(2) that were exposed to air for several days before analysis indicates that the LaC(2) is protected from degradation bythe carbon polyhedral shells of the nanoparticles. A high percentage of the carbon nanoparticles have encapsulated LaC(2) single crystals. These carbon-coated metal crystals form a new class of materials that can be protected in their pure or carbide forms and may have interesting and useful properties.


Insights on cryoprotectant toxicity from gene expression profiling of endothelial cells exposed to ethylene glycol.
Cryopreservation consists of preserving living cells or tissues generally at -80 °C or below and has many current applications in cell and tissue banking, and future potential for organ banking. Cryoprotective agents such as ethylene glycol (EG) are required for successful cryopreservation of most living systems, but have toxic side effects whose mechanisms remain largely unknown. In this work, we investigated the mechanisms of toxicity of ethylene glycol in human umbilical vein endothelial cells (HUVECs) as a model of the vascular endothelium in perfused organs. Exposing cells to 60% v/v EG for 2 h at 4 °C resulted in only a slight decrease in subsequent cell growth, suggesting only modest toxicity of EG for this cell type. Gene expression analysis with whole genome microarrays revealed signatures indicative of a generalized stress response at 24 h after EG exposure and a trend toward partial recovery at 72 h. The observed changes involved signalling pathways, glycoproteins, and genes involved in extracellular and transmembrane functions, the latter suggesting potential effects of ethylene glycol on membranes. These results continue to develop a new paradigm for understanding cryoprotectant toxicity and reveal molecular signatures helpful for future experiments in more completely elucidating the toxic effects of ethylene glycol in vascular endothelial cells and other cell types.


Consistency of Sedentary Behavior Patterns among Office Workers with Long-Term Access to Sit-Stand Workstations.
Sit-stand workstations are a popular intervention to reduce sedentary behavior (SB) in office settings. However, the extent and distribution of SB in office workers long-term accustomed to using sit-stand workstations as a natural part of their work environment are largely unknown. In the present study, we aimed to describe patterns of SB in office workers with long-term access to sit-stand workstations and to determine the extent to which these patterns vary between days and workers. SB was objectively monitored using thigh-worn accelerometers for a full week in 24 office workers who had been equipped with a sit-stand workstation for at least 10 months. A comprehensive set of variables describing SB was calculated for each workday and worker, and distributions of these variables between days and workers were examined. On average, workers spent 68% work time sitting [standard deviation (SD) between workers and between days (within worker): 10.4 and 18.2%]; workers changed from sitting to standing/walking 3.2 times per hour (SDs 0.6 and 1.2 h-1); with bouts of sitting being 14.9 min long (SDs 4.2 and 8.5 min). About one-third of the workers spent >75% of their workday sitting. Between-workers variability was significantly different from zero only for percent work time sitting, while between-days (within-worker) variability was substantial for all SB variables. Office workers accustomed to using sit-stand workstations showed homogeneous patterns of SB when averaged across several days, except for percent work time seated. However, SB differed substantially between days for any individual worker. The finding that many workers were extensively sedentary suggests that just access to sit-stand workstations may not be a sufficient remedy against SB; additional personalized interventions reinforcing use may be needed. To this end, differences in SB between days should be acknowledged as a potentially valuable source of variation.


Comparative proteome analysis of glutenin synthesis and accumulation in developing grains between superior and poor quality bread wheat cultivars.
Wheat glutenins are the major determinants of wheat quality. In this study, grains at the development stage from three wheat cultivars (Jimai 20, Jin 411 and Zhoumai 16) with different bread-making quality were harvested based on thermal times from 150 °C(d) to 750 °C(d) , and were used to investigate glutenin accumulation patterns and their relationships with wheat quality. High and low molecular weight glutenin subunits (HMW-GSs and LMW-GSs) were synthesised concurrently. No obvious correlations between HMW/LMW glutenin ratios and dough property were observed. Accumulation levels of HMW-GSs and LMW-GSs as well as 1Bx13 + 1By16 and 1Dx4 + 1Dy12 subunits were higher in superior gluten quality cultivar Jimain 20 than in poor quality cultivar Jing 411 and Zhoumai 16. According to the results of two-dimensional gel electrophoresis, six types of accumulation patterns in LMW-GSs were identified and classified. The possible relationships between individual LMW-GSs and gluten quality were established. The high accumulation level of HMW-GSs and LMW-GSs as well as 1Bx13 + 1By16 and 1Dx4 + 1Dy12 subunits contributed to the superior gluten quality of Jimai 20. Two highly expressed and 16 specifically expressed LMW glutenin subunits in Jimain 20 had positive effects on dough quality, while 17 specifically expressed subunits in Zhoumai 16 and Jing 411 appeared to have negative effects on gluten quality.


Studies of ligand binding to arrestin.
A striking homology is observed between the regions 70-83 and 361-374 of the sequence of bovine arrestin and the calcium-binding loops of calmodulin and troponin C. However, the predicted alpha-helices flanking the calcium-binding site in calmodulin and troponin C are not present in arrestin. Direct measurements therefore were made in order to assess whether arrestin can bind calcium. We found that arrestin does not bind Ca2+ at physiological ionic strength, as determined by equilibrium dialysis, gel filtration, and fluorescence spectroscopy. Rapid and quantitative precipitation of arrestin occurs with Tb3+. The precipitation is reversed by EDTA and blocked by Mg2+ but not by Ca2+. Prompted by several reports, we also investigated whether nucleotides bind to arrestin. Neither ATP nor GTP binds under the conditions tested. Binding of arrestin to photolyzed, phosphorylated rhodopsin also does not influence the binding of calcium or nucleotides.


Return to work in a cohort of low back pain patients: development and validation of a clinical prediction rule.
From the viewpoint of cost prevention, it is necessary to identify patients that are of high risk for long-term work disability, production loss and sick-leave. Secondary data analysis in a cohort of 628 workers on sick-leave between 3 and 6 weeks due to low back pain (LBP). The association of a broad set of demographic, work, LBP and psychosocial related factors on lasting return to work was studied using Cox regression analysis with backward selection. The most relevant factors were used to derive a clinical prediction rule to determine the risk of sick-leave of more than 6 months. Variable and model selection and clinical model performance were performed with bootstrapping techniques. Also the test characteristics of the clinical model were considered. Longer work absence is related to "moderate" to "poor" job satisfaction, a higher score of fear avoidance beliefs, higher pain intensity at baseline, a longer duration of complaints and being of female gender. Calibration and discrimination of the clinical prediction rule were 0.90 (slope) and 0.63 (c-index), respectively. The explained variance of 6% of the prediction rule was low and the clinical performance in terms of sensitivity, specificity, positive and negative predictive values at specific cut-off points was moderate. Our study confirmed the importance of demographic, work, LBP and psychosocial related factors on the prediction of long-term sick-leave. When these factors were used to derive a clinical prediction rule the performance was moderate. As a consequence, prudence has to be taken when using the prediction rule in practice.


Nude mouse metastatic models of human stomach cancer constructed using orthotopic implantation of histologically intact tissue.
Nude mice have been used to develop s.c. growing human stomach tumors, but these rarely metastasize. Recently, I. J. Fidler and others have developed orthotopic implantation metastatic models using cell suspensions which are inoculated into the corresponding organ of nude mice from which the tumor cells were originally derived in the human. However, recent work has indicated that disaggregated cell suspensions may not always express their full metastatic potential. In this light, we have recently developed an orthotopic implant model utilizing intact tissue such as that obtained directly from surgery. This approach has yielded high take rates and frequent metastases in colon cancer, bladder cancer, lung cancer, pancreatic cancer, and prostate cancer. We report here the application of this intact tissue orthotopic implant technique to stomach cancer resulting in the formation of metastases in 100% of the mice with extensive primary growth to the regional lymph nodes, liver, and lung. In contrast, when cell suspensions were used to inject stomach cancer cells at the same site, metastases occurred in only 6.7% of the mice with local tumor formation, emphasizing the importance of using intact tissue to allow full expression of metastatic potential. Injuring the serosa similar to that occurring in intact tissue transplantation did not increase the metastatic rate after orthotopic injection of cell suspensions of stomach tumor cells. This intact tissue orthotopic implantation model should allow development of new treatment modalities and further study of the biology of human stomach cancer.


When Is an Alveolar Type 2 Cell an Alveolar Type 2 Cell? A Conundrum for Lung Stem Cell Biology and Regenerative Medicine.
Generating mature, differentiated, adult lung cells from pluripotent cells, such as induced pluripotent stem cells and embryonic stem cells, offers the hope of both generating disease-specific in vitro models and creating definitive and personalized therapies for a host of debilitating lung parenchymal and airway diseases. With the goal of advancing lung-regenerative medicine, several groups have developed and reported on protocols using defined media, coculture with mesenchymal components, or sequential treatments mimicking lung development, to obtain distal lung epithelial cells from stem cell precursors. However, there remains significant controversy about the degree of differentiation of these cells compared with their primary counterparts, coupled with a lack of consistency or uniformity in assessing the resultant phenotypes. Given the inevitable, exponential expansion of these approaches and the probable, but yet-to-emerge second and higher generation techniques to create such assets, we were prompted to pose the question, what makes a lung epithelial cell a lung epithelial cell? More specifically for this Perspective, we also posed the question, what are the minimum features that constitute an alveolar type (AT) 2 epithelial cell? In addressing this, we summarize a body of work spanning nearly five decades, amassed by a series of "lung epithelial cell biology pioneers," which carefully describes well characterized molecular, functional, and morphological features critical for discriminately assessing an AT2 phenotype. Armed with this, we propose a series of core criteria to assist the field in confirming that cells obtained following a differentiation protocol are indeed mature and functional AT2 epithelial cells.


ERASE-Seq: Leveraging replicate measurements to enhance ultralow frequency variant detection in NGS data.
The accurate detection of ultralow allele frequency variants in DNA samples is of interest in both research and medical settings, particularly in liquid biopsies where cancer mutational status is monitored from circulating DNA. Next-generation sequencing (NGS) technologies employing molecular barcoding have shown promise but significant sensitivity and specificity improvements are still needed to detect mutations in a majority of patients before the metastatic stage. To address this we present analytical validation data for ERASE-Seq (Elimination of Recurrent Artifacts and Stochastic Errors), a method for accurate and sensitive detection of ultralow frequency DNA variants in NGS data. ERASE-Seq differs from previous methods by creating a robust statistical framework to utilize technical replicates in conjunction with background error modeling, providing a 10 to 100-fold reduction in false positive rates compared to published molecular barcoding methods. ERASE-Seq was tested using spiked human DNA mixtures with clinically realistic DNA input quantities to detect SNVs and indels between 0.05% and 1% allele frequency, the range commonly found in liquid biopsy samples. Variants were detected with greater than 90% sensitivity and a false positive rate below 0.1 calls per 10,000 possible variants. The approach represents a significant performance improvement compared to molecular barcoding methods and does not require changing molecular reagents.


The impact of image resolution on computation of fractional flow reserve: coronary computed tomography angiography versus 3-dimensional quantitative coronary angiography.
Calculation of fractional flow reserve (FFR) based on computational fluid dynamics (CFD) requires reconstruction of patient-specific coronary geometry and estimation of hyperemic flow rate. Coronary computed tomography angiography (CCTA) and invasive coronary angiography (ICA) are two dominating imaging modalities used for the geometrical reconstruction. Our aim was to investigate the impact of image resolution as inherently associated with these two imaging modalities on geometrical reconstruction and subsequent FFR calculation. Patients with mild or intermediate coronary stenoses who underwent both CCTA and ICA were included. CCTA images were acquired either by 320-row area detector CT or by 128-slice dual-source CT. Two geometrical models were reconstructed separately from CCTA and ICA, from which FFRCTA and FFRQCA were subsequently calculated using CFD simulations, applying the same hyperemic flow rate derived from the ICA images at the inlet boundaries. A total of 57 vessels in 41 patients were analyzed. Average diameter stenosis was 43.4 ± 10.8 % by 3D QCA. Reasonably good correlation between FFRCTA and FFRQCA was observed (r = 0.71, p < 0.001). The difference between FFRCTA and FFRQCA was correlated with the deviation between minimal lumen areas by CCTA and by ICA (ρ = 0.34, p = 0.01), but not with plaque volume (ρ = -0.09, p = 0.51) or calcified plaque volume (ρ = 0.01, p = 0.95). Applying the cutoff value of ≤0.8 to both FFRCTA and FFRQCA, the agreement between FFRCTA and FFRQCA in discriminating functional significant stenoses was moderate (kappa 0.47, p < 0.001). Disagreement was found in 10 (17.5 %) vessels. Acceptable correlation between FFRCTA and FFRQCA was observed, while their agreement in distinguishing functional significant stenosis was moderate. Our results suggest that image resolution has a significant impact on FFR computation.


Phosphorylation of SAS-6 by ZYG-1 is critical for centriole formation in C. elegans embryos.
Despite being essential for proper cell division, the mechanisms governing centrosome duplication are incompletely understood and represent an important open question in cell biology. Formation of a new centriole next to each existing one is critical for centrosome duplication. In Caenorhabditis elegans embryos, the proteins SPD-2, ZYG-1, SAS-6, SAS-5, and SAS-4 are essential for centriole formation, but the mechanisms underlying their requirement remain unclear. Here, we demonstrate that the kinase ZYG-1 phosphorylates the coiled-coil protein SAS-6 at serine 123 in vitro. Importantly, we show that this phosphorylation event is crucial for centriole formation in vivo. Furthermore, we establish that such phosphorylation ensures the maintenance of SAS-6 at the emerging centriole. Overall, our findings establish that phosphorylation of the evolutionarily conserved protein SAS-6 is critical for centriole formation and thus for faithful cell division.


The Ewing's sarcoma fusion protein, EWS-FLI, binds Runx2 and blocks osteoblast differentiation.
Ewing's sarcomas are highly aggressive round cell tumors of bone and soft tissues that afflict children and young adults. The majority of these tumors harbor the t(11;22) translocation and express the fusion protein EWS-FLI. Modern molecular profiling experiments indicate that Ewing's tumors originate from mesenchymal precursors in young individuals. EWS-FLI alters the morphology of mesenchymal cells and prevents lineage specification; however, the molecular mechanisms for differentiation arrest are unclear. We recently showed that EWS-FLI binds Runx2, a master regulator of osteoblast differentiation. In this report, we demonstrate that FLI sequences within EWS-FLI are responsible for interactions with Runx2. EWS-FLI blocks the expression of osteoblastic genes in a multipotent progenitor cell line that requires Runx2 to integrate bone morphogenic protein (Bmp)2 signaling while increasing proliferation and altering cell morphology. These results demonstrate that EWS-FLI blocks the ability of Runx2 to induce osteoblast specification of a mesenchymal progenitor cell. Disrupting interactions between Runx2 and EWS-FLI1 may promote differentiation of the tumor cell.


Internally generated cell assembly sequences in the rat hippocampus.
A long-standing conjecture in neuroscience is that aspects of cognition depend on the brain's ability to self-generate sequential neuronal activity. We found that reliably and continually changing cell assemblies in the rat hippocampus appeared not only during spatial navigation but also in the absence of changing environmental or body-derived inputs. During the delay period of a memory task, each moment in time was characterized by the activity of a particular assembly of neurons. Identical initial conditions triggered a similar assembly sequence, whereas different conditions gave rise to different sequences, thereby predicting behavioral choices, including errors. Such sequences were not formed in control (nonmemory) tasks. We hypothesize that neuronal representations, evolved for encoding distance in spatial navigation, also support episodic recall and the planning of action sequences.


Epithelial and stromal expression of miRNAs during prostate cancer progression.
Global microRNA (miRNA) profile may predict prostate cancer (PCa) behaviors. In this study, we examined global miRNA expression by miRNA profiling as well as specific miRNA expression levels in PCa epithelium and stroma by in situ hybridization (ISH) and correlated with various clinicopathological features. We first performed comprehensive miRNA profiling on 27 macrodissected cases of PCa by miRNA microarray. A total of 299 miRNAs were significantly dysregulated in high grade and advanced stage PCa. We demonstrated that PCa can be readily classified into high grade/stage and low-grade/stage groups by its global miRNA expression profile. Next, we examined the expression of several selected dysregulated miRNAs, including let-7c, miR-21, miR-27a, miR-30c, and miR-219, in PCa by ISH. The levels of miRNA expression in epithelial and stromal cells were scored semiquantitatively and compared with clinicopathological features, including age, race, Gleason score, stage, PSA recurrence, metastasis, hormone resistance and survival. We found that the expression of miR-30c and miR-219 were significantly down-regulated in PCa. miR-21 and miR-30c were significantly down-regulated in PCa in African Americans compared to Caucasian Americans. In addition, down-regulation of let-7c, miR-21, miR-30c, and miR-219 are associated with metastatic disease. Furthermore, down-regulation of miR-30c and let-7c are significantly associated with androgen-dependent PCa. In PCa stromal cells, let-7c downregulation is significantly associated with extraprostatic extension. Our data suggest that selected miRNAs may serve as potential biomarkers to predict cancer progression.


Inhibition of mTOR induces a paused pluripotent state.
Cultured pluripotent stem cells are a cornerstone of regenerative medicine owing to their ability to give rise to all cell types of the body. Although pluripotent stem cells can be propagated indefinitely in vitro, pluripotency is paradoxically a transient state in vivo, lasting 2-3 days around the time of blastocyst implantation. The exception to this rule is embryonic diapause, a reversible state of suspended development triggered by unfavourable conditions. Diapause is a physiological reproductive strategy widely employed across the animal kingdom, including in mammals, but its regulation remains poorly understood. Here we report that the partial inhibition of mechanistic target of rapamycin (mTOR), a major nutrient sensor and promoter of growth, induces reversible pausing of mouse blastocyst development and allows their prolonged culture ex vivo. Paused blastocysts remain pluripotent and competent-able to give rise to embryonic stem (ES) cells and live, fertile mice. We show that both naturally diapaused blastocysts in vivo and paused blastocysts ex vivo display pronounced reductions in mTOR activity, translation, histone modifications associated with gene activity and transcription. Pausing can be induced directly in cultured ES cells and sustained for weeks without appreciable cell death or deviations from cell cycle distributions. We show that paused ES cells display a remarkable global suppression of transcription, maintain a gene expression signature of diapaused blastocysts and remain pluripotent. These results uncover a new pluripotent stem cell state corresponding to the epiblast of the diapaused blastocyst and indicate that mTOR regulates developmental timing at the peri-implantation stage. Our findings have implications in the fields of assisted reproduction, regenerative medicine, cancer, metabolic disorders and ageing.


The contribution of an animal model toward uncovering biological risk factors for PTSD.
Clinical studies of posttraumatic stress disorder (PTSD) have elicited proposed risk factors for developing PTSD in the aftermath of stress exposure. Generally, these risk factors have arisen from retrospective analysis of premorbid characteristics of study populations. A valid animal model of PTSD can complement clinical studies and help to elucidate issues, such as the contribution of proposed risk factors, in ways which are not practicable in the clinical arena. Important qualities of animal models include the possibility to conduct controlled prospective studies, easy access to postmortem brains, and the availability of genetically manipulated subjects, which can be tailored to specific needs. When these qualities are further complemented by an approach which defines phenomenologic criteria to address the variance in individual response pattern and magnitude, enabling the animal subjects to be classified into definable groups for focused study, the model acquires added validity. This article presents an overview of a series of studies in such an animal model which examine the contribution of two proposed risk factors and the value of two early postexposure pharmacological manipulations on the prevalence rates of subjects displaying an extreme magnitude of behavioral response to a predator stress paradigm.


Thiol redox transitions in cell signaling: a lesson from N-acetylcysteine.
The functional status of cells is under the control of external stimuli affecting the function of critical proteins and eventually gene expression. Signal sensing and transduction by messengers to specific effectors operate by post-translational modification of proteins, among which thiol redox switches play a fundamental role that is just beginning to be understood. The maintenance of the redox status is, indeed, crucial for cellular homeostasis and its dysregulation towards a more oxidized intracellular environment is associated with aberrant proliferation, ultimately related to diseases such as cancer, cardiovascular disease, and diabetes. Redox transitions occur in sensitive cysteine residues of regulatory proteins relevant to signaling, their evolution to metastable disulfides accounting for the functional redox switch. N-acetylcysteine (NAC) is a thiol-containing compound that is able to interfere with redox transitions of thiols and, thus, in principle, able to modulate redox signaling. We here review the redox chemistry of NAC, then screen possible mechanisms to explain the effects observed in NAC-treated normal and cancer cells; such effects involve a modification of global gene expression, thus of functions and morphology, with a leitmotif of a switch from proliferation to terminal differentiation. The regulation of thiol redox transitions in cell signaling is, therefore, proposed as a new tool, holding promise not only for a deeper explanation of mechanisms, but indeed for innovative pharmacological interventions.


Functional analysis of missense mutations in Kv8.2 causing cone dystrophy with supernormal rod electroretinogram.
Mutations in KCNV2 have been proposed as the molecular basis for cone dystrophy with supernormal rod electroretinogram. KCNV2 codes for the modulatory voltage-gated potassium channel α-subunit, Kv8.2, which is incapable of forming functional channels on its own. Functional heteromeric channels are however formed with Kv2.1 in heterologous expression systems, with both α-subunit genes expressed in rod and cone photoreceptors. Of the 30 mutations identified in the KCNV2 gene, we have selected three missense mutations localized in the potassium channel pore and two missense mutations localized in the tetramerization domain for analysis. We characterized the differences between homomeric Kv2.1 and heteromeric Kv2.1/Kv8.2 channels and investigated the influence of the selected mutations on the function of heteromeric channels. We found that two pore mutations (W467G and G478R) led to the formation of nonconducting heteromeric Kv2.1/Kv8.2 channels, whereas the mutations localized in the tetramerization domain prevented heteromer generation and resulted in the formation of homomeric Kv2.1 channels only. Consequently, our study suggests the existence of two distinct molecular mechanisms involved in the disease pathology.


Afferent input and sensory function after human spinal cord injury.
Spinal cord injury (SCI) often disrupts the integrity of afferent (sensory) axons projecting through the spinal cord dorsal columns to the brain. Examinations of ascending sensory tracts, therefore, are critical for monitoring the extent of SCI and recovery processes. In this review, we discuss the most common electrophysiological techniques used to assess transmission of afferent inputs to the primary motor cortex (i.e., afferent input-induced facilitation and inhibition) and the somatosensory cortex [i.e., somatosensory evoked potentials (SSEPs), dermatomal SSEPs, and electrical perceptual thresholds] following human SCI. We discuss how afferent input modulates corticospinal excitability by involving cortical and spinal mechanisms depending on the timing of the effects, which need to be considered separately for upper and lower limb muscles. We argue that the time of arrival of afferent input onto the sensory and motor cortex is critical to consider in plasticity-induced protocols in humans with SCI. We also discuss how current sensory exams have been used to detect differences between control and SCI participants but might be less optimal to characterize the level and severity of injury. There is a need to conduct some of these electrophysiological examinations during functionally relevant behaviors to understand the contribution of impaired afferent inputs to the control, or lack of control, of movement. Thus the effects of transmission of afferent inputs to the brain need to be considered on multiple functions following human SCI.


Rupture of the pectoralis major muscle.
Nineteen cases of rupture of the pectoralis major muscle are presented. In 16 cases the rupture was repaired; in 3 cases repair was not performed. Physical findings and surgical technique are described and the anatomy of the insertion of the pectoralis major muscle is reviewed. Nine of the patients in this series sustained their injury while performing the bench press. The majority of the remaining patients were also involved in sports when injury occurred. Eleven of the ruptures were in the nondominant arm and eight in the dominant arm. All of the patients complained of weakness, pain, and deformity, with weakness being the major complaint. Following surgery and rehabilitation, all 16 patients who underwent repair were relieved of pain. The deformity was corrected in all 16 patients, although 2 patients who had a 5 year interval from repair to rupture did not achieve a completely normal contour. Thirteen of the 16 patients reported a full return to strength. The others had significant improvements. Ruptures of the pectoralis major muscle are uncommon, but not rare. Ruptures occurring under tension tear at or near the insertion onto the humerus, as in all 19 cases in this series. Distal ruptures are usually complete, despite physical signs that may be misleading. Distal tears are reparable, even after a delay to treatment. After surgical repair, the majority of patients may expect relief of pain, return of strength and normal contour, and maintenance of range of motion.


Spatial analysis of Carbon-14 dynamics in a wetland ecosystem (Duke Swamp, Chalk River Laboratories, Canada).
A detailed survey was conducted to quantify the spatial distribution of (14)C in Sphagnum moss and underlying soil collected in Duke Swamp. This wetland environment receives (14)C via groundwater pathways from a historic radioactive Waste Management Area (WMA) on Atomic Energy Canada Limited (AECL)'s Chalk River Laboratories (CRL) site. Trends in (14)C specific activities were evaluated with distance from the sampling location with the maximum (14)C specific activity (DSS-35), which was situated adjacent to the WMA and close to an area of groundwater discharge. Based on a spatial evaluation of the data, an east-to-west (14)C gradient was found, due to the influence of the WMA on (14)C specific activities in the swamp. In addition, it was possible to identify two groups of sites, each showing significant exponential declines with distance from the groundwater source area. One of the groups showed relatively more elevated (14)C specific activities at a given distance from source, likely due to their proximity to the WMA, the location of the sub-surface plume originating from the WMA, the presence of marsh and swamp habitat types, which facilitated (14)C transport to the atmosphere, and possibly, (14)C air dispersion patterns along the eastern edge of the swamp. The other group, which had lower (14)C specific activities at a given distance from the groundwater source area, included locations that were more distant from the WMA and the sub-surface plume, and contained fen habitat, which is known to act as barrier to groundwater flow. The findings suggest that proximity to source, groundwater flow patterns and habitat physical characteristics can play an important role in the dynamics of (14)C being carried by discharging groundwater into terrestrial and wetland environments.


Microfiber release from real soiled consumer laundry and the impact of fabric care products and washing conditions.
Fiber release during domestic textile washing is a cause of marine microplastic pollution, but better understanding of the magnitude of the issue and role of fabric care products, appliances and washing cycles is needed. Soiled consumer wash loads from U.K. households were found to release a mean of 114 ± 66.8 ppm (mg microfiber per kg fabric) (n = 79) fibers during typical washing conditions and these were mainly composed of natural fibers. Microfiber release decreased with increasing wash load size and hence decreasing water to fabric ratio, with mean microfiber release from wash loads in the mass range 1.0-3.5 kg (n = 57) found to be 132.4 ± 68.6 ppm, significantly (p = 3.3 x 10-8) higher than the 66.3 ± 27.0 ppm of those in the 3.5-6.0 kg range (n = 22). In further tests with similar soiled consumer wash loads, moving to colder and quicker washing cycles (i.e. 15°C for 30 mins, as opposed to 40°C for 85 mins) significantly reduced microfiber generation by 30% (p = 0.036) and reduced whiteness loss by 42% (p = 0.000) through reduced dye transfer and soil re-deposition, compared to conventional 40°C cycles. In multicycle technical testing, detergent pods were selected for investigation and found to have no impact on microfiber release compared to washing in water alone. Fabric softeners were also found to have no direct impact on microfiber release in testing under both European and North American washing conditions. Extended testing of polyester fleece garments up to a 48-wash cycle history under European conditions found that microfiber release significantly reduced to a consistent low level of 28.7 ± 10.9 ppm from eight through 64 washes. Emerging North American High-Efficiency top-loading washing machines generated significantly lower microfiber release than traditional top-loading machines, likely due to their lower water fill volumes and hence lower water to fabric ratio, with a 69.7% reduction observed for polyester fleece (n = 32, p = 7.9 x 10-6) and 37.4% reduction for polyester T-shirt (n = 32, p = 0.0032). These results conclude that consumers can directly reduce the levels of microfibers generated per wash during domestic textile washing by using colder and quicker wash cycles, washing complete (but not overfilled) loads, and (in North America) converting to High-Efficiency washing machines. Moving to colder and quicker cycles will also indirectly reduce microfiber release by extending the lifetime of clothing, leading to fewer new garments being purchased and hence lower incidence of the high microfiber release occurring during the first few washes of a new item.


Congenital muscular dystrophies and congenital myopathies.
The purpose of this review is to provide information regarding the diagnosis and natural history of some very rare disorders: congenital muscular dystrophies and congenital myopathies. Patients with these conditions share characteristics such as early onset of weakness and severe hypotonia. Other organs such as the brain, eyes, and skin may be involved. Diagnosis depends largely on recognition of phenotype, muscle biopsy, and mutation analysis. More than 30 genes have been associated with these diseases, most of which have only been recognized in the past decade. Increasing availability of DNA analysis has been important in decreasing delay in diagnosis. Patients with congenital muscular dystrophy or congenital myopathy are at high risk of complications including restrictive lung disease, orthopedic deformities, seizures, cardiomyopathy, and malignant hyperthermia. Life expectancy varies with the severity of complications. Having an accurate and specific diagnosis allows the neurologist to carry out anticipatory guidance and appropriate monitoring. New hope exists for experimental treatments for congenital muscular dystrophy and congenital myopathy as our understanding of pathogenesis evolves.


Craniopharyngioma: a comparison of tumor control with various treatment strategies.
Craniopharyngiomas have a propensity to recur after resection, potentially causing death through their aggressive local behavior in their critical site of origin. Recent data suggest that subtotal resection (STR) followed by adjuvant radiotherapy (XRT) may be an appealing substitute for gross-total resection (GTR), providing similar rates of tumor control without the morbidity associated with aggressive resection. Here, the authors summarize the published literature regarding rates of tumor control with various treatment modalities for craniopharyngiomas. The authors performed a comprehensive search of the English language literature to identify studies publishing outcome data on patients undergoing surgery for craniopharyngioma. Rates of progression-free survival (PFS) and overall survival (OS) were determined through Kaplan-Meier analysis. There were 442 patients who underwent tumor resection. Among these patients, GTR was achieved in 256 cases (58%), STR in 101 cases (23%), and STR+XRT in 85 cases (19%). The 2- and 5-year PFS rates for the GTR group versus the STR+XRT group were 88 versus 91%, and 67 versus 69%, respectively. The 5- and 10-year OS rates for the GTR group versus the STR+XRT group were 98 versus 99%, and 98 versus 95%, respectively. There was no significant difference in PFS (log-rank test) or OS with GTR (log-rank test). Given the relative rarity of craniopharyngioma, this study provides estimates of outcome for a variety of treatment combinations, as not all treatments are an option for all patients with these tumors.


Pollination and reproduction of a self-incompatible forest herb in hedgerow corridors and forest patches.
Habitat-corridors are assumed to counteract the negative impacts of habitat loss and fragmentation, but their efficiency in doing so depends on the maintenance of ecological processes in corridor conditions. For plants dispersing in linear habitats, one of these critical processes is the maintenance of adequate pollen transfer to insure seed production within the corridor. This study focuses on a common, self-incompatible forest herb, Trillium grandiflorum, to assess plant-pollinator interactions and the influence of spatial processes on plant reproduction in hedgerow corridors compared to forests. First, using pollen supplementation experiments over 2 years, we quantified the extent of pollen limitation in both habitats, testing the prediction of greater limitation in small hedgerow populations than in forests. While pollen limitation of fruit and seed set was common, its magnitude did not differ between habitats. Variations among sites, however, suggested an influence of landscape context on pollination services. Second, we examined the effect of isolation on plant reproduction by monitoring fruit and seed production, as well as pollinator activity and assemblage, in small flower arrays transplanted in hedgerows at increasing distances from forest and from each other. We detected no difference in the proportion of flowers setting fruit or in pollinator activity with isolation, but we observed some differences in pollinator assemblages. Seed set, on the other hand, declined significantly with increasing isolation in the second year of the study, but not in the first year, suggesting altered pollen transfer with distance. Overall, plants in hedgerow corridors and forests benefited from similar pollination services. In this system, plant-pollinator interactions and reproduction seem to be influenced more by variations in resource distribution over years and landscapes than by local habitat conditions.


Multimodality and the origin of a novel communication system in face-to-face interaction.
Face-to-face communication is multimodal at its core: it consists of a combination of vocal and visual signalling. However, current evidence suggests that, in the absence of an established communication system, visual signalling, especially in the form of visible gesture, is a more powerful form of communication than vocalization and therefore likely to have played a primary role in the emergence of human language. This argument is based on experimental evidence of how vocal and visual modalities (i.e. gesture) are employed to communicate about familiar concepts when participants cannot use their existing languages. To investigate this further, we introduce an experiment where pairs of participants performed a referential communication task in which they described unfamiliar stimuli in order to reduce reliance on conventional signals. Visual and auditory stimuli were described in three conditions: using visible gestures only, using non-linguistic vocalizations only and given the option to use both (multimodal communication). The results suggest that even in the absence of conventional signals, gesture is a more powerful mode of communication compared with vocalization, but that there are also advantages to multimodality compared to using gesture alone. Participants with an option to produce multimodal signals had comparable accuracy to those using only gesture, but gained an efficiency advantage. The analysis of the interactions between participants showed that interactants developed novel communication systems for unfamiliar stimuli by deploying different modalities flexibly to suit their needs and by taking advantage of multimodality when required.


Bariatric surgery to unload the stressed heart: a metabolic hypothesis.
Obesity is an independent risk factor for cardiovascular disease. Data from the Framingham Study have reported a higher incidence of heart failure in obese individuals compared with a normal cohort. The body initially copes with the abundance of fuel present in an obese milieu by storing it in adipose tissue. However, when the storage capacity is exceeded, the excess energy is taken up and stored ectopically as fat in vital organs such as the heart. Indeed, intramyocardial lipid overload is present in hearts of obese patients, as well as in hearts of animal models of obesity, and is associated with a distinct gene expression profile and cardiac dysfunction. By imposing a metabolic stress on the heart, obesity causes it to hypertrophy and ultimately to fail. Conventional measures to treat obesity include diet, exercise, and drugs. More recently, weight loss surgery (WLS) has achieved increasing prominence because of its ability to reduce the neurohumoral load, normalize metabolic dysregulation, and improve overall survival. The effects of WLS on systemic metabolic, neurohumoral, and hemodynamic parameters are well described and include an early normalization of serum glucose and insulin levels as well as reduction in blood pressure. WLS is also associated with reverse cardiac remodeling, regression of left ventricular hypertrophy, and improved left ventricular and right ventricular function. By targeting the source of the excess energy, we hypothesize that WLS improves contractile function by limiting exogenous substrate availability to the metabolically overloaded heart. These changes have also been found to be associated with increased levels of adiponectin and improved insulin sensitivity. Taken together, the sustained beneficial effects of WLS on left ventricular mass and function highlight the need to better understand the mechanism by which obesity regulates cardiovascular physiology.


Ontogeny in animal colonies: a persistent trend in the bryozoan fossil record.
The principal modes of zooid and colony growth in encrusting cheilostome bryozoans have undergone a major evolutionary transition during the last 100 million years. Comparisons of species within successive North American fossil faunas reveal a persistent trend in which one mode of growth is gradually supplanted by another. Ecological evidence from living faunas and the polyphyletic nature of this trend suggest that the transition has adaptive significance.


On a spike train probability model with interacting neural units.
We investigate an extension of the spike train stochastic model based on the conditional intensity, in which the recovery function includes an interaction between several excitatory neural units. Such function is proposed as depending both on the time elapsed since the last spike and on the last spiking unit. Our approach, being somewhat related to the competing risks model, allows to obtain the general form of the interspike distribution and of the probability of consecutive spikes from the same unit. Various results are finally presented in the two cases when the free firing rate function (i) is constant, and (ii) has a sinusoidal form.


Medial malleolar osteotomy for the correction of varus deformity during total ankle arthroplasty: results in 15 ankles.
Preoperative deformity in the frontal plane in the arthritic ankle is a risk factor for failure after total ankle arthroplasty. Medial malleolar lengthening osteotomy was developed to correct varus malalignment. From 1998 to 2005 total ankle arthroplasty combined with medial malleolar lengthening osteotomy was done in 15 ankles (13 patients) with a mean preoperative varus deformity of 14.9 (SD, 7.8) degrees. Diagnosis was arthritis with instability in 11 ankles (9 patients) and inflammatory joint disease in 4 ankles. Two mobile-bearing designs were used. Osteosynthesis of the osteotomy was done in 2 ankles; for the remaining 13 osteotomies, no fixation was used. Followup was 5 (range 2 to 8) years. Neutral alignment was obtained in all ankles. In 3 patients residual hindfoot varus remained, for which a second-stage hindfoot correction was done. Two rheumatoid ankles developed a symptom-free nonunion of the medial malleolus, all other malleolar osteotomies united. One tibial component, implanted with too much anterior slope, developed early aseptic loosening and was revised. Debridement for talar-malleolar arthritis was done in two ankles. Of the 14 ankles in followup, 12 were rated as excellent or good, one as fair. One ankle with subsidence of the talar component was rated as unsatisfactory. AOFAS score increased from 30.8 preoperative to 81.0 at followup (p < 0.01). Medial malleolar lengthening osteotomy is an easy technique for the realignment of the varus ankle at the time of total ankle arthroplasty, and served as an alternative to medial ligament release or lateral ligament reconstruction.


Healing Body and Spirit.
This drawing considers how achieving balance between allopathic, Western medical practices and spiritual practices can be critical for clinicians hoping to offer compassionate health care. Ethical and clinical challenges of consistently offering compassionate care to patients from all backgrounds requires cultivating and practicing spiritual awareness while maintaining professional boundaries.


Compliance with recommendations on outpatient antibiotic prescribing for respiratory tract infections: the case of Spain.
Inappropriate antibiotic use in primary care, such as in respiratory tract infections (RTIs), is an important cause of bacterial resistance. This study aimed at describing the current pattern of outpatient antibiotic use in acute RTIs in Spain and evaluating adherence to national recommendations. A retrospective observational study was performed including all the episodes of RTIs registered during a 1-year period in a north-eastern Spanish region. Data related to patient demography, diagnoses and antibiotic prescriptions were collected from the electronic medical history database in the region, and adherence to recommendations for antibiotic prescribing was assessed. One third of patients with a RTI were prescribed an antibiotic, with young adults (aged 15-64 years) being the most treated. High prescribing rates were observed in patients with acute otitis, sinusitis and acute tonsillitis (about 70%), whereas low rates were found in acute bronchitis (50%) and non-specific upper RTIs (24%) episodes. A high prescription of broad-spectrum agents and antibiotics not recommended as first choice was observed. In accordance with Spanish guidelines, there exists a potential over-prescribing of antibiotics for all the diagnoses studied, especially in the adult population. Moreover, the choice of antibiotics is frequently based on agents with a high risk of increasing antimicrobial resistance. Multifaceted strategies should be implemented to improve the quality of antibiotic prescribing in primary care.


Retinal gene expression changes related to IOP exposure and axonal loss in DBA/2J mice.
To determine the effects of cumulative IOP exposure and axonal damage on retinal gene expression in DBA/2 mice. DBA/2J, DBA/2J(pe) (pearl), and C57BL/6 mice from 3 to 12 months of age were used. IOP was measured with a rebound tonometer, and optic nerve (ON) damage was determined by grading of ON sections. Retinal RNA was subjected to microarray analysis. Comparisons explored the effects of cumulative IOP exposure (cIOPx) as well as ON damage (ONd) in the DBA/2J animals compared with that in the C57BL/6 and pearl mice. RT-PCR was performed to confirm some of the genes and bioinformatic analysis to identify affected gene networks. Microarrays revealed that an increasing number of genes were up- or downregulated in 9- and 12-month DBA/2J mice with various degrees of ONd. A smaller number of genes were expressed differentially between eyes with different cIOPx at the same age, from 6 months on. Expression of 1385 and 1133 genes differed between DBA/2J animals and C57BL/6 or pearl mice, respectively, and some them were confirmed by RT-PCR. Bioinformatics analysis identified functional gene networks, including members of the complement system, that appeared to be related to cIOPx, ONd, or both. Gene expression changes occur in retinas of DBA/2 mice with various amounts of cIOPx as well as ONd. Genes involved, code for proteins with diverse cellular functions and include among others the complement system. cIOPx and ONd affect common as well as unique gene sets.


Evaluation of the SLOMYCO Sensititre(®) panel for testing the antimicrobial susceptibility of Mycobacterium marinum isolates.
The agar dilution method is currently considered as the reference method for Mycobacterium marinum drug susceptibility testing (DST). As it is time-consuming, alternative methods, such as the E-test, were evaluated for M. marinum DST, but without success. The SLOMYCO Sensititre(®) panel, recently commercialized by TREK Diagnostic Systems (Cleveland, OH), can be used for DST in slow-growing mycobacteria and for antimicrobial agents recommended by the Clinical and Laboratory Standards Institute (CLSI) for M. marinum DST. The main goal of this work was to evaluate the SLOMYCO Sensititre(®) panel method for DST in M. marinum isolates from human patients and fish relative to the reference agar dilution method. The reproducibility of the minimum inhibitory concentration (MIC) determination (±1 log2 dilution) was very good for both the agar dilution method and SLOMYCO Sensititre(®) panel (>90 % agreement). The percentage essential agreement between methods varied, depending on the drug: between 97 and 75 % for ciprofloxacin, moxifloxacin, linezolid, isoniazid, clarithromycin, amikacin, rifabutin and rifampin, 74 % for trimethoprim, 72 % for doxycycline, 70 % for sulfamethoxazole, 59 % for streptomycin, 33 % for ethambutol and only 2.2 % for ethionamide. When the agar dilution and SLOMYCO Sensititre(®) panel results were converted into interpretive criteria, the category agreement was 100 % for amikacin, ciprofloxacin, clarithromycin, moxifloxacin, rifabutin, sulfamethoxazole and trimethoprim, 98 % for ethambutol and 96 % for rifampin and no agreement for doxycycline. The SLOMYCO Sensititre(®) panel method could provide a potential alternative to the reference agar dilution method, when DST in M. marinum is required, except for doxycycline.


The role of school performance in narrowing gender gaps in the formation of STEM aspirations: a cross-national study.
This study uses cross-national evidence to estimate the effect of school peer performance on the size of the gender gap in the formation of STEM career aspirations. We argue that STEM aspirations are influenced not only by gender stereotyping in the national culture but also by the performance of peers in the local school environment. Our analyses are based on the Program for International Student Assessment (PISA). They investigate whether 15-year-old students from 55 different countries expect to have STEM jobs at the age of 30. We find considerable gender differences in the plans to pursue careers in STEM occupations in all countries. Using PISA test scores in math and science aggregated at the school level as a measure of school performance, we find that stronger performance environments have a negative impact on student career aspirations in STEM. Although girls are less likely than boys to aspire to STEM occupations, even when they have comparable abilities, boys respond more than girls to competitive school performance environments. As a consequence, the aspirations gender gap narrows for high-performing students in stronger performance environments. We show that those effects are larger in countries that do not sort students into different educational tracks.


Primary neurosurgery for pediatric low-grade gliomas: a prospective multi-institutional study from the Children's Oncology Group.
Central nervous system neoplasms are the most common solid tumors in children, and more than 40% are low-grade gliomas. Variable locations, extent of resection, postoperative neurodiagnostic evaluation, and histology have confounded therapy and outcome. To investigate disease control and survival after surgery. A prospective natural history trial from 1991 to 1996 produced a subset of patients with low-grade gliomas managed by primary surgery and subsequent observation. Patients were evaluable if eligibility, tumor location, and extent of resection were confirmed by pathological diagnosis, preoperative and postoperative imaging, and the surgeon's report. Primary end points were overall survival (OS), progression-free survival (PFS), and postprogression survival. Of 726 patients enrolled, 518 were fully evaluable for analysis. The 5- and 8-year OS rates were 97% ± 0.8% and 96% ± 0.9%, respectively, and PFS rates were 80% ± 1.8% and 78% ± 2.0%. In univariate analyses, histological type, extent of residual tumor, and disease site were significantly associated with PFS and OS. In multivariate analysis, gross total resection (GTR) without residual disease was the predominant predictor of PFS. In patients with limited residual disease, 56% were free of progression at 5 years. GTR should be the goal when it can be achieved with an acceptable functional outcome. The variable rate of progression after incomplete resection highlights the need for new predictors of tumor behavior.


Understanding Australian medical student attitudes towards older people.
The aim of this article was to review the literature around Australian medical student attitudes towards older people. An Ovid cross-search and SCOPUS search were performed using keywords such as 'Attitude', 'Medical Student' and 'Aged or Older or Elderly'. Several recent studies have investigated the attitudes of Australian medical students towards older people. Baseline attitudes at two medical schools were positive. Three studies quantified attitude improvement after curriculum intervention. All the studies used US-developed instruments, which have not been validated in Australia. Qualitative studies have described mixed attitudes towards older people: negative themes included nihilism, paternalism, communication issues, greater morbidity and reduced quality of life. Positively, students placed value on clinical decision-making and critical reflection during residential aged care placements. Australian medical students' attitudes towards older people are mixed and not well understood based on quantitative measures developed for use in the US and on qualitative evidence. Future research in this area requires a reliable and locally-validated instrument.


Enhanced Wound Healing Potential of Primary Human Oral Fibroblasts and Periodontal Ligament Cells Cultured on Four Different Porcine-Derived Collagen Matrices.
Xenogenic collagen-based matrices represent an alternative to subepithelial palatal connective tissue autografts in periodontal and peri-implant soft tissue reconstructions. In the present study, we aimed to investigate the migratory, adhesive, proliferative, and wound-healing potential of primary human oral fibroblasts (hOF) and periodontal ligament cells (hPDL) in response to four commercially available collagen matrices. Non-crosslinked collagen matrix (NCM), crosslinked collagen matrix (CCM), dried acellular dermal matrix (DADM), and hydrated acellular dermal matrix (HADM) were all able to significantly enhance the ability of hPDL and hOF cells to directionally migrate toward the matrices as well as to efficiently repopulate an artificially generated wound gap covered by the matrices. Compared to NCM and DADM, CCM and HADM triggered stronger migratory response. Cells grown on CCM and HADM demonstrated significantly higher proliferative rates compared to cells grown on cell culture plastic, NCM, or DADM. The pro-proliferative effect of the matrices was supported by expression analysis of proliferative markers regulating cell cycle progression. Upregulated expression of genes encoding the adhesive molecules fibronectin, vinculin, CD44 antigen, and the intracellular adhesive molecule-1 was detected in hPDL and hOF cells cultured on each of the four matrices. This may be considered as a prerequisite for good adhesive properties of the four scaffolds ensuring proper cell-matrix and cell-cell interactions. Upregulated expression of genes encoding TGF-β1 and EGF growth factors as well as MMPs in cells grown on each of the four matrices provided support for their pro-proliferative and pro-migratory abilities. The expression of genes encoding the angiogenic factors FGF-2 and VEGF-A was dramatically increased in cells grown on DADM and HADM only, suggesting a good basis for accelerated vascularization of the latter. Altogether, our results support favorable influence of the investigated collagen matrices on the recruitment, attachment, and growth of cell types implicated in oral soft tissue regeneration. Among the four matrices, HADM has consistently exhibited stronger positive effects on the oral cellular behavior. Our data provide solid basis for future investigations on the clinical application of the collagen-based matrices in surgical periodontal therapy.


Does scapula stabilizing t-shirt help over-head athletes in shoulder discomfort? A randomized control study.
Scapular dyskinesis in pitchers can potentially lead to impairments in the optimal performance of muscles of the shoulder complex. The purpose of this prospective study was to evaluate whether the use of a scapula stabilizing t-shirt can decrease stress and discomfort in over-head baseball pitchers. Thirty-two semi-professional baseball pitchers were divided into 2 groups. Group A dressed the scapula stabilizing t-shirt during the weekly practice while group B (control group) continued to follow their training program without the t-shirt. All pitchers underwent a clinical examination at the beginning of the study (T0), 4 weeks (T1) and 8 weeks after (T2). Data collected showed a better clinical outcome at T1 which increased in T2 for pitchers in Group A, while in Group B athletes these positive results were not observed. This study underlines that scapula-stabilizing t-shirt can have positive effects on shoulder discomfort in over-head athletes.


The evolution of organellar metabolism in unicellular eukaryotes.
Metabolic innovation has facilitated the radiation of microbes into almost every niche environment on the Earth, and over geological time scales transformed the planet on which we live. A notable example of innovation is the evolution of oxygenic photosynthesis which was a prelude to the gradual transformation of an anoxic Earth into a world with oxygenated oceans and an oxygen-rich atmosphere capable of supporting complex multicellular organisms. The influence of microbial innovation on the Earth's history and the timing of pivotal events have been addressed in other recent themed editions of Philosophical Transactions of Royal Society B (Cavalier-Smith et al. 2006; Bendall et al. 2008). In this issue, our contributors provide a timely history of metabolic innovation and adaptation within unicellular eukaryotes. In eukaryotes, diverse metabolic portfolios are compartmentalized across multiple membrane-bounded compartments (or organelles). However, as a consequence of pathway retargeting, organelle degeneration or novel endosymbiotic associations, the metabolic repertoires of protists often differ extensively from classic textbook descriptions of intermediary metabolism. These differences are often important in the context of niche adaptation or the structure of microbial communities. Fundamentally interesting in its own right, the biochemical, cell biological and phylogenomic investigation of organellar metabolism also has wider relevance. For instance, in some pathogens, notably those causing some of the most significant tropical diseases, including malaria, unusual organellar metabolism provides important new drug targets. Moreover, the study of organellar metabolism in protists continues to provide critical insight into our understanding of eukaryotic evolution.


Hypoxia-independent gene expression mediated by SOX9 promotes aggressive pancreatic tumor biology.
Pancreatic cancer aggressiveness is characterized by its high capacity for local invasion, ability to promote angiogenesis, and potential to metastasize. Hypoxia is known to represent a crucial step in the development of aggressive malignant features of many human cancers. However, micrometastatic tumors are not typically subjected to hypoxic events during early stages of dissemination; therefore, it is unclear how these tumors are able to maintain their aggressive phenotype. Thus, the identification of regulators of hypoxia-related genes in aggressive/metastatic tumors represents a fundamental step for the design of future therapies to treat pancreatic cancer. To this end, transcriptomic profiles were compared between the nonmetastatic pancreatic cancer cell line FG (LMET) and its angiogenic/metastatic derivate L3.6pl (HMET) under normoxic or hypoxic conditions. Cluster analysis revealed a number of transcripts that were induced by hypoxia in nonmetastatic cancer cells. Strikingly, this cluster was determined to be constitutively activated under normoxia in the metastatic cancer cells and could not be further induced by hypoxia. A subset of these transcripts were regulated by the transcription factor SOX9 in the aggressive-metastatic cells, but driven by hypoxia-inducible factor-1α (HIF-1α) in the parental nonmetastatic cell line. Moreover, these transcripts were enriched in cancer-related networks including: WNT, CXCR4, retinoic acid, and (FAK) focal adhesion kinase, gene PTK2 signaling pathways. In functional assays, inhibition of SOX9 expression in HMET cells led to increased apoptosis and reduced migration in vitro and a significant reduction in primary tumor growth, angiogenesis, and metastasis following orthotopic tumor cell injection. At the molecular level, the control of SOX9 expression was associated with changes in the methylation status of the SOX9 promoter. Finally, SOX9 upregulation was verified in a series of tumor specimens of patients with pancreatic carcinoma. SOX9 represents a novel target for pancreatic cancer therapy.


Differentiating lipedema and Dercum's disease.
People with lipedema or Dercum's disease (DD) can have a similar distribution of excess painful nodular subcutaneous adipose tissue (SAT), making them difficult to differentiate. Case series of 94 patients with DD, 160 with lipedema and 18 with both diagnoses (Lip+DD) from a single clinic in an academic medical center to improve identification and differentiation of these disorders by comparison of clinical findings, prevalence of type 2 diabetes (DM2), hypermobility by the Beighton score and assessment of a marker of inflammation, Total complement activity (CH50). Differences between groups were by Student's t-test with α of 0.05. The Lipedema Group had significantly greater weight, body mass index (BMI), gynoid distributed nodular SAT and fibrotic and heavy tissue than the DD Group. Hypermobility was significantly higher in the Lipedema (58±0.5%) than DD Group (23±0.4%; P<0.0001). DM2 was significantly greater in the DD (16±0.2%; P=0.0007) than the Lipedema Group (6±0.2%). Average pain by an analog scale was significantly higher in the DD (6±2.5%) than the Lipedema Group (4±2.1%; P<0.0001). Fatigue and swelling were common in both groups. Easy bruising was more common in the Lipedema Group, whereas abdominal pain, shortness of breath, fibromyalgia, migraines and lipomas were more prevalent in the DD Group. The percentage of patients with elevated CH50 was significantly positive in both groups. The significantly lower prevalence of DM2 in people with lipedema compared with DD may be due to the greater amount of gynoid fat known to be protective against metabolic disorders. The high percentage of hypermobility in lipedema patients indicates that it may be a comorbid condition. The location of fat, high average daily pain, presence of lipomas and comorbid painful disorders in DD patients may help differentiate from lipedema.


General and Domain-Specific Contributions to Creative Ideation and Creative Performance.
The general objective of this study was to reexamine two views of creativity, one positing that there is a general creative capacity or talent and the other that creativity is domain-specific. These two views were compared by (a) testing correlations among measures of domain-general and domain-specific creativity and (b) examining how the general and the specific measures was each related to indices of knowledge, motivation, and personality. Participants were 147 college students enrolled in a foreign language course. Data were collected on participants' domain knowledge, motivation, and creative personality, as well as four measures representing "General or Domain-Specific Creative Ideation" or "Creative Performance and Activity". Results indicated that the four measures of creativity were correlated with one another, except for "General Performance and Activity" and "Domain-Specific Ideation." A canonical correlation indicated that knowledge, motivation, and personality were significantly correlated with the four creativity measures (Rc = .49, p < .01). Multiple regressions uncovered particular relationships consistent with the view that creativity has both general and domain-specific contributions. Limitations, such as the focus on one domain, and future directions are discussed.


Evaluation of a semiquantitative SNAP test for measurement of bile acids in dogs.
Background. Serum bile acids (SBA) are used as a routine screening tool of liver function in dogs. Serum samples are usually shipped to a referral laboratory for quantitative analysis with an enzymatic chemistry analyzer. The canine SNAP Bile Acids Test (SNAP-BAT) provides an immediate, semi-quantitative measurement of bile acid concentrations in-house. With the SNAP-BAT, bile acids concentrations of 5-30 µmol/L are quantified, and results outside of that range are classified as <5 or >30 µmol/L. Agreement of the SNAP-BAT with the enzymatic method has not been extensively investigated. Objectives. The purposes of this prospective clinical study were to assess the precision of the SNAP-BAT and determine agreement of SNAP-BAT with results from an in-house chemistry analyzer. Methods. After verifying intra-assay precision of the SNAP-BAT, a prospective analysis was performed using blood samples collected from 56 dogs suspected to have liver disease. Each sample was analyzed with an enzymatic, in-house chemistry analyzer and the SNAP-BAT. Agreement between the two methods was statistically assessed using the κ index of agreement. Results. Intra-assay variability was minimal. The κ index for agreement between the SNAP-BAT and routine chemistry analyzer was between 0.752 and 0.819, indicating substantial to near perfect agreement. Conclusions. The SNAP-BAT is a highly accurate, semi-quantitative test that yields immediate results, and has very little intra-assay variability, particularly for results >30 µmol/L.


Overexpression of cyclin DI contributes to malignant properties of esophageal tumor cells by increasing VEGF production and decreasing Fas expression.
Cyclin D1 is frequently overexpressed in human esophageal cancer. We examined the possible role of cyclin D1 overexpression on specific malignant properties of tumor cells using a series of eight human esophageal cancer cell lines that express different levels of cyclin D1. We did not find a simple correlation between levels of cyclin D1 expression and anchorage-independent growth, production of angiogenic factors, or tumorigenicity in nude mice, suggesting that other factors can influence these parameters. We did, however, obtain evidence that tumorigenicity appeared to require both the capacity for anchorage-independent growth and the production of angiogenic factors. To better assess the specific role of cyclin D1, we stably expressed an antisense cyclin D1 cDNA construct in the tumorigenic cell line TTn. This significantly decreased anchorage-independent growth and VEGF production and led to a loss of tumorigenicity in nude mice. Furthermore, these cells diplayed a marked increase in sensitivity to antitumor agents and to Fas antibody-induced apoptosis. Taken together, these findings suggest that the overexpression of cyclin D1 can confer esophageal cancer cells with enhanced malignancy through increases in anchorage-independent growth and VEGF production, and down-regulation of Fas expression, thus suggesting novel functions of the cyclin D1 protein in tumor progression.


What happened to the IUD in Ghana?
The intrauterine contraceptive device (IUD) is a safe and reversible contraceptive method that requires little effort on the part of the user. Once inserted, it offers 10 years of protection against pregnancy. However, its use in Ghana has stagnated in relation to other contraceptive methods. An exploratory study was, therefore, conducted to examine the client, provider and system characteristics that affect the demand for IUD. Data were gathered through secondary analysis, in-depth interviews, focus group discussions and simulated client survey. The stagnating demand for IUD is attributed to clients' perceptions and rumours about IUD. The fear of excessive bleeding and weight loss discourages potential users. The product design was also perceived to be unacceptable. Demand creation for the IUD has been poor and the number of providers with practical experience of insertion is insufficient. Contrary to the belief that providers' bias contributes to the decline in use, findings show that providers have a favourable attitude towards the product.


Structure and function of a ganglioside receptor for porcine rotavirus.
A ganglioside fraction isolated from pooled intestines from newborn to 4-week-old piglets, which we previously partially characterized and showed to specifically inhibit the binding of porcine rotavirus (OSU strain) to host cells (M. D. Rolsma, H. B. Gelberg, and M. S. Kuhlenschmidt, J. Virol. 68:258-268, 1994), was further purified and found to contain two major monosialogangliosides. Each ganglioside was purified to apparent homogeneity, and their carbohydrate structure was examined by high-pH anion-exchange chromatography coupled with pulsed amperometric detection and fast atom bombardment mass spectroscopy. Both gangliosides possessed a sialyllactose oligosaccharide moiety characteristic of GM3 gangliosides. Compositional analyses indicated that each ganglioside was composed of sialic acid, galactose, glucose, and sphingosine in approximately a 1:1:1:1 molar ratio. Each ganglioside differed, however, in the type of sialic acid residue it contained. An N-glycolylneuraminic acid (NeuGc) moiety was found in the more polar porcine GM3, whereas the less polar GM3 species contained N-acetylneuraminic acid (NeuAc). Both NeuGcGM3 and NeuAcGM3 displayed dose-dependent inhibition of virus binding to host cells. NeuGcGM3 was approximately two to three times more effective than NeuAcGM3 in blocking virus binding. Inhibition of binding occurred with as little as 400 pmol of NeuGcGM3/50 ng of virus (approximately 2 x 10(7) virions) and 2 x 10(6) cells/ml. Fifty percent inhibition of binding was achieved with 0.64 and 1.5 microM NeuGcGM3 and NeuAcGM3, respectively. The free oligosaccharides 3'- and 6'-sialyllactose inhibited binding 50% at millimolar concentrations, which were nearly 1,000 times the concentration of intact gangliosides required for the same degree of inhibition. Direct binding of infectious, triple-layer rotavirus particles, but not noninfectious, double-layered rotavirus particles, to NeuGcGM3 and NeuAcGM3 was demonstrated by using a thin-layer chromatographic overlay assay. NeuGcGM3 and NeuAcGM3 inhibited virus infectivity of MA-104 cells by 50% at concentrations of 3.97 and 9. 84 microM, respectively. NeuGcGM3 (700 nmol/g [dry weight] of intestine) was found to be the predominant enterocyte ganglioside (comprising 75% of the total lipid-bound sialic acid) in neonatal piglets, followed by NeuAcGM3 (200 nmol/g [dry weight] of intestine). NeuGcGM3 and NeuAcGM3 together comprised nearly 100% of the lipid-bound sialic acid in the neonatal intestine, but their quantities rapidly diminished during the first 5 weeks of life. These data support the hypothesis that porcine NeuGcGM3 and NeuAcGM3 are physiologically relevant receptors for porcine rotavirus (OSU strain). Further support for this hypothesis was obtained from virus binding studies using mutant or neuraminidase-treated cell lines. Lec-2 cells, a mutant clone of CHO cells characterized by a 90% reduction in sialyllation of its glycoconjugates, bound less than 5% of the virus compared to control cell binding. In contrast, Lec-1 cells, a mutant CHO clone characterized by a deficiency in glycosylation of N-linked oligosaccharides, still bound rotavirus. Furthermore, exogenous addition of NeuGcGM3 to the Lec-2 mutant cells restored their ability to bind rotavirus in amounts equivalent to that of their parent (CHO) cell line. In the virus-permissive MA-104 cell line, NeuGcGM3 was also able to partially restore rotavirus infectivity in neuraminidase-treated cells. These data suggest that gangliosides play a major role in recognition of host cells by porcine rotavirus (OSU strain).


Interactive memory systems in the human brain.
Learning and memory in humans rely upon several memory systems, which appear to have dissociable brain substrates. A fundamental question concerns whether, and how, these memory systems interact. Here we show using functional magnetic resonance imaging (FMRI) that these memory systems may compete with each other during classification learning in humans. The medial temporal lobe and basal ganglia were differently engaged across subjects during classification learning depending upon whether the task emphasized declarative or nondeclarative memory, even when the to-be-learned material and the level of performance did not differ. Consistent with competition between memory systems suggested by animal studies and neuroimaging, activity in these regions was negatively correlated across individuals. Further examination of classification learning using event-related FMRI showed rapid modulation of activity in these regions at the beginning of learning, suggesting that subjects relied upon the medial temporal lobe early in learning. However, this dependence rapidly declined with training, as predicted by previous computational models of associative learning.


Supplementation with Guanidinoacetic Acid in Women with Chronic Fatigue Syndrome.
A variety of dietary interventions has been used in the management of chronic fatigue syndrome (CFS), yet no therapeutic modality has demonstrated conclusive positive results in terms of effectiveness. The main aim of this study was to evaluate the effects of orally administered guanidinoacetic acid (GAA) on multidimensional fatigue inventory (MFI), musculoskeletal soreness, health-related quality of life, exercise performance, screening laboratory studies, and the occurrence of adverse events in women with CFS. Twenty-one women (age 39.3 ± 8.8 years, weight 62.8 ± 8.5 kg, height 169.5 ± 5.8 cm) who fulfilled the 1994 Centers for Disease Control and Prevention criteria for CFS were randomized in a double-blind, cross-over design, from 1 September 2014 through 31 May 2015, to receive either GAA (2.4 grams per day) or placebo (cellulose) by oral administration for three months, with a two-month wash-out period. No effects of intervention were found for the primary efficacy outcome (MFI score for general fatigue), and musculoskeletal pain at rest and during activity. After three months of intervention, participants receiving GAA significantly increased muscular creatine levels compared with the placebo group (36.3% vs. 2.4%; p < 0.01). Furthermore, changes from baseline in muscular strength and aerobic power were significantly greater in the GAA group compared with placebo (p < 0.05). Results from this study indicated that supplemental GAA can positively affect creatine metabolism and work capacity in women with CFS, yet GAA had no effect on main clinical outcomes, such as general fatigue and musculoskeletal soreness.


Preinjury statin use is associated with a higher risk of multiple organ failure after injury: a propensity score adjusted analysis.
Recent studies suggest that statin use may improve outcome in critically ill patients. This has been attributed to the pleiomorphic effect and modulation of inflammatory mediators that occurs with statin use. We sought to determine whether preinjury statin (PIS) use was associated with improved outcome in severely injured blunt trauma patients. Data were obtained from a multicenter prospective cohort study evaluating outcomes in blunt injured adults with hemorrhagic shock. Patients aged 55 years and older were analyzed. Those with isolated traumatic brain injury, cervical cord injury, and those who survived <24 hours were excluded. A propensity score predicting statin use was created using logistic regression. Cox proportional hazard regression was then used to evaluate the effects of PIS use on mortality and the development of multiple organ failure (MOF, multiple organ dysfunction syndrome >5) and nosocomial infection (NI) after adjusting for important injury characteristics and the propensity of taking PISs. Overall mortality and MOF rates for the study cohort (n = 295) were 21% and 50%, respectively. Over 24% of patients (n = 71) reported PIS use. Kaplan-Meier analysis revealed no difference in NI or mortality over time but did show a significant higher incidence of MOF in those with PIS use (p = 0.04). Regression analysis verified PIS was independently associated with an 80% higher risk of MOF (hazard ratio: 1.8; 95% confidence interval, 1.1-2.9) and was found to be one of the strongest independent risk factors for the development of MOF. PIS use was independently associated with a higher risk of MOF postinjury. These results are contrary to previous analyses. The protective effect of statins may be lost in the severely injured, and modulation of the inflammatory response may result in higher morbidity. Further studies are required to better understand the impact and potential therapeutic utility of this commonly prescribed medication both before and after injury.