Single-molecule, full-length transcript sequencing provides insight into the extreme metabolism of the ruby-throated hummingbird Archilochus colubris.
Hummingbirds oxidize ingested nectar sugars directly to fuel foraging but cannot sustain this fuel use during fasting periods, such as during the night or during long-distance migratory flights. Instead, fasting hummingbirds switch to oxidizing stored lipids that are derived from ingested sugars. The hummingbird liver plays a key role in moderating energy homeostasis and this remarkable capacity for fuel switching. Additionally, liver is the principle location of de novo lipogenesis, which can occur at exceptionally high rates, such as during premigratory fattening. Yet understanding how this tissue and whole organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation. We generated a de novo transcriptome of the hummingbird liver using PacBio full-length cDNA sequencing (Iso-Seq), yielding 8.6Gb of sequencing data, or 2.6M reads from 4 different size fractions. We analyzed data using the SMRTAnalysis v3.1 Iso-Seq pipeline, then clustered isoforms into gene families to generate de novo gene contigs using Cogent. We performed orthology analysis to identify closely related sequences between our transcriptome and other avian and human gene sets. Finally, we closely examined homology of critical lipid metabolism genes between our transcriptome data and avian and human genomes. We confirmed high levels of sequence divergence within hummingbird lipogenic enzymes, suggesting a high probability of adaptive divergent function in the hepatic lipogenic pathways. Our results leverage cutting-edge technology and a novel bioinformatics pipeline to provide a first direct look at the transcriptome of this incredible organism.


RhoB differentially controls Akt function in tumor cells and stromal endothelial cells during breast tumorigenesis.
Tumors are composed of cancer cells but also a larger number of diverse stromal cells in the tumor microenvironment. Stromal cells provide essential supports to tumor pathophysiology but the distinct characteristics of their signaling networks are not usually considered in developing drugs to target tumors. This oversight potentially confounds proof-of-concept studies and increases drug development risks. Here, we show in established murine and human models of breast cancer how differential regulation of Akt by the small GTPase RhoB in cancer cells or stromal endothelial cells determines their dormancy versus outgrowth when angiogenesis becomes critical. In cancer cells in vitro or in vivo, RhoB functions as a tumor suppressor that restricts EGF receptor (EGFR) cell surface occupancy as well as Akt signaling. However, after activation of the angiogenic switch, RhoB functions as a tumor promoter by sustaining endothelial Akt signaling, growth, and survival of stromal endothelial cells that mediate tumor neoangiogenesis. Altogether, the positive impact of RhoB on angiogenesis and progression supercedes its negative impact in cancer cells themselves. Our findings elucidate the dominant positive role of RhoB in cancer. More generally, they illustrate how differential gene function effects on signaling pathways in the tumor stromal component can complicate the challenge of developing therapeutics to target cancer pathophysiology.


Role of Synchronous Activation of Cerebellar Purkinje Cell Ensembles in Multi-joint Movement Control.
It is a longstanding question in neuroscience how elaborate multi-joint movements are coordinated coherently. Microzones of cerebellar Purkinje cells (PCs) are thought to mediate this coordination by controlling the timing of particular motor domains. However, it remains to be elucidated to what extent motor coordination deficits can be correlated with abnormalities in coherent activity within these microzones and to what extent artificially evoked synchronous activity within PC ensembles can elicit multi-joint motor behavior. To study PC ensemble correlates of limb, trunk, and tail movements, we developed a transparent disk treadmill that allows quantitative readout of locomotion and posture parameters in head-fixed mice and simultaneous cellular-resolution imaging and/or optogenetic manipulation. We show that PC ensembles in the ataxic and dystonic mouse mutant tottering have a reduced level of complex spike co-activation, which is delayed relative to movement onset and co-occurs with prolonged swing duration and reduced phase coupling of limb movements as well as with enlarged deflections of body-axis and tail movements. Using optogenetics to increase simple spike rate in PC ensembles, we find that preferred locomotion and posture patterns can be elicited or perturbed depending on the behavioral state. At rest, preferred sequences of limb movements can be elicited, whereas during locomotion, preferred gait-inhibition patterns are evoked. Our findings indicate that synchronous activation of PC ensembles can facilitate initiation and coordination of limb and trunk movements, presumably by tuning downstream systems involved in the execution of behavioral patterns.


New method of measuring water permeability of adhesive resin films.
To adapt a simple gravimetric method to measuring the permeability of adhesive resin films to liquid water, and to compare this to the water vapor permeability of the same resins. Using commercially-available permeability cups designed for industrial permeability testing, the loss of mass of water vapor or liquid water from a stainless steel cup sealed with a resin film was measured over 1-2 days. The permeabilities of Parafilm (control), Clearfil SE Bond adhesive, Xeno IV and One-Up Bond F were compared. The lowest resin film permeability was obtained with Clearfil SE Bond adhesive films. The permeabilities ofXeno IV and One-Up Bond F to liquid water were 2.76 and 3.27-fold higher (P < 0.001) than that of Clearfil SE Bond adhesive. Liquid water permeability was always 2.8-3.8-fold higher (P < 0.05) than water vapor transmission rate. It was concluded that quantitative comparisons of the permeability properties of resin films can be made gravimetrically. The large permeability cups that are available commercially may be reduced in size in the future for measuring dentin adhesive films with smaller surface areas that are less liable to contain imperfections.


High avidity chimeric monoclonal antibodies against the extracellular domains of human aquaporin-4 competing with the neuromyelitis optica autoantibody, NMO-IgG.
Most of the cases of neuromyelitis optica (NMO) are characterized by the presence of an autoantibody, NMO-IgG, which recognizes the extracellular domains of the water channel, aquaporin-4. Binding of NMO-IgG to aquaporin-4 expressed in end-feet of astrocytes leads to complement-dependent disruption of astrocytes followed by demyelination. One therapeutic option for NMO is to prevent the binding of NMO-IgG to aquaporin-4, using high-avidity, non-pathogenic-chimeric, monoclonal antibodies to this water channel. We describe here the development of such antibodies. cDNAs encoding variable regions of heavy and light chains of monoclonal antibodies against the extracellular domains of human aquaporin-4 were cloned from hybridoma total RNA and fused to those encoding constant regions of human IgG1 and Igκ respectively. Then mammalian expression vectors were constructed to establish stable cell lines secreting mature chimeric antibodies. Original monoclonal antibodies showed high avidity binding to human aquaporin-4, as determined by ELISA. Live imaging using Alexa-Fluor-555-labelled antibodies revealed that the antibody D15107 more rapidly bound to cells expressing human aquaporin-4 than others and strongly enhanced endocytosis of this water channel, while D12092 also bound rapidly to human aquaporin-4 but enhanced endocytosis to a lesser degree. Chimeric D15107 prevented complement-dependent cytotoxicity induced by NMO-IgG from patient sera in vitro. We have established non-pathogenic, high-avidity, chimeric antibodies against the extracellular domains of human aquaporin-4, which provide a novel therapeutic option for preventing the progress and recurrence of NMO/NMO spectrum disorders.


Imaging pathways: will they be well trodden or less traveled?
Evidence of inappropriate investigations is not hard to find, and these result in a range of adverse clinical and economic outcomes. An online, evidence-based education and decision support application called Diagnostic Imaging Pathways (DIP) has been developed to assist clinicians to request the most appropriate examinations in the best sequence to achieve diagnoses. The development of the clinical and academic content of DIP and its continuous review and revision involve a network of specialists and general practitioners coordinated by an editorial panel. A research fellow is employed to search the literature, evaluate the evidence, and draft pathways for review. An information technologist develops the Web site, including new functionality, as the scope of the application is expanded. Dissemination is straightforward, with DIP being freely available on the Web (http://www.imagingpathways.health.wa.gov.au). Raising awareness of DIP beyond the immediate clinical environment is challenging. This is assisted through the development of partnerships with national and international entities, which have the core purpose of encouraging evidence-based clinical practice. The greatest challenge, however, is implementation; that is, achieving compliance between requesting practice and recommendations. There is a need to embed DIP into the clinical workflow such that clinicians are guided to choose the "correct" examination. One strategy is to replace paper-based referral processes with an electronic process and to progressively link "electronic requesting" to decision support. Evaluated trials are planned to bring the strategy to the stage of "proof of concept" in Australia.


Studies of the immune response in heparin-induced thrombocytopenia.
Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize PF4/heparin complexes. Uncertainties remain regarding HIT immunobiology, including the temporal relation of antibody formation to onset of thrombocytopenia, and whether immunoglobulin class switching occurs. Using serial plasma samples from 2 heparin thromboprophylaxis trials, we determined the time of onset, antibody levels, and immunoglobulin class distributions (IgG, IgA, IgM) for 12 patients with HIT and 36 patients who formed anti-PF4/heparin antibodies, but did not develop HIT ("seropositive non-HIT controls"). In patients with HIT, anti-PF4/heparin antibodies became detectable 4 days (median) after starting heparin; antibody detection preceded the platelet count decline by 2 days (median). Patients with HIT produced higher levels of IgG antibodies, but similar IgA and IgM levels, compared with seropositive non-HIT controls. Among all 48 seroconverting patients, the first day of a positive antibody test (median, day 6) did not differ among the immunoglobulin classes. Thus, the HIT immune response does not exhibit the classic paradigm of IgM class precedence/immunoglobulin class switching; rather, relatively rapid formation of IgG antibodies is observed, sometimes with concomitant IgA and IgM formation. Compared with seropositive non-HIT controls, HIT patients develop significantly higher anti-PF4/heparin IgG levels that are detectable before the onset of thrombocytopenia.


Cross-immunity and age patterns of influenza A(H5N1) infection.
The age distribution of influenza A(H5N1) cases reported during 2006-2013 varied substantially between countries. As well as underlying demographic profiles, it is possible that cross-immunity contributed to the age distribution of reported cases: seasonal influenza A(H1N1) and avian influenza A(H5N1) share the same neuraminidase subtype, N1. Using a mechanistic model, we measured the extent to which population age distribution and heterosubtypic cross-immunity could explain the observed age patterns in Cambodia, China, Egypt, Indonesia and Vietnam. Our results support experimental evidence that prior infection with H1N1 confers partial cross-immunity to H5N1, and suggest that more than 50% of spillover events did not lead to reported cases of infection as a result. We also identified age groups that have additional risk factors for influenza A(H5N1) not captured by demography or infection history.


Colon perforation with peritonitis in an acquired immunodeficiency syndrome patient due to cytomegalovirus and amoebic colitis.
Invasive amoebiasis is rarely seen in human immunodeficiency virus (HIV)-infected individuals, even in endemic areas. By contrast, cytomegalovirus (CMV) disease is recognized as a major clinical problem in acquired immunodeficiency syndrome patients. A 34-year-old HIV-infected man with amoeba colitis, disseminated Mycobacterium avian complex and CMV infection with cecum perforation, presented with the initial symptoms of fever, shortness of breath and painful sensation when swallowing. He was treated with fluconazole, trimethoprim-sulfamethoxazole and hydrocortisone under the impression of esophageal candidiasis and Pneumocystis jiroveci pneumonia. However, diarrhea and abdominal pain developed on day 6 of hospitalization. Invasive amoebiasis and CMV colitis was diagnosed after examination of colon pathological specimens. Emergent laparotomy was performed. Right hemicolectomy with double barrel ileostomy and colostomy was done due to perforation of the cecum. Iodoquinol was given, followed by metronidazole 14 days afterwards. He underwent closure of double barrel ileostomy and colostomy 5 months later. This case illustrates the diagnostic challenge of caring for acquired immunodeficiency syndrome persons with multiple illnesses and medication use. CMV infection, amoebic colitis and possibly corticosteroid may have played a role in colon perforation in our patient.


Envisioning the future of polymer therapeutics for brain disorders.
The growing incidence of brain-related pathologies and the problems that undermine the development of efficient and effective treatments have prompted both researchers and the pharmaceutical industry to search for novel therapeutic alternatives. Polymer therapeutics (PT) display properties well suited to the treatment of neuro-related disorders, which help to overcome the many hidden obstacles on the journey to the central nervous system (CNS). The inherent features of PT, derived from drug(s) conjugation, in parallel with the progress in synthesis and analytical methods, the increasing knowledge in molecular basis of diseases, and collected clinical data through the last four decades, have driven the translation from "bench to bedside" for various biomedical applications. However, since the approval of Gliadel® wafers, little progress has been made in the CNS field, even though brain targeting represents an ever-growing challenge. A thorough assessment of the steps required for successful brain delivery via different administration routes and the consideration of the disease-specific hallmarks are essential to progress in the field. Within this review, we hope to summarize the latest developments, successes, and failures and discuss considerations on designs and strategies for PT in the treatment of CNS disorders. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.


Distinguishing regulatory DNA from neutral sites.
We explore several computational approaches to analyzing interspecies genomic sequence alignments, aiming to distinguish regulatory regions from neutrally evolving DNA. Human-mouse genomic alignments were collected for three sets of human regions: (1) experimentally defined gene regulatory regions, (2) well-characterized exons (coding sequences, as a positive control), and (3) interspersed repeats thought to have inserted before the human-mouse split (a good model for neutrally evolving DNA). Models that potentially could distinguish functional noncoding sequences from neutral DNA were evaluated on these three data sets, as well as bulk genome alignments. Our analyses show that discrimination based on frequencies of individual nucleotide pairs or gaps (i.e., of possible alignment columns) is only partially successful. In contrast, scoring procedures that include the alignment context, based on frequencies of short runs of alignment columns, dramatically improve separation between regulatory and neutral features. Such scoring functions should aid in the identification of putative regulatory regions throughout the human genome.


Bowel preparation suitable for same-day computed tomography colonography and colonoscopy.
This study was designed to evaluate whether a bowel preparation used for computed tomography (CT) colonography could also be suitable for same-day colonoscopy regardless of which test was done first. Six different endoscopists working at 3 separate hospitals evaluated 75 patients who underwent colonoscopy after receiving a bowel preparation that contained contrast material used to tag fecal and fluid material to facilitate CT colonography. This bowel preparation has been used in more than 1500 CT colonography studies. Evaluation included assessment of whether the colon was clean and dry, and whether the contrast material caused any impairment of visualization or clogging of the endoscopes. Some of the patients had first undergone CT colonography followed by same-day colonoscopy, whereas other patients had colonoscopy as their initial test. Although the contrast material was sometimes perceptible, the volumes were very small, and caused no impairment of mucosal visualization and no clogging of the endoscopes. The bowel preparation was well tolerated. Same-day CT colonography and colonoscopy with fecal tagging was technically possible. A simple, fairly low cost 1-day bowel preparation with fluid and fecal tagging is suitable for CT colonography and colonoscopy done the same day in either order. However, the preferences of individual endoscopists and difficulties with making oral contrast agents readily available are challenges to widespread adoption of a common bowel preparation regimen.


Laser photothermal radiometric instrumentation for fast in-line industrial steel hardness inspection and case depth measurements.
A contact-free, nondestructive laser photothermal radiometric instrumentation technique was developed to meet industrial demand for on-line steel hardness inspection and quality control. A series of industrial steel samples, flat or curvilinear, with different effective hardness case depths ranging between 0.21 and 1.78 mm were measured. The results demonstrated that three measurement parameters (metrics) extracted from fast swept-sine photothermal excitation and measurements, namely, the phase minimum frequency f(min), the peak or trough frequency width W, and the area S, are complementary for evaluating widely different ranges of hardness case depth: f(min) is most suitable for large case depths, and W and S for small case depths. It was also found that laser beam angular inclination with respect to the surface plane of the sample strongly affects hardness measurement resolution and that the phase frequency maximum is more reliable than the amplitude maximum for laser beam focusing on the sample surface.


Cortical complexity as a measure of age-related brain atrophy.
The structure of the human brain changes in a variety of ways as we age. While a sizeable literature has examined age-related differences in cortical thickness, and to a lesser degree, gyrification, here we examined differences in cortical complexity, as indexed by fractal dimensionality in a sample of over 400 individuals across the adult lifespan. While prior studies have shown differences in fractal dimensionality between patient populations and age-matched, healthy controls, it is unclear how well this measure would relate to age-related cortical atrophy. Initially computing a single measure for the entire cortical ribbon, i.e., unparcellated gray matter, we found fractal dimensionality to be more sensitive to age-related differences than either cortical thickness or gyrification index. We additionally observed regional differences in age-related atrophy between the three measures, suggesting that they may index distinct differences in cortical structure. We also provide a freely available MATLAB toolbox for calculating fractal dimensionality.


The "Lid" in the Streptococcus pneumoniae SrtC1 Sortase Adopts a Rigid Structure that Regulates Substrate Access to the Active Site.
Many species of Gram-positive bacteria use sortase enzymes to assemble long, proteinaceous pili structures that project from the cell surface to mediate microbial adhesion. Sortases construct highly stable structures by catalyzing a transpeptidation reaction that covalently links pilin subunits together via isopeptide bonds. Most Gram-positive pili are assembled by class C sortases that contain a "lid", a structurally unique N-terminal extension that occludes the active site. It has been hypothesized that the "lid" in many sortases is mobile and thus capable of readily being displaced from the enzyme to facilitate substrate binding. Here, we show using NMR dynamics measurements, in vitro assays, and molecular dynamics simulations that the lid in the class C sortase from Streptococcus pneumoniae (SrtC1) adopts a rigid conformation in solution that is devoid of large magnitude conformational excursions that occur on mechanistically relevant time scales. Additionally, we show that point mutations in the lid induce dynamic behavior that correlates with increased hydrolytic activity and sorting signal substrate access to the active site cysteine residue. These results suggest that the lid of the S. pneumoniae SrtC1 enzyme has a negative regulatory function and imply that a significant energetic barrier must be surmounted by currently unidentified factors to dislodge it from the active site to initiate pilus biogenesis.


A detailed gene expression study of the Miscanthus genus reveals changes in the transcriptome associated with the rejuvenation of spring rhizomes.
The Miscanthus genus of perennial C4 grasses contains promising biofuel crops for temperate climates. However, few genomic resources exist for Miscanthus, which limits understanding of its interesting biology and future genetic improvement. A comprehensive catalog of expressed sequences were generated from a variety of Miscanthus species and tissue types, with an emphasis on characterizing gene expression changes in spring compared to fall rhizomes. Illumina short read sequencing technology was used to produce transcriptome sequences from different tissues and organs during distinct developmental stages for multiple Miscanthus species, including Miscanthus sinensis, Miscanthus sacchariflorus, and their interspecific hybrid Miscanthus × giganteus. More than fifty billion base-pairs of Miscanthus transcript sequence were produced. Overall, 26,230 Sorghum gene models (i.e., ~ 96% of predicted Sorghum genes) had at least five Miscanthus reads mapped to them, suggesting that a large portion of the Miscanthus transcriptome is represented in this dataset. The Miscanthus × giganteus data was used to identify genes preferentially expressed in a single tissue, such as the spring rhizome, using Sorghum bicolor as a reference. Quantitative real-time PCR was used to verify examples of preferential expression predicted via RNA-Seq. Contiguous consensus transcript sequences were assembled for each species and annotated using InterProScan. Sequences from the assembled transcriptome were used to amplify genomic segments from a doubled haploid Miscanthus sinensis and from Miscanthus × giganteus to further disentangle the allelic and paralogous variations in genes. This large expressed sequence tag collection creates a valuable resource for the study of Miscanthus biology by providing detailed gene sequence information and tissue preferred expression patterns. We have successfully generated a database of transcriptome assemblies and demonstrated its use in the study of genes of interest. Analysis of gene expression profiles revealed biological pathways that exhibit altered regulation in spring compared to fall rhizomes, which are consistent with their different physiological functions. The expression profiles of the subterranean rhizome provides a better understanding of the biological activities of the underground stem structures that are essentials for perenniality and the storage or remobilization of carbon and nutrient resources.


Increased acetabular subchondral bone density is associated with cam-type femoroacetabular impingement.
Femoroacetabular impingement (FAI) has been associated with significant acetabular cartilage damage and subsequent degenerative arthritis. Subchondral bone, often neglected in osteoarthritis studies, may play an important role in the degenerative cascade. Hence the goal of this study was to assess acetabular subchondral bone mineral density (BMD) in subjects with asymptomatic or symptomatic cam deformities compared to normal control subjects. The relationship between BMD and the alpha angle, a quantitative measure of the deformity, was also analyzed. Patients diagnosed with symptomatic cam FAI were recruited ('Surgical') as well as subjects from the general asymptomatic population, classified from CT imaging as normal ('Control') or having a cam deformity ('Bump') based on their alpha angle measurement. There were 12 subjects in each group. All subjects underwent a CT scan with a calibration phantom. BMD was calculated in regions of interest around the acetabulum from CT image intensity and the phantom calibration. BMD was compared between groups using spine BMD as a covariate. The relationship between BMD and alpha angle was assessed by linear regression. In the antero-superior regions bone density was 15-34% higher in the Bump group (P < 0.05) and 14-38% higher in the Surgical group (P < 0.05) compared to Controls. BMD correlated positively with the alpha angle measurements (R(2) = 0.44, P < 0.001). BMD was elevated in subjects with cam-type deformities, with the severity of the deformity more correlative than symptom status. Similarities to the symptomatic group suggest that hips with an asymptomatic deformity may already be in early stages of joint degeneration.


Prognostic factors in first-ever stroke in the carotid artery territory seen within 6 hours after onset.
We sought to detect prognostic factors related to functional outcome during the first 6 hours after a first-ever stroke in the carotid artery territory. All patients with these characteristics seen during a 3-year period were included. Outcome was evaluated according to a modified Rankin scale. The following variables were examined at univariate analysis: sex, age, severity of deficit at entry and at day 7, level of consciousness at entry, time after symptom onset, history of smoking, history of hypertension, diabetes, myocardial infarction, atrial fibrillation, rheumatic heart disease, dilated cardiomyopathy, all potential cardioembolic sources, presence of a consistent lesion on computed tomography at entry and at days 5-9, and the size of such lesion. All entry criteria were met by 172 patients. Age > or = 70 years, a Canadian Neurological Scale score < 6.5 at entry and at day 7, atrial fibrillation, presence of a potential cardioembolic source, and a "large" lesion (involving more than half the cerebral lobe) on computed tomography at days 5-9 were associated with a significantly worse outcome both at 30 days and at 6 months. After multivariate analysis, a Canadian Scale score < 6.5 at entry (p < 0.0001) and atrial fibrillation (p = 0.005) were associated with a significant handicap or death at 30 days, whereas only a Canadian Scale score < 6.5 (p < 0.0001) was associated with a worse prognosis at 6 months. An association of age > or = 70 years with a worse outcome at 6 months was of borderline significance (p = 0.054). Some prognostic indicators are available during the first few hours after onset of a carotid ischemic stroke and may be useful in the stratification of patients in clinical trials. Severity of deficit is the most important indicator, whereas the presence of atrial fibrillation worsens the prognostic outlook with respect to early handicap but not mortality.


Whole genome resequencing of Botrytis cinerea isolates identifies high levels of standing diversity.
How standing genetic variation within a pathogen contributes to diversity in host/pathogen interactions is poorly understood, partly because most studied pathogens are host-specific, clonally reproducing organisms which complicates genetic analysis. In contrast, Botrytis cinerea is a sexually reproducing, true haploid ascomycete that can infect a wide range of diverse plant hosts. While previous work had shown significant genomic variation between two isolates, we proceeded to assess the level and frequency of standing variation in a population of B. cinerea. To begin measuring standing genetic variation in B. cinerea, we re-sequenced the genomes of 13 different isolates and aligned them to the previously sequenced T4 reference genome. In addition one of these isolates was resequenced from four independently repeated cultures. A high level of genetic diversity was found within the 13 isolates. Within this variation, we could identify clusters of genes with major effect polymorphisms, i.e., polymorphisms that lead to a predicted functional knockout, that surrounded genes involved in controlling vegetative incompatibility. The genotype at these loci was able to partially predict the interaction of these isolates in vegetative fusion assays showing that these loci control vegetative incompatibility. This suggests that the vegetative incompatibility loci within B. cinerea are associated with regions of increased genetic diversity. The genome re-sequencing of four clones from the one isolate (Grape) that had been independently propagated over 10 years showed no detectable spontaneous mutation. This suggests that B. cinerea does not display an elevated spontaneous mutation rate. Future work will allow us to test if, and how, this diversity may be contributing to the pathogen's broad host range.


Identification of Novel Phosphorylation Motifs Through an Integrative Computational and Experimental Analysis of the Human Phosphoproteome.
Protein phosphorylation occurs in certain sequence/structural contexts that are still incompletely understood. The amino acids surrounding the phosphorylated residues are important in determining the binding of the kinase to the protein sequence. Upon phosphorylation these sequences also determine the binding of certain domains that specifically bind to phosphorylated sequences. Thus far, such 'motifs' have been identified through alignment of a limited number of well identified kinase substrates. RESULTS: Experimentally determined phosphorylation sites from Human Protein Reference Database were used to identify 1,167 novel serine/threonine or tyrosine phosphorylation motifs using a computational approach. We were able to statistically validate a number of these novel motifs based on their enrichment in known phosphopeptides datasets over phosphoserine/threonine/tyrosine peptides in the human proteome. There were 299 novel serine/threonine or tyrosine phosphorylation motifs that were found to be statistically significant. Several of the novel motifs that we identified computationally have subsequently appeared in large datasets of experimentally determined phosphorylation sites since we initiated our analysis. Using a peptide microarray platform, we have experimentally evaluated the ability of casein kinase I to phosphorylate a subset of the novel motifs discovered in this study. Our results demonstrate that it is feasible to identify novel phosphorylation motifs through large phosphorylation datasets. Our study also establishes peptide microarrays as a novel platform for high throughput kinase assays and for the validation of consensus motifs. Finally, this extended catalog of phosphorylation motifs should assist in a systematic study of phosphorylation networks in signal transduction pathways.


Biomechanical, histological, and radiological effects of different phosphodiesterase inhibitors on femoral fracture healing in rats.
To investigate the biomechanical, histological, and radiological effects of sildenafil and pentoxifylline on femoral fracture healing in rats. Forty-eight Sprague-Dawley rats were divided into three groups equally according to the pharmacological agents to be investigated. Femoral shaft fractures were formed in the left side. Group 1 (control group), group 2, and group 3 were administered with saline, sildenafil, and pentoxifylline during the fracture healing process, respectively. Eight rats from each group were euthanized on days 15 and 30. X-ray images of the rats were taken after euthanasia for radiographical examination. Femur samples were subjected to histopathological and biomechanical (three-point bending) examinations. Radiologically, no difference between the Goldberg scores of the groups was found for day 15 ( p > 0.05), while higher Goldberg scores were obtained from group 2 than that of group 1 ( p > 0.05) and group 3 ( p < 0.05) for day 30. In the biomechanical analysis, higher mean breaking forces were found both for day 15 and day 30 from group 2 than those obtained from group 1 (for day 15 p > 0.05 and day 30 p > 0.05) and group 3 (for day 15 p < 0.05 and day 30 p < 0.01). Higher mean absorbed energy values were obtained from group 2 than those obtained from group 1 (for day 15 p > 0.05 and day 30 p < 0.05) and group 3 (for day 15 p < 0.01 and day 30 p < 0.01). A significant difference was not found between the histological scores of all groups ( p > 0.05) for day 15, while the histological score of group 1 on day 30 was found to be significantly lower than that of sildenafil and pentoxifylline groups ( p < 0.05). Sildenafil had a positive effect on fracture healing, while pentoxifylline did not provide consistent positive effect.


EGFR polymorphism as a predictor of clinical outcome in advanced lung cancer patients treated with EGFR-TKI.
Mutations in the epidermal growth factor receptor (EGFR) have been confirmed as predictors of the efficacy of treatment with EGFR-tyrosine kinase inhibitors (TKIs). We investigated whether polymorphisms of the EGFR gene were associated with clinical outcomes in non-small cell lung cancer (NSCLC) patients treated with EGFR-TKI. A polymorphic dinucleotide repeat in intron 1 [CA simple sequence repeat in intron 1(CA-SSR1)] in intron 1 and single nucleotide polymorphisms (SNP-216) in the promoter region of the EGFR gene were evaluated in 71 NSCLC patients by restriction fragment length polymorphism and DNA sequencing. The relationship between genetic polymorphisms and clinical outcomes of treatment with EGFR-TKIs was evaluated. SNP-216G/T polymorphisms were associated with the efficacy of EGFR-TKI. The response rate for the SNP-216G/T tended to be higher than that for G/G (62.5% vs. 27.4%, p=0.057). The SNP-216G/T genotype was also associated with longer progression-free survival compared with the GG genotype (16.7 months vs. 5.1 months, p=0.005). However, the length of CA-SSR1 was not associated with the efficacy of EGFR-TKI. SNP-216G/T polymorphism was a potential predictor of clinical outcomes in NSCLC patients treated with EGFR-TKI.


Orally administered whey proteins have comparable effect on C-peptide secretion in healthy subjects as standard C-peptide stimulation tests.
Our study compared total C-peptide secretion after administration of whey proteins and whey proteins in combination with glucose with results of classical tests assessing beta cell function in the pancreas of healthy individuals. Eight young, healthy (7 males, 1 female; aged 20-26 years), non-obese (BMI: 17-25.9 kg/m²) participants with normal glucose tolerance underwent six C-peptide secretion tests. Three secretion tests measured C-peptide response to orally administered substances: whey proteins only (OWT), whey proteins with glucose (OWGT) and glucose only (OGTT); while three secretion tests measured C-peptide response to intravenously administered substances: arginine (AST), glucagon (GST) and glucose (IVGTT). OWT stimulated a greater (93 %, p<0.05) C-peptide response than AST and a 64 % smaller response (p<0.05) than OGTT. OWT also showed lower variability (p<0.05) in C-peptide responses compared to OWGT and OGTT. The greatest total C-peptide response was induced by OWGT (36 % higher than glucose). OWT consistently increased C-peptide concentrations with lower individual variability, while insignificantly increasing glucose levels. Results of this study suggest that both dietology and beta-cells capacity testing could take advantage of the unique property of whey proteins to induce C-peptide secretion.


Comparative study on dispersion and interfacial properties of single walled carbon nanotube/polymer composites using Hansen solubility parameters.
Dispersion and interfacial strain transfer of single walled carbon nanotubes (SWNTs) are two major challenges for the utilization of SWNTs as reinforcements in polymer composites. Surface modifications could help change the dispersion and interfacial properties. In this study, nanocomposites were fabricated by solution blending 1 wt % SWNTs with various modification (nonmodified, nitric acid functionalized, and amine functionalized SWNTs) and three kinds of polymeric materials (polycarbonate, polyvinylidene fluoride, and epoxy). Chemical compatibilities between SWNTs and solvents or polymers are calculated by the Hansen solubility parameters (HSP) method. The dispersion of the SWNTs in solvents is evaluated by dynamic light scattering. The dispersion of SWNTs in polymers evaluated by a light optical microscope (LOM) generally agrees with the HSP prediction. The strain transfer from the matrix to SWNTs is mainly related to the dispersion, the bundle size, the residual thermal stresses on the sample, and, to lesser degree, the HSP.


The Kinase Chemogenomic Set (KCGS): An Open Science Resource for Kinase Vulnerability Identification.
We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS), current Version 1.0, is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.


A comparison of the risks of venous thromboembolic disease in association with different combined oral contraceptives.
In October 1995 in response to the results of three studies, the Committee on the Safety of Medicines advised doctors and pharmacists that oral contraceptives containing desogestrel (DSG) and gestodene (GST) were associated with around a two-fold increase in the risk of thromboembolism compared with those containing other progestogens. The objective of this study was to estimate the risk of idiopathic venous thromboembolic disease (VTE) in users of combined oral contraceptives (COCs), to compare the risk between formulations and to examine the effect of using age banding as opposed to matching by exact year of birth. A nested case control study was conducted using the General Practice Research Database. Women with a VTE event recorded between 1992 and 1997, who were treated with an anticoagulant, from consideration of their prescription records were likely to have been using a COC prescription on the day of the event and also had no exclusion factors, were deemed cases. For comparison with the previous studies, two nested case control studies were undertaken. Study 1 used controls matched by practice and year of birth. Study 2 used controls matched by practice and within 5 years age bands. We found an incidence of idiopathic VTE amongst users of combined oral contraceptives of 3.8 per 10 000 exposed women years. Incidence rates increased markedly after 35 years of age. The nested case-control study using controls matched by year of birth showed no significant difference in risk between the major COC formulations. With levonorgestrel (LNG) 150 microgram and ethinyloestradiol (EE) 30 microgram as the reference, the adjusted ORs for GST 75 microgram and EE 30 microgram was 1.3 (95% CI 0.8, 2.1), for DSG 150 microgram and EE 30 microgram it was 1.0 (95% CI 0.7, 1.7) and for DSG 150 microgram and EE 20 microgram it was 0.8 (95% CI 0.4, 1.6). Using less rigorous matching criteria, matching controls to cases within 5 years age bands, the ORs increased. When a mixed group of COCs, characterized by having LNG as the progestogen component was used as the reference category, there was an elevation in the ORs for the newer products. We found a significant association between idiopathic VTE and current smoking (OR 2.0 (1.4, 2.7)), BMI over 35 (OR 3.8 (1.8, 8.0)) and asthma (OR 1.9 (1.3, 2.9)). The OR for women who had proxy evidence of general ill health (indicated by the number of prescriptions issued) was 2.2 (1.7, 3.7). The results of this study indicate that a number of the characteristics of the women taking COCs affect the risk of VTE. There is no evidence to support the hypothesis that there is any difference in risk between COC formulations containing under 50 microg ethinyloestradiol.


Immune sensing of Aspergillus fumigatus proteins, glycolipids, and polysaccharides and the impact on Th immunity and vaccination.
The ability of the fungus Aspergillus fumigatus to activate, suppress, or subvert host immune response during life cycle in vivo through dynamic changing of cell wall structure and secretion implicates discriminative immune sensing of distinct fungal components. In this study, we have comparatively assessed secreted- and membrane-anchored proteins, glycolipids, and polysaccharides for the ability to induce vaccine-dependent protection in transplanted mice and Th cytokine production by human-specific CD4(+) T cell clones. The results show that the different fungal components are endowed with the distinct capacity to activate Th cell responses in mice and humans, with secreted proteins inducing Th2 cell activation, membrane proteins Th1/Treg, glycolipids Th17, and polysaccharides mostly IL-10 production. Of interest, the side-by-side comparison revealed that at least three fungal components (a protease and two glycosylphosphatidylinositol-anchored proteins) retained their immunodominant Th1/Treg activating potential from mice to humans. This suggests that the broadness and specificity of human T cell repertoire against the fungus could be selectively exploited with defined immunoactive Aspergillus Ags.


Effectiveness of rivaroxaban for thromboprophylaxis of prosthetic heart valves in a porcine heterotopic valve model.
Warfarin is used to reduce the risk of stroke and thromboembolic complications in patients with mechanical heart valves. Yet, despite frequent blood testing, its poor pharmacokinetic and pharmacodynamic profiles often result in variable therapeutic levels. Rivaroxaban is a direct competitive factor Xa inhibitor that is taken orally. It inhibits the active site of factor Xa without the need for the cofactor antithrombin, and thus, its mechanism of action is differentiated from that of the fractionated heparins and indirect factor Xa inhibitors. No in vivo data exist regarding the effectiveness of rivaroxaban in preventing thromboembolic complications of mechanical heart valves. We tested the hypothesis that rivaroxaban is as effective as enoxaparin for thromboprophylaxis of mechanical valves that use a previously described heterotopic aortic valve porcine model. A modified bileaflet mechanical valved conduit that bypassed the native, ligated descending thoracic aorta was implanted into 30 swine. Postoperatively, the animals were randomly assigned to groups receiving no anticoagulation (n = 10), enoxaparin at 2 mg/kg subcutaneously twice daily (n = 10) or rivaroxaban at 2 mg/kg orally twice daily (n = 10). The amount of valve thrombus was measured on post-implantation day 30 as the primary end point. Quantitative evaluation of radiolabelled platelet deposition on the valve prostheses was done and embolic and haemorrhagic events were measured as secondary end points. Animals with no anticoagulation had a thrombus mean of 759.9 mg compared with 716.8 mg with enoxaparin treatment and 209.6 mg with rivaroxaban treatment (P = 0.05 for enoxaparin vs rivaroxaban). Similarly, the mean number of platelets deposited on the valve prosthesis was lower in the rivaroxaban group (6.13 × 10(9)) than in the enoxaparin group (3.03 × 10(10)) (P = 0.03). In this study, rivaroxaban was more effective than enoxaparin for short-term thromboprophylaxis of mechanical valve prosthetics in the heterotopic aortic position. It reduced valve thrombus and platelet deposition on day 30 following implantation without increased adverse events. Future studies would provide additional support for clinical trials evaluating rivaroxaban as an alternative to warfarin for appropriately selected patients with prosthetic aortic valves.


Action control in dyads: A randomized controlled trial to promote physical activity in everyday life.
Engaging in regular physical activity requires substantial self-regulatory effort such as action control (e.g., continuously monitoring and evaluating an ongoing behavior with regard to one's standards). The present study examined the effectiveness of an ecological momentary action control intervention for promoting daily physical activity. Also, we tested whether a dyadic compared to an individual intervention displayed an additional benefit. 121 overweight and obese individuals and their partners were randomly allocated to an intervention (n = 60; information + action control text messages) or a control group (n = 61; information only). The intervention was delivered in a dyadic vs. individual version of action control. Allocation ratio was 1:1:2 for the dyadic, individual, and control groups, respectively. Daily physical activity was assessed with triaxial accelerometers during a 14-day intervention phase and a 14-day follow-up phase. Participants in the intervention group showed a higher probability (36.5%) to achieve the recommended daily activity levels (≥30 min of moderate-to-vigorous physical activity per day performed in bouts of at least 10 min) during the intervention and follow-up phase compared to those in the control group (23.0%). The intervention and control group did not differ in terms of daily moderate-to-vigorous physical activity (40.7 vs. 38.6 min per day, p = 0.623). Interventions facilitating action control via text messages seem to be an effective tool for increasing adherence to physical activity guidelines in everyday life. The comparable effects for the dyadic and individual intervention suggest that automated text messages may be just as effective as personalized messages from the romantic partner. Further investigation is needed to examine the usefulness of a dyadic conceptualizing of action control. (controlled-trials.com ISRCTN15705531).


The Variable Effect of Polyploidization on the Phenotype in Escallonia.
To induce new variation within the Escallonia genus, chromosome doubling was performed in E. rubra, E. rosea, and E. illinita, three important species within this genus of mainly evergreen woody ornamental species. Obtained tetraploids and diploid controls were analyzed for rooting capacity, leaf and flower characteristics, and plant architecture using image analysis and cold tolerance. In the present study, a breeders' collection of 23 accessions was characterized cytogenetically and described morphologically. All analyzed species and cultivars were diploid (2n = 2x = 24), with exception of E. pendula, a tetraploid. Today, breeding in Escallonia is limited to lucky finds in seedling populations and few efforts in interspecific hybridization. Three selected Escallonia species underwent an in vitro chromosome doubling with both oryzalin and trifluralin applied as either a continuous or shock treatment. The treatments successfully induced polyploids in all three species. Image analysis revealed that tetraploid E. rosea had decreased shoot length (from 3.8 to 1.3 cm), higher circularity and more dense growth habit compared to diploids. No significant changes in cold tolerance were seen. Tetraploid E. illinita did not differ in shoot length, but an increased outgrowth of axillary buds on the main axis led to denser plants. For tetraploid E. rubra, an increase in plant height (from 4.9 to 5.5 cm) was observed together with a large decrease in circularity and density due to a more polar outgrowth of branches on the main axis. E. rubra tetraploids bore larger flowers than diploids and had an increased cold tolerance (from -7.7 to -11.8°C). Leaf width and area of tetraploids increased for both E. illinita and E. rubra, while a decrease was seen in E. rosea genotypes. For all three species, the rooting capacity of the tetraploids did not differ from the diploids. We conclude that the effect of polyploidization on Escallonia was highly variable and species dependent.


Telephone survey of private patients' views on continuity of care and registration with general practice in Ireland.
The desire of patients for personal continuity of care with a General Practitioner (GP) has been well documented, but not within non-registered private patients in Ireland. This study set out to examine the attitudes and reported behaviours of private fee-paying patients towards continuity of GP care and universal registration for patients. Cross-sectional telephone survey of 400 randomly chosen fee-paying patients living within County Dublin. There is no formal system of registration with a GP for these patients. Main outcomes were attendance of respondents at primary health care facilities and their attitudes towards continuity of care and registration with a GP. Data was analysed using descriptive statistics and using parametric and non-parametric tests of association. Pearson correlation was used to quantify the association between the described variables and attitudes towards continuity and registration with a GP. Variables showing significance at the 5% level were entered into multiple linear regression models. 97% of respondents had seen a GP in the previous 5 years. The mean number of visits to the GP for respondents was 2.3 per annum. 89% of respondents had a regular GP and the mean length of time with their GP was 15.6 years. 96% preferred their personal medical care to be provided within one general practice. 16% of respondents had consulted a GP outside of their own practice in the previous year. They were more likely to be female, commute a longer distance to work or have poorer health status. 81% considered it important to be officially registered with a GP practice of their choice. Both personal and longitudinal continuity of care with a GP are important to private patients. Respondents who chose to visit GPs other than their regular GP were not easily characterised in this study and individual circumstances may lead to this behaviour. There is strong support for a system of universal patient registration within general practice.


NR4A Nuclear Receptors Target Poly-ADP-Ribosylated DNA-PKcs Protein to Promote DNA Repair.
Although poly-ADP-ribosylation (PARylation) of DNA repair factors had been well documented, its role in the repair of DNA double-strand breaks (DSBs) is poorly understood. NR4A nuclear orphan receptors were previously linked to DSB repair; however, their function in the process remains elusive. Classically, NR4As function as transcription factors using a specialized tandem zinc-finger DNA-binding domain (DBD) for target gene induction. Here, we show that NR4A DBD is bi-functional and can bind poly-ADP-ribose (PAR) through a pocket localized in the second zinc finger. Separation-of-function mutants demonstrate that NR4A PAR binding, while dispensable for transcriptional activity, facilitates repair of radiation-induced DNA double-strand breaks in G1. Moreover, we define DNA-PKcs protein as a prominent target of ionizing radiation-induced PARylation. Mechanistically, NR4As function by directly targeting poly-ADP-ribosylated DNA-PKcs to facilitate its autophosphorylation-promoting DNA-PK kinase assembly at DNA lesions. Selective targeting of the PAR-binding pocket of NR4A presents an opportunity for cancer therapy.


Contraceptive Technologies: Looking Ahead to New Approaches to Increase Options for Family Planning.
With persistently high global rates of unintended pregnancy and contraceptive nonuse, nonadherence and discontinuation, new contraceptive methods must address the needs of women and men who seek alternatives to their current options. Methods under development aim to reduce potential side effects, improve access and ease of use, ensure safety, increase secondary benefits associated with method use and expand options for both women and men. Developmental approaches employed to enhance current methods utilize new delivery systems and novel active pharmaceutical ingredients. This will improve overall user satisfaction with the methods used while expanding the number of options available to provide choice and value user autonomy in the highly diverse contraceptive markets around the world.


Association between oral habits, mouth breathing and malocclusion.
The ratio of bad habits, mouth breathing and malocclusion is an important issue in view of prevention and early treatment of disorders of the craniofacial growth. While bad habits can interfere with the position of the teeth and normal pattern of skeletal growth, on the other hand obstruction of the upper airway, resulting in mouth breathing, changes the pattern of craniofacial growth causing malocclusion. Our crosssectional study, carried out on 3017 children using the ROMA index, was developed to verify if there was a significant correlation between bad habits/mouth breathing and malocclusion. The results showed that an increase in the degree of the index increases the prevalence of bad habits and mouth breathing, meaning that these factors are associated with more severe malocclusions. Moreover, we found a significant association of bad habits with increased overjet and openbite, while no association was found with crossbite. Additionally, we found that mouth breathing is closely related to increased overjet, reduced overjet, anterior or posterior crossbite, openbite and displacement of contact points. Therefore, it is necessary to intervene early on these aetiological factors of malocclusion to prevent its development or worsening and, if already developed, correct it by early orthodontic treatment to promote eugnatic skeletal growth.


Mothers' transitions from welfare to work and the well-being of preschoolers and adolescents.
Results from a longitudinal study of 2402 low-income families during the recent unprecedented era of welfare reform suggest that mothers' transitions off welfare and into employment are not associated with negative outcomes for preschoolers (ages 2 to 4 years) or young adolescents (ages 10 to 14 years). Indeed, no significant associations with mothers' welfare and employment transitions were found for preschoolers, and the dominant pattern was also of few statistically significant associations for adolescents. The associations that did occur provided slight evidence that mothers' entry into the labor force was related to improvements in adolescents' mental health, whereas exits from employment were linked with teenagers' increased behavior problems.


Induction of macrophage C-C chemokine expression by titanium alloy and bone cement particles.
Particulate wear debris is associated with periprosthetic inflammation and loosening in total joint arthroplasty. We tested the effects of titanium alloy (Ti-alloy) and PMMA particles on monocyte/macrophage expression of the C-C chemokines, monocyte chemoattractant protein-1 (MCP-1), monocyte inflammatory protein-1 alpha (MIP-1alpha), and regulated upon activation normal T expressed and secreted protein (RANTES). Periprosthetic granulomatous tissue was analysed for expression of macrophage chemokines by immunohistochemistry. Chemokine expression in human monocytes/macrophages exposed to Ti-alloy and PMMA particles in vitro was determined by RT-PCR, ELISA and monocyte migration. We observed MCP-1 and MIP-1alpha expression in all tissue samples from failed arthroplasties. Ti-alloy and PMMA particles increased expression of MCP-1 and MIP-1alpha in macrophages in vitro in a dose- and time-dependent manner whereas RANTES was not detected. mRNA signal levels for MCP-1 and MIP-1alpha were also observed in cells after exposure to particles. Monocyte migration was stimulated by culture medium collected from macrophages exposed to Ti-alloy and PMMA particles. Antibodies to MCP-1 and MIP-1alpha inhibited chemotactic activity of the culture medium samples. Release of C-C chemokines by macrophages in response to wear particles may contribute to chronic inflammation at the bone-implant interface in total joint arthroplasty.


An economic analysis of sublobar resection versus thermal ablation for early-stage non-small-cell lung cancer.
To compare medical costs for a matched-pair cohort of Medicare patients with early-stage non-small-cell lung cancer (NSCLC) who underwent treatment with sublobar resection or thermal ablation. Patients at least 65 years of age with stage IA/IB NSCLC treated with sublobar resection or thermal ablation from 2007 to 2009 were identified from Surveillance, Epidemiology, and End Results/Medicare-linked data and matched by propensity scores. The primary outcome of interest, cost from the payer's perspective, was derived from Medicare claims data. A partitioned inverse probability-weighted estimator was used to calculate mean and median treatment-related costs and costs at 1, 3, 12, 18, and 24 months after treatment. Baseline characteristics, Kaplan-Meier survival curves, and calculated cost variables were compared between the two groups. The final matched cohort of 128 patients had similar baseline characteristics and overall survival (P = .52). Patients who underwent ablation had significantly lower treatment-related costs than those who underwent sublobar resection (P < .001). The difference in median treatment-related cost was $16,105. At 1 month, 3 months, and 12 months after treatment, cumulative costs remained significantly different (P ≤ .011). Lower cost associated with ablations performed in the outpatient setting was a major contributor to the differences between the two treatment modalities, although inpatient ablations maintained a small cost advantage over sublobar resections. Among matched Medicare patients with stage I NSCLC, thermal ablation resulted in significantly lower treatment-related costs and cumulative medical costs 1 month, 3 months, and 12 months after treatment compared with sublobar resection.


An analysis of codon bias in six red yeast species.
Red yeasts, primarily species of Rhodotorula, Sporobolomyces, and other genera of Pucciniomycotina, are traditionally considered proficient systems for lipid and terpene production, and only recently have also gained consideration for the production of a wider range of molecules of biotechnological potential. Improvements of transgene delivery protocols and regulated gene expression systems have been proposed, but a dearth of information on compositional and/or structural features of genes has prevented transgene sequence optimization efforts for high expression levels. Here, the codon compositional features of genes in six red yeast species were characterized, and the impact that evolutionary forces may have played in shaping this compositional bias was dissected by using several computational approaches. Results obtained are compatible with the hypothesis that mutational bias, although playing a significant role, cannot alone explain synonymous codon usage bias of genes. Nevertheless, several lines of evidences indicated a role for translational selection in driving the synonymous codons that allow high expression efficiency. These optimal synonymous codons are identified for each of the six species analyzed. Moreover, the presence of intragenic patterns of codon usage, which are thought to facilitate polyribosome formation, was highlighted. The information presented should be taken into consideration for transgene design for optimal expression in red yeast species.


Time Cost of Standardized Nursing Screens in the Emergency Department.
Various policies require that screening questions be asked of all patients who present to the emergency department (ED). No studies have previously examined the potential time costs of standardized screens. Our objective was to analyze the time nursing spent conducting standardized nursing screens and calculate the corresponding time cost. This was a prospective observational study of ED registered nurses (RN) performing triage assessments on adults presenting to the ED. A study author timed nurses while the RN asked five pre-selected questions from their current triage protocol. The time cost of each question was determined by multiplying the length of time spent asking the question each year by the mean hourly wage of RNs at the study hospital. (T/3,600) × V × S; T = mean time per question (in seconds); V = annual patient volume; S = mean hourly RN wage. We observed 200 triage assessments. During the triage assessments, 130 patients (65%) were asked about pneumococcal vaccine status; 161 (80.5%) about tetanus vaccine status; 184 (92%) about medication allergies; 172 (86%) about influenza vaccine; and 73 (36.5%) about recent travel. The mean time spent per question ranged from 4.37-6.26 seconds. The estimated annual time used to ask the five questions in the study ED is 590.73 hours, which equates to $20,675.50 in nursing costs per year. There are potential monetary and time costs of standardized screening questions in the ED. The values heavily impact time and cost efficiency in the ED and could be redirected to more pertinent patient care. The required screening questions often have an unclear utility on the care that the patient receives in the ED. Further studies are needed to determine cost effectiveness of required ED screenings.


Cricothyrotomy Online: Quality Assessment of Educational Videos on YouTube.
Medical learners may use YouTube® videos to prepare for procedures. Videos are convenient and readily available, but without any uploading standards, their accuracy and quality for education are uncertain. We assessed the quality of emergency cricothyrotomy videos on YouTube through an expert panel of surgeons with objective quality metrics. A YouTube® search for "emergency cricothyrotomy" was performed and results were filtered to remove animations and lectures. The 4 most-viewed videos were sent to a panel of trauma surgeons for evaluation. An educational quality (EQ) score was generated for each video based on its ability to explain the procedure indications, orient the viewer to the patient, provide accurate narration, provide clear views of procedure, identify relevant instrumentation and anatomy, and explain critical maneuvers. Reviewers were also asked if safety concerns were present and encouraged to give feedback in a free-response field. Four surgical attendings completed the survey. The median EQ score was 6 on a 7-point scale (95% CI [6, 6]). All but one of the individual parameters had a median EQ score of 6 (95% CI: indications [3, 7], orientation [5, 7], narration [6, 7], clarity [6, 7], instruments [6, 7], anatomy [6, 6], critical maneuvers [5, 6]). Safety received a lower EQ score (5.5, 95% CI [2, 6]). The most-viewed cricothyrotomy videos were rated positively by surgical attendings. Still, it is necessary to know if medical learners can distinguish high from low quality videos. If not, this suggests a need for surgical societies to create high-quality videos that can be reliably and efficiently accessed on YouTube®.


Expression of pannexin1 in the CNS of adult mouse: cellular localization and effect of 4-aminopyridine-induced seizures.
The expression pattern of pannexin1, a gene coding for a protein that forms gap junction channels, was studied as both mRNA and protein in the CNS of adult mouse. Pannexin1 was widely expressed in the CNS by neuronal cell types but not glial cells, except for Bergmann glial cells of the cerebellar cortex. Cells positive to Ca-binding proteins, principally parvalbumin, but also calbindin and calretinin, as well as glutamate decarboxylase 67 kDa isoform, were pannexin1-positive. Pannexin1 labeling was found in cells which are known to exhibit spontaneous and synchronous discharge, such as neurons of the inferior olivary complex and the reticular thalamic nucleus, and also in neurons whose electrical activity is not coupled with neighboring cells, such as motoneurons of the spinal cord. The analysis of cellular localization showed puncta that surrounded cell bodies (e.g. the pyramidal cells of hippocampus) or restricted areas inside the cell bodies (e.g. the spinal motoneurons). In Bergmann glial cells the staining was present as fine grains that covered a large part of the cellular surface. Pannexin1 stained cells that previous studies have reported as expressing connexin36, another protein forming gap junction channels. Thus, it was possible that these two proteins could be integrated in the same functions. Since connexin36 expression levels change after seizures, we examined the expression of both pannexin1 and connexin36 in cerebral cortex, hippocampus, cerebellum and brain stem at different time intervals (2, 4 and 8 h) after i.p. injection of 4-aminopyridine, which resulted in systemic seizures. The only modification of the expression levels observed in this study concerned the progressive decrement of the connexin36 in the hippocampus, while pannexin1 expression was unchanged. This finding suggested that pannexin1 and connexin36 are involved in different functional roles or that they are expressed in different cell types and that only those expressing the Cx36 are induced to apoptosis by epileptic seizures.


ω-3 Polyunsaturated Fatty Acid Biomarkers and Coronary Heart Disease: Pooling Project of 19 Cohort Studies.
The role of ω-3 polyunsaturated fatty acids for primary prevention of coronary heart disease (CHD) remains controversial. Most prior longitudinal studies evaluated self-reported consumption rather than biomarkers. To evaluate biomarkers of seafood-derived eicosapentaenoic acid (EPA; 20:5ω-3), docosapentaenoic acid (DPA; 22:5ω-3), and docosahexaenoic acid (DHA; 22:6ω-3) and plant-derived α-linolenic acid (ALA; 18:3ω-3) for incident CHD. A global consortium of 19 studies identified by November 2014. Available prospective (cohort, nested case-control) or retrospective studies with circulating or tissue ω-3 biomarkers and ascertained CHD. Each study conducted standardized, individual-level analysis using harmonized models, exposures, outcomes, and covariates. Findings were centrally pooled using random-effects meta-analysis. Heterogeneity was examined by age, sex, race, diabetes, statins, aspirin, ω-6 levels, and FADS desaturase genes. Incident total CHD, fatal CHD, and nonfatal myocardial infarction (MI). The 19 studies comprised 16 countries, 45 637 unique individuals, and 7973 total CHD, 2781 fatal CHD, and 7157 nonfatal MI events, with ω-3 measures in total plasma, phospholipids, cholesterol esters, and adipose tissue. Median age at baseline was 59 years (range, 18-97 years), and 28 660 (62.8%) were male. In continuous (per 1-SD increase) multivariable-adjusted analyses, the ω-3 biomarkers ALA, DPA, and DHA were associated with a lower risk of fatal CHD, with relative risks (RRs) of 0.91 (95% CI, 0.84-0.98) for ALA, 0.90 (95% CI, 0.85-0.96) for DPA, and 0.90 (95% CI, 0.84-0.96) for DHA. Although DPA was associated with a lower risk of total CHD (RR, 0.94; 95% CI, 0.90-0.99), ALA (RR, 1.00; 95% CI, 0.95-1.05), EPA (RR, 0.94; 95% CI, 0.87-1.02), and DHA (RR, 0.95; 95% CI, 0.91-1.00) were not. Significant associations with nonfatal MI were not evident. Associations appeared generally stronger in phospholipids and total plasma. Restricted cubic splines did not identify evidence of nonlinearity in dose responses. On the basis of available studies of free-living populations globally, biomarker concentrations of seafood and plant-derived ω-3 fatty acids are associated with a modestly lower incidence of fatal CHD.


Acceptability and Feasibility of a Low-Cost Device for Gestational Age Assessment in a Low-Resource Setting: Qualitative Study.
Ultrasound for gestational age (GA) assessment is not routinely available in resource-constrained settings, particularly in rural and remote locations. The TraCer device combines a handheld wireless ultrasound probe and a tablet with artificial intelligence (AI)-enabled software that obtains GA from videos of the fetal head by automated measurements of the fetal transcerebellar diameter and head circumference. The aim of this study was to assess the perceptions of pregnant women, their families, and health care workers regarding the feasibility and acceptability of the TraCer device in an appropriate setting. A descriptive study using qualitative methods was conducted in two public health facilities in Kilifi county in coastal Kenya prior to introduction of the new technology. Study participants were shown a video role-play of the use of TraCer at a typical antenatal clinic visit. Data were collected through 6 focus group discussions (N=52) and 18 in-depth interviews. Overall, TraCer was found to be highly acceptable to women, their families, and health care workers, and its implementation at health care facilities was considered to be feasible. Its introduction was predicted to reduce anxiety regarding fetal well-being, increase antenatal care attendance, increase confidence by women in their care providers, as well as save time and cost by reducing unnecessary referrals. TraCer was felt to increase the self-image of health care workers and reduce time spent providing antenatal care. Some participants expressed hesitancy toward the new technology, indicating the need to test its performance over time before full acceptance by some users. The preferred cadre of health care professionals to use the device were antenatal clinic nurses. Important implementation considerations included adequate staff training and the need to ensure sustainability and consistency of the service. Misconceptions were common, with a tendency to overestimate the diagnostic capability, and expectations that it would provide complete reassurance of fetal and maternal well-being and not primarily the GA. This study shows a positive attitude toward TraCer and highlights the potential role of this innovation that uses AI-enabled automation to assess GA. Clarity of messaging about the tool and its role in pregnancy is essential to address misconceptions and prevent misuse. Further research on clinical validation and related usability and safety evaluations are recommended.


Cancer mortality in Europe, 1995-1999, and an overview of trends since 1960.
Mortality data, abstracted from the World Health Organization database, are presented in tabular form for 26 cancer sites or groups of sites, plus total cancer mortality, in 36 European countries during the period 1995-1999. Trends in mortality are also given in graphic form for 23 major countries plus the European Union as a whole over the period 1960-1999. In the European Union, total cancer mortality declined by 7% for both sexes over the last 5 years considered. The fall since the late 1980s was 10% in both sexes, corresponding to the avoidance of over 90000 deaths per year, as compared to the rates of the late 1980s. For the first time, over the last few years, some leveling of mortality was reported also in the Russian Federation, the Czech Republic, Poland, Hungary and other Eastern European countries, although cancer rates in those areas remain exceedingly high. The overall favorable pattern of cancer mortality over recent years is largely driven by the decline of tobacco-related cancer mortality in men. However, important components of the trends are also the persistence of substantial falls in gastric cancer, mainly in Russia and Eastern Europe, the recent decline in intestinal cancer in both sexes and of breast cancer in women, together with the long-term falls in uterine (cervical) cancer, leukemias, Hodgkin's disease and other neoplasms amenable to advancements in diagnosis and treatment. Female lung cancer mortality has been declining in the Russian Federation, but is still rising in other areas of the continent. Thus, urgent intervention is needed to bring under control the tobacco-related lung cancer epidemic in European women before it reaches the high level observed in North America. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html


Modified DNA bearing 5(methoxycarbonylmethyl)-2'-deoxyuridine: preparation by PCR with thermophilic DNA polymerase and postsynthetic derivatization.
A thymidine analogue bearing a methyl ester at the C5 position was accepted as a substrate by the thermophilic family B DNA polymerases, KOD Dash, Pwo, and Vent(exo-), to form the corresponding PCR product, but not by the thermophilic family A DNA polymerases, Taq, Tth, and T7 thermosequenase. Modified DNA containing this analogue was prepared by PCR on a large scale with KOD Dash DNA polymerase and 5(methoxycarbonylmethyl)-2'-deoxyuridine 5'-triphosphate as a substrate. The methyl ester of the modified DNA was further allowed to react with tris(2-aminoethyl)amine or histamine by an ester-amide exchange reaction to form the corresponding derivatized DNA bearing a tris(2-aminoethyl)amine or histamine moiety. Hydrolysis of the methyl ester of the modified DNA gave a functionalized DNA bearing an anionic carboxyl group. The derivatized DNA could act as a template for the PCR with KOD Dash DNA polymerase and the natural 2'-deoxythymidine 5'-triphosphate or the modified thymidine analogue as a substrate. The postsynthetic derivatization of the modified DNA may expand the variety of structurally modified DNA produced by PCR.


Mouse embryonic stem cells have underdeveloped antiviral mechanisms that can be exploited for the development of mRNA-mediated gene expression strategy.
We have recently reported that mouse embryonic stem cells (mESCs) are deficient in expressing type I interferons (IFN) when exposed to viral infection and double-stranded RNA. In this study, we extended our investigation and demonstrated that single-stranded RNA and protein-encoding mRNA can induce strong IFN expression and cytotoxicity in fibroblasts and epithelial cells, but none of the effects associated with these antiviral responses were observed in mESCs. Our results provided additional data to support the conclusion that mESCs are intrinsically deficient in antiviral responses. While our findings represent a novel feature of mESCs that in itself is important for understanding innate immunity development, we exploited this property to develop a novel mRNA-mediated gene expression cell model. Direct introduction of synthetic mRNA to express desired genes has been shown as an effective alternative to DNA/viral vector-based gene expression. However, a major biological challenge is that a synthetic mRNA is detected as a viral RNA analog by the host cell, resulting in a series of adverse effects associated with antiviral responses. We demonstrate that the lack of antiviral responses in mESCs effectively avoids this problem. mESCs can tolerate repeated transfection and effectively express proteins from their synthetic mRNA with expected biological functions, as demonstrated by the expression of green fluorescent protein and the transcription factor Etv2. Therefore, mRNA-based gene expression could be developed into a novel ESC differentiation strategy that avoids safety concerns associated with viral/DNA-based vectors in regenerative medicine.


A commonly used rumen-protected conjugated linoleic acid supplement marginally affects fatty acid distribution of body tissues and gene expression of mammary gland in heifers during early lactation.
Conjugated linoleic acids (CLA) in general, and in particular the trans-10,cis-12 (t10,c12-CLA) isomer are potent modulators of milk fat synthesis in dairy cows. Studies in rodents, such as mice, have revealed that t10,c12-CLA is responsible for hepatic lipodystrophy and decreased adipose tissue with subsequent changes in the fatty acid distribution. The present study aimed to investigate the fatty acid distribution of lipids in several body tissues compared to their distribution in milk fat in early lactating cows in response to CLA treatment. Effects in mammary gland are further analyzed at gene expression level. Twenty-five Holstein heifers were fed a diet supplemented with (CLA groups) or without (CON groups) a rumen-protected CLA supplement that provided 6 g/d of c9,t11- and t10,c12-CLA. Five groups of randomly assigned cows were analyzed according to experimental design based on feeding and time of slaughter. Cows in the first group received no CLA supplement and were slaughtered one day postpartum (CON0). Milk samples were taken from the remaining cows in CON and CLA groups until slaughter at 42 (period 1) and 105 (period 2) days in milk (DIM). Immediately after slaughter, tissue samples from liver, retroperitoneal fat, mammary gland and M. longissimus (13th rib) were obtained and analyzed for fatty acid distribution. Relevant genes involved in lipid metabolism of the mammary gland were analyzed using a custom-made microarray platform. Both supplemented CLA isomers increased significantly in milk fat. Furthermore, preformed fatty acids increased at the expense of de novo-synthesized fatty acids. Total and single trans-octadecenoic acids (e.g., t10-18:1 and t11-18:1) also significantly increased. Fatty acid distribution of the mammary gland showed similar changes to those in milk fat, due mainly to residual milk but without affecting gene expression. Liver fatty acids were not altered except for trans-octadecenoic acids, which were increased. Adipose tissue and M. longissimus were only marginally affected by CLA supplementation. Daily supplementation with CLA led to typical alterations usually observed in milk fat depression (reduction of de novo-synthesized fatty acids) but only marginally affected tissue lipids. Gene expression of the mammary gland was not influenced by CLA supplementation.


The Influence of Na(+), K(+)-ATPase on Glutamate Signaling in Neurodegenerative Diseases and Senescence.
Decreased Na(+), K(+)-ATPase (NKA) activity causes energy deficiency, which is commonly observed in neurodegenerative diseases. The NKA is constituted of three subunits: α, β, and γ, with four distinct isoforms of the catalytic α subunit (α1-4). Genetic mutations in the ATP1A2 gene and ATP1A3 gene, encoding the α2 and α3 subunit isoforms, respectively can cause distinct neurological disorders, concurrent to impaired NKA activity. Within the central nervous system (CNS), the α2 isoform is expressed mostly in glial cells and the α3 isoform is neuron-specific. Mutations in ATP1A2 gene can result in familial hemiplegic migraine (FHM2), while mutations in the ATP1A3 gene can cause Rapid-onset dystonia-Parkinsonism (RDP) and alternating hemiplegia of childhood (AHC), as well as the cerebellar ataxia, areflexia, pescavus, optic atrophy and sensorineural hearing loss (CAPOS) syndrome. Data indicates that the central glutamatergic system is affected by mutations in the α2 isoform, however further investigations are required to establish a connection to mutations in the α3 isoform, especially given the diagnostic confusion and overlap with glutamate transporter disease. The age-related decline in brain α2∕3 activity may arise from changes in the cyclic guanosine monophosphate (cGMP) and cGMP-dependent protein kinase (PKG) pathway. Glutamate, through nitric oxide synthase (NOS), cGMP and PKG, stimulates brain α2∕3 activity, with the glutamatergic N-methyl-D-aspartate (NMDA) receptor cascade able to drive an adaptive, neuroprotective response to inflammatory and challenging stimuli, including amyloid-β. Here we review the NKA, both as an ion pump as well as a receptor that interacts with NMDA, including the role of NKA subunits mutations. Failure of the NKA-associated adaptive response mechanisms may render neurons more susceptible to degeneration over the course of aging.


The role of calcium intake in preventing bone fragility, hypertension, and certain cancers.
This paper examines the evidence that connects calcium intake and vitamin D status to bone fragility, hypertension, colon cancer, and breast cancer. Human calcium physiology, with an intestinal absorptive barrier and inefficient conservation, reflects the abundance of calcium in the primordial human food supply. The calcium intake of stone-age adults is estimated at 50 to 75 mmol/d, three to five times the median calcium intake of present-day U.S. adults. Long-term calcium restriction and/or insufficient vitamin D may promote the development of bone fragility, high blood pressure, colon cancer, and breast cancer in susceptible individuals. Conversely, improvement in calcium intake and/or in vitamin D status may help to prevent these serious health problems. At least 12 intervention studies have established the skeletal benefit of increased calcium intake among women in the late postmenopause. Other reports suggest that adequate calcium may protect against salt-sensitive and pregnancy-associated hypertension. High intakes of both dietary calcium and vitamin D are associated with reduced development of precancerous changes in colonic mucosa. Preliminary findings also suggest that vitamin D has a protective effect against breast cancer.


Solution conformation of [AF]dG opposite a -1 deletion site in a DNA duplex: intercalation of the covalently attached aminofluorene ring into the helix with base displacement of the C8-modified Syn guanine into the major groove.
This paper reports on the solution structure of the [AF]dG adduct positioned opposite a deletion site in a DNA oligomer duplex that defines the alignment of the covalent aminofluorene--C8-guanine adduct relative to the deletion site. The combined NMR molecular mechanics computational studies were undertaken on the [AF]dG adduct embedded in the d(C5-[AF]G6-C7).d(G16-G17) sequence context in a duplex containing 11 residues on the modified strand and 10 on the partner, with no base opposite the modification. The exchangeable and nonexchangeable protons of the aminofluorene moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. The solution conformation of the [AF]G.del 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bounds deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space. The aminofluorene ring of [AF]dG6 is intercalated between intact Watson-Crick dC5.dG17 and dC7.dG16 base pairs with the guanine base of [AF]dG6 in a syn alignment displaced into the major groove. The syn glycosidic torsion angle at [AF]dG6 is supported by both carbon and proton chemical shift data for the sugar resonances of the modified guanine residue. The long axis of the aminofluorene ring is parallel to the long axis of the flanking dG.dC base pairs with the AF ring undergoing rapid 180 degrees flips on the NMR time scale. The intercalation site is wedge shaped with a pronounced propeller-twisting and buckling of the dC5.dG17 base pair. The guanine base of [AF]dG6, which is positioned in the major groove, is inclined relative to the helix axis and stacks over the 5'-flanking dC5 residue in the solution structure. The intercalative-base displacement structure of the [AF]dG.del 11-mer duplex exhibits several unusually shifted proton resonances that can be readily accounted for by the ring current contributions of the guanine purine and carcinogen fluorene aromatic rings of the [AF]dG6 adduct. There are similarities between this structure of the AF-C8-dG covalent adduct positioned opposite a deletion site and the (+)-trans-anti-BP-N2-dG covalent adduct positioned opposite a deletion site in the same sequence context reported previously from this laboratory [Cosman et al. (1994) Biochemistry 33, 11507-11517]. The chromophores are intercalated into the helix opposite the deletion site with displacement of the modified guanine into the major groove in both cases.(ABSTRACT TRUNCATED AT 400 WORDS)


Perceptual category learning and visual processing: An exercise in computational cognitive neuroscience.
The field of computational cognitive neuroscience (CCN) builds and tests neurobiologically detailed computational models that account for both behavioral and neuroscience data. This article leverages a key advantage of CCN-namely, that it should be possible to interface different CCN models in a plug-and-play fashion-to produce a new and biologically detailed model of perceptual category learning. The new model was created from two existing CCN models: the HMAX model of visual object processing and the COVIS model of category learning. Using bitmap images as inputs and by adjusting only a couple of learning-rate parameters, the new HMAX/COVIS model provides impressively good fits to human category-learning data from two qualitatively different experiments that used different types of category structures and different types of visual stimuli. Overall, the model provides a comprehensive neural and behavioral account of basal ganglia-mediated learning.


Endogenous nitric oxide decreases xanthine oxidase-mediated neutrophil adherence: role of P-selectin.
The oxygen radical-producing enzyme xanthine oxidase (XO) can promote neutrophil adherence to endothelium. Recognizing that a balance often exists in inflammatory processes, we sought to determine whether XO initiates antiadherent pathways. We found that bovine pulmonary arterial endothelial cells (EC) exposed to XO released increased amounts of nitrite into the media, reflecting an increased production of nitric oxide (NO). When EC were subjected to shear stress, treatment with XO and/or the NO synthase inhibitor N omega-nitro-L-arginine (L-NNA) increased neutrophil rolling behavior and firm neutrophil adherence to EC in an additive fashion. Both rolling and adherent interactions were abolished by monoclonal antibodies directed against P-selectin. In addition, treatment of EC with XO and/or L-NNA increased both surface expression of P-selectin and release of von Willebrand factor into media. Finally, treatment of EC with the NO donor sodium nitroprusside decreased XO-mediated neutrophil rolling and adherence. We conclude that XO stimulates EC to produce NO and that NO decreases the P-selectin-dependent neutrophil adhesion initiated by XO. Such increases in endogenous NO may constitute an important negative-feedback response to the acute proadhesive effects of XO.


VH-related idiotopes detected by site-directed mutagenesis. A study induced by the failure to find CD4 anti-idiotypic antibodies mimicking the cellular receptor of HIV.
The function of the CD4 cell surface protein as coreceptor on T helper lymphocytes and as receptor for HIV makes this glycoprotein a prime target for an immune intervention with mAb. A detailed understanding of the structural determinants on the therapeutic CD4 mAb that are involved in Ag binding or are recognized by anti-idiotypic mAb (anti-Id) may be important for designing antibodies with optimal therapeutic efficacy. Seven anti-Id raised against the CD4 mAb M-T310 were selected from a large panel with the intention to obtain CD4 mimicking structures with specificity for HIV gp120. The selected anti-Id did not react with other CD4-specific mAb cross-blocking M-T310. Among these, mAb M-T404, although having the same L chain as M-T310 and a VH region sequence differing only at 14 amino acid positions, was not recognized by the anti-Id. M-T310 H chain complexed with the J558L L chain reacted with all anti-Id, thus demonstrating that the recognized idiotopes are located within the VH region. To identify the idiotopes of M-T310 seen by the anti-Id, variants of M-T404 containing one or more of the M-T310-derived substitutions were generated by oligonucleotide-directed mutagenesis. The reactivity pattern of the mutant proteins with the anti-Id demonstrated that the idiotopes reside within the complementarity determining region (CDR) 2 and CDR3 loops of the VH region. A major idiotope was defined by a single amino acid in CDR2 that was recognized by three anti-Id, whereas the four other anti-Id reacted with determinants of CDR3. Although the performed amino acid substitutions did influence the Id recognition, Ag binding was not significantly affected, suggesting that none of the anti-Id can be considered as a mimicry of the CD4 Ag.


Heat shock protein 70 increases tumorigenicity and inhibits apoptosis in pancreatic adenocarcinoma.
Pancreatic carcinoma is a malignant disease that responds poorly to chemotherapy because of its resistance to apoptosis. Heat shock proteins (Hsp) are not only cytoprotective but also interfere with the apoptotic cascade. Here, we investigated the role of Hsp70 in regulating apoptosis in pancreatic cancer cells. Hsp70 expression was increased in pancreatic cancer cells compared with normal pancreatic ductal cells. This was confirmed by increased mRNA levels for Hsp70 in human pancreatic cancer tissue compared with neighboring normal tissue from the same patient. Depletion of Hsp70 by quercetin decreased cell viability and induced apoptosis in cancer cells but not in normal pancreatic ductal cells. To show that this is a specific effect of Hsp70 on apoptosis, levels of Hsp70 were knocked down by short interfering RNA treatment, which also induced apoptosis in cancer cells as indicated by Annexin V staining and caspase activation. Daily administration of quercetin to nude mice decreased tumor size as well as Hsp70 levels in tumor tissue. These findings indicate that Hsp70 plays an important role in apoptosis and that selective Hsp70 knockdown can be used to induce apoptosis in pancreatic cancer cells.


Looking Across and Looking Beyond the Knowledge Frontier: Intellectual Distance, Novelty, and Resource Allocation in Science.
Selecting among alternative projects is a core management task in all innovating organizations. In this paper, we focus on the evaluation of frontier scientific research projects. We argue that the "intellectual distance" between the knowledge embodied in research proposals and an evaluator's own expertise systematically relates to the evaluations given. To estimate relationships, we designed and executed a grant proposal process at a leading research university in which we randomized the assignment of evaluators and proposals to generate 2,130 evaluator-proposal pairs. We find that evaluators systematically give lower scores to research proposals that are closer to their own areas of expertise and to those that are highly novel. The patterns are consistent with biases associated with boundedly rational evaluation of new ideas. The patterns are inconsistent with intellectual distance simply contributing "noise" or being associated with private interests of evaluators. We discuss implications for policy, managerial intervention, and allocation of resources in the ongoing accumulation of scientific knowledge.


Pulmonary Eosinophilic Granulomatosis with Polyangiitis Has IgG4 Plasma Cells and Immunoregulatory Features.
The immunologic mechanisms promoting eosinophilic granulomatosis with polyangiitis (EGPA) are unclear. To characterize the mechanisms underlying pulmonary EGPA, we examined and compared EGPA paraffin-embedded lung biopsies with normal lung biopsies, using immunostaining, RNA sequencing, and RT-PCR. The results revealed novel type 2 as well as immuneregulatory features. These features included basophils and increased mast cell contents; increased immunostaining for tumor necrosis factor ligand superfamily member 14; sparse mast cell degranulation; numerous forkhead box protein P3 (FoxP3)+ regulatory T cells and IgG4 plasma cells; and abundant arachidonate 15-lipoxygenase and 25-hydroxyvitamin D-1 α hydroxylase, mitochondrial. Significantly decreased 15-hydroxyprostaglandin dehydrogenase [NAD(+)], which degrades eicosanoids, was observed in EGPA samples. In addition, there was significantly increased mRNA for chemokine (C-C motif) ligands 18 and 13 and major collagen genes, IgG4-rich immune complexes coating alveolar macrophages, and increased immunostaining for phosphorylated mothers against decapentaplegic homolog 2/SMAD2, suggesting transforming growth factor-β activation. These findings suggest a novel self-promoting mechanism of activation of alveolar macrophages by arachidonate 15-lipoxygenase-derived eicosanoids to express chemokines that recruit a combined type 2/immunoregulatory immune response, which produces these eicosanoids. These results suggest that the pulmonary EGPA immune response resembles the immune response to a tissue-invasive parasite infection.


Glucocorticoid mediates water avoidance stress-sensitized colon-bladder cross-talk via RSK2/PSD-95/NR2B in rats.
Unexpected environmental and social stimuli could trigger stress. Although coping with stress is essential for survival, long-term stress impacts visceral functions, and therefore, it plays a role in the development and exacerbation of symptoms of gastrointestinal/urogenital disorders. The aim of this study is to characterize the role of corticosterone in stress-sensitized colon-bladder cross-talk, a phenomenon presumed to underlie the comorbidity of functional bowel and bladder disorders. Cystometry and protein/mRNA expression in the lumbosacral dorsal horn (L6-S1) in response to intracolonic mustard oil (MO) instillation were analyzed in female Wistar-Kyoto rats subjected to water avoidance stress (WAS; 1 h/day for 10 days) or sham stress (WAsham). Whereas it had no effect on baseline-voiding function, chronic stress upregulated plasma corticosterone concentration and dorsal horn spinal p90 ribosomal S6 kinase 2 (RSK2) protein/mRNA levels, and RSK2 immunoreactivity colocalized with NeuN-positive neurons. Intracolonic MO dose-dependently decreased intrercontraction intervals and threshold pressure, provoked spinal RSK2 and NR2B phosphorylation, and enhanced PSD-95-RSK2 and PSD-95-NR2B coupling. Intrathecal kaempferol (a RSK2 activation antagonist; 30 min before MO instillation), bilateral adrenalectomy (7 days prior the stress paradigm), and subcutaneous RU-38486 (a glucocorticoid receptor antagonist; 30 min daily before stress sessions), but not RU-28318 (a mineralocorticoid receptor antagonist), attenuated MO-induced bladder hyperactivity, protein phosphorylation, and protein-protein interactions in the WAS group. Our results suggest that stress-associated glucocorticoid release mediates WAS-dependent sensitization of colon-bladder cross-talk via the spinal RSK2/PSD-95/NR2B cascade and offer a possibility for developing pharmacological strategies for the treatment of stress-related pelvic pain.


Uterine tumour resembling ovarian sex cord tumour is an immunohistochemically polyphenotypic neoplasm which exhibits coexpression of epithelial, myoid and sex cord markers.
To describe the clinicopathological and immunohistochemical findings in four cases of uterine tumour resembling ovarian sex cord tumour (UTROSCT). Four UTROSCTs were stained with a wide range of antibodies, including epithelial (AE1/3, epithelial membrane antigen), myoid (desmin, alpha smooth muscle actin, h-caldesmon), sex cord (alpha inhibin, calretinin, melan A, CD99) and neuroendocrine (chromogranin, CD56) markers as well as hormone receptors (oestrogen receptor, progesterone receptor, androgen receptor), vimentin, CD10, WT1 and HMB45. The tumours ranged from 0.8 to 19.5 cm. Three were relatively well circumscribed intramural myometrial lesions; the other was a pedunculated mass attached to the uterine serosa. The tumours were variably composed of solid, corded, trabecular, nested, glandular and retiform arrangements of tumour cells. In three cases, cells with eccentric nuclei and abundant eosinophilic cytoplasm, resulting in a rhabdoid appearance, were a prominent feature. Three cases were diffusely positive with AE1/3 and all with epithelial membrane antigen. Positivity with myoid markers was common with 3, 4 and 1 case respectively staining with desmin, alpha smooth muscle actin and h-caldesmon; 2, 4, 1 and 2 cases respectively were positive with alpha inhibin, calretinin, melan A and CD99. All were chromogranin negative and exhibited diffuse strong staining with CD56. All were diffusely positive with oestrogen receptor, progesterone receptor, vimentin and WT1. Three cases were androgen receptor positive and all were CD10 and HMB45 negative. UTROSCT exhibits a polyphenotypic immunophenotype with coexpression of markers of epithelial, myoid and sex cord lineage as well as hormone receptors.