The membrane hyperpolarization of rat dorsolateral septal nucleus neurons is mediated by a novel nicotinic receptor.
The pharmacology, calcium dependence and G protein mediation of the membrane hyperpolarization of rat dorsolateral septal nucleus (DLSN) neurons in response to nicotinic agonists was examined to classify the nicotinic receptor mediating the response. Intracellular recording from DSLN neurons in a brain slice preparation was used to determine whether chlorisondamine, trimethaphan, cytisine or strychnine inhibited the membrane hyperpolarization in response to application of the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP). Chlorisondamine was found to block the response only at a high concentration (500 microM) although strychnine (100 microM) was without effect. Cytisine was neither an effective agonist nor an antagonist (500 microM). Surprisingly, trimethaphan appeared to act as an agonist, rather than an antagonist, with a potency and efficacy similar to that reported for nicotine at this receptor. The response was dependent on intracellular calcium stores because it persisted in the absence of extracellular calcium but was blocked by intracellular injection of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Injection of GTP gamma S into the neurons blocked the nicotinic response. Apamin, iberiotoxin and charybdotoxin reduced but did not block the response at concentrations that selectively block calcium-dependent potassium channels. These results indicate that the nicotinic response in DLSN neurons may be mediated by a metabotropic nicotinic receptor coupled to a calcium-dependent potassium channel through the activation of a G-protein and release of intracellular calcium stores. The unusual pharmacology of the nicotinic receptor on DLSN neurons indicates that it may be a novel receptor which has yet to be cloned.


Replication across O6-methylguanine by human DNA polymerase beta in vitro. Insights into the futile cytotoxic repair and mutagenesis of O6-methylguanine.
Replication in vivo across unrepaired O6-methylguanine (m6dG) lesions by mammalian DNA polymerase beta (pol beta) during short patch repair may contribute to the cytotoxicity and mutagenesis of m6dG. We have employed in vitro steady state kinetic analysis to investigate the replication of oligonucleotide templates containing site-specific m6dG by human pol beta. Our results show that m6dG is a strong but not absolute block to replication by pol beta. pol beta exhibits mixed kinetic discrimination during overall replication across dG and m6dG. pol beta preferentially inserts dTMP rather than dCMP opposite m6dG. However, pol beta extends from the dC-m6dG base pair more efficiently than from the dT-m6dG base pair. This is in strong contrast to other polymerases such as the exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I (exo-KF) that preferentially extends dT-m6dG by a factor of 10 over dC-m6dG. When both insertion and extension are considered, pol beta has a 15-fold overall preference for incorporation of the mutagenic substrate dTTP rather than the nonmutagenic substrate dCTP during replication across m6dG. This suggests that pol beta, in concert with the T:G-specific thymine DNA glycosylase, may be intricately involved in the futile cytotoxic repair induced by m6dG. Our results also suggest that replication across m6dG by pol beta may contribute to m6dG-induced G --> A transition mutations.


A role for the ETS domain transcription factor PEA3 in myogenic differentiation.
Activation of adult myoblasts called satellite cells during muscle degeneration is an important aspect of muscle regeneration. Satellite cells are believed to be the only myogenic stem cells in adult skeletal muscle and the source of regenerating muscle fibers. Upon activation, satellite cells proliferate, migrate to the site of degeneration, and become competent to fuse and differentiate. We show here that the transcription factor polyomavirus enhancer activator 3 (PEA3) is expressed in adult myoblasts in vitro when they are proliferative and during the early stages of differentiation. Overexpression of PEA3 accelerates differentiation, whereas blocking of PEA3 function delays myoblast fusion. PEA3 activates gene expression following binding to the ets motif most efficiently in conjunction with the transcription factor myocyte enhancer factor 2 (MEF2). In vivo, PEA3 is expressed in satellite cells only after muscle degeneration. Taken together, these results suggest that PEA3 is an important regulator of activated satellite cell function.


Botulinum Neurotoxin F Subtypes Cleaving the VAMP-2 Q58⁻K59 Peptide Bond Exhibit Unique Catalytic Properties and Substrate Specificities.
In the recent past, about 40 botulinum neurotoxin (BoNT) subtypes belonging to serotypes A, B, E, and F pathogenic to humans were identified among hundreds of independent isolates. BoNTs are the etiological factors of botulism and represent potential bioweapons; however, they are also recognized pharmaceuticals for the efficient counteraction of hyperactive nerve terminals in a variety of human diseases. The detailed biochemical characterization of subtypes as the basis for development of suitable countermeasures and possible novel therapeutic applications is lagging behind the increase in new subtypes. Here, we report the primary structure of a ninth subtype of BoNT/F. Its amino-acid sequence diverges by at least 8.4% at the holotoxin and 13.4% at the enzymatic domain level from all other known BoNT/F subtypes. We found that BoNT/F9 shares the scissile Q58/K59 bond in its substrate vesicle associated membrane protein 2 with the prototype BoNT/F1. Comparative biochemical analyses of four BoNT/F enzymatic domains showed that the catalytic efficiencies decrease in the order F1 > F7 > F9 > F6, and vary by up to a factor of eight. KM values increase in the order F1 > F9 > F6 ≈ F7, whereas kcat decreases in the order F7 > F1 > F9 > F6. Comparative substrate scanning mutagenesis studies revealed a unique pattern of crucial substrate residues for each subtype. Based upon structural coordinates of F1 bound to an inhibitor polypeptide, the mutational analyses suggest different substrate interactions in the substrate binding channel of each subtype.


An endoplasmic reticulum protein, calreticulin, is transported into the acrosome of rat sperm.
Recently, we purified a Ca(2+)-binding protein from rat spermatogenic cells [Biochem. Biophys. Res. Commun. 176, 135-1364, 1991]. In the present study, this protein was identified as calreticulin, which is a resident protein of the endoplasmic reticulum (ER). Immunohistochemical studies revealed that calreticulin was present in the acrosome of both round spermatids and mature sperm. However, under immunoelectron microscopy, gold-particles were seen over other subcellular structures of spermatocytes, spermatids, and Sertoli cells. When the labeling density in subcellular structures of spermatids was analyzed, the acrosome was found to be most heavily labeled and the Golgi apparatus was second. The complete amino acid sequence of calreticulin, deduced from the cDNA sequence, shares a high degree of identity with that of the analogous mouse protein. The cDNA encoded a protein of 416 amino acids, including a 17-residue NH2-terminal signal sequence. The mature protein contains a KDEL sequence as an ER signal at the COOH terminus. Sperm calreticulin contained no glycosyl moiety. Northern blot analysis of RNAs from purified populations of rat spermatogenic cells indicated that the calreticulin mRNA was present in both pre- and postmeiotic cells. Immunoblot analysis of calreticulin during developmental stages showed that calreticulin was detected in the testis between the ages of 5 and 50 days. Furthermore, purified rat calreticulin contained two Ca(2+)-binding sites, a low affinity/high capacity site and a high affinity/low capacity site. These results suggest that calreticulin, which is not specific to testis, is closely associated with spermatogenesis of rats. This ER protein may be incorporated into the acrosomal vesicle via the Golgi apparatus, without glycosylation, during spermiogenesis, and may play an important role in the regulation of cell functions such as sperm motility and acrosome reaction.


Applicability of molecular markers to determine parasitic infection origins in the animal trade: a case study from Sarcoptes mites in wildebeest.
The development of non-manipulative molecular tools to determine the origin of parasite infections in the animal trade (if infected before their export or import) is of great interest worldwide for both the animal trade industry and for animal welfare. Molecular tools have a wide range of applications, including forensic identification, wildlife preservation and conservation, veterinary public health protection, and food safety. Nonetheless, genetic markers were not reported to detect the source of infection in the animal trade. In this study we tested the applicability of molecular tools to detect the origin of Sarcoptes mite infection of wildebeest imported by the United Arab Emirate (UAE) from Tanzania. Using one multiplex of seven microsatellite markers and control samples from UAE, Kenya and Italy, we demonstrated the usefulness of the multiplex STR-typing as a molecular tool of pivotal interest to help commercialist, authorities, and conservationists, to identify the geographical origin of parasitic infections.


Variation in dengue type 2 viruses isolated in Bangkok during 1980.
Dengue type-2 viruses isolated in metropolitan Bangkok during 1980 (Bangkok/80) were characterized by oligonucleotide fingerprinting, restriction enzyme (RE) mapping and antigenic analysis using monoclonal antibody probes. Of 10 isolates analysed by oligonucleotide fingerprinting, nine were very closely related, showing 72.5% to 91.4% oligonucleotide homology. One isolate (D80-141) produced a distinctly different fingerprint (55.7% to 58.0% homology) and was less related to other Bangkok/80 dengue-2 virus isolates than to a 1964 Bangkok isolate (16681). RE mapping conducted on complementary dsDNA prepared from three Bangkok/80 isolates, strain 16681 and the prototype New Guinea C strain confirmed that D80-141 was genetically distinct. On antigenic analysis, only one of 22 monoclonal antibody probes produced against representative 1980 Bangkok dengue-2 isolates, D80-100 and D80-141, was able to distinguish between these virus strains. Monoclonal antibody 47-10/10, prepared using D80-100 virus and directed at the NS1 non-structural glycoprotein, had a significantly lower (100-fold) solid phase radioimmune assay endpoint titre for D80-141 antigen than for D80-100 antigen. By the indirect immunofluorescence assay, 47-10/10 had lower antibody endpoint titres against D80-141, the NGC strain and 13 (12%) of 110 Bangkok/80 isolates than to a control antibody preparation. These results suggest that strain D80-141 represents a second minor topotype of dengue-2 which was circulating concurrently with the major endemic topotype in Bangkok in early 1980.


A molecular timetable for apical bud formation and dormancy induction in poplar.
The growth of perennial plants in the temperate zone alternates with periods of dormancy that are typically initiated during bud development in autumn. In a systems biology approach to unravel the underlying molecular program of apical bud development in poplar (Populus tremula x Populus alba), combined transcript and metabolite profiling were applied to a high-resolution time course from short-day induction to complete dormancy. Metabolite and gene expression dynamics were used to reconstruct the temporal sequence of events during bud development. Importantly, bud development could be dissected into bud formation, acclimation to dehydration and cold, and dormancy. To each of these processes, specific sets of regulatory and marker genes and metabolites are associated and provide a reference frame for future functional studies. Light, ethylene, and abscisic acid signal transduction pathways consecutively control bud development by setting, modifying, or terminating these processes. Ethylene signal transduction is positioned temporally between light and abscisic acid signals and is putatively activated by transiently low hexose pools. The timing and place of cell proliferation arrest (related to dormancy) and of the accumulation of storage compounds (related to acclimation processes) were established within the bud by electron microscopy. Finally, the identification of a large set of genes commonly expressed during the growth-to-dormancy transitions in poplar apical buds, cambium, or Arabidopsis thaliana seeds suggests parallels in the underlying molecular mechanisms in different plant organs.


Genetic diversity of the ordinary strain of Potato virus Y (PVY) and origin of recombinant PVY strains.
The ordinary strain of Potato virus Y (PVY), PVY(O), causes mild mosaic in tobacco and induces necrosis and severe stunting in potato cultivars carrying the Ny gene. A novel substrain of PVY(O) was recently reported, PVY(O)-O5, which is spreading in the United States and is distinguished from other PVY(O) isolates serologically (i.e., reacting to the otherwise PVY(N)-specific monoclonal antibody 1F5). To characterize this new PVY(O)-O5 subgroup and address possible reasons for its continued spread, we conducted a molecular study of PVY(O) and PVY(O)-O5 isolates from a North American collection of PVY through whole-genome sequencing and phylogenetic analysis. In all, 44 PVY(O) isolates were sequenced, including 31 from the previously defined PVY(O)-O5 group, and subjected to whole-genome analysis. PVY(O)-O5 isolates formed a separate lineage within the PVY(O) genome cluster in the whole-genome phylogenetic tree and represented a novel evolutionary lineage of PVY from potato. On the other hand, the PVY(O) sequences separated into at least two distinct lineages on the whole-genome phylogenetic tree. To shed light on the origin of the three most common PVY recombinants, a more detailed phylogenetic analysis of a sequence fragment, nucleotides 2,406 to 5,821, that is present in all recombinant and nonrecombinant PVY(O) genomes was conducted. The analysis revealed that PVY(N:O) and PVY(N-Wi) recombinants acquired their PVY(O) segments from two separate PVY(O) lineages, whereas the PVY(NTN) recombinant acquired its PVY(O) segment from the same lineage as PVY(N:O). These data suggest that PVY(N:O) and PVY(N-Wi) recombinants originated from two separate recombination events involving two different PVY(O) parental genomes, whereas the PVY(NTN) recombinants likely originated from the PVY(N:O) genome via additional recombination events.


Ancient dispersal of the human fungal pathogen Cryptococcus gattii from the Amazon rainforest.
Over the past two decades, several fungal outbreaks have occurred, including the high-profile 'Vancouver Island' and 'Pacific Northwest' outbreaks, caused by Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals.


A bifunctional converter: fluorescein quenching scFv/fluorogen activating protein for photostability and improved signal to noise in fluorescence experiments.
Monoclonal antibodies are one of the most useful and ubiquitous affinity reagents used in the biological sciences. Immunostaining of fixed and live cells for microscopy or cytometry measurements frequently employs fluorescently labeled antibodies, in particular fluorescein-labeled antibodies. This dye emits light at a wavelength overlapping with cellular autofluorescence, making it difficult to measure antibody binding to proteins of relatively low copy number or in cells of high green autofluorescence. A number of high affinity fluorescein binding antibodies and antibody domains have been developed that quench the dye's fluorescence. Using a fluorescein-binding recombinant antibody domain genetically fused to a fluorogen activating protein (FAP), we demonstrate a molecular converter capable of binding and quenching fluorescein, while binding and activating a fluorogenic triarylmethane dye. This reagent converts fluorescein conjugates to far-red fluorescent probes, where cellular autofluorescence is low, improving signal-to-background of cell-based antibody binding measurements by ∼7-fold. Microscopy experiments show colocalization of both fluorescein and MG fluorescence. This dual affinity fluorescein-quenching-FAP can also be used to convert fluorescein to the red fluorescing MG fluorogen on biological molecules other than antibodies.


Conjugated linoleic acid rapidly reduces body fat content in mice without affecting energy intake.
Recent reports have demonstrated that conjugated linoleic acid (CLA) has effects on body fat accumulation. In our previous work, CLA reduced body fat accumulation in mice fed either a high-fat or low-fat diet. Although CLA feeding reduced energy intake, the results suggested that some of the metabolic effects were not a consequence of the reduced food intake. We therefore undertook a study to determine a dose of CLA that would have effects on body composition without affecting energy intake. Five doses of CLA (0.0, 0.25, 0.50, 0.75, and 1.0% by weight) were studied in AKR/J male mice (n = 12/group; age, 39 days) maintained on a high-fat diet (%fat 45 kcal). Energy intake was not suppressed by any CLA dose. Body fat was significantly lower in the 0.50, 0.75, and 1.0% CLA groups compared with controls. The retroperitoneal depot was most sensitive to the effects of CLA, whereas the epididymal depot was relatively resistant. Higher doses of CLA also significantly increased carcass protein content. A time-course study of the effects of 1% CLA on body composition showed reductions in fat pad weights within 2 wk and continued throughout 12 wk of CLA feeding. In conclusion, CLA feeding produces a rapid, marked decrease in fat accumulation, and an increase in protein accumulation, at relatively low doses without any major effects on food intake.


Risk of Salmonellosis Associated with Consumption of Chocolate in Mexico.
To assess the potential risk of salmonellosis associated with consumption of chocolate, the prevalence of Salmonella spp. was investigated in retail wrapped and nonwrapped solid chocolate in Guadalajara, Mexico. Previously, the efficiency of preenrichment and enrichment methods to isolate low numbers of Salmonella in chocolate was compared. Though not completely efficient, a preenrichment in nonfat milk without brilliant green, followed by enrichment in tetrathionate and selenite cystine broths gave better results than the other methods studied. Regarding the survey of retail chocolate, Salmonella was isolated from 2 (4.5%) of 44 samples of wrapped chocolate and from none of 56 samples of nonwrapped chocolate. The serovars identified were Salmonella agona and Salmonella derby . There were no significant differences between coliform counts or total bacterial counts obtained from wrapped and nonwrapped chocolate. The presence of Salmonella in chocolate is of concern due to the several low-infective-dose Salmonella outbreaks that have been linked to chocolate. On the other hand, a technique capable of detecting low numbers of Salmonella in chocolate is still needed.


Wildlife Conservation at a Garden Level: The Effect of Robotic Lawn Mowers on European Hedgehogs (Erinaceus europaeus).
We tested the effects of 18 models of robotic lawn mowers in collision with dead European hedgehogs and quantified the results into six damage categories. All models were tested on four weight classes of hedgehogs, each placed in three different positions. None of the robotic lawn mowers tested was able to detect the presence of dependent juvenile hedgehogs (<200 g) and all models had to touch the hedgehogs to detect them. Some models caused extensive damage to the hedgehog cadavers, but there were noteworthy differences in the degree of harm inflicted, with some consistently causing no damage. Our results showed that the following technical features significantly increased the safety index of the robotic lawn mowers: pivoting blades, skid plates, and front wheel drive. Based on these findings, we encourage future collaboration with the manufacturers of robotic lawn mowers to improve the safety for hedgehogs and other garden wildlife species.


Approaches to integrating genetic data into ecological networks.
As molecular tools for assessing trophic interactions become common, research is increasingly focused on the construction of interaction networks. Here, we demonstrate three key methods for incorporating DNA data into network ecology and discuss analytical considerations using a model consisting of plants, insects, bats and their parasites from the Costa Rica dry forest. The simplest method involves the use of Sanger sequencing to acquire long sequences to validate or refine field identifications, for example of bats and their parasites, where one specimen yields one sequence and one identification. This method can be fully quantified and resolved and these data resemble traditional ecological networks. For more complex taxonomic identifications, we target multiple DNA loci, for example from a seed or fruit pulp sample in faeces. These networks are also well resolved but gene targets vary in resolution and quantification is difficult. Finally, for mixed templates such as faecal contents of insectivorous bats, we use DNA metabarcoding targeting two sequence lengths (157 and 407 bp) of one gene region and a MOTU, BLAST and BIN association approach to resolve nodes. This network type is complex to generate and analyse, and we discuss the implications of this type of resolution on network analysis. Using these data, we construct the first molecular-based network of networks containing 3,304 interactions between 762 nodes of eight trophic functions and involving parasitic, mutualistic and predatory interactions. We provide a comparison of the relative strengths and weaknesses of these data types in network ecology.


Simple method of measuring scattering losses in optical fibers.
A simple method of measuring the scattering losses of optical fibers was developed. The method permits the measurement of the scattering-loss spectra by use of photon counting. Measurement is based on right-angle scattering, which is dominated by Rayleigh scattering, a material-intrinsic loss. A reference fiber for which the scattering loss is known is used to cancel out the unknown factors that are dependent on the optical setup. The scattering-loss measurement was demonstrated by use of two different low-loss fibers and was found to agree with predetermined figures to within 10% over the wavelength range 0.44-1.0 microm. Finally, the method was applied to a new high-numerical-aperture optical fiber to find its material scattering loss.


Are the Eatwell Guide and Nutrient Profiling Models Consistent in the UK?
A nutrient profiling model (NPM) was developed in 2005 in the UK to regulate the marketing of foods to children. It was revised in 2018, but the new version has not been finalised. The Eatwell Guide (EWG) is the UK's official food-based dietary guidelines. The aim of this study was to evaluate the agreement between the 2005 and 2018 versions of the NPM and the EWG. Using recent National Diet and Nutrition Surveys, we estimated the healthiness of individual diets based on an EWG dietary score and a NPM dietary index. We then compared the percentage of agreement and Cohen's kappa for each combination of the EWG score and NPM index across the range of observed values for the 2005 and 2018 versions. A total of 3028 individual diets were assessed. Individuals with a higher (i.e., healthier) EWG score consumed a diet with, on average, a lower (i.e., healthier) NPM index both for the 2005 and 2018 versions. Overall, there was good agreement between the EWG score and the NPM dietary index at assessing the healthiness of representative diets of the UK population, when a low cut-off for the NPM dietary index was used, irrespective of the version. This suggests that dietary advice to the public is broadly aligned with NPM-based food policies and vice-versa.


Randomized Controlled Trial of Ondansetron vs. Prochlorperazine in Adults in the Emergency Department.
To compare the effectiveness of ondansetron and prochlorperazine to treat vomiting. Secondary objectives were the effectiveness of ondansetron and prochlorperazine to treat nausea and their tolerability. This was a prospective, randomized, active controlled, double-blinded study. Using a convenience sample, patients were randomized to either intravenous ondansetron 4mg (n=32) or prochlorperazine 10mg (n=32). The primary outcome was the percentage of patients with vomiting at 0-30, 31-60, and 61-120 minutes after the administration of ondansetron or prochlorperazine. Secondary outcomes were nausea assessed by a visual analog scale (VAS) at baseline, 0-30, 31-60, and 61-120 minutes after the administration of ondansetron or prochlorperazine and the percentage of patients with adverse effects (sedation, headache, akathisia, dystonia) to either drug. We performed statistical analyses on the VAS scales at each time point and did a subgroup analysis to examine if nausea scores were affected if the patient had vomited at baseline. The primary identified cause for nausea and vomiting was flu-like illness or gastroenteritis (19%). The number of patients experiencing breakthrough vomiting at 0-30, 31-60, and 61-120 minutes was similar between groups for these time periods; however, more patients receiving ondansetron experienced vomiting overall (7 [22%] vs. 2[3.2%] patients, p=not significant). Nausea scores at baseline and 0-30 minutes were severe and similar between groups; however, at 31-60 and 61-120 minutes, patients receiving prochlorperazine had better control of nausea (24.9 vs. 43.7 mm, p=0.03; 16.8 vs. 34.3 mm, p=0.05). Sedation scores were similar between groups. There were no cases of extrapyramidal symptoms as assessed by the treating physician and there were four cases of akathisia (prochlorperazine=3 [9%], ondansetron=1[3%]). Prochlorperazine and ondansetron appear to be equally effective at treating vomiting in the emergency department.


Turkey ovomucoid third domain inhibits eight different serine proteinases of varied specificity on the same ...Leu18-Glu19 ... reactive site.
We show that eight different serine proteinases--bovine chymotrypsins A and B, porcine pancreatic elastase I, proteinase K, Streptomyces griseus proteinases A and B, and subtilisins BPN' and Carlsberg--interact with turkey ovomucoid third domain at the same Leu18-Glu19 peptide bond, the reactive site of the inhibitor. Turkey ovomucoid third domain was converted to modified (the reactive site peptide bond hydrolyzed) form as documented by sequencing. Complexes of all eight enzymes both with virgin and with modified inhibitor were prepared. All 16 complexes were subjected to kinetically controlled dissociation, and all 16 produced predominantly virgin (greater than 90%) inhibitor, thus proving our point. During this investigation, we found that both alpha-chymotrypsin and especially S. griseus proteinase B convert virgin to modified turkey ovomucoid third domain, even in the pH range 1-2, a much lower pH than we expected. We have also measured rate constants kon and kon* for the association of virgin and modified turkey ovomucoid third domain with several serine proteinases. The kon/kon* ratio is 4.8 X 10(6) for chymotrypsin, but it is only 1.5 for subtilisin Carlsberg. A number of generalizations concerning reactive sites of protein proteinase inhibitor are proposed and discussed.


Accuracy of point-of-care ultrasound for diagnosis of skull fractures in children.
To determine the test performance characteristics for point-of-care ultrasound performed by clinicians compared with computed tomography (CT) diagnosis of skull fractures. We conducted a prospective study in a convenience sample of patients ≤21 years of age who presented to the emergency department with head injuries or suspected skull fractures that required CT scan evaluation. After a 1-hour, focused ultrasound training session, clinicians performed ultrasound examinations to evaluate patients for skull fractures. CT scan interpretations by attending radiologists were the reference standard for this study. Point-of-care ultrasound scans were reviewed by an experienced sonologist to evaluate interobserver agreement. Point-of-care ultrasound was performed by 17 clinicians in 69 subjects with suspected skull fractures. The patients' mean age was 6.4 years (SD: 6.2 years), and 65% of patients were male. The prevalence of fracture was 12% (n = 8). Point-of-care ultrasound for skull fracture had a sensitivity of 88% (95% confidence interval [CI]: 53%-98%), a specificity of 97% (95% CI: 89%-99%), a positive likelihood ratio of 27 (95% CI: 7-107), and a negative likelihood ratio of 0.13 (95% CI: 0.02-0.81). The only false-negative ultrasound scan was due to a skull fracture not directly under a scalp hematoma, but rather adjacent to it. The κ for interobserver agreement was 0.86 (95% CI: 0.67-1.0). Clinicians with focused ultrasound training were able to diagnose skull fractures in children with high specificity.


Aging successfully: the importance of physical activity in maintaining health and function.
Physicians caring for middle-aged and older patients frequently overlook the importance of regular physical activity. Exercise on a routine basis is an important component of successful aging. It has been shown that many age-related declines in musculoskeletal function can be markedly reduced by participation in some form of regular exercise. Examination of records from masters athletic competitions has been used to determine the true rate of age-related functional declines in highly trained, healthy individuals and further supports these findings. Declines in physical abilities for masters athletes are gradual, which suggests that for many, the potential for participation in competitive athletics can persist well into the seventh decade of life. Recent studies indicate that health gains can be achieved with relatively low volumes of exercise. Indeed, the greatest benefit is seen when one "goes from doing nothing to doing something." Current data suggest that a cumulative total of 30 to 50 minutes of aerobic exercise a day performed 3 to 5 days a week and one set of resistance exercises targeting the major muscle groups twice a week can produce significant health benefits. The aerobic conditioning requirement need not be a formal or structured activity, but can be satisfied through regular participation in many common physical tasks (e.g., walking, gardening, housekeeping). Musculoskeletal injuries are a major cause of noncompliance with any exercise regimen and are especially debilitating for older individuals. Prompt recognition of injury and treatment that emphasizes alternative conditioning exercises and minimizes "downtime" are especially important. Orthopaedists should be aware of the pattern of musculoskeletal injuries seen in this population, so as to assist their patients in avoiding these problems.


Low Peripheral T Follicular Helper Cells in Perinatally HIV-Infected Children Correlate With Advancing HIV Disease.
T follicular helper (Tfh) cells are crucial for B cell differentiation and antigen-specific antibody production. Dysregulation of Tfh-mediated B cell help weakens B cell responses in HIV infection. Moreover, Tfh cells in the lymph node and peripheral blood comprise a significant portion of the latent HIV reservoir. There is limited data on the effects of perinatal HIV infection on Tfh cells in children. We examined peripheral Tfh (pTfh) cell frequencies and phenotype in HIV-infected children and their associations with disease progression, immune activation, and B cell differentiation. In a Kenyan cohort of 76 perinatally HIV-infected children, comprised of 43 treatment-naïve (ART-) and 33 on antiretroviral therapy (ART+), and 42 healthy controls (HIV-), we identified memory pTfh cells, T cell activation markers, and B cell differentiation states using multi-parameter flow cytometry. Soluble CD163 and intestinal fatty acid-binding protein plasma levels were quantified by ELISA. ART- children had reduced levels of pTfh cells compared with HIV- children that increased with antiretroviral therapy. HIV+ children had higher programmed cell death protein 1 (PD-1) expression on pTfh cells, regardless of treatment status. Low memory pTfh cells with elevated PD-1 levels correlated with advancing HIV disease status, indicated by increasing HIV viral loads and T cell and monocyte activation, and decreasing %CD4 and CD4:CD8 ratios. Antiretroviral treatment, particularly when started at younger ages, restored pTfh cell frequency and eliminated correlations with disease progression, but failed to lower PD-1 levels on pTfh cells and their associations with CD4 T cell percentages and activation. Altered B cell subsets, with decreased naïve and resting memory B cells and increased activated and tissue-like memory B cells in HIV+ children, correlated with low memory pTfh cell frequencies. Last, HIV+ children had decreased proportions of CXCR5+ CD8 T cells that associated with low %CD4 and CD4:CD8 ratios. Low memory pTfh cell frequencies with high PD-1 expression in HIV+ children correlate with worsening disease status and an activated and differentiated B cell profile. This perturbed memory pTfh cell population may contribute to weak vaccine and HIV-specific antibody responses in HIV+ children. Restoring Tfh cell capacity may be important for novel pediatric HIV cure and vaccine strategies.


An expedient synthesis of flexible nucleosides via a regiocontrolled enzymatic glycosylation of functionalized imidazoles.
A versatile two-step synthesis of C4- and C5-arylated 2'-deoxyribosylimidazoles was elaborated using enzymatic N-transglycosylation followed by microwave-assisted Pd-catalysed arylation reactions. We report herein the reaction conditions that permit managing regioselectivity (N3 versus N1-isomers) in the enzymatic glycosylation of 4-iodoimidazole using the nucleoside N-deoxyribosyltransferase from L. leichmannii. Regiocontrolled glycosylation was also observed among several other imidazole derivatives studied, providing simple access to isomers not readily accessible by chemical routes. Finally, a series of flexible nucleosides was obtained in one step from 4- or 5-iodo-imidazole nucleosides by the Suzuki-Miyaura cross-coupling reaction with (hetero)aryl-boronic acids in aqueous media. Moreover, this chemoenzymatic approach is compatible with a one-pot two-step process affording a straightforward access to a broad array of potential anticancer and antiviral drugs as well as new DNA building blocks.


Healthcare-professional patients' conceptions of being ill and hospitalised - a phenomenographic study.
To describe the variation of conceptions of being ill and hospitalised, from the perspective of healthcare-professional patients. Previous literature focuses on either physicians' or nurses' experiences of being a patient, without aiming at determining a variation of ways of understanding that phenomena. Nor have we been able to identify any study reporting other healthcare professionals' experiences. This study has an inductive descriptive design. Qualitative interviews with healthcare professionals (n = 16), who had been hospitalised for at least two days. Phenomenographic data analysis was conducted. The feelings of security were based on knowledge, insight and trust, and acceptance of the healthcare system. Being exposed and totally dependent due to illness provoked feelings of vulnerability and insecurity. The patients used their knowledge to achieve participation in the care. The more severe they perceived their illness to be, the less they wanted to participate and the more they expressed a need for being allowed to surrender control. The patients' ideal picture of care was sometimes disrupted, and based on their experience, they criticised care and made suggestions that could contribute to general care improvements. Healthcare-professional patients have various conceptions of being ill and hospitalised. Based on the general nature of the many needs expressed, we believe that the insights provided in this study can be transferred so as to also be valid for lay patients. Possibly, an overhaul of routines for discharge planning and follow-up, and adopting a person-centred approach to care, can resolve some of the identified shortcomings. The results can be used for the purpose of developing knowledge for healthcare professions and for educational purposes.


Integration column: artificial ECM: expanding the cell biology toolbox in 3D.
Many crucial cellular processes in our tissues are governed by complex, spatio-temporally regulated interactions between cells and their extracellular matrix (ECM). These interactions can be studied using well-established 3D in vitro model systems such as collagen gels or Matrigel generated from native ECM macromolecular components. Recent advances in the molecular design of 'smart' synthetic biomaterials have generated artificial ECM (aECM) that mimic some of the key structural and biochemical characteristics of their naturally derived counterparts and, thanks to their synthetic origin, promise to overcome some complexities of the latter. Here I will discuss emerging approaches in aECM design, hopefully inspiring cell biologists to apply these systems to address their specific biological question.


Metabolomic/lipidomic profiling of COVID-19 and individual response to tocilizumab.
The current pandemic emergence of novel coronavirus disease (COVID-19) poses a relevant threat to global health. SARS-CoV-2 infection is characterized by a wide range of clinical manifestations, ranging from absence of symptoms to severe forms that need intensive care treatment. Here, plasma-EDTA samples of 30 patients compared with age- and sex-matched controls were analyzed via untargeted nuclear magnetic resonance (NMR)-based metabolomics and lipidomics. With the same approach, the effect of tocilizumab administration was evaluated in a subset of patients. Despite the heterogeneity of the clinical symptoms, COVID-19 patients are characterized by common plasma metabolomic and lipidomic signatures (91.7% and 87.5% accuracy, respectively, when compared to controls). Tocilizumab treatment resulted in at least partial reversion of the metabolic alterations due to SARS-CoV-2 infection. In conclusion, NMR-based metabolomic and lipidomic profiling provides novel insights into the pathophysiological mechanism of human response to SARS-CoV-2 infection and to monitor treatment outcomes.


Modelling of killer T-cell and cancer cell subpopulation dynamics under immuno- and chemotherapies.
Each patient's cancer has a unique molecular makeup, often comprised of distinct cancer cell subpopulations. Improved understanding of dynamic processes between cancer cell populations is therefore critical for making treatment more effective and personalized. It has been shown that immunotherapy increases the survival of melanoma patients. However, there remain critical open questions, such as timing and duration of immunotherapy and its added benefits when combined with other types of treatments. We introduce a model for the dynamics of active killer T-cells and cancer cell subpopulations. Rather than defining the cancer cell populations based on their genetic makeup alone, we consider also other, non-genetic differences that make the cell populations either sensitive or resistant to a therapy. Using the model, we make predictions of possible outcomes of the various treatment strategies in virtual melanoma patients, providing hypotheses regarding therapeutic efficacy and side-effects. It is shown, for instance, that starting immunotherapy with a denser treatment schedule may enable changing to a sparser schedule later during the treatment. Furthermore, combination of targeted and immunotherapy results in a better treatment effect, compared to mono-immunotherapy, and a stable disease can be reached with a patient-tailored combination. These results offer better understanding of the competition between T-cells and cancer cells, toward personalized immunotherapy regimens.


I Am Men's Health: Generating Adherence to HIV Pre-Exposure Prophylaxis (PrEP) in Young Men of Color Who Have Sex with Men.
In 2012, the US Food and Drug Administration approved Truvada as a pre-exposure prophylaxis (PrEP) for adults at risk of HIV. PrEP is highly effective at preventing HIV when taken daily, but no gold standard exists for consistently administering PrEP to populations at highest risk. The "I Am Men's Health" program used an innovative methodology to generate adherence to PrEP in 23 mostly young men who have sex with men of color (yMSMc), during a 28-week period from February to September 2013. Adherence was measured using weekly medication pickup rates. The average age of the participants was 21 years, and the majority were black and lived below the poverty line. Time on PrEP ranged from 1 to 28 weeks (2723 person-days), and the weighted average adherence was 73%. The methodology used in this study was preliminarily effective at generating adherence to PrEP among high-risk yMSMc in a community setting and may help inform large-scale future HIV prevention interventions.


Biological evaluation of bismuth non-steroidal anti-inflammatory drugs (BiNSAIDs): stability, toxicity and uptake in HCT-8 colon cancer cells.
Recent studies showed that the metal-coordinated non-steroidal anti-inflammatory drug (NSAID), copper indomethacin, reduced aberrant crypt formation in the rodent colon cancer model, while also exhibiting gastrointestinal sparing properties. In the present study, the stability and biological activity of three BiNSAIDs of the general formula [Bi(L)3]n, where L=diflunisal (difl), mefenamate (mef) or tolfenamate (tolf) were examined. NMR spectroscopy of high concentrations of BiNSAIDs (24h in cell medium, 37°C) indicated that their structural stability and interactions with cell medium components were NSAID specific. Assessment of cell viability using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium]bromide (MTT) assay showed that the toxicity ranking of the BiNSAIDs paralleled those of the respective free NSAIDs: diflH<mefH<tolfH. While the IC50 values of the BiNSAIDs (ranging between 16 and 81μM) were lower than the free NSAIDs, it was apparent that the toxicity of the BiNSAIDs was due to the molar ratio of the three NSAID molecules contained in the BiNSAIDs, with the exception of [Bi(difl)3]. The highest cellular bismuth content was observed following treatment with [Bi(tolf)3]. Since NMR studies indicated that [Bi(tolf)3] was the most stable BiNSAID and that cellular uptake of bismuth correlated with structural stability it appears that bismuth uptake is assisted by the NSAID. Microprobe SR-XRF imaging showed that the intracellular fate of bismuth was independent of the specific BiNSAID treatment whereby all BiNSAID-treated cells showed bismuth accumulation in the cytoplasm within 24-h exposure. The size and location of the hot spots (0.3-5.8μm(2)), were consistent with cellular organelles such as lysosomes.


Defining the effect and mediators of two knowledge translation strategies designed to alter knowledge, intent and clinical utilization of rehabilitation outcome measures: a study protocol [NCT00298727].
A substantial number of valid outcome measures have been developed to measure health in adult musculoskeletal and childhood disability. Regrettably, national initiatives have merely resulted in changes in attitude, while utilization remains unacceptably low. This study will compare the effectiveness and mediators of two different knowledge transfer (KT) interventions in terms of their impact on changing knowledge and behavior (utilization and clinical reasoning) related to health outcome measures. Physical and occupational therapists (n = 144) will be recruited in partnership with the national professional associations to evaluate two different KT interventions with the same curriculum: 1) Stakeholder-Hosted Interactive Problem-Based Seminar (SHIPS), and 2) Online Problem-Based course (e-PBL). SHIPS will consist of face-to-face problem-based learning (PBL) for 2 1/2 days with outcome measure developers as facilitators, using six problems generated in consultation with participants. The e-PBL will consist of a 6-week web-based course with six generic problems developed by content experts. SHIPS will be conducted in three urban centers in Canada. Participants will be block-allocated by a minimization procedure to either of the two interventions to minimize any prognostic differences. Trained evaluators at each site will conduct chart audits and chart-stimulated recall. Trained interviewers will conduct semi-structured interviews focused on identifying critical elements in KT and implementing practice changes. Interviews will be transcribed verbatim. Baseline predictors including demographics, knowledge, attitudes/barriers regarding outcome measures, and Readiness to Change will be assessed by self-report. Immediately post-intervention and 6 months later, these will be re-administered. Primary qualitative and quantitative evaluations will be conducted 6-months post-intervention to assess the relative effectiveness of KT interventions and to identify elements that contribute to changing clinical behavior. Chart audits will determine the utilization of outcome measures (counts). Incorporation of outcome measures into clinical reasoning will be assessed using an innovative technique: chart-stimulated recall. A strategy for optimal transfer of health outcome measures into practice will be developed and shared with multiple disciplines involved in primary and specialty management of musculoskeletal and childhood disability.


Patients' attitudes towards "do not attempt resuscitation" status.
The decision of "do not attempt resuscitation" (DNAR) in the event of cardiopulmonary arrest is usually made when the patients are critically ill and cannot make an informed choice. Although, various professional bodies have published guidelines, little is know about the patients' own views regarding DNAR discussion. The aim of this study was to determine patients' attitudes regarding discussing DNAR before they are critically ill. A prospective study was performed in a general out patients department. A questionnaire was distributed to consecutive outpatients along with an explanatory leaflet in the adult outpatient clinic. 364 patients completed the questionnaire (response rate 77%). 90% of respondents wanted all patients to be asked regarding DNAR decision at some point during a hospital admission. The majority would not find a DNAR discussion distressing. Only 10% would find it upsetting, however, 48% of these still wanted a discussion. 37% of respondents wanted to discuss DNAR decisions on admission; 32% in outpatients; 17% at consent for surgery, 14% when they are critically ill. 87% of respondents would not object to their relatives being involved in making decisions about their resuscitation status. However, only 12% of the subjects in the study had been involved in discussing the resuscitation status of a relative and 21% would not be comfortable to discuss a relative's resuscitation status. Although 33% of patients preferred their resuscitation status to simply be documented within their clinical notes, 77% wanted it to be more easily accessible. This study suggests that contrary to current practice most patients want to discuss their DNAR status prior to becoming critically ill. This includes half of the small number that find it distressing to discuss. Although most patients are comfortable with relatives being involved in discussing DNAR, a significant proportion do not want their relatives to be asked. Furthermore, once a decision has been made, the majority of patients want it to be more accessible than current practice allows.


Link prediction in a MeSH co-occurrence network: preliminary results.
Literature-based discovery (LBD) refers to automatic discovery of implicit relations from the scientific literature. Co-occurrence associations between biomedical concepts are commonly used in LBD. These co-occurrences can be represented as a network that consists of a set of nodes representing concepts and a set of edges representing their relationships (or links). In this paper we propose and evaluate a methodology for link prediction of implicit connections in a network of co-occurring Medical Subject Headings (MeSH®). The proposed approach is complementary to, and may augment, existing LBD methods. Link prediction was performed using Jaccard and Adamic-Adar similarity measures. The preliminary results showed high prediction performance, with area under the ROC curve of 0.78 and 0.82 for the two similarity measures, respectively.


A pilot study of increasing dose intensity of epirubicin and ifosfamide in patients with small cell lung cancer by using recombinant granulocyte colony-stimulating factor.
The aim of this prospective study was to investigate the feasibility of increasing the dose intensity of chemotherapy in patients with small cell lung cancer (SCLC) by using recombinant human granulocyte colony-stimulating factor (r-metHuG-CSF). Seventeen previously untreated patients (11 male, 6 female) were treated with ifosfamide (5.0 g/m2) and epirubicin (80 mg/m2) in two successive cohorts. Eight patients received chemotherapy every 2 weeks and r-metHuG-CSF 5 microg/kg given subcutaneously daily for 10 days (cohort A), and nine patients received chemotherapy at 10-day intervals with r-metHuG-CSF 5 microg/kg subcutaneously given daily for 7 days (cohort B). The relative dose intensity compared with the conventional 3-weekly regimen was 1.5 and 2.1 for cohorts A and B, respectively. Neutropenia-associated fever complicated two and five treatment courses in cohorts A and B, respectively. There were five episodes of grade 3/4 thrombocytopenia. There were no treatment delays in cohort A and one cycle was delayed in cohort B. One patient from each cohort was withdrawn due to toxicity. Grade 3/4 non-haematological toxicity, other than alopecia, was not observed. This study confirms that it is feasible to increase the relative dose intensity of ifosfamide and epirubicin in patients with SCLC to 2.1 by using r-metHuG-CSF and shortening the interval between treatment cycles.


Chromatin as an expansive canvas for chemical biology.
Chromatin is extensively chemically modified and thereby acts as a dynamic signaling platform controlling gene function. Chromatin regulation is integral to cell differentiation, lineage commitment and organism development, whereas chromatin dysregulation can lead to age-related and neurodegenerative disorders as well as cancer. Investigating chromatin biology presents a unique challenge, as the issue spans many disciplines, including cell and systems biology, biochemistry and molecular biophysics. In recent years, the application of chemical biology methods for investigating chromatin processes has gained considerable traction. Indeed, chemical biologists now have at their disposal powerful chemical tools that allow chromatin biology to be scrutinized at the level of the cell all the way down to the single chromatin fiber. Here we present recent examples of how this rapidly expanding palette of chemical tools is being used to paint a detailed picture of chromatin function in organism development and disease.


Familial Recurrent Myocarditis Triggered by Exercise in Patients With a Truncating Variant of the Desmoplakin Gene.
Background Variants of the desmosomal protein desmoplakin are associated with arrhythmogenic cardiomyopathy, an important cause of ventricular arrhythmias in children and young adults. Disease penetrance of desmoplakin variants is incomplete and variant carriers may display noncardiac, dermatologic phenotypes. We describe a novel cardiac phenotype associated with a truncating desmoplakin variant, likely causing mechanical instability of myocardial desmosomes. Methods and Results In 2 young brothers with recurrent myocarditis triggered by physical exercise, screening of 218 cardiomyopathy-related genes identified the heterozygous truncating variant p.Arg1458Ter in desmoplakin. Screening for infections yielded no evidence of viral or nonviral infections. Myosin and troponin I autoantibodies were detected at high titers. Immunohistology failed to detect any residual DSP protein in endomyocardial biopsies, and none of the histologic criteria of arrhythmogenic cardiomyopathy were fulfilled. Cardiac magnetic resonance imaging revealed no features associated with right ventricular arrhythmogenic cardiomyopathy, but multifocal subepicardial late gadolinium enhancement was present in the left ventricles of both brothers. Screening of adult cardiomyopathy cohorts for truncating variants identified the rare genetic variants p.Gln307Ter, p.Tyr1391Ter, and p.Tyr1512Ter, suggesting that over subsequent decades critical genetic/exogenous modifiers drive pathogenesis from desmoplakin truncations toward different end points. Conclusions The described novel phenotype of familial recurrent myocarditis associated with a desmoplakin truncation in adolescents likely represents a serendipitously revealed subtype of arrhythmogenic cardiomyopathy. It may be caused by a distinctive adverse effect of the variant desmoplakin upon the mechanical stability of myocardial desmosomes. Variant screening is advisable to allow early detection of patients with similar phenotypes.


Bubbles in the Heart: A Case of Venous Air Thromboembolism.
Venous air embolism (VAE) due to central venous catheter (CVC) placement is a rare but preventable complication which is potentially fatal. We describe a case highlighting unique patient characteristics which increase the risk of developing VAE. A sixty-year-old gentleman was admitted to the hospital with dyspnea and altered mental status. His comorbidities include cancer of the neck and tongue, currently in remission, and schizophrenia. On presentation, he was found to be in acute respiratory failure, due to pneumonia, and required mechanical ventilation. Following extubation, his CVC was removed from the right internal jugular vein. While ambulating around the unit, he experienced a coughing fit and dizziness. He rapidly developed cardiopulmonary collapse requiring re-intubation and vasopressor support. Chest x-ray demonstrated a radiolucent column along the lateral aspect of the right neck. Due to concern for VAE, an echocardiogram was performed, revealing multiple air-bubbles in the right and left chambers of the heart. Our patient was predisposed to developing VAE due to the extensive radiation induced skin changes, from his cancer treatment, on the neck and upper thorax. This resulted in loss of underlying subcutaneous tissue and decreased skin pliability. He had a large, open puncture wound at the catheter site on his neck, probably resulting in air entry. Anxiety and agitation, due to schizophrenia, made it difficult to maintain our patient in a supine or Trendelenburg position following CVC removal. This case highlights the importance of recognizing patient factors that may increase the risk of VAE.


Utility of C-arm CT in patients with hepatocellular carcinoma undergoing transhepatic arterial chemoembolization.
To evaluate the utility of C-arm computed tomography (CT) on treatment algorithms in patients undergoing transhepatic arterial chemoembolization for hepatocellular carcinoma (HCC). From March 2008 to July 2008, 84 consecutive patients with HCC underwent 100 consecutive transhepatic arterial chemoembolizations with iodized oil. Unenhanced and iodinated contrast medium-enhanced C-arm CT with planar and three-dimensional imaging were performed in addition to conventional digital subtraction angiography (DSA) in all patients. The effect on diagnosis and treatment was determined by testing the hypotheses that C-arm CT, in comparison to DSA, provides (a) improved lesion detection, (b) expedient identification and mapping of arterial supply to a tumor, (c) improved characterization of a lesion to allow confident differentiation of HCC from pseudolesions such as arterioportal shunts, and (d) an improved evaluation of treatment completeness. The effect of C-arm CT was analyzed on the basis of information provided with C-arm CT that was not provided or readily apparent at DSA. C-arm CT was technically successful in 93 of the 100 procedures (93%). C-arm CT provided information not apparent or discernible at DSA in 30 of the 84 patients (36%) and resulted in a change in diagnosis, treatment planning, or treatment delivery in 24 (28%). The additional information included, amongst others, visualization of additional or angiographically occult tumors in 13 of the 84 patients (15%) and identification of incomplete treatment in six (7.1%). C-arm CT is a useful collaborative tool in patients undergoing transhepatic arterial chemoembolization and can affect patient care in more than one-fourth of patients.


GLUT12 deficiency during early development results in heart failure and a diabetic phenotype in zebrafish.
Cardiomyopathies-associated metabolic pathologies (e.g., type 2 diabetes and insulin resistance) are a leading cause of mortality. It is known that the association between these pathologies works in both directions, for which heart failure can lead to metabolic derangements such as insulin resistance. This intricate crosstalk exemplifies the importance of a fine coordination between one of the most energy-demanding organs and an equilibrated carbohydrate metabolism. In this light, to assist in the understanding of the role of insulin-regulated glucose transporters (GLUTs) and the development of cardiomyopathies, we have developed a model for glut12 deficiency in zebrafish. GLUT12 is a novel insulin-regulated GLUT expressed in the main insulin-sensitive tissues, such as cardiac muscle, skeletal muscle, and adipose tissue. In this study, we show that glut12 knockdown impacts the development of the embryonic heart resulting in abnormal valve formation. Moreover, glut12-deficient embryos also exhibited poor glycemic control. Glucose measurements showed that these larvae were hyperglycemic and resistant to insulin administration. Transcriptome analysis demonstrated that a number of genes known to be important in cardiac development and function as well as metabolic mediators were dysregulated in these larvae. These results indicate that glut12 is an essential GLUT in the heart where the reduction in glucose uptake due to glut12 deficiency leads to heart failure presumably due to the lack of glucose as energy substrate. In addition, the diabetic phenotype displayed by these larvae after glut12 abrogation highlights the importance of this GLUT during early developmental stages.


Agonistic behavior and environmental enrichment of cats communally housed in a shelter.
To evaluate the presence of a dominance rank in a group of cats and the relation between agonistic behavior and the use of resources, including environmental enrichment, in these cats. Observational analytic study. 27 neutered cats in a shelter in Sao Paulo, Brazil. The cats were video recorded for 4 consecutive days to obtain baseline data. Subsequently, a puzzle feeder was added as an enrichment device every other day over 8 days, for a total of 4 days with enrichment. Cats were also video recorded on these days. All pretreatment and posttreatment agonistic behaviors and interactions with the puzzle feeder were recorded by reviewing the videotapes. 143 agonistic encounters were recorded, of which 44 were related to resources and 99 were not. There were insufficient agonistic interactions to determine a dominance rank. Presence or absence of the puzzle feeder did not affect the rate of aggression. There was no significant effect of weight, sex, or coat color on the rate of aggression, and aggressive behavior did not correlate with time spent with the puzzle feeder. Twenty-three of the 27 cats interacted with the puzzle feeder. In a stable group of communally housed cats, environmental enrichment did not cause increased aggression as a result of competition for the source of enrichment. Because environmental enrichment increases the opportunity to perform exploratory behaviors, it may improve the welfare of groups of cats maintained long-term in shelters, sanctuaries, or multicat households.


Coprescription of levodopa with antipsychotics in a population of 84,596 psychiatric inpatients from 1994 to 2008.
Patients on levodopa therapy frequently require additional antipsychotic pharmacotherapy. However, consideration must be given to antagonistic interactions on dopamine receptors between levodopa and antipsychotics, and efficacy and safety of such combinations. We therefore aimed to explore the practice and rationale of coprescription between levodopa and antipsychotics in psychiatric patients. A descriptive retrospective study based on cross-sectional prescription data repeatedly collected from psychiatric inpatients through the international Drug Safety in Psychiatry (AMSP) program between 1994 and 2008 was undertaken. Within a population of 84 596 psychiatric patients the prevalence of levodopa therapy was 1.0% (n=886). Among those patients on levodopa therapy 59.6% (n=528) also received antipsychotics. Quetiapine coprescription increased after its first marketing in 2000 to 45.9% in 2008. Coprescription of clozapine and olanzapine decreased from up to 25 and 22%, respectively, before to less than 10% after the introduction of quetiapine. Coprescribing of other antipsychotics remained approximately stable with average prevalences between 6 and less than 1%. Quetiapine has now replaced clozapine as the most frequently coprescribed neuroleptic in psychiatric patients with levodopa therapy. This is in accordance with recent data indicating a low potential for clinically relevant interactions with levodopa and efficacy against psychosis in levodopa-treated patients. The combined use of antipsychotics other than quetiapine and clozapine with levodopa is less common and generally not supported by appropriate evidence.


Maternal alcohol drinking pattern during pregnancy and the risk for an offspring with an isolated congenital heart defect and in particular a ventricular septal defect or an atrial septal defect.
This cohort study examines the possible association between maternal alcohol intake, including binge drinking, during pregnancy, and the subsequent risk of having a child with an isolated congenital heart defect and, more specifically, with the isolated form of ventricular septal defect (VSD) or of an atrial septal defect (ASD). Participants were 80,346 pregnant women who were enrolled into the Danish National Birth Cohort in 1996-2002 and gave birth to a live-born singleton without any chromosome anomalies. Twice during pregnancy these women were asked about their intake of alcohol. Few (if any) women with an excessive/abusive intake of alcohol were enrolled into the Danish National Birth Cohort. Through linkage with the National Hospital Discharge Registry, we identified 477 infants with a diagnosis of isolated congenital heart defect registered at any time during their first 3½-years of life; they included 198 infants with a VSD and 145 with an ASD. Neither the number of episodes of binge drinking nor binge drinking during three different developmental periods was associated with VSD or ASD. Women drinking ½-1½, 2, and 3+ drinks of alcohol per week had adjusted prevalence ratios of delivering an infant with a VSD of 1.22 (95% CI = 0.90-1.66); 1.38 (95% CI = 0.83-2.28); and 1.10 (95% CI = 0.54-2.23), respectively. The test for trend was 0.29. Prenatal exposure to low-to-moderate levels of alcohol on a weekly basis or occasional binge drinking during the early part of pregnancy was not statistical significantly associated with the prevalence of isolated VSD and ASD in offspring.


SORBS1 gene, a new candidate for diabetic nephropathy: results from a multi-stage genome-wide association study in patients with type 1 diabetes.
The genetic determinants of diabetic nephropathy remain poorly understood. We aimed to identify novel susceptibility genes for diabetic nephropathy. We performed a genome-wide association study using 1000 Genomes-based imputation to compare type 1 diabetic nephropathy cases with proteinuria and with or without renal failure with control patients who have had diabetes for more than 15 years and no evidence of renal disease. None of the single nucleotide polymorphisms (SNPs) tested in a discovery cohort composed of 683 cases and 779 controls reached genome-wide statistical significance. The 46 top hits (p < 10(-5)) were then sought for first-stage analysis in the Genetics of Kidneys in Diabetes US (US-GoKinD) study, an independent population of 820 cases and 885 controls. Two SNPs in strong linkage disequilibrium with each other and located in the SORBS1 gene were consistently and significantly (p < 10(-4)) associated with diabetic nephropathy. The minor rs1326934-C allele was less frequent in cases than in controls (0.34 vs 0.43) and was associated with a decreased risk for diabetic nephropathy (OR 0.70; 95% CI 0.60, 0.82). However, this association was not observed in a second stage with two additional diabetic nephropathy cohorts, the All Ireland-Warren 3-Genetics of Kidneys in Diabetes UK and Republic of Ireland (UK-ROI; p = 0.15) and the Finnish Diabetic Nephropathy (FinnDiane; p = 0.44) studies, totalling 2,142 cases and 2,494 controls. Altogether, the random-effect meta-analysed rs1326934-C allele OR for diabetic nephropathy was 0.83 (95% CI 0.72, 0.96; p = 0.009). These data suggest that SORBS1 might be a gene involved in diabetic nephropathy.


Accurate high throughput alignment via line sweep-based seed processing.
Accurate and fast aligners are required to handle the steadily increasing volume of sequencing data. Here we present an approach allowing performant alignments of short reads (Illumina) as well as long reads (Pacific Bioscience, Ultralong Oxford Nanopore), while achieving high accuracy, based on a universal three-stage scheme. It is also suitable for the discovery of insertions and deletions that originate from structural variants. We comprehensively compare our approach to other state-of-the-art aligners in order to confirm its performance with respect to accuracy and runtime. As part of our algorithmic scheme, we introduce two line sweep-based techniques called "strip of consideration" and "seed harmonization". These techniques represent a replacement for chaining and do not rely on any specially tailored data structures. Additionally, we propose a refined form of seeding on the foundation of the FMD-index.


A double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge.
Severe acute respiratory syndrome coronavirus (SARS-CoV) is an important emerging virus that is highly pathogenic in aged populations and is maintained with great diversity in zoonotic reservoirs. While a variety of vaccine platforms have shown efficacy in young-animal models and against homologous viral strains, vaccine efficacy has not been thoroughly evaluated using highly pathogenic variants that replicate the acute end stage lung disease phenotypes seen during the human epidemic. Using an adjuvanted and an unadjuvanted double-inactivated SARS-CoV (DIV) vaccine, we demonstrate an eosinophilic immunopathology in aged mice comparable to that seen in mice immunized with the SARS nucleocapsid protein, and poor protection against a nonlethal heterologous challenge. In young and 1-year-old animals, we demonstrate that adjuvanted DIV vaccine provides protection against lethal disease in young animals following homologous and heterologous challenge, although enhanced immune pathology and eosinophilia are evident following heterologous challenge. In the absence of alum, DIV vaccine performed poorly in young animals challenged with lethal homologous or heterologous strains. In contrast, DIV vaccines (both adjuvanted and unadjuvanted) performed poorly in aged-animal models. Importantly, aged animals displayed increased eosinophilic immune pathology in the lungs and were not protected against significant virus replication. These data raise significant concerns regarding DIV vaccine safety and highlight the need for additional studies of the molecular mechanisms governing DIV-induced eosinophilia and vaccine failure, especially in the more vulnerable aged-animal models of human disease.


The effects of sandostatin (Octreotide, SMS 201-995) infusion on splanchnic and hepatic blood flow in an experimental model of hepatic metastases.
Manipulation of hepatic blood flow may improve drug delivery to hepatic tumour. Somatostatin and its long acting analogues are known to elicit effects upon hepatic and splanchnic blood flow in experimental animals and patients with portal hypertension. This study investigates the effects of SMS 201-995 (sandostatin) infusion on hepatic, splanchnic and tumour blood flow in an experimental model of liver metastases. Hepatic tumour was induced by the intraportal inoculation of 10(6) HSN sarcoma cells and blood flow measured using the dual reference microsphere method before and after infusion of SMS 201-995. There was a significant decrease in hepatic arterial flow and a significant increase in the tumour:liver blood flow ratio associated with a marked reduction in blood flow to normal hepatic parenchyma. Portal venous inflow and tumour blood flow were not significantly affected. SMS 201-995 infusion may lead to preferential delivery of concomitantly injected cytotoxic drugs to hepatic tumour. In addition, the reduction in growth of hepatic tumour may be due to a reduction in nutritive, arterial blood flow to hepatic tumour.


Relative Age, Maturation and Physical Biases on Position Allocation in Elite-Youth Soccer.
This study assessed the contribution of relative age, anthropometry, maturation, and physical fitness characteristics on soccer playing position (goalkeeper [GK], central-defender [CD], lateral-defender [LD], central-midfield [CM], lateral-midfielder [LM], and forward [FWD]) for 465 elite-youth players (U13-U18's). U13-14 CD were relatively older than LD and CM (likely small effects). CD and GK were generally taller and heavier (likely small to very-likely moderate effects) than other players at each developmental stage and were advanced maturers at U13-14 (very-likely small to likely moderate effects). GK had inferior agility (very-likely small to likely moderate effects), endurance (very-likely small to likely moderate effects), and sprint capacities (likely small-moderate effects) vs. outfield positions at U13-14, but deficits in anaerobic phenotypes were diminished in U15-16 and U17-18. Position specific fitness characteristics were distinguished at U15-16 (likely small) and U17-18 (likely moderate), where LM were faster than their central counterparts. In summary, relative age, maturation and anthropometric characteristics appear to bias the allocation of players into key defensive roles from an early development stage, whereas position-specific physical attributes do not become apparent until the latter stages of talent development in outfield players. Given the inter-individual trajectories of physical development according to biological maturation, playing position allocation might be considered 'plastic' by selectors, until complete-maturity is achieved.


Which unmet needs contribute to behavior problems in persons with advanced dementia?
The Unmet Needs Model states that problem behaviors of people with dementia result from unmet needs stemming from a decreased ability to communicate those needs and to provide for oneself. The purpose of this study is to describe the unmet needs of persons with dementia exhibiting behavior problems. Eighty-nine residents with dementia from six Maryland nursing homes were assessed by research assistants and nursing assistants for their unmet needs using multiple assessment tools. Three unmet needs per resident were identified on average, with informants rating boredom/sensory deprivation, loneliness/need for social interaction, and need for meaningful activity as the most prevalent needs. Discomfort was associated with higher levels of verbally agitated behaviors (e.g., complaining). Based on results and independent ratings of pain, the authors estimate notable under-detection of discomfort and pain by both types of informants. The study demonstrates methodologies for uncovering unmet needs among persons with dementia and highlights the importance of developing programs that address those unmet needs, especially social and activity needs of nursing home residents. The detection of pain, and possibly that of discomfort, may require a different methodology.


Randomized trial comparing saquinavir soft gelatin capsules versus indinavir as part of triple therapy (CHEESE study).
To compare efficacy and tolerability of saquinavir soft gelatin capsule (SQV-SGC) formulation and indinavir, both given as part of a triple drug regimen containing zidovudine and lamivudine, in HIV-1-infected individuals. Randomized, open label, multicentre study. A total of 70 patients who were antiretroviral-naive and who had a CD4 cell count < 500 x 10(6)/I and/or > 10000 HIV RNA copies/ml plasma and/or HIV-related symptoms. Subjects were assigned randomly to zidovudine 200 mg three times per day plus lamivudine 150 mg twice per day plus either SQV-SGC 1200 mg three times per day (SQV-SGC group) or indinavir 800 mg three times per day (indinavir group). Data are presented for all patients up to week 24. Mean baseline CD4 cell counts (+/- SE) were 301+/-29 x 10(6) cells/l and 310 +/-43 x 10(6) cells/l in the SQV-SGC and indinavir groups, respectively. The log10 median baseline HIV RNA load was 5.00 copies/ml in the SQV-SGC group and 4.98 copies/ml in the indinavir group. No difference in antiretroviral effect between the treatment arms could be demonstrated. Intention-to-treat analysis (last observation carried forward [LOCF]) at week 24 revealed that RNA levels decreased to < 50 copies/ml in 74.3% of patients in the SQV-SGC group and in 71.4% of the patients in the indinavir group (P = 0.78). In the on-treatment analysis the proportion of patients < 50 copies/ml at week 24 was 88.0% in the SQV-SGC group and 84.6% in the indinavir group (P = 0.725). Intriguingly, the mean increase of CD4 cells in the first 24 weeks was 162+/-20 x 10(6) cells/l in the SQV-SGC group and 89+/-21 x 10(6) cells/l in the indinavir group (P = 0.01), but preliminary data indicate that this difference in CD4 cell count gain may disappear after 24 weeks of treatment. Both regimens were generally well tolerated. During the first 24 weeks of the study, we found no difference in antiviral potency between the indinavir group and the SQV-SGC group. A significantly higher CD4 response in the SQV-SGC group was observed.


Assessing the quality of the dyadic relationship in first-time parents: development of a new instrument.
The purpose of this research was to psychometrically evaluate the Dyadic Adjustment Scale (DAS), modified for use with new, first-time parents by extending the items of communication, sensuality, and sexuality. A total of 820 Swedish respondents, 6 months after the birth of their first child, participated in the study. Psychometric evaluation was conducted with factor analysis. The obtained factor structure was tested with multitrait analysis program. Thirty-three items were found to fit into a five-factor solution, explaining 50% of the total variance. Descriptive data revealed that most new parents were satisfied with their intimate relationship in general, but dissatisfied with their sexual lives. The modified DAS, now called the Quality of Dyadic Relationship Instrument includes 33 items and seems to be a useful, updated measurement for assessing quality of the intimate relationship in new first-time parents.


Family-based activity settings of children in a low-income African context.
There has been an overwhelming call to improve the understanding of how children develop within an African context as Euro-American definitions of competence have been uncritically adopted as the norm for children in Africa. The activities that children engage in within the family setting are seen as important to understand how children develop within context. The use of activity settings is closely aligned with a strengths-based perspective of family-centred practice and contributes to improved sustainability of intervention. This study that was conducted in Soweto, South Africa, aims to describe activity settings that typically developing young children in low-income African contexts participate in. A descriptive design using structured interviews was utilised to obtain information about activity settings that children aged 3-5 years and 11 months engaged in. Structured interviews with 90 caregivers were conducted. Findings show that children participate in a variety of activities with varied participation levels. The types of activities are dependent on the context and perceptions of caregivers. These findings draw attention to understanding activities that children engage in within the family context.


Differential induction of the ZENK gene in the avian forebrain and song control circuit after metrazole-induced depolarization.
ZENK is an immediate early gene (IEG) that encodes a transcription factor protein, and its induction has been proposed as a necessary step in the cellular process underlying long-term memory formation. We have previously shown that ZENK is induced in adult songbird brain by the sound of birdsong, but, interestingly, induction did not occur in several areas known to respond to song stimuli. Conceivably, the ZENK gene may be repressed in these areas in adult birds. As a further test of this hypothesis, we administered metrazole, a strong GABAergic antagonist that leads to widespread excitation in the brain. Following metrazole, ZENK mRNA increases more than 10-fold throughout most of the telencephalon in both canaries and zebra finches, and primarily in neurons. In contrast, ZENK induction is much lower or absent in the archistriatum, the primary telencephalic sensory-recipient areas (including auditory field L), and the three telencephalic androgen receptor-containing song nuclei (high vocal center, lateral magnocellular nucleus of the anterior neostriatum, and the robust nucleus of the archistriatum). We did not observe any differences in ZENK induction patterns in juvenile versus adult zebra finches, or fall versus spring male canaries. Together with our previous studies of induction by song, these results suggest that in specific parts of the forebrain, including most of the song control system, IEG expression is subject to different constraints than in the rest of the forebrain. Understanding the molecular basis for this differential gene regulation may prove invaluable in understanding the organization of the song control circuit and the avian telencephalon.


Implantation of radiotelemetry transmitters yielding data on ECG, heart rate, core body temperature and activity in free-moving laboratory mice.
The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a manageable size and relatively easy to house. These factors, together with the availability of a wealth of spontaneous and experimentally induced mutants, make laboratory mice ideally suited to a wide variety of research areas. In cardiovascular, pharmacological and toxicological research, accurate measurement of parameters relating to the circulatory system of laboratory animals is often required. Determination of heart rate, heart rate variability, and duration of PQ and QT intervals are based on electrocardiogram (ECG) recordings. However, obtaining reliable ECG curves as well as physiological data such as core body temperature in mice can be difficult using conventional measurement techniques, which require connecting sensors and lead wires to a restrained, tethered, or even anaesthetized animal. Data obtained in this fashion must be interpreted with caution, as it is well known that restraining and anesthesia can have a major artifactual influence on physiological parameters. Radiotelemetry enables data to be collected from conscious and untethered animals. Measurements can be conducted even in freely moving animals, and without requiring the investigator to be in the proximity of the animal. Thus, known sources of artifacts are avoided, and accurate and reliable measurements are assured. This methodology also reduces interanimal variability, thus reducing the number of animals used, rendering this technology the most humane method of monitoring physiological parameters in laboratory animals. Constant advancements in data acquisition technology and implant miniaturization mean that it is now possible to record physiological parameters and locomotor activity continuously and in realtime over longer periods such as hours, days or even weeks. Here, we describe a surgical technique for implantation of a commercially available telemetry transmitter used for continuous measurements of core body temperature, locomotor activity and biopotential (i.e. onelead ECG), from which heart rate, heart rate variability, and PQ and QT intervals can be established in freeroaming, untethered mice. We also present pre-operative procedures and protocols for post-operative intensive care and pain treatment that improve recovery, well-being and survival rates in implanted mice.


Prediction of lung cancer risk in a Chinese population using a multifactorial genetic model.
Lung cancer is a complex polygenic disease. Although recent genome-wide association (GWA) studies have identified multiple susceptibility loci for lung cancer, most of these variants have not been validated in a Chinese population. In this study, we investigated whether a genetic risk score combining multiple. Five single-nucleotide polymorphisms (SNPs) identified in previous GWA or large cohort studies were genotyped in 5068 Chinese case-control subjects. The genetic risk score (GRS) based on these SNPs was estimated by two approaches: a simple risk alleles count (cGRS) and a weighted (wGRS) method. The area under the receiver operating characteristic (ROC) curve (AUC) in combination with the bootstrap resampling method was used to assess the predictive performance of the genetic risk score for lung cancer. Four independent SNPs (rs2736100, rs402710, rs4488809 and rs4083914), were found to be associated with a risk of lung cancer. The wGRS based on these four SNPs was a better predictor than cGRS. Using a liability threshold model, we estimated that these four SNPs accounted for only 4.02% of genetic variance in lung cancer. Smoking history contributed significantly to lung cancer (P < 0.001) risk [AUC = 0.619 (0.603-0.634)], and incorporated with wGRS gave an AUC value of 0.639 (0.621-0.652) after adjustment for over-fitting. This model shows promise for assessing lung cancer risk in a Chinese population. Our results indicate that although genetic variants related to lung cancer only added moderate discriminatory accuracy, it still improved the predictive ability of the assessment model in Chinese population.


Exploration and exploitation of Victorian science in Darwin's reading notebooks.
Search in an environment with an uncertain distribution of resources involves a trade-off between exploitation of past discoveries and further exploration. This extends to information foraging, where a knowledge-seeker shifts between reading in depth and studying new domains. To study this decision-making process, we examine the reading choices made by one of the most celebrated scientists of the modern era: Charles Darwin. From the full-text of books listed in his chronologically-organized reading journals, we generate topic models to quantify his local (text-to-text) and global (text-to-past) reading decisions using Kullback-Liebler Divergence, a cognitively-validated, information-theoretic measure of relative surprise. Rather than a pattern of surprise-minimization, corresponding to a pure exploitation strategy, Darwin's behavior shifts from early exploitation to later exploration, seeking unusually high levels of cognitive surprise relative to previous eras. These shifts, detected by an unsupervised Bayesian model, correlate with major intellectual epochs of his career as identified both by qualitative scholarship and Darwin's own self-commentary. Our methods allow us to compare his consumption of texts with their publication order. We find Darwin's consumption more exploratory than the culture's production, suggesting that underneath gradual societal changes are the explorations of individual synthesis and discovery. Our quantitative methods advance the study of cognitive search through a framework for testing interactions between individual and collective behavior and between short- and long-term consumption choices. This novel application of topic modeling to characterize individual reading complements widespread studies of collective scientific behavior.


Detailed contact data and the dissemination of Staphylococcus aureus in hospitals.
Close proximity interactions (CPIs) measured by wireless electronic devices are increasingly used in epidemiological models. However, no evidence supports that electronically collected CPIs inform on the contacts leading to transmission. Here, we analyzed Staphylococcus aureus carriage and CPIs recorded simultaneously in a long-term care facility for 4 months in 329 patients and 261 healthcare workers to test this hypothesis. In the broad diversity of isolated S. aureus strains, 173 transmission events were observed between participants. The joint analysis of carriage and CPIs showed that CPI paths linking incident cases to other individuals carrying the same strain (i.e. possible infectors) had fewer intermediaries than predicted by chance (P < 0.001), a feature that simulations showed to be the signature of transmission along CPIs. Additional analyses revealed a higher dissemination risk between patients via healthcare workers than via other patients. In conclusion, S. aureus transmission was consistent with contacts defined by electronically collected CPIs, illustrating their potential as a tool to control hospital-acquired infections and help direct surveillance.


"Transdifferentiation" of C6 glial cells in culture.
The activities of cyclic nucleotide phosphohydrolase, an enzyme marker for oligodendrocytes, and glutamine synthetase, an enzyme marker for astrocytes, were studied at early (21 to 26) and late (82 to 88) cell passages. The activity of cyclic nucleotide phosphohydrolase was markedly high and that of glutamine synthetase was low in the early passages, but this relation was reversed in the late passages. These findings suggest a "transdifferentiation" of C6 glial cells with passage in culture.


Carbon monoxide: to boldly go where NO has gone before.
The discovery that nitric oxide (NO) has powerful vasoactive properties identical to those of endothelial-derived relaxing factor spawned a vast body of research investigating the physiological actions of small gas molecules. NO, which arises endogenously through the action of nitric oxide synthase (NOS) enzymes, is a highly reactive gas that plays important roles in the regulation of vascular and immune function. Carbon monoxide (CO), a similar yet much more chemically stable gas, occurs in nature as a product of the oxidation or combustion of organic materials. CO also arises in cells and tissues as a byproduct of heme oxygenase (HO) activity, which degrades heme to biliverdin-IXalpha. Like NO, CO acts as a vasorelaxant and may regulate other vascular functions such as platelet aggregation and smooth muscle proliferation. CO has also been implicated as a neurotransmitter in the central nervous system. HO-1, the inducible form of HO, confers cytoprotection against oxidative stress in vitro and in vivo. CO, when applied at low concentration, exerts potent cytoprotective effects mimicking those of HO-1 induction, including down-regulation of inflammation and suppression of apoptosis. Many of the effects of CO depend on the activation of guanylate cyclase, which generates guanosine 3',5'-monophosphate (cGMP), and the modulation of mitogen-activated protein kinase (MAPK) signaling pathways. This review highlights new advances in the interaction of CO with cellular signaling processes.


Mitochondrial gene expression in Saccharomyces cerevisiae. Proteolysis of nascent chains in isolated yeast mitochondria optimized for protein synthesis.
We demonstrate here that mitochondrial translation products synthesized by isolated yeast mitochondria are subject to rapid proteolysis. The loss of label from mitochondrial peptides synthesized in vitro comes from two distinct pools of peptides: one that is rapidly degraded (t1/2 of minutes) and one that is much more resistant to proteolysis (t1/2 of hours). As the length of the incubation period increases, the percentage of labelled peptides in the rapidly-turning-over pool decreases and cannot be detected after 60 min of incubation. This proteolysis is inhibited by chloramphenicol and is dependent on the presence of ATP. The loss of label during the chase occurs from fully completed translation products. The proteolysis observed here markedly affects measurements of rates of mitochondrial protein synthesis in isolated yeast mitochondria. In earlier work, in which proteolysis was not considered, mitochondrial translation was thought to stop after 20-30 min of incubation. In the present study, by taking proteolysis into account, we demonstrate that the rate of translation in isolated mitochondria is actually constant for nearly 60 min and then decreases to near zero by 80 min of incorporation. These findings have allowed us to devise a procedure for measuring the 'true' rate of translation in isolated mitochondria. In addition, they suggest that mitochondrial translation products which normally assemble with nuclear-encoded gene products into multimeric enzyme complexes are unstable without their nuclear-encoded counterparts.


Flavonoids redirect PIN-mediated polar auxin fluxes during root gravitropic responses.
The rate, polarity, and symmetry of the flow of the plant hormone auxin are determined by the polar cellular localization of PIN-FORMED (PIN) auxin efflux carriers. Flavonoids, a class of secondary plant metabolites, have been suspected to modulate auxin transport and tropic responses. Nevertheless, the identity of specific flavonoid compounds involved and their molecular function and targets in vivo are essentially unknown. Here we show that the root elongation zone of agravitropic pin2/eir1/wav6/agr1 has an altered pattern and amount of flavonol glycosides. Application of nanomolar concentrations of flavonols to pin2 roots is sufficient to partially restore root gravitropism. By employing a quantitative cell biological approach, we demonstrate that flavonoids partially restore the formation of lateral auxin gradients in the absence of PIN2. Chemical complementation by flavonoids correlates with an asymmetric distribution of the PIN1 protein. pin2 complementation probably does not result from inhibition of auxin efflux, as supply of the auxin transport inhibitor N-1-naphthylphthalamic acid failed to restore pin2 gravitropism. We propose that flavonoids promote asymmetric PIN shifts during gravity stimulation, thus redirecting basipetal auxin streams necessary for root bending.


Penetration of a topically applied nonsteroidal anti-inflammatory drug into local tissues and synovial fluid of dogs.
To investigate penetration of a topically applied nonsteroidal anti-inflammatory drug (NSAID) into tissues and synovial fluid. 5 Greyhounds. Dogs were anesthetized and microdialysis probes placed in the dermis and gluteal muscle over each coxofemoral (hip) joint. Methylsalicylate (MeSA) was applied topically over the left hip joint. Dialysate and plasma (blood samples from the cephalic and femoral veins) were obtained during the subsequent 5 hours. Dogs were euthanatized, and tissue samples and synovial fluid were collected and analyzed for salicylic acid (SA) and MeSA by use of high-pressure liquid chromatography. SA and MeSA concentrations increased rapidly (< 30 minutes after application) in dialysate obtained from treated dermis. Salicylic acid also appeared in plasma within 30 minutes and reached a plateau concentration after 2 hours, although combined drug concentrations (SA plus MeSA) in plasma obtained from femoral vein samples were twice those measured in plasma obtained from the cephalic vein (SA only). Treated muscle had a progressive decrease in NSAID concentration with increasing depth (SA and MeSA), but it was significantly higher than the concentration in untreated muscle. Substantial amounts of SA and MeSA were also measured in synovial fluid of treated joints. Topically applied NSAIDs can penetrate deeply into tissues and synovial fluid. Local concentrations higher than circulating systemic concentrations are suggestive that direct diffusion and local blood redistribution are contributing to this effect. Systemic blood concentrations may be inadequate to describe regional kinetics of topically applied drugs.


Epidemiology of insomnia: what we know and what we still need to learn.
Epidemiologists have published more than 50 studies of insomnia based on data collected in various representative community-dwelling samples or populations. These surveys provide estimates of the prevalence of insomnia according to four definitions: insomnia symptoms, insomnia symptoms with daytime consequences, sleep dissatisfaction and insomnia diagnoses. The first definition, based on insomnia criteria as defined by the DSM-IV, recognizes that about one-third of a general population presents at least one of them. The second definition shows that, when daytime consequences of insomnia are taken into account, the prevalence is between 9% and 15%. The third definition represents 8-18% of the general population. The last definition, more precise and corresponding to a decision-making diagnosis, sets the prevalence at 6% of insomnia diagnoses according to the DSM-IV classification. These four definitions of insomnia have higher prevalence rates in women than in men. The prevalence of insomnia symptoms generally increases with age, while the rates of sleep dissatisfaction and diagnoses have little variation with age. Numerous factors can initiate or maintain insomnia. Mental disorders and organic diseases are the factors that have been the most frequently studied. The association between insomnia and major depressive episodes has been constantly reported: individuals with insomnia are more likely to have a major depressive illness. Longitudinal studies have shown that the persistence of insomnia is associated with the appearance of a new depressive episode. Future epidemiological studies should focus on the natural evolution of insomnia. Epidemiological genetic links of insomnia are yet to be studied.


Biomarker evidence for a major preservation pathway of sedimentary organic carbon.
Hydrogenation processes leading from biomolecules to fossil biomarkers in anoxic sediments are crucial for the preservation of organic matter. However, these processes are still poorly understood. The present identification of several reduced carotenoids in recent sediments attests that these processes operate at the earliest stages of diagenesis without structural or stereochemical specificity, implying a nonbiological reduction pathway. Sulfur species (e.g., H2S) are the hydrogen donors involved in such reduction, as demonstrated with laboratory experiments. These reactions allow the preservation of abundant organic carbon in the rock record.


Evaluation of urodynamic parameters after sling surgery in men with post-prostatectomy urinary incontinence.
To evaluate urodynamic alterations after sub-urethral sling surgery (SSU) in patients with post-prostatectomy urinary incontinence (PPUI). We evaluated data of 22 patients submitted to radical prostatectomy (RP) or transurethral resection of prostate (TURP) that presented post-surgical urinary incontinence and were treated with SSU implant in a pilot study previously performed in our institution. Those patients with PPUI were evaluated by urodynamic exam (UD) before and after surgery, and the parameters were compared, including uroflow, cystometry and micturition study. Exclusion criteria included patients without pre-operatory urodynamic study, those with urethral stenosis, those not healed of prostate cancer, patients without clinical conditions to be submitted to urodynamic study and those with severe neurological diseases or that refused to sign the consent form. Results were analyzed statistically by Fisher, Wilcoxon or Mann-Whitney tests. During free uroflow, none parameters showed any statistical significant differences. During cystometry, there were also no statistical differences and the same was observed at pressure versus flow study; the exception was at maximal flow detrusor pressure (PdetQmax), that was lower at post-operatory (p=0.028). In relation to the presence of urinary dysfunctions associated to PPUI, we observed a significant reduction of detrusor overactivity (p=0.035) in relation to pre-operatory period. SSU surgery significantly reduced detrusor overactivity and PdetQMax; however, there were no alterations of other evaluated urodynamic parameters.