Integrated clinical and omics approach to rare diseases: novel genes and oligogenic inheritance in holoprosencephaly.
Holoprosencephaly is a pathology of forebrain development characterized by high phenotypic heterogeneity. The disease presents with various clinical manifestations at the cerebral or facial levels. Several genes have been implicated in holoprosencephaly but its genetic basis remains unclear: different transmission patterns have been described including autosomal dominant, recessive and digenic inheritance. Conventional molecular testing approaches result in a very low diagnostic yield and most cases remain unsolved. In our study, we address the possibility that genetically unsolved cases of holoprosencephaly present an oligogenic origin and result from combined inherited mutations in several genes. Twenty-six unrelated families, for whom no genetic cause of holoprosencephaly could be identified in clinical settings [whole exome sequencing and comparative genomic hybridization (CGH)-array analyses], were reanalysed under the hypothesis of oligogenic inheritance. Standard variant analysis was improved with a gene prioritization strategy based on clinical ontologies and gene co-expression networks. Clinical phenotyping and exploration of cross-species similarities were further performed on a family-by-family basis. Statistical validation was performed on 248 ancestrally similar control trios provided by the Genome of the Netherlands project and on 574 ancestrally matched controls provided by the French Exome Project. Variants of clinical interest were identified in 180 genes significantly associated with key pathways of forebrain development including sonic hedgehog (SHH) and primary cilia. Oligogenic events were observed in 10 families and involved both known and novel holoprosencephaly genes including recurrently mutated FAT1, NDST1, COL2A1 and SCUBE2. The incidence of oligogenic combinations was significantly higher in holoprosencephaly patients compared to two control populations (P < 10-9). We also show that depending on the affected genes, patients present with particular clinical features. This study reports novel disease genes and supports oligogenicity as clinically relevant model in holoprosencephaly. It also highlights key roles of SHH signalling and primary cilia in forebrain development. We hypothesize that distinction between different clinical manifestations of holoprosencephaly lies in the degree of overall functional impact on SHH signalling. Finally, we underline that integrating clinical phenotyping in genetic studies is a powerful tool to specify the clinical relevance of certain mutations.


Micro-environmental cross-talk in an organotypic human melanoma-in-skin model directs M2-like monocyte differentiation via IL-10.
Preclinical assessment of novel therapies to fight cancer requires models that reflect the human physiology and immune response. Here, we established an in vitro three-dimensional (3D) reconstructed organotypic human melanoma-in-skin (Mel-RhS) model to investigate cellular and molecular features of tumor formation over a period of 6 weeks. Tumor nests developed over time at the epidermal-dermal junction and spread towards the dermis, in places disrupting the basement membrane. This coincided with secretion of matrix metalloproteinase 9 (MMP-9) by melanoma cells. These features resemble the initial stages of invasive melanoma. Interestingly, while the SK-MEL-28 cell line did not secrete detectable levels of interleukin-10 (IL-10) in traditional two-dimensional monolayers, it did express IL-10 in the 3D Mel-RhS, as did the surrounding keratinocytes and fibroblasts. This cellular cross-talk-induced secretion of IL-10 in the Mel-RhS indicated the generation of an immune suppressive microenvironment. Culture supernatants from Mel-RhS interfered with monocyte-to-dendritic-cell differentiation, leading to the development of M2-like macrophages, which was in part prevented by antibody-mediated IL-10 blockade. Indeed, high-dimensional single-cell analysis revealed a shift within the monocyte population away from a CD163+PD-L1+ M2-like phenotype upon IL-10 blockade. Thus, the 3D configuration of the Mel-RhS model revealed a role for IL-10 in immune escape through misdirected myeloid differentiation, which would have been missed in classical monolayer cultures.


Endovascular surgery: the future without limits.
The new millennium brings with it tremendous advances in technology and information sharing. Various medical, surgical, and engineering specialities are becoming more focused, thus enabling experts in these fields to gain insight and understanding previously unappreciated by those who came before us. Economic and technologic evolution will eliminate some of the drawbacks of endovascular therapy, such as persistent neck remnants. It is likely that drug delivery systems exist that would allow maximal benefit with little or no systemic adverse effects. It is likely that devices are available that could minimize various procedural complications. We as future neurosurgeons must persist in the quest to find collaborations that will promote cross-fertilization and cross-pollination among cardiologists, neurosurgeons, physicists, radiologists, neurologists, engineers, and other scientists so that the technology can be translated to reality. It is clear that, in time, transcatheter techniques will replace many operations for the treatment of cerebrovascular pathologies. Let us not allow ourselves the luxury of time.


Simultaneously-evoked auditory potentials (SEAP): A new method for concurrent measurement of cortical and subcortical auditory-evoked activity.
Recent electrophysiological work has evinced a capacity for plasticity in subcortical auditory nuclei in human listeners. Similar plastic effects have been measured in cortically-generated auditory potentials but it is unclear how the two interact. Here we present Simultaneously-Evoked Auditory Potentials (SEAP), a method designed to concurrently elicit electrophysiological brain potentials from inferior colliculus, thalamus, and primary and secondary auditory cortices. Twenty-six normal-hearing adult subjects (mean 19.26 years, 9 male) were exposed to 2400 monaural (right-ear) presentations of a specially-designed stimulus which consisted of a pure-tone carrier (500 or 600 Hz) that had been amplitude-modulated at the sum of 37 and 81 Hz (depth 100%). Presentation followed an oddball paradigm wherein the pure-tone carrier was set to 500 Hz for 85% of presentations and pseudo-randomly changed to 600 Hz for the remaining 15% of presentations. Single-channel electroencephalographic data were recorded from each subject using a vertical montage referenced to the right earlobe. We show that SEAP elicits a 500 Hz frequency-following response (FFR; generated in inferior colliculus), 80 (subcortical) and 40 (primary auditory cortex) Hz auditory steady-state responses (ASSRs), mismatch negativity (MMN) and P3a (when there is an occasional change in carrier frequency; secondary auditory cortex) in addition to the obligatory N1-P2 complex (secondary auditory cortex). Analyses showed that subcortical and cortical processes are linked as (i) the latency of the FFR predicts the phase delay of the 40 Hz steady-state response, (ii) the phase delays of the 40 and 80 Hz steady-state responses are correlated, and (iii) the fidelity of the FFR predicts the latency of the N1 component. The SEAP method offers a new approach for measuring the dynamic encoding of acoustic features at multiple levels of the auditory pathway. As such, SEAP is a promising tool with which to study how relationships between subcortical and cortical processes change through early development and auditory learning as well as by hearing loss and aging.


Comparison of phase-resolved functional lung (PREFUL) MRI derived perfusion and ventilation parameters at 1.5T and 3T in healthy volunteers.
The purpose of this study is to evaluate the influence of different field strengths on perfusion and ventilation parameters, SNR and CNR derived by PREFUL MRI using predefined sequence parameters. Data sets of free breathing 2d FLASH lung MRI were acquired from 15 healthy subjects at 1.5T and 3T (Magnetom Avanto and Skyra, Siemens Healthcare, Erlangen, Germany) with a maximum period of 3 days in between. The processed functional parameters regional ventilation (RVent), perfusion (Q), quantified perfusion (QQuant), perfusion defect percentage (QDP), ventilation defect percentage (VDP) and ventilation-perfusion match (VQM) were compared for systematic differences. Signal- and contrast-to-noise ratio (SNR and CNR) of both acquisitions were analyzed. RVent, Q, VDP, SNR and CNR presented no significant differences between 1.5T and 3T. QQuant (1.5T vs. 3T, P = 0.04), and QDP (1.5T vs. 3T, P≤0.01) decreased significantly at 3T. Consequently, VQM increased significantly (1.5T vs. 3T, P≤0.01). Skewness and kurtosis of the Q-values increased significantly at 3T (P≤0.01). The mean Sørensen-Dice coefficients between both series were 0.91 for QDP and 0.94 for VDP. The Bland-Altman analysis of both series showed mean differences of 4.29% for QDP, 1.23% for VDP and -5.15% for VQM. Using the above-mentioned parameters for three-day repeatability at two different scanners and field strengths, the retrospective power calculation showed, that a sample size of 15 can detect differences of 3.7% for QDP, of 2.9% for VDP and differences of 2.6% for VQM. Significant differences in QDP may be related to field inhomogeneities, which is expressed by increasing skewness and kurtosis at 3T. QQuant reveals only poor reproducibility between 1.5T and 3T. RVent, Q, VDP, SNR and CNR were not altered significantly at the used sequence parameters. Healthy participants with minimal defects present high spatial agreement of QDP and VDP.


The Association between Serum Bilirubin and Kernicterus Spectrum Disorder: A Systematic Review and Meta-Analysis.
Total serum bilirubin (TSB) is used in managing neonates with jaundice, but clear evidence on its association with major outcomes is lacking. We evaluated the association between TSB and kernicterus spectrum disorder (KSD). We searched PubMed, EMBASE, and CENTRAL till July 2021. Two authors independently selected relevant cohort studies, extracted data (CHARMS checklist), assessed risk of bias (RoB) (QUIPS tool), and rated certainty-of-evidence (Grades of Recommendation, Assessment, Development, and Evaluation). We pooled adjusted odds ratio (aOR) (random-effect) via generic inverse variance methods. From 2,826 records retrieved, we included 37 studies (n = 648,979). Fifteen studies had low, 16 moderate, and 6 high RoB, with majority having concerns on confounder adjustment and statistical analysis. Twenty-two studies contributed meta-analysis data, and 15 were summarized narratively. TSB appears associated with KSD in infants with certain risk factors (aOR 1.10, 95% CI: 1.07-1.13; 5 studies [n = 4,484]). However, TSB (aOR 1.10, 95% CI: 0.98-1.23; 1 study [n = 34,533]) or hyperbilirubinemia (aOR 1.00, 95% CI: 0.51-1.95; 2 studies [n = 56,578]) have no clear association with kernicterus or neurological diagnosis in overall neonatal population (moderate-certainty-evidence). One study shows that infants with hyperbilirubinemia appear likelier to develop attention-deficit disorder (aOR 1.90, 95% CI: 1.10-3.28) and autistic spectrum disorder (aOR 1.60, 95% CI: 1.03-2.49, n = 56,019) (low-certainty-evidence). Certain clinical factors appear associated with KSD, although very few studies contributed to the analyses. Despite the importance of this question, there is insufficient high-quality evidence on the independent prognostic value of TSB for adverse neurodevelopmental outcomes in most neonatal populations. Future studies should incorporate all known risk factors alongside TSB in a multivariable analysis to improve certainty-of-evidence.


Mothers with living children and children with living mothers: the role of fertility and mortality in the period 1911-2050.
Whether an individual has one or more surviving children at a given age and/or a surviving parent depends both on fertility (not only number of children, but also timing of childbearing) and mortality. We consider the contribution of changes in mortality and fertility to past and likely future trends on availability of such kin. The proportion of people aged 60 with a mother alive will more than double between those born in 1911 and 1970, and the figure will increase for at least the next 30 years. While there are increasing concerns about the availability of informal care for elderly people from children in the next quarter century or so, in fact we conclude that a higher proportion of elderly people are likely to have a surviving child than for any generation ever born in Britain, and will have more than any foreseeable future cohort.


Cannabinoids for the treatment of dementia.
Following the discovery of an endogenous cannabinoid system and the identification of specific cannabinoid receptors in the central nervous system, much work has been done to investigate the main effects of these compounds. There is increasing evidence that the cannabinoid system may regulate neurodegenerative processes such as excessive glutamate production, oxidative stress and neuroinflammation. Neurodegeneration is a feature common to the various types of dementia and this has led to interest in whether cannabinoids may be clinically useful in the treatment of people with dementia. Recent studies have also shown that cannabinoids may have more specific effects in interrupting the pathological process in Alzheimer's disease. To determine from available research whether cannabinoids are clinically effective in the treatment of dementia. The Specialized Register of the Cochrane Dementia and Cognitive Improvement Group (CDCIG), The Cochrane Library, MEDLINE, EMBASE, PsycINFO, CINAHL and LILACS were searched on 11 April 2008 using the terms: cannabis or cannabinoid* or endocannabinoid* or cannabidiol or THC or CBD or dronabinol or delta-9-tetrahydrocannabinol or marijuana or marihuana or hashish. The CDCIG Specialized Register contains records from all major health care databases (The Cochrane Library, MEDLINE, EMBASE, PsycINFO, CINAHL, LILACS) as well as from many clinical trials registries and grey literature sources. All double-blind and single (rater)-blind randomized placebo controlled trials assessing the efficacy of cannabinoids at any dose in the treatment of people with dementia. Two reviewers independently examined the retrieved studies for inclusion according to the selection criteria. They then independently assessed the methodological quality of selected trials and extracted data where possible. Only one study met the inclusion criteria. The data in the study report were presented in such a way that they could not be extracted for further analysis and there was insufficient quantitative data to validate the results. This review finds no evidence that cannabinoids are effective in the improvement of disturbed behaviour in dementia or in the treatment of other symptoms of dementia. More randomized double-blind placebo controlled trials are needed to determine whether cannabinoids are clinically effective in the treatment of dementia.


NF-kappaB mediates the transcription of mouse calsarcin-1 gene, but not calsarcin-2, in C2C12 cells.
The calsarcins comprise a novel family of muscle-specific calcineurin-interaction proteins that play an important role in modulating both the function and substrate specificity of calcineurin in muscle cells. The expression of calsarcin-1 (CS-1) is restricted to slow-twitch skeletal muscle fibres, whereas that of both calsarcin-2 (CS-2) and calsarcin-3 (CS-3) is enriched in fast-twitch fibres. However, the transcriptional control of this selective expression has not been previously elucidated. Our real-time RT-PCR analyses suggest that the expression of CS-1 and CS-2 is increased during the myogenic differentiation of mouse C2C12 cells. Promoter deletion analysis further suggests that an NF-kappaB binding site within the CS-1 promoter is responsible for the up-regulation of CS-1 transcription, but no similar mechanism was evident for CS-2. These findings are further supported by the results of EMSA analysis, as well as by overexpression and inhibition experiments in which NF-kappaB function was blocked by treatment with its inhibitor, PDTC. In addition, the overexpression of NFATc4 induces both the CS-1 and CS-2 promoters, whereas MEF2C only activates CS-1. Our present data suggest that NF-kappaB is required for the transcription of mouse CS-1 but not CS-2, and that the regulation of the calsarcins is mediated also by the NFAT and MEF2 transcription factors. These results provide new insights into the molecular mechanisms governing transcription in specific muscle fibre cells. The calsarcins may also serve as a valuable mechanistic tool to better understand the regulation of calcineurin signalling during muscle differentiation.


Tocotrienols for normalisation of hepatic echogenic response in nonalcoholic fatty liver: a randomised placebo-controlled clinical trial.
Nonalcoholic fatty liver disease (NAFLD) is one of the commonest liver disorders. Obesity, insulin resistance, lipid peroxidation and oxidative stress have been identified amongst the possible hits leading to the onset and progression of this disease. Nutritional evaluation of NAFLD patients showed a lower-than-recommended intake of vitamin E. Vitamin E is a family of 8 isoforms, 4 tocopherols and 4 tocotrienols. Alpha-tocopherol has been widely investigated in liver diseases, whereas no previous clinical trial has investigated tocotrienols for NAFLD. Aim of the study was to determine the effects of mixed tocotrienols, in normalising the hepatic echogenic response in hypercholesterolaemic patients with ultrasound-proven NAFLD. Eighty-seven untreated hypercholesterolaemic adults with ultrasound-proven NAFLD were enrolled and randomised into control group (n = 44) and tocotrienols group (n = 43). The treatment, either mixed tocotrienols 200 mg twice daily or placebo, had a 1-year duration.Normalisation of hepatic echogenic response, being the trial primary aim, was used in sample size calculations. The data were assessed according to intention to treat principle as primary outcome. Per protocol analysis was also carried out as secondary outcome measurement. Thirty and 34 participants concluded the study in the tocotrienols and placebo group respectively. Alpha-tocopherol levels were within the normal range for all subjects. As primary outcome, the normalisation of hepatic echogenic response was significantly higher for the tocotrienols treated group compared to the placebo group in the intention to treat analysis (P = 0.039; 95% CI = 0.896-6.488). As secondary objective, the per protocol assessment also showed significant rate of remission (P = 0.014; 95% CI = 1.117-9.456). Worsening of NAFLD grade was recorded in two patients in the placebo group, but none in the group treated with tocotrienols. No adverse events were reported for both groups. This is the first clinical trial that showed the hepatoprotective effects of mixed palm tocotrienols in hypercholesterolemic adults with NAFLD.


Using genomics to study how chromatin influences gene expression.
A postgenome challenge is to understand how the code in DNA is converted into the biological processes underlying various cell fates. By establishing the appropriate technical tools, we are moving from an era in which such questions have been asked by studying individual genes to one in which large domains, whole chromosomes, and the entire human genome can be investigated. These developments will allow us to study in parallel the transcriptional program and components of the epigenetic program (nuclear position, timing of replication, chromatin structure and modification, DNA methylation) to determine the hierarchy and order of events required to switch genes on and off during differentiation and development.


Environment determines evolutionary trajectory in a constrained phenotypic space.
Constraints on phenotypic variation limit the capacity of organisms to adapt to the multiple selection pressures encountered in natural environments. To better understand evolutionary dynamics in this context, we select Escherichia coli for faster migration through a porous environment, a process which depends on both motility and growth. We find that a trade-off between swimming speed and growth rate constrains the evolution of faster migration. Evolving faster migration in rich medium results in slow growth and fast swimming, while evolution in minimal medium results in fast growth and slow swimming. In each condition parallel genomic evolution drives adaptation through different mutations. We show that the trade-off is mediated by antagonistic pleiotropy through mutations that affect negative regulation. A model of the evolutionary process shows that the genetic capacity of an organism to vary traits can qualitatively depend on its environment, which in turn alters its evolutionary trajectory.


Pollen limitation and reproductive assurance in Antillean Gesnerieae: a specialists vs. generalist comparison.
Pollen limitation of female fecundity is widespread among angiosperms, a signal that pollinators frequently fail to transfer pollen to fertilize all ovules. Recent surveys have suggested that pollen limitation is associated with floral specialization. This study uses a group of Antillean Gesneriaceae with contrasting pollination systems (bat, hummingbird, and generalist) to assess the premise that plants with specialized pollination systems and infrequent pollinator visitation experience greater pollen limitation of fruit and seed set than their generalist congeners. Alternatively, specialists may possess mechanisms that reduce pollen limitation, such as autonomous self-pollination. A survey of autonomous self-pollination conducted on 13 Gesneria and Rhytidophyllum species during 2006-2008 revealed no significant association between reproductive assurance mechanisms and pollination system specialization. However, high levels of potential autonomous self-pollination were only found among specialized hummingbird-pollinated species. A comparison of fruit and seed set between emasculated and unmanipulated flowers provided evidence for autonomous selfing acting as a reproductive assurance mechanism in three out of four ornithophilous species. Furthermore, the Puerto Rican population of G. reticulata relies almost exclusively on self-pollination for reproduction. Two-year pollen supplementation experiments conducted on nine Gesnerieae species from the Dominican Republic and Puerto Rico provided evidence for significant pollen limitation associated with pollination specialization including both bat- and hummingbird-pollinated Gesnerieae; no pollen limitation was detected in any of the four generalist species. No pollen limitation was detected either in two ornithophilous Gesneria species with low hummingbird visitation and high levels of autonomous self-pollination. This study provides support for the idea that generalized pollination systems may, in some cases, buffer against fluctuations in the pollinator environment. However, the use of reproductive assurance mechanisms allows the maintenance of highly specialized pollination systems in pollinator depauperate environments.


Hormonal and reproductive factors and pancreatic cancer risk: a prospective cohort study.
We examined pancreatic cancer risk in association with hormonal and reproductive factors in a prospective cohort study of 89,835 Canadian women, aged 40-59 at recruitment, who were enrolled in the National Breast Screening Study (NBSS). Linkages to national cancer and mortality databases yielded data on cancer incidence and deaths of all causes, respectively, with follow-up ending between 1998 and 2000. Cox proportional hazards models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for the association between hormonal and reproductive factors and risk of pancreatic cancer. During a mean of 16.4 years of follow-up, we observed 187 incident pancreatic cancer cases. Compared with women who were premenopausal at baseline, postmenopausal women were at significantly increased risk of pancreatic cancer (odds ratio = 2.44, 95% confidence interval [CI] = 1.45-4.09). Age at first livebirth, parity, age at menarche, use of oral contraceptive, and use of hormone replacement therapy (HRT) were not associated with altered pancreatic cancer risk in our study population. However, among parous women, risk increased with increasing parity. Other than the increased risk among postmenopausal women, the present study provides little support for associations with hormonal factors. Additional prospective data are needed.


Stimulation of 3D osteogenesis by mesenchymal stem cells using a nanovibrational bioreactor.
Bone grafts are one of the most commonly transplanted tissues. However, autologous grafts are in short supply, and can be associated with pain and donor-site morbidity. The creation of tissue-engineered bone grafts could help to fulfil clinical demand and provide a crucial resource for drug screening. Here, we show that vibrations of nanoscale amplitude provided by a newly developed bioreactor can differentiate a potential autologous cell source, mesenchymal stem cells (MSCs), into mineralized tissue in 3D. We demonstrate that nanoscale mechanotransduction can stimulate osteogenesis independently of other environmental factors, such as matrix rigidity. We show this by generating mineralized matrix from MSCs seeded in collagen gels with stiffness an order of magnitude below the stiffness of gels needed to induce bone formation in vitro. Our approach is scalable and can be compatible with 3D scaffolds.


Inositol- and folate-resistant neural tube defects in mice lacking the epithelial-specific factor Grhl-3.
The neural tube defects (NTDs) spina bifida and anencephaly are widely prevalent severe birth defects. The mouse mutant curly tail (ct/ct) has served as a model of NTDs for 50 years, even though the responsible genetic defect remained unrecognized. Here we show by gene targeting, mapping and genetic complementation studies that a mouse homolog of the Drosophila grainyhead (grh) gene, grainyhead-like-3 (Grhl3), is a compelling candidate for the gene underlying the curly tail phenotype. The NTDs in Grhl3-null mice are more severe than those in the curly tail strain, as the Grhl3 alleles in ct/ct mice are hypomorphic. Spina bifida in ct/ct mice is folate resistant, but its incidence can be markedly reduced by maternal inositol supplementation periconceptually. The NTDs in Grhl3-/- embryos are also folate resistant, but unlike those in ct/ct mice, they are resistant to inositol. These findings suggest that residual Grhl3 expression in ct/ct mice may be required for inositol rescue of folate-resistant NTDs.


Stochasticity in space, persistence in time: genetic heterogeneity in harbour populations of the introduced ascidian Styela plicata.
Spatio-temporal changes in genetic structure among populations provide crucial information on the dynamics of secondary spread for introduced marine species. However, temporal components have rarely been taken into consideration when studying the population genetics of non-indigenous species. This study analysed the genetic structure of Styela plicata, a solitary ascidian introduced in harbours and marinas of tropical and temperate waters, across spatial and temporal scales. A fragment of the mitochondrial gene Cytochrome Oxidase subunit I (COI) was sequenced from 395 individuals collected at 9 harbours along the NW Mediterranean coast and adjacent Atlantic waters (> 1,200 km range) at two time points 5 years apart (2009 and 2014). The levels of gene diversity were relatively low for all 9 locations in both years. Analyses of genetic differentiation and distribution of molecular variance revealed strong genetic structure, with significant differences among many populations, but no significant differences among years. A weak and marginally significant correlation between geographic distance and gene differentiation was found. Our results revealed spatial structure and temporal genetic homogeneity in S. plicata, suggesting a limited role of recurrent, vessel-mediated transport of organisms among small to medium-size harbours. Our study area is representative of many highly urbanized coasts with dense harbours. In these environments, the episodic chance arrival of colonisers appears to determine the genetic structure of harbour populations and the genetic composition of these early colonising individuals persists in the respective harbours, at least over moderate time frames (five years) that encompass ca. 20 generations of S. plicata.


A single-chain-variable-fragment fluorescence biosensor activates fluorogens from dissimilar chemical families.
Current advancements in biological protein discovery utilize bi-partite methods of fluorescence detection where chromophore and scaffold are uncoupled. One such technology, called fluorogen-activating proteins (FAPs), consists of single-chain-variable-fragments (scFvs) selected against small organic molecules (fluorogens) that are non-fluorescent in solution, but highly fluorescent when bound to the scFv. In unusual circumstances a scFv may activate similar fluorogens from a single chemical family. In this report we identified a scFv biosensor with fluorescence activity against multiple fluorogens from two structurally dissimilar families. In-vitro analysis revealed highly selective scFv-ligand interactions at sub-micromolar ranges. Additionally, each scFv-fluorogen complex possesses unique excitation and emission spectra, which allows broader detection limits from the biosensor. Further analysis indicated that ligand activation, regardless of chemical family, occurs at a common scFv binding region that proves flexible, yet selective for fluorogen binding. As a protein reporter at the surface of mammalian cells, the scFv revealed bright signal detection and minimal background. Additionally, when tagged to a G-protein-coupled receptor, we observed agonist dependent signaling leading to protein traffic from cell surface to endosomes via multi-color fluorescence tracking. In summary, this report unveils a noncanonical scFv biosensor with properties of high ligand affinity and multi-channel fluorescence detection, which consequently offers expanded opportunities for cellular protein discovery.


Identification of genetically homozygous rapid and slow acetylators of drugs and environmental carcinogens among established inbred rabbit strains.
Liver and gut mucosa N-acetyltransferase (NAT) cytosol (105,000 x g) was prepared from selected lines of New Zealand White rapid and slow acetylator rabbits bred and housed at the University of Michigan, and from inbred and partially inbred rabbits obtained from The Jackson Laboratory. Liver NAT activity was determined with p-aminobenzoic acid, p-aminosalicyclic acid, procainamide, sulfamethazine, isoniazid and 2-aminofluorene as substrates. Gut mucosal NAT activity was determined with 2-aminofluorene. A gene dose-response relationship was observed for both liver NAT and gut mucosa NAT with all substrates tested. Highest levels were always observed in homozygous rapid acetylator inbred strains (B/J, III/J, IIIC/J, III/DwJ, IIIEP/J and IIIVO/J), lower levels in obligate heterozygous rapid acetylator rabbits and lowest levels in homozygous slow acetylator inbred (ACEP/J, III/cdJ, IIIVO/ahJ, and IIIVO/vptJ) and outbred rabbits. The differences in magnitude of liver NAT activity level between acetylator genotypes was dependent on the substrate employed, progressively increasing in the following order: p-aminobenzoic acid, p-aminosalicyclic acid, procainamide, sulfamethazine, isoniazid, 2-aminofluorene. The determination of kinetic constants for liver p-aminosalicyclic acid NAT activity indicated a 2-fold difference in apparent Vmax between rapid acetylator genotypes and a 30-fold difference between rapid and slow acetylator phenotypes. In addition, the apparent Km for p-aminosalicyclic acid was significantly lower in the slow acetylators than in the rapid acetylators.


At the centennial of Michaelis and Menten, competing Michaelis-Menten steps explain effect of GLP-1 on blood-brain transfer and metabolism of glucose.
Glucagon-like peptide-1 (GLP-1) is a potent insulinotropic incretin hormone with both pancreatic and extrapancreatic effects. Studies of GLP-1 reveal significant effects in regions of brain tissue that regulate appetite and satiety. GLP-1 mimetics are used for the treatment of type 2 diabetes mellitus. GLP-1 interacts with peripheral functions in which the autonomic nervous system plays an important role, and emerging pre-clinical findings indicate a potential neuroprotective role of the peptide, for example in models of stroke and in neurodegenerative disorders. A century ago, Leonor Michaelis and Maud Menten described the steady-state enzyme kinetics that still apply to the multiple receptors, transporters and enzymes that define the biochemical reactions of the brain, including the glucose-dependent impact of GLP-1 on blood-brain glucose transfer and metabolism. This MiniReview examines the potential of GLP-1 as a molecule of interest for the understanding of brain energy metabolism and with reference to the impact on brain metabolism related to appetite and satiety regulation, stroke and neurodegenerative disorders. These effects can be understood only by reference to the original formulation of the Michaelis-Menten equation as applied to a chain of kinetically controlled steps. Indeed, the effects of GLP-1 receptor activation on blood-brain glucose transfer and brain metabolism of glucose depend on the glucose concentration and relative affinities of the steps both in vitro and in vivo, as in the pancreas.


Tomographic imaging via spectral encoding of spatial frequency.
Three-dimensional optical tomographic imaging plays an important role in biomedical research and clinical applications. We introduce spectral tomographic imaging (STI) via spectral encoding of spatial frequency principle that not only has the capability for visualizing the three-dimensional object at sub-micron resolution but also providing spatially-resolved quantitative characterization of its structure with nanoscale accuracy for any volume of interest within the object. The theoretical basis and the proof-of-concept numerical simulations are presented to demonstrate the feasibility of spectral tomographic imaging.


Ab initio GGA+U study of oxygen evolution and oxygen reduction electrocatalysis on the (001) surfaces of lanthanum transition metal perovskites LaBO₃ (B = Cr, Mn, Fe, Co and Ni).
In this work, we performed density functional theory (DFT) calculations with inclusion of Hubbard U corrections for the transition metal d-electrons, to investigate stability and electrocatalytic activities of the oxygen evolution reaction (OER) and oxygen reduction reaction (ORR) for the ABO3 (A = La; B = Cr, Mn, Fe, Co, and Ni) (001) surfaces. We showed surface binding energies of relevant ORR/OER species are coupled strongly to surface polarity and local oxidation states, giving large (∼1 eV scale per adsorbate) differences in binding between (001) AO and BO2 surfaces, where the more oxidized BO2 bare surfaces in general exhibit weak coverage dependence, while the more reduced AO bare surfaces of the LaCrO3, LaMnO3, and LaFeO3 perovskites with lower d-electron filling show strong/moderate coverage dependences. We then predicted that surface coverage can play a key role in determining surface stability, and when coverage effects are included the AO and BO2(001) surfaces have either similar stability or the AO surface is more stable, as found for 1 monolayer HO* covered AO surfaces of LaCrO3 and LaFeO3 under ORR conditions and 1 monolayer O* covered LaNiO3 AO surface under OER conditions. For the (001) AO surfaces with strong coverage dependent surface adsorption, we predicted a decrease in ORR overpotential of 1-2 V with proper treatment of coverage effects as compared to those of the bare surface simulations. Our results indicated that the GGA+U method and proper treatment of coverage effects more accurately predict ORR and OER overpotentials relative to experimental values as compared to the GGA method and bare surfaces. The overall ORR activity trends vs. the LaBO3 series were predicted to be Co > Mn ≈ Ni > Fe > Cr.


Patient and social work factors related to successful placement of long-term psychiatric in-patients from a specialist psychiatric hospital in South Africa.
The slow discharge of long-term psychiatry patients from Weskoppies Hospital into the community has not matched the national and international drive towards deinstitutionalisation. This article investigates patient and social work factors related to successful community placement, in the context of limited community care facilities. Thirty-six long-term patients who were successfully placed outside of the hospital during a seven month period were compared to 235 unplaced long-term patients in terms of demographic and clinical variables. Social work services were analysed in terms of which patients received the most interventions, and the most common type of interventions. The most significant patient factors associated with successful placement were: female patients; medium-to-high level of functioning; having involved relatives living far away; a low frequency of behavioural problems (especially of cannabis abuse, verbal or physical aggression, uncontrolled sexual activity), and agitation or restlessness. These patient factors were mirrored in the social work services rendered to the long-term patients during the study period: The recipients were mostly female, in open wards (higher-functioning); and the social services utilised were mostly related to planning for placement and patient support. The lack of community care facilities in the Pretoria area that are able to care for the more difficult long-term psychiatry patients, limits successful placement and increases the burden of hospital based social workers. The problem cannot be resolved at a hospital level and needs to be addressed in the context of provincial and national health departments.


Changes of higher order chromatin arrangements during major genome activation in bovine preimplantation embryos.
Gene-dense chromosome territories (CTs) are typically located more interior, gene-poor CTs more peripheral in mammalian cell nuclei. Here, we show that this gene-density correlated CT positioning holds for the most gene-rich and gene-poor bovine chromosomes 19 and 20, respectively, in bovine fibroblast and lymphocyte nuclei. In order to determine the period at which this non-random CT order is established during development, we performed fluorescence in situ hybridization, on three-dimensionally preserved bovine preimplantation embryos generated by in vitro fertilization and investigated the distribution of BTA 19 and 20 CTs. Radial arrangements of CTs 19 and 20 were the same up to the 8-cell stage. At the 10- to 16-cell stage, however, a significant difference became apparent with CTs 19 localized more internally and CTs 20 more peripherally. Since major genome activation in bovine embryos occurs at the 8- to 16-cell stage, our findings demonstrate a temporal correlation between transcriptional activation and a major rearrangement of chromatin topography in blastomere nuclei.


The potential of distance-based thresholds and character-based DNA barcoding for defining problematic taxonomic entities by CO1 and ND1.
The mitochondrial CO1 gene (cytochrome c oxidase I) is a widely accepted metazoan barcode region. In insects, the mitochondrial NADH dehydrogenase subunit 1 (ND1) gene region has proved to be another suitable marker especially for the identification of lower level taxonomic entities such as populations and sister species. To evaluate the potential of distance-based thresholds and character-based DNA barcoding for the identification of problematic species-rich taxa, both markers, CO1 and ND1, were used as test parameters in odonates. We sequenced and compared gene fragments of CO1 and ND1 for 271 odonate individuals representing 51 species, 22 genera and eight families. Our data suggests that (i) the combination of the CO1 and ND1 fragment forms a better identifier than a single region alone; and (ii) the character-based approach provides higher resolution than the distance-based method in Odonata especially in closely related taxonomic entities.


Comparison of a Smartphone-Based Photographic Method with Face-to-Face Caries Assessment: A Mobile Teledentistry Model.
This study sought to evaluate the efficacy of a mobile teledentistry approach using a smartphone camera for remote screening of dental caries. An image acquisition Android App was created to facilitate the acquisition and transmission of dental images to a store-and-forward based telemedicine server. One hundred participants who were attending routine checkups at dental clinics were enrolled in 2014. Following a face-to-face oral screening by a screener (dentist), images of patients' teeth were obtained using a smartphone camera. These images, along with patient information, were then transmitted from the Android App to the server through the Internet for later independent assessment by two charters (off-site dentists). The assessments of these charters were then compared to the benchmark face-to-face caries assessment. Sensitivity values for the photographic method when compared to the benchmark face-to-face caries assessment were moderate, and ranged from 60% to 63%. Weighted kappa (K) as a measure of intragrader agreement for the photographic assessment was estimated as almost perfect (K = 0.84). The intergrader agreement for the photographic method compared to the face-to-face caries assessment ranged from moderate to substantial (K = 0.54-0.66). Despite some limitations, the mobile teledentistry approach has shown the potential to detect occlusal caries from photographs taken by a smartphone camera with an acceptable diagnostic performance compared to traditional face-to-face screening. This study suggests that telemedicine and cellular phone technology can be combined to create an inexpensive and reliable screening tool.


A Fluorescent, Reagentless Biosensor for ATP, Based on Malonyl-Coenzyme A Synthetase.
A fluorescent reagentless biosensor for ATP has been developed, based on malonyl-coenzyme A synthetase from Rhodopseudomonas palustris as the protein scaffold and recognition element. Two 5-iodoacetamidotetramethylrhodamines were covalently bound to this protein to provide the readout. This adduct couples ATP binding to a 3.7-fold increase in fluorescence intensity with excitation at 553 nm and emission at 575 nm. It measures ATP concentrations with micromolar sensitivity and is highly selective for ATP relative to ADP. Its ability to monitor enzymatic ATP production or depletion was demonstrated in steady-state kinetic assays in which ATP is a product or substrate, respectively.


Autolytic debridement and management of bacterial load with an occlusive hydroactive deressing impregnated with polyhexamethylene biguanide.
Debridement and control of bacterial load are key-points of wound care. The aim of this study is to evaluate the effectiveness of autolytic debridement and management of bacterial load (bioburden) of an occlusive hydro-active dressing impregnated with polyhexamethylene biguanide (PHMB). We used an occlusive dressing of hydro-active for leg ulcers' treatment (TenderWet-plus® - Hartmann). Inclusion criteria were: presence of a fibrinous ulcer with multiple etiopathogenesis, in absence of clinical infection, no hypersecretion and diameter within 5 cm. Swab for bacterial bioburden control were made at start of the study, after 3 and 7 days. We made also a measurement of the amount of granulation tissue and of pain with VAS scale; bacterial bioburden control inside the dressing was made too. 28 leg ulcers were treated (12 venous, 5 diabetic, 8 thraumatic, 3 mixed) in 25 patients. The ulcer has been completely covered with fibrin in almost all cases and the bacterial load was represented mainly by Staphylococcus aureus (80% of the cases) with a load within 10.000UFC/plate in 81,5% of the cases. Control at 3 and 7 days have shown the appearance of granulaton tissue up to 67,8% of the wound surface. Bacterial bioburden (load) remained constant in 50% of the cases, decreased in 15%, increased in 35% of the cases. TenderWet-plus® has proven to be effective and quick in autolytic debridement and at the same time be able to handle the bacterial load in most cases. It can then be used for home treatment.


Effects of soy protein on alcoholic liver disease in rats undergoing ethanol withdrawal.
This investigation attempted to clarify the effects of soy protein on alcoholic liver disease (ALD) in rats undergoing ethanol withdrawal. Alcoholic liver disease was induced in rats by administration of a low-carbohydrate ethanol liquid diet for 12 weeks, after which the ethanol was withdrawn and the rats were divided into two experimental groups: a control group (EC group) and a soy protein group (EP group) for 4 weeks. After the 12-week ALD-inducing period, the ethanol group had significantly higher hepatic lipid accumulation, oxidative stress and inflammation. We found that the EP group had significantly lower hepatic lipids, malondialdehyde, tumor necrosis factor-α, interleukin (IL)-1β, IL-6, hydroxyproline levels and myeloperoxidase activity compared to the EC group. Moreover, the fecal total cholesterol and total lipids were higher in the EP group. Expression of the hepatic cytochrome P450 2E1 (CYP2E1) protein in the EP group was significantly lower than that in the EC group, and the hepatic peroxisome proliferator-activated receptor (PPAR) α and cytochrome P450 4A (CYP4A) protein expressions in the EP group were significantly higher than those in the EC group. In the histopathological analysis, we also found that soy protein ameliorated fat accumulation in the liver. These results suggest that soy protein may improve alcohol-induced lipid accumulation, oxidative stress and inflammation by decreasing proinflammatory cytokines and CYP2E1 protein expression and by increasing PPARα and CYP4A protein expressions and fecal lipid excretion, thereby producing beneficial effects on ALD during ethanol withdrawal.


Increasing Compliance With an Antibiotic Prophylaxis Guideline to Prevent Pediatric Surgical Site Infection: Before and After Study.
To evaluate an intervention for improving antibiotic prophylaxis (AP) guideline compliance to prevent surgical site infections in children. Although appropriate AP reduces surgical site infection, and guidelines improve quality of care, changing practice is difficult. To facilitate behavioral change, various barriers need to be addressed. A multidisciplinary task force at a pediatric hospital developed an evidence-based AP guideline. Subsequently, the guideline was posted in operating rooms and the online formulary, only recommended antibiotics were available in operating rooms, incoming trainees received orientation, antibiotic verification was included in time-out, computerized alerts were set for inappropriate postoperative prophylaxis, and surgeons received e-mails when guideline was not followed. AP indication and administration were documented for surgical procedures in July 2008 (preintervention), September 2011 (postintervention), and April-May 2013 (follow-up). Compliance was defined as complete--appropriate antibiotic, dose, timing, redosing, and duration when prophylaxis was indicated; partial--appropriate drug and timing when prophylaxis was indicated; and appropriate use--complete compliance when prophylaxis was indicated, no antibiotics when not indicated. Compliance at preintervention and follow-up was compared using χ(2) tests. AP was indicated in 43.9% (187/426) and 62.0% (124/200) of surgical procedures at preintervention and follow-up, respectively. There were significant improvements in appropriate antibiotic use (51.6%-67.0%; P < 0.001), complete (26.2%-53.2%; P < 0.001) and partial compliance (73.3%-88.7%, P = 0.001), correct dosage (77.5%-90.7%; P = 0.003), timing (83.3%-95.8%; P = 0.001), redosing (62.5%-95.8%, P = 0.003), and duration (47.1%-65.3%; P < 0.002). A multifaceted intervention improved compliance with a pediatric AP guideline.


Resveratrol prevents the wasting disorders of mechanical unloading by acting as a physical exercise mimetic in the rat.
Long-term spaceflight induces hypokinesia and hypodynamia, which, along microgravity per se, result in a number of significant physiological alterations, such as muscle atrophy, force reduction, insulin resistance, substrate use shift from fats to carbohydrates, and bone loss. Each of these adaptations could turn to serious health deterioration during the long-term spaceflight needed for planetary exploration. We hypothesized that resveratrol (RES), a natural polyphenol, could be used as a nutritional countermeasure to prevent muscle metabolic and bone adaptations to 15 d of rat hindlimb unloading. RES treatment maintained a net protein balance, soleus muscle mass, and soleus muscle maximal force contraction. RES also fully maintained soleus mitochondrial capacity to oxidize palmitoyl-carnitine and reversed the decrease of the glutathione vs. glutathione disulfide ratio, a biomarker of oxidative stress. At the molecular level, the protein content of Sirt-1 and COXIV in soleus muscle was also preserved. RES further protected whole-body insulin sensitivity and lipid trafficking and oxidation, and this was likely associated with the maintained expression of FAT/CD36, CPT-1, and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in muscle. Finally, chronic RES supplementation maintained the bone mineral density and strength of the femur. For the first time, we report a simple countermeasure that prevents the deleterious adaptations of the major physiological functions affected by mechanical unloading. RES could thus be envisaged as a nutritional countermeasure for spaceflight but remains to be tested in humans.


Requirement for reduced, unliganded hemoprotein for the hemoglobin- and myoglobin-mediated biotransformation of glyceryl trinitrate.
The biotransformation of glyceryl trinitrate (GTN) by hemoglobin (Hb) and myoglobin (Mb) was assessed using solutions of various forms of the hemoproteins, viz., the oxy-, deoxy-, carbonmonoxy- and met-forms. After incubation with these, GTN loss was observed only with the deoxy-forms of Hb and Mb. The stoichiometry and products of [14C]GTN biotransformation by deoxy-Hb and deoxy-Mb were determined by measuring the formation of [14C]glyceryl dinitrate [14C]GDN), met-Hb (or met-Mb) and inorganic nitrite anion. Biotransformation of GTN involved the oxidation of 2 mol of heme iron (II) per mol of GTN biotransformed to GDN and inorganic nitrite anion. In addition to the formation of GDN, 1 to 2.5% of the radioactivity could not be extracted from the incubation samples. The ratio of 1,2-GDN/1,3-GDN formed during incubation of deoxy-Hb with GTN was 11:1, which indicated a high degree of regioselectivity for the denitration of the nitrate ester group in position 1 or position 3 of GTN. The metabolite ratio obtained for deoxy-Mb incubation with GTN (1,2-GDN/1,3-GDN,3:1) was less than that for deoxy-Hb, which indicated less regioselectivity for the deoxy-Mb-mediated denitration reaction. This could reflect differences in the topography of the heme pocket of the two hemoproteins and steric differences in the GTN-hemoprotein interaction.


Adolescents' perceptions of inpatient postpartum nursing care.
The authors used a transcendental phenomenological approach to describe adolescent mothers' satisfactory and unsatisfactory inpatient postpartum nursing care experiences. They analyzed data from 14 in-depth interviews and found that adolescent mothers' satisfaction is dependent on their perceptions of the nurse's ability to place them "at ease." Nursing care qualities that contributed to satisfactory experiences included nurses' sharing information about themselves, being calm, demonstrating confidence in mothers, speaking to adolescent and adult mothers in the same way, and anticipating unstated needs. Nursing care was perceived to be unsatisfactory when it was too serious, limited to the job required, or different from care to adult mothers, or when nurses failed to recognize individual needs. In extreme cases, unsatisfactory experiences hindered development of an effective nurse-client relationship. These findings illustrate the value of qualitative inquiry for understanding patients' satisfaction with care, can be used for self-reflection, and have implications for nursing education programs.


Safety margins and adaptive capacity of vegetation to climate change.
Vegetation is composed of many individual species whose climatic tolerances can be integrated into spatial analyses of climate change risk. Here, we quantify climate change risk to vegetation at a continental scale by calculating the safety margins for warming and drying (i.e., tolerance to projected change in temperature and precipitation respectively) across plants sharing 100 km × 100 km grid cells (locations). These safety margins measure how much warmer, or drier, a location could become before its 'typical' species exceeds its observed climatic limit. We also analyse the potential adaptive capacity of vegetation to temperature and precipitation change (i.e., likelihood of in situ persistence) using median precipitation and temperature breadth across all species in each location. 47% of vegetation across Australia is potentially at risk from increases in mean annual temperature (MAT) by 2070, with tropical regions most vulnerable. Vegetation at high risk from climate change often also exhibited low adaptive capacity. By contrast, 2% of the continent is at risk from reductions in annual precipitation by 2070. Risk from precipitation change was isolated to the southwest of Western Australia where both the safety margin for drier conditions in the typical species is low, and substantial reductions in MAP are projected.


Resolution of a meningococcal disease outbreak from whole-genome sequence data with rapid Web-based analysis methods.
The increase in the capacity and reduction in cost of whole-genome sequencing methods present the imminent prospect of such data being used routinely in real time for investigations of bacterial disease outbreaks. For this to be realized, however, it is necessary that generic, portable, and robust analysis frameworks be available, which can be readily interpreted and used in real time by microbiologists, clinicians, and public health epidemiologists. We have achieved this with a set of analysis tools integrated into the PubMLST.org website, which can in principle be used for the analysis of any pathogen. The approach is demonstrated with genomic data from isolates obtained during a well-characterized meningococcal disease outbreak at the University of Southampton, United Kingdom, that occurred in 1997. Whole-genome sequence data were collected, de novo assembled, and deposited into the PubMLST Neisseria BIGSdb database, which automatically annotated the sequences. This enabled the immediate and backwards-compatible classification of the isolates with a number of schemes, including the following: conventional, extended, and ribosomal multilocus sequence typing (MLST, eMLST, and rMLST); antigen gene sequence typing (AGST); analysis based on genes conferring antibiotic susceptibility. The isolates were also compared to a reference isolate belonging to the same clonal complex (ST-11) at 1,975 loci. Visualization of the data with the NeighborNet algorithm, implemented in SplitsTree 4 within the PubMLST website, permitted complete resolution of the outbreak and related isolates, demonstrating that multiple closely related but distinct strains were simultaneously present in asymptomatic carriage and disease, with two causing disease and one responsible for the outbreak itself.


An ancient founder mutation in PROKR2 impairs human reproduction.
Congenital gonadotropin-releasing hormone (GnRH) deficiency manifests as absent or incomplete sexual maturation and infertility. Although the disease exhibits marked locus and allelic heterogeneity, with the causal mutations being both rare and private, one causal mutation in the prokineticin receptor, PROKR2 L173R, appears unusually prevalent among GnRH-deficient patients of diverse geographic and ethnic origins. To track the genetic ancestry of PROKR2 L173R, haplotype mapping was performed in 22 unrelated patients with GnRH deficiency carrying L173R and their 30 first-degree relatives. The mutation's age was estimated using a haplotype-decay model. Thirteen subjects were informative and in all of them the mutation was present on the same ~123 kb haplotype whose population frequency is ≤10%. Thus, PROKR2 L173R represents a founder mutation whose age is estimated at approximately 9000 years. Inheritance of PROKR2 L173R-associated GnRH deficiency was complex with highly variable penetrance among carriers, influenced by additional mutations in the other PROKR2 allele (recessive inheritance) or another gene (digenicity). The paradoxical identification of an ancient founder mutation that impairs reproduction has intriguing implications for the inheritance mechanisms of PROKR2 L173R-associated GnRH deficiency and for the relevant processes of evolutionary selection, including potential selective advantages of mutation carriers in genes affecting reproduction.


Malaria mortality in Africa and Asia: evidence from INDEPTH health and demographic surveillance system sites.
Malaria continues to be a major cause of infectious disease mortality in tropical regions. However, deaths from malaria are most often not individually documented, and as a result overall understanding of malaria epidemiology is inadequate. INDEPTH Network members maintain population surveillance in Health and Demographic Surveillance System sites across Africa and Asia, in which individual deaths are followed up with verbal autopsies. To present patterns of malaria mortality determined by verbal autopsy from INDEPTH sites across Africa and Asia, comparing these findings with other relevant information on malaria in the same regions. From a database covering 111,910 deaths over 12,204,043 person-years in 22 sites, in which verbal autopsy data were handled according to the WHO 2012 standard and processed using the InterVA-4 model, over 6,000 deaths were attributed to malaria. The overall period covered was 1992-2012, but two-thirds of the observations related to 2006-2012. These deaths were analysed by site, time period, age group and sex to investigate epidemiological differences in malaria mortality. Rates of malaria mortality varied by 1:10,000 across the sites, with generally low rates in Asia (one site recording no malaria deaths over 0.5 million person-years) and some of the highest rates in West Africa (Nouna, Burkina Faso: 2.47 per 1,000 person-years). Childhood malaria mortality rates were strongly correlated with Malaria Atlas Project estimates of Plasmodium falciparum parasite rates for the same locations. Adult malaria mortality rates, while lower than corresponding childhood rates, were strongly correlated with childhood rates at the site level. The wide variations observed in malaria mortality, which were nevertheless consistent with various other estimates, suggest that population-based registration of deaths using verbal autopsy is a useful approach to understanding the details of malaria epidemiology.


Single-chip electron spin resonance detectors operating at 50GHz, 92GHz, and 146GHz.
We report on the design and characterization of single-chip electron spin resonance (ESR) detectors operating at 50GHz, 92GHz, and 146GHz. The core of the single-chip ESR detectors is an integrated LC-oscillator, formed by a single turn aluminum planar coil, a metal-oxide-metal capacitor, and two metal-oxide semiconductor field effect transistors used as negative resistance network. On the same chip, a second, nominally identical, LC-oscillator together with a mixer and an output buffer are also integrated. Thanks to the slightly asymmetric capacitance of the mixer inputs, a signal at a few hundreds of MHz is obtained at the output of the mixer. The mixer is used for frequency down-conversion, with the aim to obtain an output signal at a frequency easily manageable off-chip. The coil diameters are 120μm, 70μm, and 45μm for the U-band, W-band, and the D-band oscillators, respectively. The experimental frequency noises at 100kHz offset from the carrier are 90Hz/Hz1/2, 300Hz/Hz1/2, and 700Hz/Hz1/2 at 300K, respectively. The ESR spectra are obtained by measuring the frequency variations of the single-chip oscillators as a function of the applied magnetic field. The experimental spin sensitivities, as measured with a sample of α,γ-bisdiphenylene-β-phenylallyl (BDPA)/benzene complex, are 1×108spins/Hz1/2, 4×107spins/Hz1/2, 2×107spins/Hz1/2 at 300K, respectively. We also show the possibility to perform experiments up to 360GHz by means of the higher harmonics in the microwave field produced by the integrated single-chip LC-oscillators.


Effect of exposure to hydrogen peroxide on the virulence of Escherichia coli.
To eliminate pathogenic bacteria, the host presents conditions that are stressful for bacteria. Oxidative stress arises when the concentration of pro-oxidants like hydrogen peroxide (H2O2 ) and superoxide anion increases to a level over the basal defence capacity of the cell. In the present study, we studied the effect of oxidative stress on the production of certain virulence factors by Escherichia coli . E. coli was exposed to oxidative stress by growing in the presence of different concentrations of H2O2 . The effect of oxidative stress on the expression of surface hydrophobicity, adherence, haemolysin production, serum resistance and phagocytosis was studied. Oxidative stress caused a significant decrease in the expression of all the virulence factors of E. coli . Synthesis of virulence factors can be significantly altered by oxidative stress and such changes may affect the pathogenicity of E. coli.


The impact of physician-owned specialty orthopaedic hospitals on surgical volume and case complexity in competing hospitals.
Published studies of physician-owned specialty hospitals have typically examined the impact of these hospitals on disparities, quality, and utilization at a national level. Our objective was to examine the impact of newly opened physician-owned specialty orthopaedic hospitals on individual competing general hospitals. We used Medicare Part A administrative data to identify all physician-owned specialty orthopaedic hospitals performing total hip arthroplasty (THA) and total knee arthroplasty (TKA) between 1991 and 2005. We identified newly opened specialty hospitals in three representative markets (Durham, NC, Kansas City, and Oklahoma City) and assessed their impact on surgical volume and patient case complexity for the five competing general hospitals located closest to each specialty hospital. The average general hospital maintained THA and TKA volume following the opening of the specialty hospitals. The average general hospital also did not experience an increase in patient case complexity. Thus, based on these three markets, we found no clear evidence that entry of physician-owned specialty orthopaedic hospitals resulted in declines in THA or TKA volume or increases in patient case complexity for the average competing general hospital.


Structural modeling of heteromeric protein complexes from disassembly pathways and ion mobility-mass spectrometry.
Structure determination of macromolecular protein assemblies remains a challenge for well-established methods. Here, we provide an assessment of an emerging structural technique, ion mobility-mass spectrometry (IM-MS), and examine the use of collision cross-sections (CCSs), derived from IM-MS, as restraints for structure characterization of heteromeric protein assemblies. Using 15 complexes selected from the Protein Data Bank, we validate the use of low-resolution models by comparing their CCSs with those calculated for all-atom structures. We then select six heteromeric complexes, disrupting them in solution to form subcomplexes. Experimental and calculated CCSs reveal close similarity for 18 of the 21 (sub)complexes. Exploring the use of CCS as a restraint, we incorporate it into a scoring function and show good correlation between the score and similarity to the native structure for heteromers, especially when an additional symmetry restraint was introduced.


Active tuberculosis in Indochinese refugees in British Columbia.
The incidence of active tuberculosis in 8692 Indochinese refugees admitted to British Columbia between 1979 and 1981 was reviewed. In the first 3 months after entry into the province the rate was extremely high--estimated at 1890/100 000 (126 times the provincial average). A large proportion of these cases were of primary or minimal pulmonary tuberculosis. However, although the proportion of cases of minimal pulmonary tuberculosis was twice the provincial average, the proportion of these cases that were confirmed by culture was only one third the provincial average; this suggests some overdiagnosis in this period. In the subsequent 21 months of residence the incidence of active tuberculosis was also high, at 353/100 000, which was more than 20 times the provincial average. The distribution of cases by severity was closer to the provincial distribution in this period, but advanced disease accounted for a far smaller proportion of cases in both periods than it did in 1980 in the entire province.


Heuristics guide the implementation of social preferences in one-shot Prisoner's Dilemma experiments.
Cooperation in one-shot anonymous interactions is a widely documented aspect of human behaviour. Here we shed light on the motivations behind this behaviour by experimentally exploring cooperation in a one-shot continuous-strategy Prisoner's Dilemma (i.e. one-shot two-player Public Goods Game). We examine the distribution of cooperation amounts, and how that distribution varies based on the benefit-to-cost ratio of cooperation (b/c). Interestingly, we find a trimodal distribution at all b/c values investigated. Increasing b/c decreases the fraction of participants engaging in zero cooperation and increases the fraction engaging in maximal cooperation, suggesting a role for efficiency concerns. However, a substantial fraction of participants consistently engage in 50% cooperation regardless of b/c. The presence of these persistent 50% cooperators is surprising, and not easily explained by standard models of social preferences. We present evidence that this behaviour is a result of social preferences guided by simple decision heuristics, rather than the rational examination of payoffs assumed by most social preference models. We also find a strong correlation between play in the Prisoner's Dilemma and in a subsequent Dictator Game, confirming previous findings suggesting a common prosocial motivation underlying altruism and cooperation.


Deliberative process in sharing information with different audiences: Eye-tracking correlates.
Research on conversational pragmatics demonstrates how interlocutors tailor the information they share depending on the audience. Previous research showed that, in informal contexts, speakers often provide several alternative answers, whereas in formal contexts, they tend to give only a single answer; however, the psychological underpinnings of these effects remain obscure. To investigate this answer selection process, we measured participants' eye movements in different experimentally modelled social contexts. Participants answered general knowledge questions by providing responses with either single (one) or plural (three) alternatives. Then, a formal (job interview) or informal (conversation with friends) context was presented and participants decided either to report or withdraw their responses after considering the given social context. Growth curve analysis on the eye movements indicates that the selected response option attracted more eye movements. There was a discrepancy between the answer selection likelihood and the proportion of fixations to the corresponding option-but only in the formal context. These findings support a more elaborate decision-making processes in formal contexts. They also suggest that eye movements do not necessarily accompany the options considered in the decision-making processes.


Dynamic assessment and prediction of learning potential in clients with unilateral neglect.
We investigated the application of dynamic assessment in examining learning potential for adults with right hemisphere stroke and unilateral neglect. Forty adults with right cerebrovascular accident were randomly assigned to a control or dynamic group. Both groups were administered standard pretests and posttests of unilateral neglect. The dynamic group received cues, strategy training, and feedback during an object search task. Multivariate analysis of covariance indicated significant differences between groups on the object search task, with reduced unilateral neglect in the dynamic group. Greater initiation of left-sided search, strategy use, and near and intermediate transfer of learning were observed in the dynamic group. The brief dynamic assessment, developed for this study, provided an opportunity to observe the client's ability to incorporate strategy training and feedback across visual search tasks. This assessment approach has the potential to characterize learning profiles and to guide intervention decisions.


Programmed cell death-1 deficiency exacerbates T cell activation and atherogenesis despite expansion of regulatory T cells in atherosclerosis-prone mice.
T cell activation represents a double-edged sword in atherogenesis, as it promotes both pro-inflammatory T cell activation and atheroprotective Foxp3(+) regulatory T cell (Treg) responses. Here, we investigated the role of the co-inhibitory receptor programmed cell death-1 (PD-1) in T cell activation and CD4(+) T cell polarization towards pro-atherogenic or atheroprotective responses in mice. Mice deficient for both low density lipoprotein receptor and PD-1 (Ldlr(-/-)Pd1(-/-)) displayed striking increases in systemic CD4(+) and CD8(+) T cell activation after 9 weeks of high fat diet feeding, associated with an expansion of both pro-atherogenic IFNγ-secreting T helper 1 cells and atheroprotective Foxp3+ Tregs. Importantly, PD-1 deficiency did not affect Treg suppressive function in vitro. Notably, PD-1 deficiency exacerbated atherosclerotic lesion growth and entailed a massive infiltration of T cells in atherosclerotic lesions. In addition, aggravated hypercholesterolemia was observed in Ldlr(-/-)Pd1(-/-) mice. In conclusion, we here demonstrate that although disruption of PD-1 signaling enhances both pro- and anti-atherogenic T cell responses in Ldlr(-/-) mice, pro-inflammatory T cell activation prevails and enhances dyslipidemia, vascular inflammation and atherosclerosis.


Estrogen regulates the rate of bone turnover but bone balance in ovariectomized rats is modulated by prevailing mechanical strain.
Estrogen deficiency induced bone loss is associated with increased bone turnover in rats and humans. The respective roles of increased bone turnover and altered balance between bone formation and bone resorption in mediating estrogen deficiency-induced cancellous bone loss was investigated in ovariectomized rats. Ovariectomy resulted in increased bone turnover in the distal femur. However, cancellous bone was preferentially lost in the metaphysis, a site that normally experiences low strain energy. No bone loss was observed in the epiphysis, a site experiencing higher strain energy. The role of mechanical strain in maintaining bone balance was investigated by altering the strain history. Mechanical strain was increased and decreased in long bones of ovariectomized rats by treadmill exercise and functional unloading, respectively. Functional unloading was achieved during orbital spaceflight and following unilateral sciatic neurotomy. Increasing mechanical loading reduced bone loss in the metaphysis. In contrast, decreasing loading accentuated bone loss in the metaphysis and resulted in bone loss in the epiphysis. Finally, administration of estrogen to ovariectomized rats reduced bone loss in the unloaded and prevented loss in the loaded limb following unilateral sciatic neurotomy in part by reducing indices of bone turnover. These results suggest that estrogen regulates the rate of bone turnover, but the overall balance between bone formation and bone resorption is influenced by prevailing levels of mechanical strain.


Moulding calixarenes for biomacromolecule targeting.
After their successful use as a preorganized platform for the preparation of receptors for metal ions and small neutral molecules over the last 15 years, calixarenes are enjoying a renaissance of popularity as scaffolds for ligands that are able to efficiently and selectively target macromolecules such as proteins/enzymes, nucleic acids and lipids. This feature article summarizes the peculiar factors characterizing the calixarene structure and properties, as well as outlines the main rules that can be used to turn such macrocycles into efficient and successful ligands for these classes of biomacromolecules. Factors that affect the multivalent properties of calixarenes, such as the size, conformation and stereochemical presentation of binding groups or their amphiphilicity and hybrid character, are described in detail with the use of a few selected examples from the literature. Perspectives and applications of these ligands in bionanotechnology and nanomedicine, such as protein sensing and inhibition, gene-delivery, targeted drug-delivery and cell imaging, are also discussed.


Local biotic environment shapes the spatial scale of bacteriophage adaptation to bacteria.
The ecological, epidemiological, and evolutionary consequences of host-parasite interactions are critically shaped by the spatial scale at which parasites adapt to hosts. The scale of interaction between hyperparasites and their parasites is likely to be influenced by the host of the parasite and potentially likely to differ among within-host environments. Here we examine the scale at which bacteriophages adapt to their host bacteria by studying natural isolates from the surface or interior of horse chestnut leaves. We find that phages are more infective to bacteria from the same tree relative to those from other trees but do not differ in infectivity to bacteria from different leaves within the same tree. The results suggest that phages target common bacterial species, including an important plant pathogen, within plant host tissues; this result has important implications for therapeutic phage epidemiology. Furthermore, we show that phages from the leaf interior are more infective to their local hosts than phages from the leaf surface are to theirs, suggesting either increased resistance of bacteria on the leaf surface or increased phage adaptation within the leaf. These results highlight that biotic environment can play a key role in shaping the spatial scale of parasite adaptation and influencing the outcome of coevolutionary interactions.


Frequency-bin entangled comb of photon pairs from a Silicon-on-Insulator micro-resonator.
We present a quantum-mechanical theory to describe narrowband photon-pair generation via four-wave mixing in a Silicon-on-Insulator (SOI) micro-resonator. We also provide design principles for efficient photon-pair generation in an SOI micro-resonator through extensive numerical simulations. Microring cavities are shown to have a much wider dispersion-compensated frequency range than straight cavities. A microring with an inner radius of 8 μm can output an entangled photon comb of 21 pairwise-correlated peaks (42 comb lines) spanning from 1.3 μm to 1.8 μm. Such on-chip quantum photonic devices offer a path toward future integrated quantum photonics and quantum integrated circuits.


The effect of fibrin glue in preventing staple-line leak after sleeve gastrectomy. An experimental study in rats.
To evaluate the effect of fibrin glue on staple-line leak after sleeve gastrectomy. Fourteen adult wistar rats 300 gr were randomized into two groups: Control group (n=7) and study group (n=7). All the rats underwent sleeve gastrectomy using lineer stapler. In the study group, fibrin glue was used to reinforce the staple-line. The rats were sacrificed 7 days after surgery. The stomach was resected, submerged in saline and exposed to excess pressure to obtain a burst pressure value. The gastric staple line was evaluated histopathologically according to the Ehrlich Hunt scale. The results of the two groups were compared. The mean Ehrlich-Hunt scores for inflammation, fibroblastic activity and neo-angiogenesis were similar between the groups (p>0.05). Collagen deposition was significantly higher in study group (3.42±0.53) when compared with control group (2.57±0.78) (p=0.035). The mean burst pressure was 137.8±8.5 mmHg for control group and 135.0±8.1 mmHg for study group (p=0.536). Reinforcement of the staple-line with fibrin glue has no effect on the burst pressure after sleeve gastrectomy. More studies are needed to evaluate the precautions against leak after sleeve gastrectomy.


Statistics in physiology and pharmacology: a slow and erratic learning curve.
1. Learning how to apply statistical analyses to the results of experimental or clinical studies may take a lifetime of trial (and sometimes error), as it has done in the author's case. There is no evidence that biomedical investigators of the present generation are on a steeper learning curve. Gross misunderstandings of the purpose and functions of statistical analysis are apparent in applications to research grant-giving bodies and ethics committees, in manuscripts submitted to journals and sometimes in published papers. 2. Although estimation of minimal group (sample) size for a given power is an essential step in planning clinical studies, it seems to be used rarely in laboratory experimental work. This is despite exhortations to restrict the number of animals used to a minimum. 3. Most investigators use hypothesis testing to analyse their results, but their understanding of the meaning of the resultant P-values is slight. 4. A flaw found almost universally in biomedical manuscripts is to make multiple inferences from the results of a single study. The goal of statistical analysis is to maintain the familywise type I error rate (risk of false-positive inference) at a predetermined level (usually 5%). But, when multiple inferences are made from the same experiment, the risk of false-positive error is inflated. There are two solutions to this problem: (i) use a multiple comparison procedure to control the familywise type I error rate; and (ii) test a single, global hypothesis. 5. Biomedical investigators have been quick to acquire computer statistics software and to use it to analyse their experiments. However, they have been slow to recognize the limitations of this software. These include: (i) inadequate documentation of routines, so that neither the user nor the reader of published papers can be sure how the tests have been executed; (ii) flawed algorithms for the execution of statistical procedures; and (iii) failure to recognize that the best software for their purposes is that which takes them just beyond their statistical horizons. 6. The obvious solution to these difficulties is to recruit a biomedical statistician into every research group, at a relatively trivial cost. However, properly qualified biostatisticians are in desperately short supply in Australia. It follows that research groups, national grant-giving agencies and academic institutions must make provision for the proper training and subsequent employment of biostatisticians.


Idealized computational models for auditory receptive fields.
We present a theory by which idealized models of auditory receptive fields can be derived in a principled axiomatic manner, from a set of structural properties to (i) enable invariance of receptive field responses under natural sound transformations and (ii) ensure internal consistency between spectro-temporal receptive fields at different temporal and spectral scales. For defining a time-frequency transformation of a purely temporal sound signal, it is shown that the framework allows for a new way of deriving the Gabor and Gammatone filters as well as a novel family of generalized Gammatone filters, with additional degrees of freedom to obtain different trade-offs between the spectral selectivity and the temporal delay of time-causal temporal window functions. When applied to the definition of a second-layer of receptive fields from a spectrogram, it is shown that the framework leads to two canonical families of spectro-temporal receptive fields, in terms of spectro-temporal derivatives of either spectro-temporal Gaussian kernels for non-causal time or a cascade of time-causal first-order integrators over the temporal domain and a Gaussian filter over the logspectral domain. For each filter family, the spectro-temporal receptive fields can be either separable over the time-frequency domain or be adapted to local glissando transformations that represent variations in logarithmic frequencies over time. Within each domain of either non-causal or time-causal time, these receptive field families are derived by uniqueness from the assumptions. It is demonstrated how the presented framework allows for computation of basic auditory features for audio processing and that it leads to predictions about auditory receptive fields with good qualitative similarity to biological receptive fields measured in the inferior colliculus (ICC) and primary auditory cortex (A1) of mammals.


Search for technicolor particles produced in association with a W Boson at CDF.
We present a search for the technicolor particles rho{T} and pi_{T} in the process pp-->rho{T}-->Wpi{T} at a center of mass energy of sqrt[s]=1.96 TeV. The search uses a data sample corresponding to approximately 1.9 fb{-1} of integrated luminosity accumulated by the CDF II detector at the Fermilab Tevatron. The event signature we consider is W-->lnu and pi{T}-->bb, bc or bu depending on the pi{T} charge. We select events with a single high-p{T} electron or muon, large missing transverse energy, and two jets. Jets corresponding to bottom quarks are identified with multiple b-tagging algorithms. The observed number of events and the invariant mass distributions are consistent with the standard model background expectations, and we exclude a region at 95% confidence level in the rho{T}-pi{T} mass plane. As a result, a large fraction of the region m(rho{T})=180-250 GeV/c{2} and m(pi{T})=95-145 GeV/c{2} is excluded.


Perforin and Fas cytolytic pathways coordinately shape the selection and diversity of CD8+-T-cell escape variants of influenza virus.
Antigenic variation is a viral strategy exploited to promote survival in the face of the host immune response and represents a major challenge for efficient vaccine development. Influenza viruses are pathogens with high transmissibility and mutation rates, enabling viral escape from immunity induced by prior infection or vaccination. Intense selection from neutralizing antibody drives antigenic changes in the surface glycoproteins, resulting in emergence of new strains able to reinfect hosts immune to previously circulating viruses. CD8+ cytotoxic T cells (CTLs) also provide protective immunity from influenza virus infection and may contribute to the antigenic evolution of influenza viruses. Utilizing mice transgenic for an influenza virus NP366-374 peptide-specific T-cell receptor, we demonstrated that the respiratory tract is a suitable site for generation of escape variants of influenza virus selected by CTL in vivo. In this report the contributions of the perforin and Fas pathways utilized by influenza virus-specific CTLs in viral clearance and selection of CTL escape variants have been evaluated. While transgenic CTLs deficient in either perforin- or Fas-mediated pathways are efficient in initial pulmonary viral control, variant virus emergence was observed in all the mice studied, although the spectrum of viral CTL escape variants selected varied profoundly. Thus, a less-restricted repertoire of escape variants was observed in mice with an intact perforin cytotoxic pathway compared with a limited variant diversity in perforin pathway-deficient mice, although maximal variant diversity was observed in mice having both Fas and perforin pathways intact. We conclude that selection of viral CTL escape variants reflects coordinate action between the tightly controlled perforin/granzyme pathway and the more promiscuous Fas/FasL pathway.


Increased deposition of chondroitin/dermatan sulfate glycosaminoglycan and upregulation of β1,3-glucuronosyltransferase I in pulmonary fibrosis.
Pulmonary fibrosis (PF) is characterized by increased deposition of proteoglycans (PGs), in particular core proteins. Glycosaminoglycans (GAGs) are key players in tissue repair and fibrosis, and we investigated whether PF is associated with changes in the expression and structure of GAGs as well as in the expression of β1,3-glucuronosyltransferase I (GlcAT-I), a rate-limiting enzyme in GAG synthesis. Lung biopsies from idiopathic pulmonary fibrosis (IPF) patients and lung tissue from a rat model of bleomycin (BLM)-induced PF were immunostained for chondroitin sulfated-GAGs and GlcAT-I expression. Alterations in disaccharide composition and sulfation of chondroitin/dermatan sulfate (CS/DS) were evaluated by fluorophore-assisted carbohydrate electrophoresis (FACE) in BLM rats. Lung fibroblasts isolated from control (saline-instilled) or BLM rat lungs were assessed for GAG structure and GlcAT-I expression. Disaccharide analysis showed that 4- and 6-sulfated disaccharides were increased in the lungs and lung fibroblasts obtained from fibrotic rats compared with controls. Fibrotic lung fibroblasts and transforming growth factor-β(1) (TGF-β(1))-treated normal lung fibroblasts expressed increased amounts of hyaluronan and 4- and 6-sulfated chondroitin, and neutralizing anti-TGF-β(1) antibody diminished the same. TGF-β(1) upregulated GlcAT-I and versican expression in lung fibroblasts, and signaling through TGF-β type I receptor/p38 MAPK was required for TGF-β(1)-mediated GlcAT-I and CS-GAG expression in fibroblasts. Our data show for the first time increased expression of CS-GAGs and GlcAT-I in IPF, fibrotic rat lungs, and fibrotic lung fibroblasts. These data suggest that alterations of sulfation isomers of CS/DS and upregulation of GlcAT-I contribute to the pathological PG-GAG accumulation in PF.


Improvements in functional capacity from Nordic walking: a randomized-controlled trial among elderly people.
This study examined the effects of an instructed structured Nordic walking (NW) exercise program on the functional capacity of older sedentary people. Volunteers were randomly assigned to an NW group (68.2 ± 3.8 yr old) or control group (69.9 ± 3.0 yr old). Before and at the end of the 9-wk intervention, functional tests and 2-dimensional ground-reaction-force (GRF) patterns of normal (1.40 m/s) and fast (1.94 m/s) walking speeds were measured. The intervention included a 60-min supervised NW session on an inside track twice a week for 9 wk. The mean changes in functional tests differed between groups significantly. Gait analyses showed no significant differences between the groups on any GRF parameters for walking speed either before or after the intervention. The study showed that NW has favorable effects on functional capacity in older people and is a suitable form of exercise for them.


Proton-nuclear-magnetic-resonance studies of serum, plasma and urine from fasting normal and diabetic subjects.
Resonances for the ketone bodies 3-D-hydroxybutyrate, acetone and acetoacetate are readily detected in serum, plasma and urine samples from fasting and diabetic subjects by 1H n.m.r. spectroscopy at 400 MHz. Besides the simultaneous observation of metabolites, the major advantage of n.m.r. is that little or no pretreatment of samples is required. N.m.r. determinations of 3-D-hydroxybutyrate, acetoacetate, lactate, valine and alanine were compared with determinations made with conventional assays at six 2-hourly intervals after insulin withdrawal from a diabetic subject. The n.m.r. results closely paralleled those of other assays although, by n.m.r., acetoacetate levels continued to rise rather than reaching a plateau 4h after insulin withdrawal. The 3-D-hydroxybutyrate/acetoacetate ratio in urine during withdrawal gradually increased to the value observed in plasma (3.0 +/- 0.2) as determined by n.m.r. The acetoacetate/acetone ratio in urine (17 +/- 6) was much higher than in plasma (2.5 +/- 0.7). Depletion of a mobile pool of fatty acids in plasma during fasting, as seen by n.m.r., paralleled that seen during insulin withdrawal. These fatty acids were thought to be largely in chylomicrons, acylglycerols and lipoproteins, and were grossly elevated in plasma samples from a non-insulin-dependent diabetic and in cases of known hyperlipidaemia.


The pore of voltage-gated potassium ion channels is strained when closed.
Voltage-gated potassium channels form potassium-selective pores in cell membranes. They open or close in response to changes in the transmembrane potential and are essential for generating action potentials, and thus for the functioning of heart and brain. While a mechanism for how these channels close has been proposed, it is not clear what drives their opening. Here we use free energy molecular dynamics simulations to show that work must be done on the pore to reduce the kink in the pore-lining (S6) α-helices, thereby forming the helix bundle crossing and closing the channel. Strain is built up as the pore closes, which subsequently drives opening. We also determine the effect of mutating the PVPV motif that causes the kink in the S6 helix. Finally, an approximate upper limit on how far the S4 helix is displaced as the pore closes is estimated.


Nanostructured multilayer polyelectrolyte films with silver nanoparticles as antibacterial coatings.
Ultrathin polyelectrolyte films containing silver nanoparticles appear to be a promising material for antimicrobial coatings used in the medical area. The present work is focused on the formation of multilayer polyelectrolyte films using: polyethyleneimine (PEI) as polycation, Poly(sodium 4-styrenesulfonate) (PSS) as polyanions and negatively charged silver nanoparticles (AgNPs), which led to the polyelectrolyte-silver nanocomposite coatings. The film thickness and mass were measured by ellipsometry and quartz crystal microbalance with dissipation monitoring (QCM-D) and the structure and morphology of films were visualized using scanning electron microscopy (SEM). Systematic increase of the UV-Vis absorption confirmed formation of the consecutive layers of the film. The analysis of bacteria cell adhesion to films surface was done by the luminometry measurement. Three gram-negative bacterial strains with strong adhesive properties were used in this study: Escherichia coli, Aeromonas hydrophila, and Asaia lannenesis. It was found that nanocomposite films have antimicrobial properties, which makes them very interesting for a number of practical applications, e.g. for the prevention of microbial colonization on treated surfaces.


The role of interferon in spontaneous cell-mediated cytotoxicity in pigs.
Specific release of 51Cr and the production of interferon (IFN) increased in parallel in a spontaneous cell-mediated cytotoxicity (SCMC) assay in which uninfected PK-15 cells or PK-15 cells persistently infected with transmissible gastroenteritis virus (PK-15-TGE cells) were used as targets, and peripheral blood lymphocytes (PBL) from a young adult pig were used as effector cells. Higher levels of both specific 51Cr release and IFN were obtained in the assays containing PK-15-TGE cells. Co-cultivation of PBL from newborn piglets with PK-15-TGE cells yielded similar levels of IFN to those produced by co-cultivation of adult PBL and PK-15-TGE cells, but lower levels of IFN were produced by co-cultivation with uninfected PK-15 cells. Pretreatment of adult PBL with IFN augmented their SCMC effector activity for both PK-15 and PK-15-TGE cells in the 51Cr release assay. Pretreatment of the PK-15-TGE target cells with IFN did not affect their release of 51Cr in the SCMC reaction, while IFN pretreatment of PK-15 targets protected them against SCMC. In a single cell cytotoxicity assay the effects of IFN pretreatment on the effector adult PBL and on the PK-15 and PK-15-TGE target cells were confirmed, and SCMC incompetent PBL from neonatal piglets were rendered cytotoxic by pretreatment with IFN. PBL from newborn piglets bound to either target cell with the same frequency as PBL from SCMC competent adult pigs, and IFN pretreatment of either effector or target cells had no effect on target-binding frequency.


Osteoporosis intervention in ambulatory patients with previous hip fracture: a multicentric, nationwide Italian survey.
Our study investigated the patterns of treatment and adherence to prescribed therapies in 2,191 ambulatory patients with previous hip osteoporotic fractures at 207 participating orthopedic centers throughout Italy. All patients who came to the attention of the involved orthopedic surgeons were administered a questionnaire investigating: age, sex, height, weight, date of admission and length of stay in the hospital, other previous clinical fractures, bone density or biochemical testing concerning mineral metabolism, treatment with bone-active drugs in the six months before the fracture, treatment after discharge from the hospital, continuous use of prescribed drugs, pain at the site of hip surgery, and comorbidity. A multivariate logistic regression model was applied, considering a subset of the variables in the questionnaire, in order to determine the factors that significantly influenced discontinuation of treatment after hip fracture. Among the patients, 88.1% were female and 86.2% of the subjects were older than 65. The mean length of hospital stay for hip fracture was 19.0+/-25.3 days. At the time of interview, the mean time elapsed since hospitalization was 542.9+/-1,197.3 days. A previous clinical fracture was referred by 20.2% of patients. Before hip fracture occurrence, 52.8% of patients had never received any kind of treatment, and this figure reached 80% if we also included those who had taken only calcium and/or vitamin D. Corresponding proportions after fracture were 22% and 31.3%, respectively. Finally, 52% of patients had stopped treatment given for osteoporosis after a mean period of 1.4 years. According to the results of the logistic regression, increasing age, pain [odds ratio (OR): 1.36; 95% confidence interval (CI): 1.21-1.65] and no use of diagnostic tests (OR: 2.46; CI: 1.79-3.37) showed a positive effect on the probability of quitting the medication. On the other hand, being female reduces by half (OR: 0.49; CI: 0.37-0.45) the probability of quitting medication. Our data showed a low rate of primary prevention, a still insufficient post-fracture therapy, along with a high rate of early discontinuation of osteoporosis medication in patients with previous hip fracture.


Representation and detection of deformable shapes.
We describe some techniques that can be used to represent and detect deformable shapes in images. The main difficulty with deformable template models is the very large or infinite number of possible nonrigid transformations of the templates. This makes the problem of finding an optimal match of a deformable template to an image incredibly hard. Using a new representation for deformable shapes, we show how to efficiently find a global optimal solution to the nonrigid matching problem. The representation is based on the description of objects using triangulated polygons. Our matching algorithm can minimize a large class of energy functions, making it applicable to a wide range of problems. We present experimental results of detecting shapes in medical images and images of natural scenes. Our method does not depend on initialization and is very robust, yielding good matches even in images with high clutter. We also consider the problem of learning a nonrigid shape model for a class of objects from examples. We show how to learn good models while constraining them to be in the form required by the matching algorithm.


Caustic merit function for optical design.
The two caustic surfaces, which are in turn the loci of the image points, are formed in the image region by each element of the source of an optical system. Lens optimization is achieved herein by adjusting parameters so that the caustic surfaces formed by rays from selected points of the object coalesce and converge to the corresponding Gaussian image points. A merit function, specifying the sum of the distances between each caustic surface and the corresponding Gaussians image, is defined and minimized subject to the constraints that the system focal length and physical length are constant and that the third-order aberrations of the system are zero. An optimization procedure, based on minimizing the caustic merit function, is used to design a singlet, doublet, and triplet lens system.