CO2 Hydration Shell Structure and Transformation.
The hydration-shell of CO2 is characterized using Raman multivariate curve resolution (Raman-MCR) spectroscopy combined with ab initio molecular dynamics (AIMD) vibrational density of states simulations, to validate our assignment of the experimentally observed high-frequency OH band to a weak hydrogen bond between water and CO2. Our results reveal that while the hydration-shell of CO2 is highly tetrahedral, it is also occasionally disrupted by the presence of entropically stabilized defects associated with the CO2-water hydrogen bond. Moreover, we find that the hydration-shell of CO2 undergoes a temperature-dependent structural transformation to a highly disordered (less tetrahedral) structure, reminiscent of the transformation that takes place at higher temperatures around much larger oily molecules. The biological significance of the CO2 hydration shell structural transformation is suggested by the fact that it takes place near physiological temperatures.


Response of pest control by generalist predators to local-scale plant diversity: a meta-analysis.
Disentangling the effects of plant diversity on the control of herbivores is important for understanding agricultural sustainability. Recent studies have investigated the relationships between plant diversity and arthropod communities at the landscape scale, but few have done so at the local scale. We conducted a meta-analysis of 32 papers containing 175 independent measures of the relationship between plant diversity and arthropod communities. We found that generalist predators had a strong positive response to plant diversity, that is, their abundance increased as plant diversity increased. Herbivores, in contrast, had an overall weak and negative response to plant diversity. However, specialist and generalist herbivores differed in their response to plant diversity, that is, the response was negative for specialists and not significant for generalists. While the effects of scale remain unclear, the response to plant diversity tended to increase for specialist herbivores, but decrease for generalist herbivores as the scale increased. There was no clear effect of scale on the response of generalist predators to plant diversity. Our results suggest that the response of herbivores to plant diversity at the local scale is a balance between habitat and trophic effects that vary according to arthropod specialization and habitat type. Synthesis and applications. Positive effects of plant diversity on generalist predators confirm that, at the local scale, plant diversification of agroecosystems is a credible and promising option for increasing pest regulation. Results from our meta-analysis suggest that natural control in plant-diversified systems is more likely to occur for specialist than for generalist herbivores. In terms of pest management, our results indicate that small-scale plant diversification (via the planting of cover crops or intercrops and reduced weed management) is likely to increase the control of specialist herbivores by generalist predators.


An anatomical and biomechanical comparison of anteromedial and anterolateral approaches for tibial tunnel of posterior cruciate ligament reconstruction: evaluation of the widening effect of the anterolateral approach.
An anterolateral approach to the tibial tunnel of posterior cruciate ligament reconstruction is used to reduce the sharpness of the graft-tunnel angle, the so-called killer turn effect. However, with the anterolateral approach, the tunnel might be widened into an ovoid shape because of the small angle between the tunnel and the anterolateral cortex. The fixation strength of the posterior cruciate ligament graft in the tibial tunnel will be weaker in the anterolateral approach compared with the anteromedial approach. Controlled laboratory study. Twenty paired cadaveric tibias were used. Tibial tunnels were made using following approaches: an anteromedial approach for 10 tibias and an anterolateral approach for 10 tibias. The anterior cortex-tunnel angle and the diameter of the tunnel entrance were measured by 2-dimensional computed tomographic scans. After fixation of the Achilles tendon allograft with a biodegradable screw, the maximal strength of the graft at failure was measured using a materials testing machine. The mean cortex-tunnel angle was 47.5 degrees +/- 9.3 degrees in the anteromedial approach group and 28.3 degrees +/- 7.4 degrees in the anterolateral approach group. The mean long diameter of the tunnels in the anteromedial approach group was 10.6 +/- 1.0 mm and in the anterolateral approach group it was 14.0 +/- 1.5 mm. These two parameters showed statistically significant differences between the 2 groups (P < .01). The mean maximum load at failure for the anteromedial approach group was 385.4 +/- 139.7 N, and for the anterolateral approach group it was 225.1 +/- 144.1 N. This difference was statistically significant (P = .021). The anterolateral approach resulted in a tunnel with a wider entrance, a more acute cortex-tunnel angle, and a lower maximal load at failure compared with tunnels created using the anteromedial approach. The use of additional fixation methods, such as post ties or ligament washers and screws, should be considered when using an anterolateral approach for tibial tunnel of posterior cruciate ligament reconstruction.


Autosomal dominant nocturnal frontal lobe epilepsy. A video-polysomnographic and genetic appraisal of 40 patients and delineation of the epileptic syndrome.
A number of clinical and aetiological studies have been performed, during the last 30 years, on patients with abnormal nocturnal motor and behavioural phenomena. The aetiological conclusions of these studies were often conflicting, suggesting either an epileptic or a non-epileptic origin. Among the clinical characteristics of these patients, the familial clustering was one thoroughly accepted. A nocturnal familial form of frontal lobe epilepsy (autosomal dominant nocturnal frontal lobe epilepsy, ADNFLE), often misdiagnosed as parasomnia, has been recently described in some families. In one large Australian kindred, a missense mutation in the second transmembrane domain of the neuronal nicotinic acetylcholine receptor alpha 4 subunit (CHRNA4) gene, located on chromosome 20 q13.2-13.3, has been reported to be associated with nocturnal frontal lobe epilepsy. We performed an extensive clinical and video-polysomnographic study in 40 patients complaining of repeated abnormal nocturnal motor and/or behavioural phenomena, from 30 unrelated Italian families. Thirty-eight patients had an electroclinical picture strongly suggesting the diagnosis of ADNFLE. They had a wide clinical spectrum, ranging from nocturnal enuresis to sleep-related violent behaviour, thus including all the main features of the so-called 'typical' parasomnias. The video-polysomnographic recording confirmed the wide spectrum of abnormal manifestations, including sudden awakenings with dystonic/ dyskinetic movements (in 42.1% of patients), complex behaviours (13.2%) and sleep-related violent behaviour (5.3%). The EEG findings showed ictal epileptiform abnormalities predominantly over frontal areas in 31.6% of patients. In another 47.4% of patients the EEG showed ictal rhythmic slow activity over anterior areas. Only 18.4% of the patients had already received a correct diagnosis of epilepsy. In 73.3% of the patients treated with anti-epileptic drugs the seizures were readily controlled. Pedigree analysis on 28 of the families was consistent with autosomal dominant transmission with reduced penetrance (81%). DNAs from 20 representative affected individuals were sequenced in order to check for the presence of the missense mutation in the CHRNA4 gene found in the Australian kindred affected by ADNFLE. Nucleotide sequence analysis did not reveal the presence of this mutation, but it did confirm the presence of two other base substitutions, not leading to amino acid changes. These two intragenic polymorphisms, together with a closely linked restriction fragment length polymorphism at the D20S20 locus, have been used for linkage analysis of ADNFLE to the terminal region of the long arm of chromosome 20 in five compliant families. The results allowed us to exclude linkage of ADNFLE to this chromosomal region in these families, thus confirming the locus heterogeneity of the disorder. Large and full video-polysomnographical studies are of the utmost importance in order to clarify the real prevalence of both nocturnal frontal lobe epilepsy and parasomnias, and to provide a correct therapy.


Daphnia as a Sentinel Species for Environmental Health Protection: A Perspective on Biomonitoring and Bioremediation of Chemical Pollution.
Despite available technology and the knowledge that chemical pollution damages human and ecosystem health, chemical pollution remains rampant, ineffectively monitored, rarely prevented, and only occasionally mitigated. We present a framework that helps address current major challenges in the monitoring and assessment of chemical pollution by broadening the use of the sentinel species Daphnia as a diagnostic agent of water pollution. And where prevention has failed, we propose the application of Daphnia as a bioremediation agent to help reduce hazards from chemical mixtures in the environment. By applying "omics" technologies to Daphnia exposed to real-world ambient chemical mixtures, we show improvements at detecting bioactive components of chemical mixtures, determining the potential effects of untested chemicals within mixtures, and identifying targets of toxicity. We also show that using Daphnia strains that naturally adapted to chemical pollution as removal agents of ambient chemical mixtures can sustainably improve environmental health protection. Expanding the use of Daphnia beyond its current applications in regulatory toxicology has the potential to improve both the assessment and the remediation of environmental pollution.


Alamandine attenuates arterial remodelling induced by transverse aortic constriction in mice.
Aims: The renin-angiotensin system (RAS) plays an important role in the pathophysiology of vascular diseases, especially as a mediator of inflammation and tissue remodelling. Alamandine (Ala1-angiotensin-(1-7)) is a new biologically active peptide from the RAS, interacting with Mas-related G-protein-coupled receptor member D. Although a growing number of studies reveal the cardioprotective effects of alamandine, there is a paucity of data on its participation in vascular remodelling associated events. In the present study, we investigated the effects of alamandine on ascending aorta remodelling after transverse aortic constriction (TAC) in mice. Methods and results: C57BL/6J male mice were divided into the following groups: Sham (sham-operated), TAC (operated) and TAC+ALA (operated and treated with alamandine-HPβCD (2-Hydroxypropyl-β-cyclodextrin), 30 μg/kg/day, by gavage). Oral administration of alamandine for 14 days attenuated arterial remodelling by decreasing ascending aorta media layer thickness and the cells density in the adventitia induced by TAC. Alamandine administration attenuated ascending aorta fibrosis induced by TAC, through a reduction in the following parameters; total collagen deposition, expression collagen III and transforming growth factor-β (TGF-β) transcripts, matrix metalloproteinases (MMPs) activity and vascular expression of MMP-2. Importantly, alamandine decreased vascular expression of proinflammatory genes as CCL2, tumour necrosis factor α (TNF-α) and interleukin-1β (IL-1β), and was able to increase expression of MRC1 and FIZZ1, pro-resolution markers, after TAC surgery. Conclusion: Alamandine treatment attenuates vascular remodelling after TAC, at least in part, through anti-fibrotic and anti-inflammatory effects. Hence, this work opens new avenues for the use of this heptapeptide also as a therapeutic target for vascular disease.


Matrix Metalloproteinase-sensitive Multistage Nanogels Promote Drug Transport in 3D Tumor Model.
Physiological barriers inside of tumor tissue often result in poor interstitial penetration and heterogeneous intratumoral distribution of nanoparticle-based drug delivery systems (DDS). Novel, matrix metalloproteinase (MMP)-sensitive peptide-crosslinked nanogels (pNGs) as multistage DDS are reported with a beneficial size reduction property to promote the process of deep tissue penetration. Methods: The presented pNGs are based on a dendritic polyglycerol (dPG) scaffold crosslinked by a modified MMP-sensitive fluorogenic peptide. The crosslinker integrates degradability in response to proteases present in the tumor microenvironment. Surfactant-free, inverse nanoprecipitation is employed to prepare the nanogels using strain-promoted click chemistry. The size and crosslinking density of the pNGs are controlled by the functionalization degree of dPG with cyclooctyne groups and by the peptide crosslinker fraction. The intrinsic reporter moiety of the crosslinker was used to study the influence of pNG compositions on the degradation profile. The therapeutic drug Doxorubicin was conjugated through a pH-sensitive linkage to dPG to form a multistage DDS. The penetration behavior of the pNGs was studied using agarose matrix and multicellular tumor spheroids (MCTS). Results: Nanogel sizes were controlled in the range of 150-650 nm with narrow size distributions and varying degrees of crosslinking. The pNGs showed stability in PBS and cell media but were readily degraded in the presence of MMP-7. The crosslinking density influenced the degradation kinetic mediated by MMP-7 or cells. Stable conjugation of DOX at physiological pH and controlled drug release at acidic pH were observed. The digestions of nanogels lead to a size reduction to polymer-drug fragments which efficiently penetrated into agarose gels. Moreover, the degradable multistage pNGs demonstrated deeper penetration into MCTS as compared to their non-degradable counterparts. Thus, degradable pNGs were able to deliver their cargo and efficiently reduce the cell viability in MCTS. Conclusion: The triggered size reduction of the pNGs by enzymatic degradation can facilitate the infiltration of the nanocarrier into dense tissue, and thereby promote the delivery of its cargo.


Chronic migraine in the population: burden, diagnosis, and satisfaction with treatment.
To evaluate the disability profile and patterns of treatment and health care use for chronic migraine (CM) in the general population, in contrast to episodic migraine. We identified 24,000 headache sufferers, drawn from more than 165,000 individuals representative of the US population. This sample has been followed up with annual surveys using validated questionnaires for the diagnosis of episodic migraine and CM. As a part of the survey, subjects were asked to report the specific medications currently used for their most severe headaches, as well as level of satisfaction with treatment. Our sample consisted of 520 individuals with CM and 9,424 with episodic migraine. Over a 3-month period, more than half of the individuals with CM missed at least 5 days of household work, compared with 24.3% of those with episodic migraine (p < 0.001). Reduced productivity in household work for at least 5 days over 3 months was reported by 58.1% and 18.2% (p < 0.001); at least 5 days of missed family activities was reported by 36.9% and 9.5% (p < 0.001). The majority of the CM sufferers (87.6%) had previously sought care to discuss their headaches with a health professional. Migraine-specific acute treatments were used by 31.6% of respondents with CM and 24.8% with episodic migraine. Around 48% of the individuals with CM were satisfied with their acute therapies. Just 33.3% of those with CM were currently using preventive medications. Chronic migraine (CM) is more disabling than episodic migraine in the population. Although most individuals with CM sought medical care for this disorder, the majority did not receive specific acute or preventive medications.


Survival After Pelvic Exenteration for Cervical Cancer: A National Cancer Database Study.
To determine overall survival (OS) and factors associated with OS after pelvic exenteration for cervical cancer. Women with cervical cancer who underwent exenteration (n = 517) were identified from the 1998 to 2011 National Cancer Database. Kaplan-Meier and multivariate Cox proportional-hazards survival analyses were performed to test for associations of potential explanatory variables with OS. Analyzed confounders included age, insurance status, income, distance from home to treatment center, stage, exenteration type, surgical margin status, and treatment with adjuvant radiation and/or chemotherapy. Among the entire cohort with clinical follow-up (n = 313), median OS was 24 months. Stage (P = 2.5 × 10), lymph node status (P = 1.3 × 10), insurance status (P = 1.5 × 10), and histologic type (P = 0.04) were significantly associated with OS by the log-rank test. Unadjusted median OS was 24.2 and 61.8 months for women with squamous and adenocarcinoma histologies, respectively. By multivariate Cox regression, age, insurance status, stage, margin status, and adjuvant radiation were associated with OS. Histology was not independently associated with OS on multivariate regression. Among women with node-negative disease, median OS was 73.2 months. Exenteration may be curative for more than half of women with node-negative cervical cancer. Stage, insurance status, lymph node status, and surgical margin are independently associated with differential OS after exenteration.


Quantification of biofilms in a sub-surface flow wetland and their role in nutrient removal.
Subsurface flow wetlands contain gravel or sand substrates through which the wastewater flows vertically or horizontally. The aims of this study were, firstly, to quantify biofilm development associated with different size gravel in sections of a subsurface flow wetland with and without plants, and secondly, to conduct laboratory experiments to examine the role of biofilms in nutrient removal. Techniques to quantify biofilm included: bacterial cell counts, EPS and total protein extraction. Based on comparative gravel sample volume, only EPS was greater on the smaller 5 mm gravel particles. There was no significant difference between biofilm growth in sections with and without plants. Two vertical flow laboratory-scale reactors, one containing fresh wetland gravel, the other containing autoclaved gravel, were constructed to determine nutrient transformations. The autoclaved gravel in the "sterile" reactor rapidly became colonised with biofilm. Both reactors were dosed with two types of influent. Initially the influent contained 7.25 mg/L NO3-N and 0.3 mg/L NH4-N; the biofilm reactor removed most of the ammonium and nitrite but nitrate concentrations were only reduced by 20%. In the "sterile" reactor there was negligible removal of ammonium and nitrite indicating little nitrification, however nitrate was reduced by 72%, possibly due to assimilatory nitrate reduction associated with new biofilm development. When the influent contained 3 mg/L NO3-N and 16 mg/L NH4-N almost 100% removal and transformation of NH4-N occurred in both reactors providing an effluent high in NO3-N. Organic P was reduced but inorganic soluble P increased possibly due to mineralisation.


Adolescent peer relationships and behavior problems predict young adults' communication on social networking websites.
This study examined online communication on social networking web pages in a longitudinal sample of 92 youths (39 male, 53 female). Participants' social and behavioral adjustment was assessed when they were ages 13-14 years and again at ages 20-22 years. At ages 20-22 years, participants' social networking website use and indicators of friendship quality on their web pages were coded by observers. Results suggested that youths who had been better adjusted at ages 13-14 years were more likely to be using social networking web pages at ages 20-22 years, after statistically controlling for age, gender, ethnicity, and parental income. Overall, youths' patterns of peer relationships, friendship quality, and behavioral adjustment at ages 13-14 years and at ages 20-22 years predicted similar qualities of interaction and problem behavior on their social networking websites at ages 20-22 years. Findings are consistent with developmental theory asserting that youths display cross-situational continuity in their social behaviors and suggest that the conceptualization of continuity may be extended into the online domain.


Proteins of the glycine decarboxylase complex in the hydrogenosome of Trichomonas vaginalis.
Trichomonas vaginalis is a unicellular eukaryote that lacks mitochondria and contains a specialized organelle, the hydrogenosome, involved in carbohydrate metabolism and iron-sulfur cluster assembly. We report the identification of two glycine cleavage H proteins and a dihydrolipoamide dehydrogenase (L protein) of the glycine decarboxylase complex in T. vaginalis with predicted N-terminal hydrogenosomal presequences. Immunofluorescence analyses reveal that both H and L proteins are localized in hydrogenosomes, providing the first evidence for amino acid metabolism in this organelle. All three proteins were expressed in Escherichia coli and purified to homogeneity. The experimental Km of L protein for the two H proteins were 2.6 microM and 3.7 microM, consistent with both H proteins serving as substrates of L protein. Analyses using purified hydrogenosomes showed that endogenous H proteins exist as monomers and endogenous L protein as a homodimer in their native states. Phylogenetic analyses of L proteins revealed that the T. vaginalis homologue shares a common ancestry with dihydrolipoamide dehydrogenases from the firmicute bacteria, indicating its acquisition via a horizontal gene transfer event independent of the origins of mitochondria and hydrogenosomes.


Genetic consequences of mass human chemotherapy for Schistosoma mansoni: population structure pre- and post-praziquantel treatment in Tanzania.
Recent shifts in global health policy have led to the implementation of mass drug administration (MDA) for neglected tropical diseases. Here we show how population genetic analyses can provide vital insights into the impact of such MDA on endemic parasite populations. We show that even a single round of MDA produced a genetic bottleneck with reductions in a range of measures of genetic diversity of Schistosoma mansoni. Phylogenetic analyses and indices of population differentiation indicated that schistosomes collected in the same schools in different years were more dissimilar than those from different schools collected within either of the study's 2 years, in addition to distinguishing re-infection from non-clearance (that might indicate putatively resistant parasites) from within those children infected at both baseline and follow-up. Such unique results illustrate the importance of genetic monitoring and examination of long lived multi-cellular parasites such as these under novel or increased chemotherapeutic selective pressures.


Patterns of white matter damage are non-random and associated with cognitive function in secondary progressive multiple sclerosis.
In multiple sclerosis (MS), white matter damage is thought to contribute to cognitive dysfunction, which is especially prominent in secondary progressive MS (SPMS). While studies in healthy subjects have revealed patterns of correlated fractional anisotropy (FA) across white matter tracts, little is known about the underlying patterns of white matter damage in MS. In the present study, we aimed to map the SPMS-related covariance patterns of microstructural white matter changes, and investigated whether or not these patterns were associated with cognitive dysfunction. Diffusion MRI was acquired from 30 SPMS patients and 32 healthy controls (HC). A tensor model was fitted and FA maps were processed using tract-based spatial statistics (TBSS) in order to obtain a skeletonised map for each subject. The skeletonised FA maps of patients only were decomposed into 18 spatially independent components (ICs) using independent component analysis. Comprehensive cognitive assessment was conducted to evaluate five cognitive domains. Correlations between cognitive performance and (1) severity of FA abnormalities of the extracted ICs (i.e. z-scores relative to FA values of HC) and (2) IC load (i.e. FA covariance of a particular IC) were examined. SPMS patients showed lower FA values of all examined patterns of correlated FA (i.e. spatially independent components) than HC (p < 0.01). Tracts visually assigned to the supratentorial commissural class were most severely damaged (z = - 3.54; p < 0.001). Reduced FA was significantly correlated with reduced IC load (i.e. FA covariance) (r = 0.441; p < 0.05). Lower mean FA and component load of the supratentorial projection tracts and limbic association tracts classes were associated with worse cognitive function, including executive function, working memory and verbal memory. Despite the presence of white matter damage, it was possible to reveal patterns of FA covariance across SPMS patients. This could indicate that white matter tracts belonging to the same cluster, and thus with similar characteristics, tend to follow similar trends during neurodegeneration. Furthermore, these underlying FA patterns might help to explain cognitive dysfunction in SPMS.


Ultraviolet Photoluminescence of Carbon Nanospheres and its Surface Plasmon-Induced Enhancement.
Ultraviolet (UV) light can be used in versatile applications ranging from photoelectronic devices to biomedical imaging. In the development of new UV light sources, in this study, stable UV emission at ≈350 nm is unprecedentedly obtained from carbon nanospheres (CNSs). The origin of the UV fluorescence is comprehensively investigated via various characterization methods, including Raman and Fourier transform infrared analyses, with comparison to the visible emission of carbon nanodots. Based on the density functional calculations, the UV fluorescence is assigned to the carbon nanostructures bonded to bridging O atoms and dangling -OH groups. Moreover, a twofold enhancement in the UV emission is acquired for Au-carbon core-shell nanospheres (Au-CNSs). This remarkable modification of the UV emission is primarily ascribed to charge transfer between the CNSs and the Au surface.


GeneticStudio: a suite of programs for spatial analysis of genetic-marker data.
The analysis of genetic marker data is increasingly being conducted in the context of the spatial arrangement of strata (e.g. populations) necessitating a more flexible set of analysis tools. GeneticStudio consists of four interacting programs: (i) Geno a spreadsheet-like interface for the analysis of spatially explicit marker-based genetic variation; (ii) Graph software for the analysis of Population Graph and network topologies, (iii) Manteller, a general purpose for matrix analysis program; and (iv) SNPFinder, a program for identifying single nucleotide polymorphisms. The GeneticStudio suite is available as source code as well as binaries for OSX and Windows and is distributed under the GNU General Public License.


IROA: International Register of Open Abdomen, preliminary results.
No definitive data about open abdomen (OA) epidemiology and outcomes exist. The World Society of Emergency Surgery (WSES) and the Panamerican Trauma Society (PTS) promoted the International Register of Open Abdomen (IROA). A prospective observational cohort study including patients with an OA treatment. Data were recorded on a web platform (Clinical Registers®) through a dedicated website: www.clinicalregisters.org. Four hundred two patients enrolled. Adult patients: 369 patients; Mean age: 57.39±18.37; 56% male; Mean BMI: 36±5.6. OA indication: Peritonitis (48.7%), Trauma (20.5%), Vascular Emergencies/Hemorrhage (9.4%), Ischemia (9.1%), Pancreatitis (4.2%),Post-operative abdominal-compartment-syndrome (3.9%), Others (4.2%). The most adopted Temporary-abdominal-closure systems were the commercial negative pressure ones (44.2%). During OA 38% of patients had complications; among them 10.5% had fistula. Definitive closure: 82.8%; Mortality during treatment: 17.2%. Mean duration of OA: 5.39(±4.83) days; Mean number of dressing changes: 0.88(±0.88). After-closure complications: (49.5%) and Mortality: (9%). No significant associations among TACT, indications, mortality, complications and fistula. A linear correlationexists between days of OA and complications (Pearson linear correlation = 0.326 p<0.0001) and with the fistula development (Pearson = 0.146 p= 0.016). Pediatric patients: 33 patients. Mean age: 5.91±(3.68) years; 60% male. Mortality: 3.4%; Complications: 44.8%; Fistula: 3.4%. Mean duration of OA: 3.22(±3.09) days. Temporary abdominal closure is reliable and safe. The different techniques account for different results according to the different indications. In peritonitis commercial negative pressure temporary closure seems to improve results. In trauma skin-closure and Bogotà-bag seem to improve results. ClinicalTrials.gov NCT02382770.


Functional characterization of CD8(+) antigen-specific cytotoxic T lymphocytes after enrichment based on cytokine secretion: comparison with the MHC-tetramer technology.
Cell therapy with antigen-specific T cells holds promise for various diseases including cancer and viral infections. The powerful enrichment procedure based on major histocompatibility complex (MHC)-tetramers, however, is of limited applicability so far. Therefore, the recently developed cell surface affinity matrix technology that allows direct identification and enrichment of life antigen-specific T cells based on cytokine secretion was evaluated in this respect. To this end, CD8(+) T cells directed against the HLA-A(*)0201-restricted melanoma-associated peptide Melan-A (aa26-35) were generated by combining stimulation of peptide-pulsed autologous dendritic cells (DC) with antigen-independent expansion with anti-CD3/anti-CD28 monoclonal antibodies (MoAb). Antigen-specific cytotoxic T lymphocyte (CTL) were detected based on stimulation-induced interferon (IFN)-gamma and interleukin (IL)-4 secretion and enriched > 100-fold using the cell surface affinity matrix technology. The resulting IFN-gamma- and IL-4-secreting CTL lines contained > 80% and > 70% cytokine positive T cells, respectively. They exhibited a cytotoxic activity against Melan-A expressing target cells that was significantly higher as compared to nonpurified CTL. Direct staining of enriched CTL with HLA-A2-Melan-A-tetramers revealed a high correlation between the results obtained from the cell surface affinity matrix technology and those obtained from tetrameric complexes. Altogether, our study demonstrates that cytokine-driven enrichment based on the cell surface affinity matrix technology enables selective isolation of functionally active antigen-specific CTL that may be used for an adoptive T cell transfer in immunotherapy.


In vivo alterations in cardiac metabolism and function in the spontaneously hypertensive rat heart.
The aim of this work was to use hyperpolarized carbon-13 ((13)C) magnetic resonance (MR) spectroscopy and cine MR imaging (MRI) to assess in vivo cardiac metabolism and function in the 15-week-old spontaneously hypertensive rat (SHR) heart. At this time point, the SHR displays hypertension and concentric hypertrophy. One of the cellular adaptations to hypertrophy is a reduction in β-oxidation, and it has previously been shown that in response to hypertrophy the SHR heart switches to a glycolytic/glucose-oxidative phenotype. Cine-MRI (magnetic resonance imaging) was used to assess cardiac function and degree of cardiac hypertrophy. Wistar rats were used as controls. SHRs displayed functional changes in stroke volume, heart rate, and late peak-diastolic filling alongside significant hypertrophy (a 56% increase in left ventricular mass). Using hyperpolarized [1-(13)C] and [2-(13)C]pyruvate, an 85% increase in (13)C label flux through pyruvate dehydrogenase (PDH) was seen in the SHR heart and (13)C label incorporation into citrate, acetylcarnitine, and glutamate pools was elevated in proportion to the increase in PDH flux. These findings were confirmed using biochemical analysis of PDH activity and protein expression of PDH regulatory enzymes. Functional and structural alterations in the SHR heart are consistent with the hypertrophied phenotype. Our in vivo work indicates a preference for glucose metabolism in the SHR heart, a move away from predominantly fatty acid oxidative metabolism. Interestingly, (13)C label flux into lactate was unchanged, indicating no switch to an anaerobic glycolytic phenotype, but rather an increased reliance on glucose oxidation in the SHR heart.


Reduced glucose uptake precedes insulin signaling defects in adipocytes from heterozygous GLUT4 knockout mice.
Decreased GLUT4 expression, impaired insulin receptor (IR), IRS-1, and pp60/IRS-3 tyrosine phosphorylation are characteristics of adipocytes from insulin-resistant animal models and obese NIDDM humans. However, the sequence of events leading to the development of insulin signaling defects and the significance of decreased GLUT4 expression in causing adipocyte insulin resistance are unknown. The present study used male heterozygous GLUT4 knockout mice (GLUT4(+/-)) as a novel model of diabetes to study the development of insulin signaling defects in adipocytes with the progression of whole body insulin resistance and diabetes. Male GLUT4(+/-) mice with normal fed glycemia and insulinemia (N/N), normal fed glycemia and hyperinsulinemia (N/H), and fed hyperglycemia with hyperinsulinemia (H/H) exist at all ages. The expression of GLUT4 protein and the maximal insulin-stimulated glucose transport was 50% decreased in adipocytes from all three groups. Insulin signaling was normal in N/N adipose cells. From 35 to 70% reductions in insulin-stimulated tyrosine phosphorylation of IR, IRS-1, and pp60/IRS-3 were noted with no changes in the cellular content of IR, IRS-1, and p85 in N/H adipocytes. Insulin-stimulated protein tyrosine phosphorylation was further decreased to 12-23% in H/H adipose cells accompanied by 42% decreased IR and 80% increased p85 expression. Insulin-stimulated, IRS-1-associated PI3 kinase activity was decreased by 20% in N/H and 68% reduced in H/H GLUT4(+/-) adipocytes. However, total insulin-stimulated PI3 kinase activity was normal in H/H GLUT4(+/-) adipocytes. Taken together, these results strongly suggest that hyperinsulinemia triggers a reduction of IR tyrosine kinase activity that is further exacerbated by the appearance of hyperglycemia. However, the insulin signaling cascade has sufficient plasticity to accommodate significant changes in specific components without further reducing glucose uptake. Furthermore, the data indicate that the cellular content of GLUT4 is the rate-limiting factor in mediating maximal insulin-stimulated glucose uptake in GLUT4(+/-) adipocytes.


Digital optical counter using directional coupler switches.
We describe the design and implementation of a bit-serial, four-bit, binary optical counter. The counter was designed and simulated using a digital optical simulation program developed for this purpose. It consists of five switches, a 4-bit fiber loop memory to store the count, four splitters, and fibers to interconnect the components. The counter is presently limited to a clock rate of 50 MHz because of the propagation delay in the single-bit time feedback loop. As designed, the same hardware may be used to count any even number of bits simply by changing the lengths of two fiber loops. The counter is unique in that it does not employ latches or other synchronizing memory elements, rather relying on a time-of-flight architecture. We describe the system issues involved in construction of the counter as well as the novel requirements on the switch drive electronics. We then outline the issues still to be addressed for the current counter and conclude with suggested design alternatives to improve its operation and increase its clock rate.


Immediate loading of implants with 3-unit fixed partial dentures: a 12-month clinical study.
The aim of the present clinical trial was to evaluate the 12-month success rate of titanium dental implants placed in the posterior mandible and immediately loaded with 3-unit fixed partial dentures. Patients with missing mandibular premolars and molars were enrolled in this study. To be included in the study, the implants had to show good primary stability. Implant stability was measured with resonance frequency analysis using the Osstell device (Integration Diagnostics). Implants were included in the study when the stability quotient (ISQ) exceeded 62. Clinical measurements, such as width of keratinized tissue, ISQ, and radiographic assessment of peri-implant bone crest levels, were performed at baseline and at the 12-month follow-up. The comparison between the baseline and the 12-month visits was performed with the Student t test for paired data (statistically significant at a level of alpha = 0.05). Forty implants with a sandblasted, large grit, acid-etched (SLA) surface (Straumann) were placed in 20 patients. At 12 months, only 1 implant had been lost because of an acute infection. The remaining 39 implants were successful, resulting in a 1-year success rate of 97.5%. Neither peri-implant bone levels, measured radiographically, nor implant stability changed significantly from baseline to the 12-month follow-up (P > .05). The immediate functional loading of implants placed in this case series study resulted in a satisfactory success rate. The findings from this clinical study showed that the placement of SLA transmucosal implants in the mandibular area and their immediate loading with 3-unit fixed partial dentures may be a safe and successful procedure.


Direct recognition of hepatocyte-expressed MHC class I alloantigens is required for tolerance induction.
Adeno-associated viral vector-mediated (AAV-mediated) expression of allogeneic major histocompatibility complex class I (MHC class I) in recipient liver induces donor-specific tolerance in mouse skin transplant models in which a class I allele (H-2Kb or H-2Kd) is mismatched between donor and recipient. Tolerance can be induced in mice primed by prior rejection of a donor-strain skin graft, as well as in naive recipients. Allogeneic MHC class I may be recognized by recipient T cells as an intact molecule (direct recognition) or may be processed and presented as an allogeneic peptide in the context of self-MHC (indirect recognition). The relative contributions of direct and indirect allorecognition to tolerance induction in this setting are unknown. Using hepatocyte-specific AAV vectors encoding WT allogeneic MHC class I molecules, or class I molecules containing a point mutation (D227K) that impedes direct recognition of intact allogeneic MHC class I by CD8+ T cells without hampering the presentation of processed peptides derived from allogeneic MHC class I, we show here that tolerance induction depends upon recognition of intact MHC class I. Indirect recognition alone yielded a modest prolongation of subsequent skin graft survival, attributable to the generation of CD4+ Tregs, but it was not sufficient to induce tolerance.


Mobile Delivery of Health Information for People with Mild Cognitive Impairment.
The design of a smartphone application (app) for promoting healthy lifestyle choices has been investigated for people with mild cognitive impairment. The app was designed to provide health-related messages and assist users to keep track of activities such as walking, eating and drinking water. A reward scheme with gold, silver and bronze awards was incorporated as a means of assisting user motivation. Responses to the app and user feedback were gathered for purposes of evaluation and improvement. Outcomes indicate that the approach has some potential and could have good implications for encouraging positive health-related behaviour change in this population, hence prompting further investigation and development of the concept.


Physicians' reasons for not entering their patients in a randomized controlled trial in Japan.
Physicians' not entering their patients can jeopardize the success of a randomized controlled trial (RCT). We used a survey to investigate the possible reasons why physicians who initially agreed to collaborate did not recruit any patients for an RCT being conducted in Japan. A total of 167 questionnaires were sent out and 122 responses were received. Main reasons for not entering patients were: concern about the detrimental effects on the doctor-patient relationship (51.8%), patients' refusal (47.5%), complicated registration and follow-up procedures (34.9%), and not feeling comfortable recruiting their own patients (32.4%). Multivariate logistic regression made it clear that physicians who thought that registering their own patients would damage the doctor-patient relationship and who expected the RCT would fail were more likely to be uncomfortable entering their own patients. Moreover, physicians aged 50 years or older, who felt uncomfortable recruiting their own patients, and saw no advantage in participating in the trial, were more likely to view the enrolment and follow-up procedures as cumbersome. We conclude that training and a manual for obtaining informed consent and a face-to-face demonstration of patient registration/follow-up procedures for the potential participants are prerequisites for increasing physician participation in RCTs in Japan.