A nucleic acid strand displacement system for the multiplexed detection of tuberculosis-specific mRNA using quantum dots.
The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar range using only a standard laboratory plate reader. We demonstrate the utility of our QD-based system for the detection of two genes selected from a microarray-derived tuberculosis-specific gene expression signature. Levels of up- and downregulated gene transcripts comprising this signature can be combined to give a disease risk score, making the signature more amenable for use as a diagnostic marker. Our QD-based approach to detect these transcripts could pave the way for novel diagnostic assays for tuberculosis.


Early mouse embryo development: could epigenetics influence cell fate determination?
It is generally assumed that the developmental program of embryogenesis relies on epigenetic mechanisms. However, a mechanistic link between epigenetic marks and cell fate decisions had not been established so far. In a recent article, Torres-Padilla and colleagues show that epigenetic information and, more precisely, histone arginine methylation mediated by CARM1 could contribute to cell fate decisions in the mouse 4-cell-stage embryo. It provides the first indications that global epigenetic information influences allocation of pluripotent cells toward the first cell lineages.


Genome-wide association study of schizophrenia using microsatellite markers in the Japanese population.
To search for schizophrenia susceptibility loci, we carried out a case-control study using 28601 microsatellite markers distributed across the entire genome. To control the highly multiple testing, we designed three sequential steps of screening using three independent sets of pooled samples, followed by the confirmatory step using an independent sample set (>2200 case-control pairs). The first screening using pooled samples of 157 case-control pairs showed 2966 markers to be significantly associated with the disorder (P<0.05). After the second and the third screening steps using pooled samples of 150 pairs each, 374 markers remained significantly associated with the disorder. We individually genotyped all screening samples using a total of 1536 tag single nucleotide polymorphisms (SNPs) located in the vicinity of ~200 kb from the 59 positive microsatellite markers. Of the 167 SNPs that replicated the significance, we selected 31 SNPs on the basis of the levels of P values for the confirmatory association test using an independent-sample set. The best association signal was observed in rs13404754, located in the upstream region of SLC23A3. We genotyped six additional SNPs in the vicinity of rs13404754. Significant associations were observed in rs13404754, rs6436122, and rs1043160 in the cumulative samples (2617 cases and 2698 controls) (P=0.005, 0.035, and 0.011, respectively). These SNPs are located in the linkage disequilibrium block of 20 kb in size containing SLC23A3, CNPPD1, and FAM134A genes. Genome-wide association study using microsatellite markers suggested SLC23A3, CNPPD1, and FAM134A genes as candidates for schizophrenia susceptibility in the Japanese population.


Left-handers look before they leap: handedness influences reactivity to novel Tower of Hanoi tasks.
A sample of 203 task naïve left- and right-handed participants were asked to complete a combination of the 3- and 4-disk Towers of Hanoi (ToH), manipulating novelty and complexity. Self-reported state anxiety and latency to respond (initiation time) were recorded before each ToH. Novelty had a major effect on initiation time, particularly for left-handers. Left-handers had a longer latency to start and this was significantly longer on the first trial. Irrespective of hand-preference, initiation time reduced on the second trial, however, this was greatest for left-handers. Condition of task did not systematically influence initiation time for right handers, but did for left-handers. State anxiety was influenced by task novelty and complexity in a more complicated way. During the first trial, there was a significant handedness × number of disks interaction with left-handers having significantly higher state anxiety levels before the 3-disk ToH. This suggests that the initial reaction to this task for left-handers was not simply due to perceived difficulty. On their second trial, participants completing a novel ToH had higher state anxiety scores than those completing a repeated version. Overall, left-handers had a larger reduction in their state anxiety across trials. Relating to this, the expected strong positive correlation between state and trait anxiety was absent for left-handed females in their first tower presentation, but appeared on their second. This was driven by low trait anxiety individuals showing a higher state anxiety response in the first (novel) trial, supporting the idea that left-handed females respond to novelty in a way that is not directly a consequence of their trait anxiety. A possible explanation may be stereotype threat influencing the behavior of left-handed females.


Arabidopsis hot2 encodes an endochitinase-like protein that is essential for tolerance to heat, salt and drought stresses.
The Arabidopsis hot2 mutant was originally identified based on its lack of thermotolerance, but pleiotropic abnormal phenotypes are also exhibited under normal conditions, including semi-dwarfism, ethylene overproduction and aberrant cell shape with incomplete cell walls. Here we present additional characterization of the hot2 mutant, and the map-based cloning of HOT2. Mutants of hot2 had an aberrant tolerance to salt and drought stresses, and accumulated high levels of Na(+) in cells under either normal or NaCl stress conditions. Expression of the stress-inducible COR15A and KIN1 gene in hot2 mutants in response to increased NaCl concentrations was normal. HOT2 encoded a chitinase-like protein (AtCTL1) that has not previously been shown to be involved in tolerance to salt stress. Ten-day-old seedlings of wild-type plants exhibited constitutive expression of the AtCTL1 transcript, the level of which was unaffected by treatment with NaCl, mannitol or mild heat. These observations provide genetic evidence that a chitinase-like protein prevents the overaccumulation of Na(+) ions, thereby contributing to the salt tolerance in Arabidopsis. A possible role for this chitinase-like protein in Arabidopsis tolerance to abiotic stress is discussed.


Posterior lumbar interbody fusion using nonresorbable poly-ether-ether-ketone versus resorbable poly-L-lactide-co-D,L-lactide fusion devices: a prospective, randomized study to assess fusion and clinical outcome.
A prospective randomized clinical study. OBJECTIVE.: To assess fusion, clinical outcome, and complications. Resorbable poly-L- lactide-co-D,L-lactide (PLDLLA) cages intended to aid spinal interbody fusion have been introduced into clinical practice within the last decade. Although early case series show promising results with respect to fusion rate, worries persist with regards to efficacy and potential risks of early failure of these implants. Despite widespread clinical application this is the first randomized prospective study to assess clinical and radiologic outcomes of PLDLLA cage compared with a traditionally applicable counterpart. Twenty-six patients were randomly assigned to undergo instrumented posterior lumbar interbody fusion whereby either a nonresorbable poly-ether-ether-ketone (PEEK) cage or a resorbable PLDLLA cage was implanted to aid fusion. Fusion rate, subsidence, and clinical outcome based on visual analog scale scores for leg pain and back pain, as well as Oswestry Disability Index and SF-36 questionnaires were documented and analyzed. Complications and adverse events were recorded. Fusion rate was significantly higher with the PEEK cage compared with PLDLLA cage (Fisher exact test, P = 0.0302). Rate of subsidence was significantly higher with the PLDLLA cage compared to PEEK cage (Fisher exact test, P = 0.0414). The PEEK group demonstrated greater improvement in the Oswestry Disability Index compared with the PLDLLA group (Fisher exact test, P = 0.1414). Two cases of mild to moderate osteolysis were seen in the PLDLLA group. Our results strongly suggest that PLDLLA cage proffers a lower rate of fusion compared with PEEK cage, and also confirms that the efficacy of PLDLLA cage in enhancing interbody spinal fusion is yet to be established. The higher rate of subsidence and occurrence of osteolysis seen in association with PLDLLA cages in this study remain worrisome.


Using stochastic models calibrated from nanosecond nonequilibrium simulations to approximate mesoscale information.
We demonstrate how the surrogate process approximation (SPA) method can be used to compute both the potential of mean force along a reaction coordinate and the associated diffusion coefficient using a relatively small number (10-20) of bidirectional nonequilibrium trajectories coming from a complex system. Our method provides confidence bands which take the variability of the initial configuration of the high-dimensional system, continuous nature of the work paths, and thermal fluctuations into account. Maximum-likelihood-type methods are used to estimate a stochastic differential equation (SDE) approximating the dynamics. For each observed time series, we estimate a new SDE resulting in a collection of SPA models. The physical significance of the collection of SPA models is discussed and methods for exploiting information in the population of estimated SPA models are demonstrated and suggested. Molecular dynamics simulations of potassium ion dynamics inside a gramicidin A channel are used to demonstrate the methodology, although SPA-type modeling has also proven useful in analyzing single-molecule experimental time series [J. Phys. Chem. B 113, 118 (2009)].


Effect of single high dose infusions of aminohydroxypropylidene diphosphonate on hypercalcaemia caused by cancer.
Single intravenous infusions of 30 mg aminohydroxypropylidene diphosphonate were given to 16 patients who had malignant hypercalcaemia to assess host tolerance and the effect on serum calcium concentration. Ten of these patients also received intravenous rehydration or corticosteroids, or both. The serum calcium concentrations decreased significantly after treatment with aminohydroxypropylidene diphosphonate. Ten patients became normocalcaemic (normal range, adjusted for serum albumin, 2.25-2.75 mmol/l), two became hypocalcaemic, three showed decreases in serum calcium concentrations of more than 0.75 mmol/l, and one showed a decrease of more than 0.55 mmol/l. Only one patient had a minimum concentration greater than 2.77 mmol/l. Aminohydroxypropylidene diphosphonate was effective in metastatic and non-metastatic hypercalcaemia, and its hypocalcaemic effect was prolonged in some cases. There were no appreciable side effects. Single high dose infusions of aminohydroxypropylidene diphosphonate could replace conventional daily lower dose infusions, but the optimum frequency of high dose infusions remains to be determined.


Chest Compression Fraction and Factors influencing it.
Chest compression fraction (CCF) is the cumulative time spent providing chest compressions divided by the total time taken for the entire resuscitation. Targeting a CCF of at least 60% is intended to limit interruptions in compressions and maximize coronary perfusion during resuscitation. We aimed to identify the mean CCF and its relationship with various factors affecting it. Patients presenting to the emergency department in cardiac arrest at a single center were prospectively included in this study. Resuscitation was provided by trained health-care providers. The feedback device Cprmeter2™ was placed on the patient's sternum at the beginning of resuscitation. The total time taken for the entire resuscitation was noted by the device and CCF calculated. The mean CCF was analyzed using descriptive statistics and was found to be 71.60% ± 7.52%. The total duration of resuscitation (R = -0.55, P = < 0.001, min-max, 2.02-34.31, mean 12.25 ± 6.54), number of people giving chest compressions (R = -0.48, P = < 0.001, min-max, 1-6, mean 4.04 ± 1.12), and total number of team members in resuscitation (R = -0.50, P = < 0.001, min-max, 4-10, mean 6.65 ± 1.32) had negative correlation with CCF. Diurnal variation (day, n = 35; mean 69.20% ± 7% and night, n = 20; mean 75.80% ± 5.6%, P = 0.001) and patients receiving defibrillation (receiving n = 10 mean 67.00% ± 4.11% and not receiving n = 45 mean 72.62 ± 7.42%, P = 0.005) were found to significantly affect CCF. The mean CCF for cardiac arrest patients was well within the targets of guideline recommendation. CCF decreased when resuscitation lasted longer, during daytime when the defibrillator was used, the total team members increased, and also when the number of people giving chest compressions increased. CCF during resuscitation may improve if there is a focus on improving these factors and requires validation in multicentric settings.


Computational simulation of the input-output relationship in hippocampal pyramidal cells.
The precise mapping of how complex patterns of synaptic inputs are integrated into specific patterns of spiking output is an essential step in the characterization of the cellular basis of network dynamics and function. Relative to other principal neurons of the hippocampus, the electrophysiology of CA1 pyramidal cells has been extensively investigated. Yet, the precise input-output relationship is to date unknown even for this neuronal class. CA1 pyramidal neurons receive laminated excitatory inputs from three distinct pathways: recurrent CA1 collaterals on basal dendrites, CA3 Schaffer collaterals, mostly on oblique and proximal apical dendrites, and entorhinal perforant pathway on distal apical dendrites. We implemented detailed computer simulations of pyramidal cell electrophysiology based on three-dimensional anatomical reconstructions and compartmental models of available biophysical properties from the experimental literature. To investigate the effect of synaptic input on axosomatic firing, we stochastically distributed a realistic number of excitatory synapses in each of the three dendritic layers. We then recorded the spiking response to different stimulation patterns. For all dendritic layers, synchronous stimuli resulted in trains of spiking output and a linear relationship between input and output firing frequencies. In contrast, asynchronous stimuli evoked non-bursting spike patterns and the corresponding firing frequency input-output function was logarithmic. The regular/irregular nature of the input synaptic intervals was only reflected in the regularity of output inter-burst intervals in response to synchronous stimulation, and never affected firing frequency. Synaptic stimulations in the basal and proximal apical trees across individual neuronal morphologies yielded remarkably similar input-output relationships. Results were also robust with respect to the detailed distributions of dendritic and synaptic conductances within a plausible range constrained by experimental evidence. In contrast, the input-output relationship in response to distal apical stimuli showed dramatic differences from the other dendritic locations as well as among neurons, and was more sensible to the exact channel densities.


Andrographolide enhances nuclear factor-kappaB subunit p65 Ser536 dephosphorylation through activation of protein phosphatase 2A in vascular smooth muscle cells.
Recent studies have demonstrated that transcription factor nuclear factor (NF)-κB inhibition may contribute to the protective anti-inflammatory actions of andrographolide, an abundant component of plants of the genus Andrographis. However, the precise mechanism by which andrographolide inhibits NF-κB signaling remains unclear. We thus investigated the mechanism involved in andrographolide suppression of NF-κB signaling in rat vascular smooth muscle cells (VSMCs) exposed to proinflammatory stimuli, LPS, and IFN-γ. Andrographolide was shown to suppress LPS/IFN-γ-induced inducible nitric-oxide synthase and matrix metalloprotease 9 expression in rat VSMCs. Andrographolide also inhibited LPS/IFN-γ-induced p65 nuclear translocation, DNA binding activity, p65 Ser(536) phosphorylation, and NF-κB reporter activity. However, IKK phosphorylation and downstream inhibitory κBα phosphorylation and degradation were not altered by the presence of andrographolide in LPS/IFN-γ-stimulated VSMCs. These andrographolide inhibitory actions could be prevented by selective inhibition of neutral sphingomyelinase and protein phosphatase 2A (PP2A). Furthermore, andrographolide was demonstrated to increase ceramide formation and PP2A activity in VSMCs and to inhibit neointimal formation in rat carotid injury models. These results suggest that andrographolide caused neutral sphingomyelinase-mediated ceramide formation and PP2A activation to dephosphorylate p65 Ser(536), leading to NF-κB inactivation and subsequent inducible nitric-oxide synthase down-regulation in rat VSMCs stimulated by LPS and IFN-γ.


Fungal functioning in a pine forest: evidence from a ¹⁵N-labeled global change experiment.
• We used natural and tracer nitrogen (N) isotopes in a Pinus taeda free air CO₂ enrichment (FACE) experiment to investigate functioning of ectomycorrhizal and saprotrophic fungi in N cycling. • Fungal sporocarps were sampled in 2004 (natural abundance and (15) N tracer) and 2010 (tracer) and δ(15)N patterns were compared against litter and soil pools. • Ectomycorrhizal fungi with hydrophobic ectomycorrhizas (e.g. Cortinarius and Tricholoma) acquired N from the Oea horizon or deeper. Taxa with hydrophilic ectomycorrhizas acquired N from the Oi horizon (Russula and Lactarius) or deeper (Laccaria, Inocybe, and Amanita). (15)N enrichment patterns for Cortinarius and Amanita in 2010 did not correspond to any measured bulk pool, suggesting that a persistent pool of active organic N supplied these two taxa. Saprotrophic fungi could be separated into those colonizing pine cones (Baeospora), wood, litter (Oi), and soil (Ramariopsis), with δ(15)N of taxa reflecting substrate differences. (15)N enrichment between sources and sporocarps varied across taxa and contributed to δ(15)N patterns. • Natural abundance and (15)N tracers proved useful for tracking N from different depths into fungal taxa, generally corresponded to literature estimates of fungal activity within soil profiles, and provided new insights into interpreting natural abundance δ(15)N patterns.


Outsiders in nursing education: cultural sensitivity in clinical education.
Cultural competence is a stated value of nursing and nursing education. However, some institutional and traditional practices in nursing education can unintentionally impede nurses from achieving cultural competence. Both the literature and interviews with nurse educators show that despite educators' intentions to treat all students the same, nontraditional students may feel singled out and may in fact be singled out for closer scrutiny because of their difference from the demographic norms of nursing students. To ensure that the nursing profession reflects the composition of the patient population it serves, nurse educators must first acknowledge the Eurocentric culture of nursing education and, then, work to change the environment in which students are recruited, learn, and take on the role of beginning practicing nurses.


omega-Conotoxin block of N-type calcium channels in frog and rat sympathetic neurons.
Block of N-type Ca channels by omega-conotoxin GVIA (CgTx) was studied in freshly dissociated bullfrog and rat sympathetic neurons. With 2-5 mM Ba as charge carrier, CgTx blocked almost all of the high-threshold Ca channel current recorded in the presence of nimodipine (3 microM) to block L-type Ca channels. Toxin block reversed slowly (time constant approximately 1 hr) in frog cells and even more slowly in rat cells. CgTx block was faster and more potent in rat cells than frog cells. The rate of block was proportional to CgTx concentration, consistent with 1:1 binding of CgTx to channels. When the external Ba concentration was increased, the development of block was slower, consistent with competition between CgTx and Ba for a binding site. The recovery from block was somewhat faster in higher external Ba. Some cells had significant current remaining in saturating concentrations of nimodipine and CgTx, especially with high Ba concentrations in the external solution. The current resistant to nimodipine and CgTx was activated at lower depolarizations than the CgTx-sensitive current and had faster activation and inactivation kinetics, but unlike low-threshold T-type current, the resistant current had rapidly decaying tail currents.


CO2 cerebrovascular reactivity as a function of perfusion pressure--a modelling study.
A mathematical model is described that demonstrated the properties of cerebral vascular resistance and compliance expressed as a function of cerebral perfusion pressure (CPP) and arterial CO2 partial pressure (PaCO2). The hypercapnic induced shift of the lower limit of autoregulation to a higher range of CPP, as shown by this model, is a useful characteristic that facilitates the differentiation between normal and impaired autoregulation described previously in experimental studies. Dynamic properties of cerebrovascular circulation derived from the relationship between pulse wave of CBF waveform and CPP have been analysed at different levels of PaCO2-phenomenon, being often described as dependence of blood flow velocity pulsatility index on the autoregulatory reserve. The model was also used to interpret interhemispheric asymmetry of CBF reactivity to changes in arterial concentration of CO2 in patients with carotid artery stenosis.


A multi-scale approach for percolation transition and its application to cement setting.
Shortly after mixing cement grains with water, a cementitious fluid paste is formed that immediately transforms into a solid form by a phenomenon known as setting. Setting actually corresponds to the percolation of emergent network structures consisting of dissolving cement grains glued together by nanoscale hydration products, mainly calcium-silicate-hydrates. As happens in many percolation phenomena problems, the theoretical identification of the percolation threshold (i.e. the cement setting) is still challenging, since the length scale where percolation becomes apparent (typically the length of the cement grains, microns) is many times larger than the nanoscale hydrates forming the growing spanning network. Up to now, the long-lasting gap of knowledge on the establishment of a seamless handshake between both scales has been an unsurmountable obstacle for the development of a predictive theory of setting. Herein we present a true multi-scale model which concurrently provides information at the scale of cement grains (microns) and at the scale of the nano-hydrates that emerge during cement hydration. A key feature of the model is the recognition of cement setting as an off-lattice bond percolation process between cement grains. Inasmuch as this is so, the macroscopic probability of forming bonds between cement grains can be statistically analysed in smaller local observation windows containing fewer cement grains, where the nucleation and growth of the nano-hydrates can be explicitly described using a kinetic Monte Carlo Nucleation and Growth model. The most striking result of the model is the finding that only a few links (~12%) between cement grains are needed to reach setting. This directly unveils the importance of explicitly including nano-texture on the description of setting and explains why so low amount of nano-hydrates is needed for forming a spanning network. From the simulations, it becomes evident that this low amount is least affected by processing variables like the water-to-cement ratio and the presence of large quantities of nonreactive fillers. These counter-intuitive predictions were verified by ex-professo experiments that we have carried out to check the validity of our model.


Analysis of the genomic region containing the tammar wallaby (Macropus eugenii) orthologues of MHC class III genes.
Major histocompatibility complex (MHC) molecules are central to development and regulation of the immune system in all jawed vertebrates. MHC class III cytokine genes from the tumor necrosis factor core family, including tumor necrosis factor (TNF) and lymphotoxin alpha and beta (LTA, LTB), are well studied in human and mouse. Orthologues have been identified in several other eutherian species and the cDNA sequences have been reported for a model marsupial, the tammar wallaby. Comparative genomics can help to determine gene function, to understand the evolution of a gene or gene family, and to identify potential regulatory regions. We therefore cloned the genomic region containing the tammar LTB, TNF, and LTA orthologues by "genome walking", using primers designed from known tammar sequences and regions conserved in other species. We isolated two tammar BAC clones containing all three genes. These tammar genes show similar intergenic distances and the same transcriptional orientation as in human and mouse. Gene structures and sequences are also very conserved. By comparing the tammar, human and mouse genomic sequences we were able to identify candidate regulatory regions for these genes in mammals. Full length sequencing of BACs containing the three genes has been partially completed, and reveals the presence of a number of other tammar MHC III orthologues in this region.


Pedicled, vascularized occipital bone graft to supplement atlantoaxial arthrodesis for the treatment of pseudoarthrosis.
Atlantoaxial pseudoarthrosis is a challenging postoperative complication. The use of a local, vascularized bone graft, without free tissue transfer, to support a revision atlantoaxial fusion has not been previously described. We report the first surgical patient who received a semispinalis capitis muscle pedicled, occipital bone graft for supplementation of a revision atlantoaxial arthrodesis. A 72-year-old female had a failed atlantoaxial fusion and developed neck pain from continued instability and fractured hardware. The fixation and fusion were revised and supplemented with a novel, pedicled occipital bone graft. A craniectomy was performed in the occipital bone while still attached to the semispinalis capitis muscle to provide graft vascularity. This graft was rotated inferiorly from the skull base to the C1 arch and C2 spinous process in order to supplement a revision atlantoaxial arthrodesis. The patient had excellent clinical recovery over 18-month clinical follow up. The bone graft harvesting and rotation were performed safely and without complication. The 6-month postoperative CT scan showed partial fusion into the graft. This novel surgical technique leverages the advantages of vascularized structural autograft without adding extensive time or morbidity to the procedure as observed in free-tissue transfers. It is a safe and useful salvage technique to supplement revision atlantoaxial fusion surgeries.


IL1/ETAF activity and undetectable IL1 beta mRNA and minimal IL1 alpha mRNA levels in normal adult heat-separated epidermis.
Interleukin 1 (IL 1), a cytokine expressed from a number of cell types, plays a vital part in immune and inflammatory processes. Epidermal IL1-like activity, also known as epidermal cell thymocyte-activating factor (ETAF), is associated with whole skin, epidermis, Langerhans cells and cultured keratinocytes and keratinocyte cell lines. We show that preparations of heat-separated epidermis possess abundant IL1/ETAF activity as indicated by PGE/collagenase induction in cultured synovial cells and proliferation of mouse thymocytes. However, using cDNA probes to interleukin-1 alpha and beta we show that freshly isolated heat-separated epidermis from healthy adult donors contains only a slight detectable level of IL1 alpha mRNA by dot blot analysis and no IL1 beta and IL1 alpha mRNA by Northern blot analysis. The implications of these observations will be discussed.


Effect of Basic Fibroblast Growth Factor on Xenogeneic Islets in Subcutaneous Transplantation-A Murine Model.
Subcutaneous pockets provide an extrahepatic transplant site for islet grafting to treat type 1 diabetes. However, a hypoxic environment may cause central necrosis to islets and lead to graft failure. Our previous studies focused on a pre-treated subcutaneous site with basic fibroblast growth factor (bFGF) for the formation of vascular bed. In addition to neovascularization, bFGF was also shown to protect islets against oxidative stress and chemical-induced damage in vitro. Accordingly, we propose that subcutaneous islet transplantation with a bFGF-slow releasing device simultaneously can improve islet survival in vivo. A bFGF-impregnated collagen sheet was implanted in the right back of a streptozotocin-induced diabetic mouse for neovascularization. After 10 days, the sheet was removed and the rat islet-embedding gel within the immune-isolation device was transplanted (2-time operation [OP]). In another group, the diabetic mice received bFGF-impregnated gel with rat islets within the immune-isolation device simultaneously (1-time OP). Diabetic mice in 2-time OP group experienced a decrease in their non-fasting blood glucose level for a period of 10 days, and the glucose levels were lower than those of untreated diabetic mice post-implantation. However, the mice in the 1-time OP group remained hyperglycemic post-operation and showed no improvements in body weight or the area under curve in intraperitoneal glucose tolerance test. Furthermore, mice in the 2-time OP had relatively higher serum insulin levels with improved renal and metabolic biomarkers. Our findings suggest that bFGF had no beneficial effect on a 1-time operation in subcutaneous islet transplantation.


The neural systems for perceptual updating.
In a constantly changing environment we must adapt to both abrupt and gradual changes to incoming information. Previously, we demonstrated that a distributed network (including the anterior insula and anterior cingulate cortex) was active when participants updated their initial representations (e.g., it's a cat) in a gradually morphing picture task (e.g., now it's a rabbit; Stöttinger et al., 2015). To shed light on whether these activations reflect the proactive decisions to update or perceptual uncertainty, we introduced two additional conditions. By presenting picture morphs twice we controlled for uncertainty in perceptual decision making. Inducing an abrupt shift in a third condition allowed us to differentiate between a proactive decision in uncertainty-driven updating and a reactive decision in surprise-based updating. We replicated our earlier result, showing the robustness of the effect. In addition, we found activation in the anterior insula (bilaterally) and the mid frontal area/ACC in all three conditions, indicative of the importance of these areas in updating of all kinds. When participants were naïve as to the identity of the second object, we found higher activations in the mid-cingulate cortex and cuneus - areas typically associated with task difficulty, in addition to higher activations in the right TPJ most likely reflecting the shift to a new perspective. Activations associated with the proactive decision to update to a new interpretation were found in a network including the dorsal ACC known to be involved in exploration and the endogenous decision to switch to a new interpretation. These findings suggest a general network commonly engaged in all types of perceptual decision making supported by additional networks associated with perceptual uncertainty or updating provoked by either proactive or reactive decision making.


Antilipoxygenase activity of copper complexes of aminoalkanoate type.
Lipoxygenases (EC 188.8.131.52, LOX) catalyze the hydroperoxidation of polyunsaturated fatty acids (PUFA). This reaction leads to the production of conjugated hydro peroxide dienes of PUFA. In animals, LOX generate leukotriens (LT) and lipoxins, which belong to inflammatory mediators. It is believed that restricting LT synthesis by inhibition of LOX would have therapeutic utility for the treatment of a variety of inflammatory conditions (e.g. asthma, rheumatoid arthritis, psoriasis). The process of production and elimination of radical intermediates in vitro can be monitored by determination of LOX activity. We assessed the concentration of PUFA hydroperoxides in our system with LOX-catalyzed enzyme reaction spectrophotometrically. The inhibition of LOX activity (LOX from cytosol fraction of rat lungs) with selected N-salicylideneaminoalkanoatocopper(II) complexes was tested. In our experiments, all compounds tested showed an inhibitory effect on LOX catalyzed hydroperoxidation of PUFA. Cu(II) (from CuSO(4).5H(2)O dilution) ions also inhibited this enzyme reaction, but all compounds studied had a 10 times higher anti-LOX activity. The most effective of these complexes was monohydrate aqua-(N-salicylidene-L-a-alaninato)copper(II) complex [Cu(sal-L-alpha-ala)(H(2)O)].H(2)O with IC(50) 1.86 x 10(-4) mol/l. These complexes are known for their anti-phlogistic and radioprotective properties and they can be classified as potential anti-radical compounds. The structure of these complexes is similar to the active site of Cu,Zn-SOD (superoxide dismutase) and superoxide radical scavenger activity of these complexes is known. We found that these copper complexes were capable to inhibit LOX activity, which may be related with their anti-radical properties.


Determining User Intent of Partly Dynamic Shoulder Tasks in Individuals With Chronic Stroke Using Pattern Recognition.
Stroke remains the leading cause of long-term disability in the US. Although therapy can achieve limited improvement of paretic arm use and performance, weakness and abnormal muscle synergies-which cause unintentional elbow, wrist, and finger flexion during shoulder abduction-contribute significantly to limb disuse and compound rehabilitation efforts. Emerging wearable exoskeleton technology could provide powered abduction support for the paretic arm, but requires a clinically feasible, robust control scheme capable of differentiating multiple shoulder degrees-of-freedom. This study examines whether pattern recognition of sensor data can accurately identify user intent for 9 combinations of 1- and 2- degree-of-freedom shoulder tasks. Participants with stroke (n = 12) used their paretic and non-paretic arms, and healthy controls (n = 12) used their dominant arm to complete tasks on a lab-based robot involving combinations of abduction, adduction, and internal and external rotation of the shoulder. We examined the effect of arm (paretic, non-paretic), load level (25% vs 50% maximal voluntary torque), and dataset (electromyography, load cell, or combined) on classifier performance. Results suggest that paretic arm, lower load levels, and using load cell or EMG data alone reduced classifier accuracy. However, this method still shows promise. Further work will examine classifier-user interaction during active control of a robotic device and optimization/minimization of sensors.


Utility of Noncontrast Magnetic Resonance Imaging for Detection of Recurrent Vestibular Schwannoma.
Gadolinium-enhanced T1-weighted magnetic resonance imaging (MRI) (T1WI) is the conventional imaging technique of choice to detect vestibular schwannoma (VS) recurrence or regrowth, despite suboptimal specificity secondary to enhancing postoperative changes. Furthermore, recent concerns regarding the accumulation of gadolinium in body tissues have led for a call to reduce the number of contrast-enhanced examinations. The objective of the current study is to evaluate the diagnostic accuracy of high-resolution noncontrast (three-dimensional [3D] T2) MRI relative to gadolinium-enhanced T1WI in the detection of VS recurrence after resection. Following Institutional Review Board approval, 13 consecutive postoperative patients with VS recurrence or regrowth were identified from a prospectively maintained clinical database in which recurrence was determined by progression on serial postoperative MRI examination. Three blinded neuroradiologists retrospectively evaluated a total of 41 postoperative MRI examinations from these patients using only gadolinium-enhanced T1WI and 3D T2 MRI for recurrence or regrowth. Interobserver agreement, differences in detection between the two sequences, and the sensitivity and specificity of 3D T2 MRI were assessed. Fifteen of the 41 postoperative MRIs demonstrated progression, as determined by examiner consensus on the gadolinium-enhanced T1WI. Agreement, measured using Krippendorff's alpha, was 0.82 for the 3D T2 images and 0.83 for the contrast-enhanced T1WI. All the three examiners demonstrated no difference in the detection of progression between the two sequences (McNemar's test p values 0.69 for examiner 1, 0.63 for examiner 2, and 0.99 for examiner 3). The sensitivity of 3D T2 was 0.78 (CI 0.60-0.96), while the specificity was 0.94 (CI 0.86-1.00). Noncontrast high-resolution 3D T2 MRI seems sufficient to assess for recurrence or regrowth after VS resection. The results of this study have implications for reducing cost, time, and adverse events associated with gadolinium administration in this population requiring serial follow-up examinations. These promising, yet preliminary findings warrant confirmation with a larger prospective cohort of patients.


Field performance of transgenic citrus trees: assessment of the long-term expression of uidA and nptII transgenes and its impact on relevant agronomic and phenotypic characteristics.
The future of genetic transformation as a tool for the improvement of fruit trees depends on the development of proper systems for the assessment of unintended effects in field-grown GM lines. In this study, we used eight transgenic lines of two different citrus types (sweet orange and citrange) transformed with the marker genes β-glucuronidase (uidA) and neomycin phosphotransferase II (nptII) as model systems to study for the first time in citrus the long-term stability of transgene expression and whether transgene-derived pleiotropic effects occur with regard to the morphology, development and fruit quality of orchard-grown GM citrus trees. The stability of the integration and expression of the transgenes was confirmed in 7-year-old, orchard-grown transgenic lines by Southern blot analysis and enzymatic assays (GUS and ELISA NPTII), respectively. Little seasonal variation was detected in the expression levels between plants of the same transgenic line in different organs and over the 3 years of analysis, confirming the absence of rearrangements and/or silencing of the transgenes after transferring the plants to field conditions. Comparisons between the GM citrus lines with their non-GM counterparts across the study years showed that the expression of these transgenes did not cause alterations of the main phenotypic and agronomic plant and fruit characteristics. However, when comparisons were performed between diploid and tetraploid transgenic citrange trees and/or between juvenile and mature transgenic sweet orange trees, significant and consistent differences were detected, indicating that factors other than their transgenic nature induced a much higher phenotypic variability. Our results indicate that transgene expression in GM citrus remains stable during long-term agricultural cultivation, without causing unexpected effects on crop characteristics. This study also shows that the transgenic citrus trees expressing the selectable marker genes that are most commonly used in citrus transformation were substantially equivalent to the non-transformed controls with regard to their overall agronomic performance, as based on the use of robust and powerful assessment techniques. Therefore, future studies of the possible pleiotropic effects induced by the integration and expression of transgenes in field-grown GM citrus may focus on the newly inserted trait(s) of biotechnological interest.


Roles of Biopsychosocial Factors in the Development of Breast Cancer.
The aim of this study was to determine the roles of biopsychosocial risk factors in the development of breast cancer. This hospital-based case-control study included 491 women with breast cancer (study group) and 512 women who did not have cancer or other serious diseases (control group). Biological, psychological, and social risk factors were compared between the two groups. Data were collected using the semi-structured interview, the Stress Assessment Form, and the Coping Strategy Indicator to assess these factors. When the significantly different biopsychosocial variables between the study and the control groups were evaluated together, independent breast cancer risk factors were found as follows: a stressor experienced in the last 5 years, age 40 years and older, inadequate social support perception, use of avoidance coping strategy, being a housewife, having a family history of cancer, and having a body mass index ≥25. This study showed a relationship between breast cancer risk and manageable variables (obesity, stressor and coping strategy, social support, and employment status), age and family history of cancer, which are biopsychosocial factors. Biopsychosocial aspects are becoming a greater part of many different healthcare systems.


Associations between organochlorine pesticides and cognition in U.S. elders: National Health and Nutrition Examination Survey 1999-2002.
There is limited evidence about whether background exposure to organochlorine pesticides is related to impairment of cognitive function in general populations. This study was performed to investigate cross-sectional associations between serum concentrations of organochlorine pesticides and cognitive function, a predictor of dementia, among U.S. elders without overt dementia. Study subjects were 644 elders aged 60-85, participating in the National Health and Nutrition Examination Survey 1999-2002. We selected 6 organochlorine pesticides (p,p'-dichlorodiphenyltrichloroethane (DDT), p,p'-dichlorodiphenyldichloroethylene (DDE), trans-nonachlor, oxychlordane, heptachlor epoxide, and β-hexachlorocyclohexane) which were commonly detected in current general population. Cognitive function was assessed with the Digit-Symbol Substitution Test. All 6 compounds showed statistically significant or marginally significant inverse associations with cognitive score after adjusting for covariates including education levels. The strongest association was observed with p,p'-DDT. With the outcome of low cognitive score defined as <25th percentile, elders in the highest quartile of p,p'-DDT, p,p'-DDE, and β-hexachlorocyclohexane had 2 to 3 times higher risks than those in the lowest quartile. In particular, when their concentrations were further divided with the cutoff points of 90th and 95th percentiles, p,p'-DDT in the highest 5th percentile showed 6.5 (95% confidence interval: 2.6-16.3) times higher risk of low cognitive score. On the other hand, non-persistent pesticides like organophosphates or pyrethroid showed little association with this cognitive score. The potential role of background exposure to organochlorine pesticides in the development of dementia should be explored in future prospective studies and in-vitro/in-vivo experimental studies.


Adequate dietary protein is associated with better physical performance among post-menopausal women 60-90 years.
Sarcopenia, the involuntary loss of skeletal muscle with age, affects up to one-quarter of older adults. Evidence indicates a positive association between dietary protein intake and lean muscle mass and strength among older persons, but information on dietary protein's effect on physical performance in older adults has received less attention. Cross-sectional observational analysis of the relationship of dietary protein on body composition and physical performance. Clinical research center. 387 healthy women aged 60 - 90 years (mean 72.7 ± 7.0 y). Measures included body composition (fat-free mass, appendicular skeletal mass and fat mass) via dual x-ray absorptiometry (DXA), physical performance (Physical Performance Test [PPT] and Short Physical Performance Battery [SPPB]), handgrip strength, Physical Activity Scale in the Elderly (PASE), quality of life measure (SF-8), falls, fractures, nutrient and macromolecule intake (four-day food record). Independent samples t-tests determined mean differences between the above or below RDA protein groups. Analysis of covariance was used to control for body mass index (BMI) between groups when assessing physical performance, physical activity and health-related quality of life. The subjects consumed an average of 72.2 g protein/day representing 1.1 g protein/kg body weight/day. Subjects were categorized as below the recommended daily allowance (RDA) for protein (defined as less than 0.8 g protein/kg) or at or above the RDA (equal to or higher than 0.8 g protein/kg). Ninety-seven subjects (25%) were in the low protein group, and 290 (75%) were in the higher protein group. Women in the higher protein group had lower body mass, including fat and lean mass, and fat-to-lean ratio than those in the lower-protein group (p <0.001). Composite scores of upper and lower extremity strength were impaired in the group with low protein intake; SPPB score was 9.9±1.9 compared to 10.6±1.6 in those with higher protein intake and PPT was 19.8± 2.9 compared to 20.9± 2.1 in the low and higher protein groups, respectively. The results were attenuated by correction for BMI, but remained significant. The physical component of the SF-8 was also lower in the low protein group but did not remain significant when controlling for BMI. No significant differences were found in hand grip strength or reported physical activity. Healthy, older postmenopausal women consumed, on average, 1.1 g/kg/d protein, although 25% consumed less than the RDA. Those in the low protein group had higher body fat and fat-to-lean ratio than those who consumed the higher protein diet. Upper and lower extremity function was impaired in those who consumed a low protein diet compared to those with a higher protein intake. Protein intake should be considered when evaluating the multi-factorial loss of physical function in older women.


RAGE activation in macrophages and development of experimental diabetic polyneuropathy.
It is suggested that activation of receptor for advanced glycation end products (RAGE) induces proinflammatory response in diabetic nerve tissues. Macrophage infiltration is invoked in the pathogenesis of diabetic polyneuropathy (DPN), while the association between macrophage and RAGE activation and the downstream effects of macrophages remain to be fully clarified in DPN. This study explored the role of RAGE in the pathogenesis of DPN through the modified macrophages. Infiltrating proinflammatory macrophages impaired insulin sensitivity, atrophied the neurons in dorsal root ganglion, and slowed retrograde axonal transport (RAT) in the sciatic nerve of type 1 diabetic mice. RAGE-null mice showed an increase in the population of antiinflammatory macrophages, accompanied by intact insulin sensitivity, normalized ganglion cells, and RAT. BM transplantation from RAGE-null mice to diabetic mice protected the peripheral nerve deficits, suggesting that RAGE is a major determinant for the polarity of macrophages in DPN. In vitro coculture analyses revealed proinflammatory macrophage-elicited insulin resistance in the primary neuronal cells isolated from dorsal root ganglia. Applying time-lapse recording disclosed a direct impact of proinflammatory macrophage and insulin resistance on the RAT deficits in primary neuronal cultures. These results provide a potentially novel insight into the development of RAGE-related DPN.


No Evidence That Abstract Structure Learning Disrupts Novel-Event Learning in 8- to 11-Month-Olds.
Although infants acquire specific information (e.g., motion of a specific toy) and abstract information (e.g., likelihood of events repeating), it is unclear whether extraction of abstract information interferes with specific learning. In the present study, 8- to 11-month-old infants were shown four audio-visual movies, either with a mixed or uniform presentation structure. Learning of abstract information was operationally defined as the looking time to changes in presentation structure of the movies (mixed vs. uniform blocks), and learning of specific information was defined as the looking time to changes in content in the four movies (object properties and identities). We found evidence of both specific and abstract learning, but did not find evidence that extraction of the presentation structure (i.e., abstract learning) impacts specific learning of the events. We discuss the implications of the costs and benefits of the interaction between abstract and specific learning for infants.


Population structure and drug resistance patterns of emerging non-PCV-13 Streptococcus pneumoniae serotypes 22F, 15A, and 8 isolated from adults in Ontario, Canada.
The introduction of pneumococcal conjugate vaccines has led to the emergence of non-vaccine serotypes, which contributed to invasive pneumococcal disease in Canada and worldwide. A significant increase in the prevalence of non-13-valent pneumococcal conjugate vaccine (PCV-13)-included serotypes 22F, 15A, and 8 was observed from 2009 to 2013 in Ontario (all p values<0.01). In this study, whole genome sequencing was conducted on the 25 isolates of serotype 22F, seven of 15A and 10 of 8 to investigate the population structure and antibiotic resistance. All seven serotype 15A isolates were found to be multidrug resistant. From whole genome analysis, we observed recombination events among serotypes 22F, 15A and 8 populations. Serotype 22F (ST433) has emerged into two sub-populations, with 28% (7/25) exhibiting recombination events, and five also acquiring macrolide resistance as a result of recombination. This study enhances the knowledge on the molecular evolution of emerging non-PCV-13 vaccine serotype 22F, including acquisition of resistance genes through recombination events. It underpins the importance of whole genome sequencing in studying Streptococcus pneumoniae population structures and dynamics, and its utility in molecular surveillance.


Rehabilitating the severely disabled: the foreign experience.
This paper sets forth a model for examining the relationships between fourteen policy and politicoeconomic variables, and the social benefits and costs of rehabilitation. Based on discussions in early 1977 with scholars, ministry officials, trade unionists, and politicians in several northwestern European countries, as well as on documentation relating to the rapid growth of disability expenditures and the factors thought to influence it, some "lessons" are presented for policymakers in the United States and other countries. In general, current trends are seen as depressing the post-service earnings of individual rehabilitants, limiting the stabilizing effects of rehabilitation on labor market turnover, and increasing available time for unpaid work in the home and elsewhere. Unequal intergovernmental cost sharing in the provision of benefits and services, it is argued, seems likely to promote inefficient allocation of scarce rehabilitation resources with negative consequences for goal attainment. The high rates of inflation which prevail in the United States and in many northwestern European countries are causing the immediate costs of providing rehabilitation services to rise and simultaneously increasing the opportunity costs of spending for rehabilitation. The net effect of these cost increases is a reduction in the overall benefit/cost ratio that results from investments in rehabilitation. A number of predictions are made about how the United States will shape its disability and rehabilitation policies in the course of the next twenty years.


Polyamine depletion stabilizes p53 resulting in inhibition of normal intestinal epithelial cell proliferation.
The p53 nuclear phosphoprotein plays a critical role in transcriptional regulation of target genes involved in growth arrest and apoptosis. The natural polyamines, including spermidine, spermine, and their precursor putrescine, are required for cell proliferation, and decreasing cellular polyamines inhibits growth of the small intestinal mucosa. In the current study, we investigated the mechanisms of regulation of p53 gene expression by cellular polyamines and further determined the role of the gene product in the process of growth inhibition after polyamine depletion. Studies were conducted both in vivo and in vitro using rats and the IEC-6 cell line, derived from rat small intestinal crypt cells. Levels for p53 mRNA and protein, transcription and posttranscription of the p53 gene, and cell growth were examined. Depletion of cellular polyamines by treatment with alpha-difluoromethylornithine (DFMO) increased p53 gene expression and caused growth inhibition in the intact small intestinal mucosa and the cultured cells. Polyamine depletion dramatically increased the stability of p53 mRNA as measured by the mRNA half-life but had no effect on p53 gene transcription in IEC-6 cells. Induction of p53 mRNA levels in DFMO-treated cells was paralleled by an increase in the rate of newly synthesized p53 protein. The stability of p53 protein was also increased after polyamine depletion, which was associated with a decrease in Mdm2 expression. When polyamine-deficient cells were exposed to exogenous spermidine, a decrease in p53 gene expression preceded an increase in cellular DNA synthesis. Inhibition of the p53 gene expression by using p53 antisense oligodeoxyribonucleotides significantly promoted cell growth in the presence of DFMO. These findings indicate that polyamines downregulate p53 gene expression posttranscriptionally and that growth inhibition of small intestinal mucosa after polyamine depletion is mediated, at least partially, through the activation of p53 gene.


Clonotype analysis of cytomegalovirus-specific cytotoxic T lymphocytes.
Cytotoxic T lymphocytes (CTL) control the replication of human cytomegalovirus (CMV). Previous studies assessed the clonotypic composition of CTL specific for individual immunodominant peptides within a certain HLA context. Such an approach has inherent limitations and may not assess the true clonal CTL response in vivo. Here, the clonotypic composition of CMV-specific CTL was determined in HLA-A2, CMV-seropositive kidney transplant recipients and healthy blood donors after stimulation of peripheral blood mononuclear cells with either a pp65 whole-peptide pool or a single immunodominant peptide. Even after stimulation with the whole peptide pool, CMV-specific CTL remained monoclonal or oligoclonal. Regarding intraindividual variation, the CDR3 motifs of the dominant clones were identical to those observed in CTL generated by the single immunodominant peptide. Sequencing of the CDR3 regions demonstrated significant interindividual variation; however, structural homology was observed for immunodominant clonotypes in three individuals. In conclusion, the highly focused T cell receptor repertoire found after stimulation with either a single immunodominant peptide or a peptide pool demonstrates a pivotal role for immunodominant epitopes in the generation of a clonal repertoire. These results provide new insights into the regulation of CMV clonal dominance and may contribute to the design and monitoring of adoptive immunotherapy.


Effects of an 8-Week Protein Supplementation Regimen with Hyperimmunized Cow Milk on Exercise-Induced Organ Damage and Inflammation in Male Runners: A Randomized, Placebo Controlled, Cross-Over Study.
Prolonged strenuous exercise may induce inflammation, cause changes in gastrointestinal permeability, and lead to other unfavorable biological changes and diseases. Nutritional approaches have been used to prevent exercise-induced inflammatory responses and gastrointestinal disorders. Hyperimmunized milk, obtained by immunizing cows against specific antigens, promotes the development of immunity against pathogens, promotes anti-inflammatory effects, and protects intestinal function. Immune protein (IMP) is a concentrated product of hyperimmunized milk and is a more promising means of supplementation to protect against acute infections and inflammation. To determine whether IMP has protective properties against exercise-induced gastrointestinal dysfunction and inflammation, we examined biochemical markers, intestinal damage markers, and pro-/anti-inflammatory profiles of young male runners using a randomized, placebo controlled, cross-over design. Urine samples were collected and used for measurements of creatinine, N-acetyl-β-d-glucosaminidase, osmotic pressure, and specific gravity. Titin was measured as a muscle damage marker. Further, urine concentrations of complement 5a, calprotectin, fractalkine, myeloperoxidase, macrophage colony-stimulating factor, monocyte chemotactic protein-1, intestinal fatty acid binding protein (I-FABP), interferon (IFN)-γ, interleukin (IL)-1β, IL-1 receptor antagonist, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, and tumor necrosis factor (TNF)-α were measured by enzyme-linked immunosorbent assays. We demonstrated that urine osmotic pressure, urine specific gravity, I-FABP, IFN-γ, IL-1β, and TNF-α were reduced by 8 weeks of IMP supplementation, indicating that IMP may have potential in preventing strenuous exercise-induced renal dysfunction, increased intestinal permeability, and inflammation. Thus, IMP supplementation may be a feasible nutritional approach for the prevention of unfavorable exercise-induced symptoms.


Effect of gentamicin administration on the neuromuscular blockade induced by atracurium in cats.
Atracurium besylate, a nondepolarizing neuromuscular blocking agent, was administered as an infusion to 8 anesthetized cats in which neuromuscular blockade was assessed, using the train-of-four response. Once 50% depression of the first-twitch (T1) response was achieved, the infusion was held constant for 60 minutes before being discontinued and the recovery time was determined. The time for recovery was recorded as the time for the train-of-four (T4 ratio) to increase from 50% to 75%. After recovery, atracurium infusion was reinstituted and the cats were again maintained for 60 minutes at 50% depression. A single bolus of gentamicin sulfate (2.0 mg/kg of body weight) was administered IV, and the infusion was continued for another 60 minutes before it was discontinued and the time for recovery was recorded. Within 1 minute of gentamicin administration, the mean +/- SD T1 response decreased from 49 +/- 5% to 33 +/- 8% of baseline and the T4 ratio decreased from 28 +/- 19% to 14 +/- 11%. Peak effect occurred at 5 minutes, with a T1 response of 29 +/- 6% of baseline and a T4 ratio of 13 +/- 12%. By 60 minutes after gentamicin administration, the T1 response had increased to 38 +/- 7% of baseline and the T4 ratio had increased to 21 +/- 13%. The time for recovery significantly (P less than 0.03) increased from 9.9 +/- 3.4 minutes during the control study to 18.1 +/- 10.7 minutes during the gentamicin study. In this study, gentamicin potentiated the neuromuscular blockade induced by atracurium and increased the recovery time. Residual blockade, observed after gentamicin administration was reversed with edrophonium.


M are better than one: an ensemble-based motif finder and its application to regulatory element prediction.
Identifying regulatory elements in genomic sequences is a key component in understanding the control of gene expression. Computationally, this problem is often addressed by motif discovery, where the goal is to find a set of mutually similar subsequences within a collection of input sequences. Though motif discovery is widely studied and many approaches to it have been suggested, it remains a challenging and as yet unresolved problem. We introduce SAMF (Solution-Aggregating Motif Finder), a novel approach for motif discovery. SAMF is based on a Markov Random Field formulation, and its key idea is to uncover and aggregate multiple statistically significant solutions to the given motif finding problem. In contrast to many earlier methods, SAMF does not require prior estimates on the number of motif instances present in the data, is not limited by motif length, and allows motifs to overlap. Though SAMF is broadly applicable, these features make it particularly well suited for addressing the challenges of prokaryotic regulatory element detection. We test SAMF's ability to find transcription factor binding sites in an Escherichia coli dataset and show that it outperforms previous methods. Additionally, we uncover a number of previously unidentified binding sites in this data, and provide evidence that they correspond to actual regulatory elements. Supplementary data are available at Bioinformatics online.


Hepatitis C virus notification rates in Australia are highest in socioeconomically disadvantaged areas.
Poor access to health services is a significant barrier to achieving the World Health Organization's hepatitis C virus (HCV) elimination targets. We demonstrate how geospatial analysis can be performed with commonly available data to identify areas with the greatest unmet demand for HCV services. We performed an Australia-wide cross-sectional analysis of 2015 HCV notification rates using local government areas (LGAs) as our unit of analysis. A zero-inflated negative binomial regression was used to determine associations between notification rates and socioeconomic/demographic factors, health service and geographic remoteness area (RA) classification variables. Additionally, component scores were extracted from a principal component analysis (PCA) of the healthcare service variables to provide rankings of relative service coverage and unmet demand across Australia. Among LGAs with non-zero notifications, higher rates were associated with areas that had increased socioeconomic disadvantage, more needle and syringe services (incidence rate ratio [IRR] 1.022; 95%CI 1.001, 1.044) and more alcohol and other drug services (IRR 1.019; 1.005, 1.034). The distribution of PCA component scores indicated that per-capita healthcare service coverage was lower in areas outside of major Australian cities. Areas outside of major cities also contained 94% of LGAs in the lowest two socioeconomic quintiles, as well as 35% of HCV notifications despite only representing 29% of the population. As countries aim for HCV elimination, routinely collected data can be used to identify geographical areas for priority service delivery. In Australia, the unmet demand for HCV treatment services is greatest in socioeconomically disadvantaged and non-metropolitan areas.


Carbohydrate Intake in Early Childhood and Body Composition and Metabolic Health: Results from the Generation R Study.
High sugar intake in childhood has been linked to obesity. However, the role of macronutrient substitutions and associations with metabolic health remain unclear. We examined associations of carbohydrate intake and its subtypes with body composition and metabolic health among 3573 children participating in a population-based cohort in the Netherlands. Intake of total carbohydrate, monosaccharides and disaccharides, and polysaccharides at age 1 year was assessed with a food-frequency questionnaire. We repeatedly measured children's height and weight to calculate BMI between their ages of 1 and 10 years. At ages 6 and 10 years, fat and fat-free mass were measured with dual-energy X-ray-absorptiometry and blood concentrations of triglycerides, cholesterol, and insulin were obtained. For all outcomes, we calculated age and sexspecific SD-scores. In multivariable-adjusted linear mixed models, we found no associations of intake of carbohydrates or its subtypes with children's BMI or body composition. A higher intake of monosaccharides and disaccharides was associated with higher triglyceride concentrations (0.02 SDS per 10 g/d, 95% CI: 0.01, 0.04). Higher monosaccharide and disaccharide intake was also associated with lower HDL-cholesterol (-0.03 SDS, 95% CI: -0.04; -0.01), especially when it replaced polysaccharides. Overall, our findings suggest associations of higher monosaccharide and disaccharide intake in early childhood with higher triglyceride and lower HDL-cholesterol concentrations, but do not support associations with body composition.


High-risk patients with ST-elevation myocardial infarction derive greatest absolute benefit from primary percutaneous coronary intervention: results from the Primary Coronary Angioplasty Trialist versus thrombolysis (PCAT)-2 collaboration.
Meta-analyses of randomized trials show that primary percutaneous coronary intervention (PPCI) results in lower mortality than fibrinolytic therapy in patients with myocardial infarction. We investigated which categories of patients with myocardial infarction would benefit most from the strategy of PPCI and, thus, have lowest numbers needed to treat to prevent a death. Individual patient data were obtained from 22 (n = 6,763) randomized trials evaluating efficacy and safety of PPCI versus fibrinolysis. A risk score was developed and validated to estimate the probability of 30-day death in individuals. Patients were then divided in quartiles according to risk. Subsequent analyses were performed to evaluate if the treatment effect was modified by estimated risk. Overall, 446 patients (6.6%) died within 30 days after randomization. The mortality risk score contained clinical characteristics, including the time from symptom onset to randomization. The c-index was 0.76, and the Hosmer-Lemeshow test was nonsignificant, reflecting adequate discrimination and calibration. Patients randomized to PPCI had lower mortality than did patients randomized to fibrinolysis (5.3% vs 7.9%, adjusted odds ratio 0.63, 95% CI 0.42-0.84, P < .001). The interaction between risk score and allocated treatment interaction term had no significant contribution (P = .52) to the model, indicating that the relative mortality reduction by PPCI was similar at all levels of estimated risk. In contrast, the absolute risk reduction was strongly related to estimated risk at baseline: the numbers needed to treat to prevent a death by PPCI versus fibrinolysis was 516 in the lowest quartile of estimated risk compared with only 17 in the highest quartile. Primary percutaneous coronary intervention is consistently associated with a strong relative reduction in 30-day mortality, irrespective of patient baseline risk, and should therefore be considered as the first choice reperfusion strategy whenever feasible. If access to percutaneous coronary intervention is >2 hours, fibrinolysis remains a legitimate option in low-risk patients because of the small absolute risk reduction by PPCI in this particular cohort.


Water vapor absorption coefficients in the 8-13-microm spectral region: a critical review.
Measurements of water vapor absorption coefficients in the thermal IR atmospheric window (8-13 microm) during the past 20 years obtained by a variety of techniques are reviewed for consistency and are compared with computed values based on the AFGL spectral data tapes. The methods of data collection considered were atmospheric long path absorption with a CO(2) laser or a broadband source and filters, a White cell and a CO(2) laser or a broadband source and a spectrometer, and a spectrophone with a CO(2) laser. Advantages and disadvantages of each measurement approach are given as a guide to further research. Continuum absorption has apparently been measured accurately to about the 5-10% level in five of the measurements reported. However, the effect of oxygen broadening has not been fully considered, since most laboratory measurements were made using nitrogen buffering. Oxygen could lead to a small reduction in the adopted value of the water vapor continuum absorption coefficient in air. Also, the temperature dependence does not seem to have been measured well for temperatures <20 degrees C. The rotational and v(2) line absorption coefficients do not appear to have been determined well in this spectral region except at CO(2) laser line frequencies, because the agreement between such measurements and the AFGL spectral data tapes is generally not good.


Age-related changes in trunk neuromuscular activation patterns during a controlled functional transfer task include amplitude and temporal synergies.
While healthy aging is associated with physiological changes that can impair control of trunk motion, few studies examine how spinal muscle responses change with increasing age. This study examined whether older (over 65 years) compared to younger (20-45 years) adults had higher overall amplitude and altered temporal recruitment patterns of trunk musculature when performing a functional transfer task. Surface electromyograms from twelve bilateral trunk muscle (24) sites were analyzed using principal component analysis, extracting amplitude and temporal features (PCs) from electromyographic waveforms. Two PCs explained 96% of the waveform variance. Three factor ANOVA models tested main effects (group, muscle and reach) and interactions for PC scores. Significant (p<.0125) group interactions were found for all PC scores. Post hoc analysis revealed that relative to younger adults, older adults recruited higher agonist and antagonistic activity, demonstrated continuous activation levels in specific muscle sites despite changing external moments, and had altered temporal synergies within abdominal and back musculature. In summary both older and younger adults recruit highly organized activation patterns in response to changing external moments. Differences in temporal trunk musculature recruitment patterns suggest that older adults experience different dynamic spinal stiffness and loading compared to younger adults during a functional lifting task.


Derivation and validation of a machine learning record linkage algorithm between emergency medical services and the emergency department.
Linking emergency medical services (EMS) electronic patient care reports (ePCRs) to emergency department (ED) records can provide clinicians access to vital information that can alter management. It can also create rich databases for research and quality improvement. Unfortunately, previous attempts at ePCR and ED record linkage have had limited success. In this study, we use supervised machine learning to derive and validate an automated record linkage algorithm between EMS ePCRs and ED records. All consecutive ePCRs from a single EMS provider between June 2013 and June 2015 were included. A primary reviewer matched ePCRs to a list of ED patients to create a gold standard. Age, gender, last name, first name, social security number, and date of birth were extracted. Data were randomly split into 80% training and 20% test datasets. We derived missing indicators, identical indicators, edit distances, and percent differences. A multivariate logistic regression model was trained using 5-fold cross-validation, using label k-fold, L2 regularization, and class reweighting. A total of 14 032 ePCRs were included in the study. Interrater reliability between the primary and secondary reviewer had a kappa of 0.9. The algorithm had a sensitivity of 99.4%, a positive predictive value of 99.9%, and an area under the receiver-operating characteristic curve of 0.99 in both the training and test datasets. Date-of-birth match had the highest odds ratio of 16.9, followed by last name match (10.6). Social security number match had an odds ratio of 3.8. We were able to successfully derive and validate a record linkage algorithm from a single EMS ePCR provider to our hospital EMR.


Self-protection against gliotoxin--a component of the gliotoxin biosynthetic cluster, GliT, completely protects Aspergillus fumigatus against exogenous gliotoxin.
Gliotoxin, and other related molecules, are encoded by multi-gene clusters and biosynthesized by fungi using non-ribosomal biosynthetic mechanisms. Almost universally described in terms of its toxicity towards mammalian cells, gliotoxin has come to be considered as a component of the virulence arsenal of Aspergillus fumigatus. Here we show that deletion of a single gene, gliT, in the gliotoxin biosynthetic cluster of two A. fumigatus strains, rendered the organism highly sensitive to exogenous gliotoxin and completely disrupted gliotoxin secretion. Addition of glutathione to both A. fumigatus Delta gliT strains relieved gliotoxin inhibition. Moreover, expression of gliT appears to be independently regulated compared to all other cluster components and is up-regulated by exogenous gliotoxin presence, at both the transcript and protein level. Upon gliotoxin exposure, gliT is also expressed in A. fumigatus Delta gliZ, which cannot express any other genes in the gliotoxin biosynthetic cluster, indicating that gliT is primarily responsible for protecting this strain against exogenous gliotoxin. GliT exhibits a gliotoxin reductase activity up to 9 microM gliotoxin and appears to prevent irreversible depletion of intracellular glutathione stores by reduction of the oxidized form of gliotoxin. Cross-species resistance to exogenous gliotoxin is acquired by A. nidulans and Saccharomyces cerevisiae, respectively, when transformed with gliT. We hypothesise that the primary role of gliotoxin may be as an antioxidant and that in addition to GliT functionality, gliotoxin secretion may be a component of an auto-protective mechanism, deployed by A. fumigatus to protect itself against this potent biomolecule.


5-fu metabolism in cancer and orally-administrable 5-fu drugs.
5-Fluorouracil (5-FU) is a key anticancer drug that for its broad antitumor activity, as well as for its synergism with other anticancer drugs, has been used to treat various types of malignancies. In chemotherapeutic regimens, 5-FU has been combined with oxaliplatin, irinotecan and other drugs as a continuous intravenous infusion. Recent clinical chemotherapy studies have shown that several of the regimens with oral 5-FU drugs are not inferior compared to those involving continuous 5-FU infusion chemotherapy, and it is probable that in some regimens continuous 5-FU infusion can be replaced by oral 5-FU drugs. Historically, both the pharmaceutical industry and academia in Japan have been involved in the development of oral 5-FU drugs, and this review will focus on the current knowledge of 5-FU anabolism and catabolism, and the available information about the various orally-administrable 5-FU drugs, including UFT, S-1 and capecitabine. Clinical studies comparing the efficacy and adverse events of S-1 and capecitabine have been reported, and the accumulated results should be utilized to optimize the treatment of cancer patients. On the other hand, it is essential to elucidate the pharmacokinetic mechanism of each of the newly-developed drugs, to correctly select the drugs for each patient in the clinical setting, and to further develop optimized drug derivatives.


Characteristics of Toxic Keratopathy, an In Vivo Confocal Microscopy Study.
Toxic keratopathy (TK) involves complex clinical manifestations and is difficult to differentiate from other ocular surface diseases by conventional slit-lamp examination. The challenge faced by clinicians in confidently diagnosing TK cannot be underestimated. This study aimed to explore the microstructural characteristics and diagnostic parameters by in vivo confocal microscopy (IVCM) in TK. In this prospective, cross-sectional, comparative study, slit-lamp and IVCM examinations were performed on 20 normal eyes and 54 eyes with TK. Based on slit-lamp imaging, TK subjects were divided into four groups: superficial punctate keratitis (n = 10 eyes), pseudodendritic keratitis (n = 14 eyes), ulcerative keratitis (UK; n = 16 eyes), and ring keratitis (RK; n = 14 eyes). The microstructural characteristics of TK were described according to the following IVCM parameters: basal cell (BC) density, dendritiform cell (DC) density, DC size, corneal nerve fiber (CNF) length, nerve tortuosity, and keratocyte reflectivity. A receiver operating characteristic (ROC) curve model was also formulated to compare the predictive power of BC density, DC density, and CNF length. TK eyes showed significantly higher values for DC density (45.8 cells/mm2; range, 25.0-100.0) compared with normal eyes (24.0 cells/mm2; range, 20.8-32.3; P = 0.013; DC size (111.0 µm2; range, 92.0-137.8) compared with normal eyes (63.7 µm2; range, 47.7-70.3; P < 0.001); nerve tortuosity (0.08; range, 0.05-0.09) compared with normal eyes (0.04; range, 0.02-0.04; P < 0.001); and keratocyte reflectivity. BC density and CNF length values were found to be significantly less than those for normal controls (both P < 0.001). In all subgroups, CNF length appeared to be significantly lower than that of controls (all P < 0.001), and DC density was only statistically significantly higher in the UK (P = 0.003) and RK (P < 0.001) groups. Corneal fluorescein staining had no correlation with the analyzed IVCM parameters (all P ˃ 0.05). However, the increase in DC density and DC size showed negative correlations to CNF length (density: r = -0.325, P < 0.005; size: r = -0.493, P < 0.005), as well as positive correlations to duration and frequency of topical eye drops and DC size (density: r = 0.361, P < 0.05; size: r = 0.581, P < 0.05). A ROC curve showed that CNF length had the strongest predictive power, with the estimated area under the curve being 0.992 ± 0.008. Lower BC density and CNF length, greater DC density and DC size, and greater keratocyte reflectivity were the microstructural characteristics of TK. The role of subbasal nerve, inflammatory response, and limbal stem cells in the progression of TK and the appropriate treatment of different TK stages are future research directions. The evaluation of basal cells, subbasal nerve, and dendritiform cells is helpful to our understanding of the pathological process of TK.


Age, season and spatio-temporal factors affecting the prevalence of Echinococcus multilocularis and Taenia taeniaeformis in Arvicola terrestris.
Taenia taeniaeformis and the related zoonotic cestode Echinococcus multilocularis both infect the water vole Arvicola terrestris. We investigated the effect of age, spatio-temporal and season-related factors on the prevalence of these parasites in their shared intermediate host. The absolute age of the voles was calculated based on their eye lens weights, and we included the mean day temperature and mean precipitation experienced by each individual as independent factors. Overall prevalences of E. multilocularis and T. taeniaeformis were 15.1% and 23.4%, respectively, in 856 A. terrestris trapped in the canton Zürich, Switzerland. Prevalences were lower in young (≤ 3 months: E. multilocularis 7.6%, T. taeniaeformis 17.9%) than in older animals (>7 months: 32.6% and 34.8%). Only 12 of 129 E. multilocularis-infected voles harboured protoscoleces. Similar proportions of animals with several strobilocerci were found in T. taeniaeformis infected voles of <5 months and ≥5 months of age (12.8% and 11.9%). Multivariate analyses revealed strong spatio-temporal variations in prevalences of E. multilocularis. In one trapping area, prevalences varied on an exceptional high level of 40.6-78.5% during the whole study period. Low temperatures significantly correlated with the infection rate whereas precipitation was of lower importance. Significant spatial variations in prevalences were also identified for Taenia taeniaeformis. Although the trapping period and the meteorological factors temperature and precipitation were included in the best models for explaining the infection risk, their effects were not significant for this parasite. Our results demonstrate that, besides temporal and spatial factors, low temperatures contribute to the risk of infection with E. multilocularis. This suggests that the enhanced survival of E. multilocularis eggs under cold weather conditions determines the level of infection pressure on the intermediate hosts and possibly also the infection risk for human alveolar echincoccosis (AE). Therefore, interventions against the zoonotic cestode E. multilocularis by deworming foxes may be most efficient if conducted just before and during winter.


p53 Deletion or Hotspot Mutations Enhance mTORC1 Activity by Altering Lysosomal Dynamics of TSC2 and Rheb.
The activity of mammalian target of rapamycin complex 1 (mTORC1) is frequently enhanced in carcinomas, an effect thought to contribute to the malignant phenotype. Here, it is demonstrated that either deletion or mutation of TP53 in colon or lung carcinoma cells substantially enhances mTORC1 kinase activity by an effect downstream of and independent of AMPK. Mechanistically, it was determined that loss or mutation of p53 decreased expression of TSC2 and Sestrin2 (SESN2). Complementation of p53 null cells with TSC2 or Sestrin2 reduced mTORC1 activity to levels found in p53 wild-type (wt) cells, whereas their genetic depletion enhanced mTORC1 activity in p53 wt cells. However, the primary causal event in enhanced mTORC1 activity upon loss of p53 appeared to be a diminished distribution of TSC2 to lysosomal membranes containing mTOR. Subsequently, there was increased Rheb in the lysosomal compartment, and a higher mTOR association with Raptor. Transfection of TSC2 into p53 null cells replaced TSC2 and diminished Rheb at the lysosome, recapitulating cells with wt p53. In contrast, transfection of Sestrin2 decreased mTOR in lysosomes, but the lower levels of Sestrin2 in p53 null cells did not change lysosomal mTOR. In summary, loss of the transcriptional activity of p53, either by deletion or by key mutations in the DNA-binding domain, diminishes expression of TSC2 and Sestrin2, thus, shifting membrane-bound TSC2 out of lysosomal membranes, increasing lysosomal Rheb and increasing the kinase activity of mTORC1. This study establishes that loss of p53 function decreases lysosomal TSC2 and increases lysosomal Rheb resulting in hyperactive mTORC1, findings that are consistent with a more malignant phenotype.


Adaptive Pulvinar Circuitry Supports Visual Cognition.
The pulvinar is the largest thalamic nucleus in primates and one of the most mysterious. Endeavors to understand its role in vision have focused on its abundant connections with the visual cortex. While its connectivity mapping in the cortex displays a broad topographic organization, its projections are also marked by considerable convergence and divergence. As a result, the pulvinar is often regarded as a central forebrain hub. Moreover, new evidence suggests that its comparatively modest input from structures such as the retina and superior colliculus may critically shape the functional organization of the visual cortex, particularly during early development. Here we review recent studies that cast fresh light on how the many convergent pathways through the pulvinar contribute to visual cognition.


Modeling and experimental investigations of Fano resonances in free-standing LiNbO3 photonic crystal slabs.
In this paper the Fano resonance in a free-standing LiNbO(3) photonic crystal slab is demonstrated. We present a numerical analysis and experimental measurements with free space illumination where the dependence of slab thickness, radius of air holes and lattice types are investigated. The unique property of polarization dependence for LiNbO(3) photonic crystal slabs is also analyzed, and we show that the transmission spectra exhibit significant sensitivity (~25nm) to polarization. A monolithic free-standing LiNbO(3) photonic crystal slab was fabricated using ion beam enhanced etching (IBEE) technology. Measurement results of the reflection spectra agree with the numerical analysis.


Significant differences between the G+C content of synonymous codons in orthologous genes and the genomic G+C content.
The relationship between the overall G+C content of the genome (GC) and the GC content at the third codon positions (GC3) of genes, which we refer to as a GC3-plot, was examined using 15 currently available complete genome sequences. A remarkably linear relationship was found between these two quantities, confirming previous observations of a strong positive correlation in the GC3-plot. In order to conduct a more detailed analysis of the GC3-plot, we examined the GC3 content by separating orthologous codons into three categories: synonymously different codons (namely identical amino acids, IA), different amino acids (DA), and identical codons (IC), for a pairwise comparison of two closely related species. When we took pairwise species comparisons between Mycoplasma genitalium (Mg) and Mycoplasma pneumoniae (Mp) and between Mycobacterium tuberculosis (Mt) and Mycobacterium leprae (Ml) as examples, we found that for Mp and Ml, the GC3 for IA deviated the most from the linear expectation in the GC3-plot, whereas for Mg and Mt the deviation was minimal. These findings suggest that the major changes of GC content took place in Mp and Ml, but not in Mg and Mt. This analysis also enables us to predict the future direction of the evolutionary changes of the genomic GC content.


An assessment of acoustic contrast between long and short vowels using convex hulls.
An alternative to the spectral overlap assessment metric (SOAM), first introduced by Wassink [(2006). J. Acoust. Soc. Am. 119(4), 2334-2350], is introduced. The SOAM quantifies the intra- and inter-language differences between long-short vowel pairs through a comparison of spectral (F1, F2) and temporal properties modeled with best fit ellipses (F1 × F2 space) and ellipsoids (F1 × F2 × duration). However, the SOAM ellipses and ellipsoids rely on a Gaussian distribution of vowel data and a dense dataset, neither of which can be assumed in endangered languages or languages with limited available data. The method presented in this paper, called the Vowel Overlap Assessment with Convex Hulls (VOACH) method, improves upon the earlier metric through the use of best-fit convex shapes. The VOACH method reduces the incorporation of "empty" data into calculations of vowel space. Both methods are applied to Numu (Oregon Northern Paiute), an endangered language of the western United States. Calculations from the VOACH method suggest that Numu is a primary quantity language, a result that is well aligned with impressionistic analyses of spectral and durational data from the language and with observations by field researchers.


Clinical and Patient-reported Outcomes of a Zirconia Oral Implant: Three-year Results of a Prospective Cohort Investigation.
The objective of this study was to determine the clinical, radiographic, and patient-reported outcomes of a 1-piece alumina-toughened zirconia implant restored with single crowns (SCs) or 3-unit fixed dental prostheses (FDPs) after 3 y of observation. Forty patients received 53 implants, placed in a 1-stage operation with immediate temporization. Finally, 50 implants were restored with 24 SCs and 13 FDPs. To evaluate peri-implant bone loss, standardized radiographs were taken at implant insertion, at final restoration delivery, and after 1 and 3 y. Additionally, several soft tissue parameters and patient-reported outcome measures were evaluated. Linear mixed models with random intercept for each patient and patients as clusters were used to compare subgroups. Three patients did not receive a SC due to early implant loss, and 1 patient died. As a result, 36 patients with 49 implants were followed-up for 3 y, giving a cumulative survival rate of 94.2%. The average marginal bone loss amounted to 0.79 mm (SCs, 0.47 mm; FDPs, 1.07 mm; P < 0.001). After the delivery of the final prosthetic restoration, further bone loss was not statistically significant (0.09 mm; P = 0.700). Probing depth, clinical attachment level, and modified bleeding index increased significantly at the implant sites, whereas gingival recession decreased significantly. Compared with the pretreatment questionnaires, the patient-reported outcome measures showed a permanently improved perception of function, aesthetics, sense, speech and self-esteem. The survival rate of the investigated ceramic implant system seems to be comparable to reported survival rates of titanium implants when immediately restored. The recorded parameters suggest its potential for clinical utilization.


High TCL1 levels are a marker of B-cell receptor pathway responsiveness and adverse outcome in chronic lymphocytic leukemia.
Although activation of the B-cell receptor (BCR) signaling pathway is implicated in the pathogenesis of chronic lymphocytic leukemia (CLL), its clinical impact and the molecular correlates of such response are not clearly defined. T-cell leukemia 1 (TCL1), the AKT modulator and proto-oncogene, is differentially expressed in CLL and linked to its pathogenesis based on CD5(+) B-cell expansions arising in TCL1-transgenic mice. We studied here the association of TCL1 levels and its intracellular dynamics with the in vitro responses to BCR stimulation in 70 CLL cases. The growth kinetics after BCR engagement correlated strongly with the degree and timing of induced AKT phospho-activation. This signaling intensity was best predicted by TCL1 levels and the kinetics of TCL1-AKT corecruitment to BCR membrane activation complexes, which further included the kinases LYN, SYK, ZAP70, and PKC. High TCL1 levels were also strongly associated with aggressive disease features, such as advanced clinical stage, higher white blood cell counts, and shorter lymphocyte doubling time. Higher TCL1 levels independently predicted an inferior clinical outcome (ie, shorter progression-free survival, P < .001), regardless of therapy regimen, especially for ZAP70(+) tumors. We propose TCL1 as a marker of the BCR-responsive CLL subset identifying poor prognostic cases where targeting BCR-associated kinases may be therapeutically useful.


Diagnostic and mutational spectrum of progressive osseous heteroplasia (POH) and other forms of GNAS-based heterotopic ossification.
Progressive osseous heteroplasia (POH) is a rare, disabling disease of heterotopic ossification (HO) that progresses from skin and subcutaneous tissues into deep skeletal muscle. POH occurs in the absence of multiple developmental features of Albright hereditary osteodystrophy (AHO) or hormone resistance, clinical manifestations that are also associated with GNAS inactivation. However, occasional patients with AHO and pseudohypoparathyroidism 1a/c (PHP1a/c; AHO features plus hormone resistance) have also been described who have progressive HO. This study was undertaken to define the diagnostic and mutational spectrum of POH and progressive disorders of HO, and to distinguish them from related disorders in which HO remains confined to the skin and subcutaneous tissues. We reviewed the charts of 111 individuals who had cutaneous and subcutaneous ossification. All patients were assessed for eight characteristics: age of onset of HO, presence and location of HO, depth of HO, type of HO, progression of HO, features of AHO, PTH resistance, and GNAS mutation analysis. We found, based on clinical criteria, that POH and progressive HO syndromes are at the severe end of a phenotypic spectrum of GNAS-inactivating conditions associated with extra-skeletal ossification. While most individuals with superficial or progressive ossification had mutations in GNAS, there were no specific genotype-phenotype correlations that distinguished the more progressive forms of HO (e.g., POH) from the non-progressive forms (osteoma cutis, AHO, and PHP1a/c).


Safety of CD34+ Hematopoietic Stem Cells and CD4+ T Lymphocytes Transduced with LVsh5/C46 in HIV-1 Infected Patients with High-Risk Lymphoma.
Although the risk of developing lymphoma has decreased in the highly active antiretroviral therapy era, this cancer remains the major cause of mortality in HIV-infected patients. Autologous hematopoietic stem cell transplantation (ASCT) outcome does not differ for HIV-infected versus HIV-uninfected patients. We propose to develop a new treatment for HIV-associated high-risk lymphoma based on autologous transplantation of two genetically modified products: CD4+ T lymphocytes and CD34+ hematopoietic stem cells (HSPCs). The cells will be transduced ex vivo with the Cal-1 lentiviral vector encoding for both a short hairpin RNA (shRNA) against CCR5 (sh5) and the HIV-1 fusion inhibitor C46. The transduced cells will be resistant to HIV infection by two complementary mechanisms: impaired binding of the virus to the cellular CCR5 co-receptor and decreased fusion of the virus as C46 interacts with gp41 and inhibits HIV infection. This phase I/II pilot study, also entitled GENHIV, will involve two French participating centers: Saint Louis Hospital and Necker Hospital in Paris. We plan to enroll five HIV-1-infected patients presenting with high-risk lymphoma and require a treatment with ASCT. The primary objective of this study is to evaluate the safety, feasibility, and success of engraftment of Cal-1 gene-transduced CD4+ T lymphocytes and CD34+ HSPCs.


Improved contrast in multispectral phase images derived from magnetic resonance exams of multiple sclerosis patients.
We describe a method to extract data from multispectral MR exams of patients with Multiple Sclerosis (MS). The technique produces images of "spectral phase" (SP) relative to a reference tissue. SP images allow retrospective suppression of signal in the reference tissue, while maintaining high spatial resolution. Image quality in SP images was determined from MR exams of 5 MS patients selected at random from a clinical trial underway at our institute. Exams consisting of proton density weighted (PDw), T2 weighted (T2w), T1 weighted (T1w), and gadolinium-DTPA enhanced T1w (GAD) images were acquired from each patient. The MR exams were corrected for intensity nonuniformity, then filtered with an algorithm based upon anisotropic diffusion, to reduce noise. Principal component (PC) images and SP images relative to cerebrospinal fluid (SP(CSF)), normal appearing white matter (SP(NAWM)), gray matter (SP(GM)), and temporalis muscle (SP(MUS)) were then calculated. Contrast between tissues and MS lesions in the MR and derived images was then determined by measuring the signal-difference-to-noise ratio (dSNR) between tissues. Our new SP images provided better tissue contrast than the original MR, filtered MR, and PC images. Contrast improved between CSF and NAWM (from 19.5 to 56), CSF and GM (from 15 to 36), GM and NAWM (from 8 to 14), MS lesions and CSF (from 16 to 35), and between MS lesions and NAWM (from 24 to 47). (Maximum contrast in the original MR images compared to maximum contrast in the SP images.) The additional contrast in SP images may aid the quantification and analysis of lesion activity in MR exams of MS patients.


Cannabis use disorder is associated with greater illness severity in tobacco smoking patients with bipolar disorder.
Cannabis use disorders (CUD) may influence the course of bipolar disorder (BD), but key confounding factors such as tobacco smoking have not been adequately addressed. This study examined whether CUD was associated with a more severe illness course in tobacco smoking BD patients. A sample of French and Norwegian tobacco smoking patients with BD I and II (N=642) was investigated. DSM-IV diagnoses and other characteristics were obtained through personal interviews using structured questionnaires. The association between CUD and illness course was assessed in regression analyses. In bivariate analyses, CUD was associated with earlier BD onset, higher frequency of manic (in BD I) and depressive episodes and hospitalizations per illness year, and a higher occurrence of psychotic episodes. After controlling for potential confounders, the relationships with earlier BD onset (B=-5.60 95% CI=-7.65 to -3.64), and increased rates of manic episodes (OR=1.93, 95% CI: 1.15 to 3.23) and hospitalizations (OR=2.93, 95% CI: 1.85 to 4.64) remained statistically significant. Despite the multivariate approach, differences between the two samples may lead to spurious findings related to hidden confounders. Substance use and mood episode information was collected retrospectively, and potential birth cohort effects could not be controlled for. Studies have found associations between tobacco smoking and poorer outcomes in BD. In this study on tobacco smoking BD patients we report an association between CUD and illness severity, suggesting that CUD exacerbates the disease evolution independently of tobacco smoking. Specific treatment and prevention programs addressing CUD in BD patients are warranted.


Vacuole Integrity Maintained by DUF300 Proteins Is Required for Brassinosteroid Signaling Regulation.
Brassinosteroid (BR) hormone signaling controls multiple processes during plant growth and development and is initiated at the plasma membrane through the receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1) together with co-receptors such as BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1). BRI1 abundance is regulated by endosomal recycling and vacuolar targeting, but the role of vacuole-related proteins in BR receptor dynamics and BR responses remains elusive. Here, we show that the absence of two DUF300 domain-containing tonoplast proteins, LAZARUS1 (LAZ1) and LAZ1 HOMOLOG1 (LAZ1H1), causes vacuole morphology defects, growth inhibition, and constitutive activation of BR signaling. Intriguingly, tonoplast accumulation of BAK1 was substantially increased and appeared causally linked to enhanced BRI1 trafficking and degradation in laz1 laz1h1 plants. Since unrelated vacuole mutants exhibited normal BR responses, our findings indicate that DUF300 proteins play distinct roles in the regulation of BR signaling by maintaining vacuole integrity required to balance subcellular BAK1 pools and BR receptor distribution.