Metabolic engineering of Clostridium cellulolyticum for the production of n-butanol from crystalline cellulose.
Sustainable alternatives for the production of fuels and chemicals are needed to reduce our dependency on fossil resources and to avoid the negative impact of their excessive use on the global climate. Lignocellulosic feedstock from agricultural residues, energy crops and municipal solid waste provides an abundant and carbon-neutral alternative, but it is recalcitrant towards microbial degradation and must therefore undergo extensive pretreatment to release the monomeric sugar units used by biofuel-producing microbes. These pretreatment steps can be reduced by using microbes such as Clostridium cellulolyticum that naturally digest lignocellulose, but this limits the range of biofuels that can be produced. We therefore developed a metabolic engineering approach in C. cellulolyticum to expand its natural product spectrum and to fine tune the engineered metabolic pathways. Here we report the metabolic engineering of C. cellulolyticum to produce n-butanol, a next-generation biofuel and important chemical feedstock, directly from crystalline cellulose. We introduced the CoA-dependent pathway for n-butanol synthesis from C. acetobutylicum and measured the expression of functional enzymes (using targeted proteomics) and the abundance of metabolic intermediates (by LC-MS/MS) to identify potential bottlenecks in the n-butanol biosynthesis pathway. We achieved yields of 40 and 120 mg/L n-butanol from cellobiose and crystalline cellulose, respectively, after cultivating the bacteria for 6 and 20 days. The analysis of enzyme activities and key intracellular metabolites provides a robust framework to determine the metabolic flux through heterologous pathways in C. cellulolyticum, allowing further improvements by fine tuning individual steps to improve the yields of n-butanol.


Shared synaptic pathophysiology in syndromic and nonsyndromic rodent models of autism.
The genetic heterogeneity of autism poses a major challenge for identifying mechanism-based treatments. A number of rare mutations are associated with autism, and it is unclear whether these result in common neuronal alterations. Monogenic syndromes, such as fragile X, include autism as one of their multifaceted symptoms and have revealed specific defects in synaptic plasticity. We discovered an unexpected convergence of synaptic pathophysiology in a nonsyndromic form of autism with those in fragile X syndrome. Neuroligin-3 knockout mice (a model for nonsyndromic autism) exhibited disrupted heterosynaptic competition and perturbed metabotropic glutamate receptor-dependent synaptic plasticity, a hallmark of fragile X. These phenotypes could be rescued by reexpression of neuroligin-3 in juvenile mice, highlighting the possibility of reverting neuronal circuit alterations in autism after the completion of development.


Systems modeling of the role of interleukin-21 in the maintenance of effector CD4+ T cell responses during chronic Helicobacter pylori infection.
The development of gastritis during Helicobacter pylori infection is dependent on an activated adaptive immune response orchestrated by T helper (Th) cells. However, the relative contributions of the Th1 and Th17 subsets to gastritis and control of infection are still under investigation. To investigate the role of interleukin-21 (IL-21) in the gastric mucosa during H. pylori infection, we combined mathematical modeling of CD4(+) T cell differentiation with in vivo mechanistic studies. We infected IL-21-deficient and wild-type mice with H. pylori strain SS1 and assessed colonization, gastric inflammation, cellular infiltration, and cytokine profiles. Chronically H. pylori-infected IL-21-deficient mice had higher H. pylori colonization, significantly less gastritis, and reduced expression of proinflammatory cytokines and chemokines compared to these parameters in infected wild-type littermates. These in vivo data were used to calibrate an H. pylori infection-dependent, CD4(+) T cell-specific computational model, which then described the mechanism by which IL-21 activates the production of interferon gamma (IFN-γ) and IL-17 during chronic H. pylori infection. The model predicted activated expression of T-bet and RORγt and the phosphorylation of STAT3 and STAT1 and suggested a potential role of IL-21 in the modulation of IL-10. Driven by our modeling-derived predictions, we found reduced levels of CD4(+) splenocyte-specific tbx21 and rorc expression, reduced phosphorylation of STAT1 and STAT3, and an increase in CD4(+) T cell-specific IL-10 expression in H. pylori-infected IL-21-deficient mice. Our results indicate that IL-21 regulates Th1 and Th17 effector responses during chronic H. pylori infection in a STAT1- and STAT3-dependent manner, therefore playing a major role controlling H. pylori infection and gastritis. Importance: Helicobacter pylori is the dominant member of the gastric microbiota in more than 50% of the world's population. H. pylori colonization has been implicated in gastritis and gastric cancer, as infection with H. pylori is the single most common risk factor for gastric cancer. Current data suggest that, in addition to bacterial virulence factors, the magnitude and types of immune responses influence the outcome of colonization and chronic infection. This study uses a combined computational and experimental approach to investigate how IL-21, a proinflammatory T cell-derived cytokine, maintains the chronic proinflammatory T cell immune response driving chronic gastritis during H. pylori infection. This research will also provide insight into a myriad of other infectious and immune disorders in which IL-21 is increasingly recognized to play a central role. The use of IL-21-related therapies may provide treatment options for individuals chronically colonized with H. pylori as an alternative to aggressive antibiotics.


Multiplexing mutation rate assessment: determining pathogenicity of Msh2 variants in Saccharomyces cerevisiae.
Despite the fundamental importance of mutation rate as a driving force in evolution and disease risk, common methods to assay mutation rate are time-consuming and tedious. Established methods such as fluctuation tests and mutation accumulation experiments are low-throughput and often require significant optimization to ensure accuracy. We established a new method to determine the mutation rate of many strains simultaneously by tracking mutation events in a chemostat continuous culture device and applying deep sequencing to link mutations to alleles of a DNA-repair gene. We applied this method to assay the mutation rate of hundreds of Saccharomyces cerevisiae strains carrying mutations in the gene encoding Msh2, a DNA repair enzyme in the mismatch repair pathway. Loss-of-function mutations in MSH2 are associated with hereditary nonpolyposis colorectal cancer, an inherited disorder that increases risk for many different cancers. However, the vast majority of MSH2 variants found in human populations have insufficient evidence to be classified as either pathogenic or benign. We first benchmarked our method against Luria-Delbrück fluctuation tests using a collection of published MSH2 missense variants. Our pooled screen successfully identified previously characterized nonfunctional alleles as high mutators. We then created an additional 185 human missense variants in the yeast ortholog, including both characterized and uncharacterized alleles curated from ClinVar and other clinical testing data. In a set of alleles of known pathogenicity, our assay recapitulated ClinVar's classification; we then estimated pathogenicity for 157 variants classified as uncertain or conflicting reports of significance. This method is capable of studying the mutation rate of many microbial species and can be applied to problems ranging from the generation of high-fidelity polymerases to measuring the frequency of antibiotic resistance emergence.


Treatment of early recurrent prostate cancer with 1,25-dihydroxyvitamin D3 (calcitriol)
Substantial experimental and epidemiological data indicate that 1,25-dihydroxyvitamin D3 (calcitriol) has potent antiproliferative effects on human prostate cancer cells. We performed an open label, nonrandomized pilot trial to determine whether calcitriol therapy is safe and efficacious for early recurrent prostate cancer. Our hypothesis was that calcitriol therapy slows the rate of rise of prostate specific antigen (PSA) compared with the pretreatment rate. After primary treatment with radiation or surgery recurrence was indicated by rising serum PSA levels documented on at least 3 occasions. Seven subjects completed 6 to 15 months of calcitriol therapy, starting with 0.5 microg. calcitriol daily and slowly increasing to a maximum dose of 2.5 microg. daily depending on individual calciuric and calcemic responses. Each subject served as his own control, comparing the rate of PSA rise before and after calcitriol treatment. As determined by multiple regression analysis, the rate of PSA rise during versus before calcitriol therapy significantly decreased in 6 of 7 patients, while in the remaining man a deceleration in the rate of PSA rise did not reach statistical significance. Overall the decreased rate of PSA rise was statistically significant (p = 0.02 Wilcoxon signed rank test). Dose dependent hypercalciuria limited the maximal calcitriol therapy given (range 1.5 to 2.5 microg. daily). This pilot study provides preliminary evidence that calcitriol effectively slows the rate of PSA rise in select cases, although dose dependent calciuric side effects limit its clinical usefulness. The development of calcitriol analogues with decreased calcemic side effects is promising, since such analogues may be even more effective for treating prostate cancer.


Extending the dynamic range of biomedical micro-computed tomography for application to geomaterials.
X-ray computed tomography (CT) can non-destructively examine objects by producing three-dimensional images of their internal structure. Although the availability of biomedical micro-CT offers the increased access to scanners, CT images of dense objects are susceptible to artifacts particularly due to beam hardening. This study proposes and evaluates a simple semi-empirical correction method for beam hardening and scatter that can be applied to biomedical scanners. Novel calibration phantoms of varying diameters were designed and built from aluminum and poly[methyl-methacrylate]. They were imaged using two biomedical micro-CT scanners. Absorbance measurements made through different phantom sections were fit to polynomial and inversely exponential functions and used to determine linearization parameters. Corrections based on the linearization equations were applied to the projection data before reconstruction. Correction for beam hardening was achieved when applying both scanners with the correction methods to all test objects. Among them, applying polynomial correction method based on the aluminum phantom provided the best improvement. Correction of sample data demonstrated a high agreement of percent-volume composition of dense metallic inclusions between using the Bassikounou meteorite from the micro-CT images (13.7%) and previously published results using the petrographic thin sections (14.6% 8% metal and 6.6% troilite). Semi-empirical linearization of X-ray projection data with custom calibration phantoms allows accurate measurements to be obtained on the radiodense samples after applying the proposed correction method on biomedical micro-CT images.


Good agreement between questionnaire and administrative databases for health care use and costs in patients with osteoarthritis.
Estimating costs is essential to the economic analysis of health care programs. Health care costs are often captured from administrative databases or by patient report. Administrative records only provide a partial representation of health care costs and have additional limitations. Patient-completed questionnaires may allow a broader representation of health care costs; however the validity and feasibility of such methods have not been firmly established. This study was conducted to assess the validity and feasibility of using a patient-completed questionnaire to capture health care use and costs for patients with osteoarthritis, and to compare the research costs of the data-capture methods. We designed a patient questionnaire and applied it in a clinical trial. We captured equivalent data from four administrative databases. We evaluated aspects of the questionnaire's validity using sensitivity and specificity, Lin's concordance correlation coefficient (ρ(c)), and Bland-Altman comparisons. The questionnaire's response rate was 89%. Acceptable sensitivity and specificity levels were found for all types of health care use. The numbers of visits and the majority of medications reported by patients were in agreement with the database-derived estimates (ρ(c) > 0.40). Total cost estimates from the questionnaire agreed with those from the databases. Patient-reported co-payments agreed with administrative records with respect to GP office transactions, but not pharmaceutical co-payments. Research costs for the questionnaire-based method were less than one-third of the costs for the databases method. A patient-completed questionnaire is feasible for capturing health care use and costs for patients with osteoarthritis, and data collected using it mostly agree with administrative databases. Caution should be exercised when applying unit costs and collecting co-payment data.


Naringenin regulates production of matrix metalloproteinases in the knee-joint and primary cultured articular chondrocytes and alleviates pain in rat osteoarthritis model.
Inflammation of cartilage is a primary symptom for knee-joint osteoarthritis. Matrix metalloproteinases (MMPs) are known to play an important role in the articular cartilage destruction related to osteoarthritis. Naringenin is a plant-derived flavonoid known for its anti-inflammatory properties. We studied the effect of naringenin on the transcriptional expression, secretion and enzymatic activity of MMP-3 in vivo in the murine monosodium iodoacetate (MIA) osteoarthritis model. The assessment of pain behavior was also performed in the MIA rats. The destruction of knee-joint tissues was analyzed microscopically. Moreover, the effect of naringenin was also studied in vitro in IL-1β activated articular chondrocytes. The transcriptional expression of MMP-3, MMP-1, MMP-13, thrombospondin motifs (ADAMTS-4) and ADAMTS-5 was also studied in primary cultured chondrocytes of rats. Naringenin caused significant reduction in pain behavior and showed marked improvement in the tissue morphology of MIA rats. Moreover, a significant inhibition of MMP-3 expression in MIA rats was observed upon treatment with naringenin. In the in vitro tests, naringenin caused a significant reduction in the transcriptional expression, secretion and enzymatic activity of the studied degradative enzymes. The NF-κB pathway was also found to be inhibited upon treatment with naringenin in vitro. Overall, the study suggests that naringenin alleviated pain and regulated the production of matrix-metalloproteinases via regulation of NF-κB pathway. Thus, naringenin could be a potent therapeutic option for the treatment of osteoarthritis.


Stereoselective Copolymerization of Unprotected Polar and Nonpolar Styrenes by an Yttrium Precursor: Control of Polar-Group Distribution and Mechanism.
Styrene underwent unprecedented coordination-insertion copolymerization with naked polar monomers (ortho-/meta-/para-methoxystyrene) in the presence of a pyridyl methylene fluorenyl yttrium catalyst. High activity (1.26×106 g molY-1 h-1 ) and excellent syndioselectivity were observed, and high-molecular-weight copolymers (24.6×104 g mol-1 ) were obtained. The insertion rate of the polar monomers could be adjusted in the full range of 0-100 % simply by changing the loading of the polar styrene monomer. Strikingly, the copolymers had tapered, gradient, and even random sequence distributions, depending on the position of the polar methoxy group on the phenyl ring and thus on its mode of coordination to the active metal center, as shown by tracking the polymerization process and DFT calculations.


Organic anion transporting polypeptide 2B1 - More than a glass-full of drug interactions.
The importance of uptake transporters in determining drug disposition is increasingly appreciated. While the focus of regulatory agencies worldwide has been on the hepatic organic anion transporting polypeptides (OATPs)-1B1 and-1B3, there is another isoform of the OATP sub-family, OATP2B1, which should be considered equally relevant. Unlike the other members of the OATP sub-family, OATP2B1 is expressed in multiple organs in humans, including in the intestine and the liver. Similar to other OATPs, OATP2B1 mediates the hepatic and intestinal uptake of many drugs and endogenous compounds. The importance of OATP2B1 in the disposition of many drugs is highlighted by the growing recognition of its role in significant in vivo drug-drug or food-drug interactions. The dramatic changes in drug exposure attributable to inhibition of OATP2B1 highlight the importance of developing a better understanding of the clinical role of OATP2B1. This review aims to provide a thorough summary of the current understanding of the pharmacogenetics, regulation, expression and abundance of OATP2B1 in humans, as well as its clinical relevance in drug-drug and food-drug interactions.


Isolation and partial characterization of a 110-kD dimer actin-binding protein.
Two Triton-insoluble fractions were isolated from Acanthamoeba castellanii. The major non-membrane proteins in both fractions were actin (30-40%), myosin II (4-9%), myosin I (1-5%), and a 55-kD polypeptide (10%). The 55-kD polypeptide did not react with antibodies against tubulins from turkey brain, paramecium, or yeast. All of these proteins were much more concentrated in the Triton-insoluble fractions than in the whole homogenate or soluble supernatant. The 55-kD polypeptide was extracted with 0.3 M NaCl, fractionated by ammonium sulfate, and purified to near homogeneity by DEAE-cellulose and hydroxyapatite chromatography. The purified protein had a molecular mass of 110 kD and appeared to be a homodimer by isoelectric focusing. The 110-kD dimer bound to F-actin with a maximal binding stoichiometry of 0.5 mol/mol of actin (1 mol of 55-kD subunit/mol of actin). Although the 110-kD protein enhanced the sedimentation of F-actin, it did not affect the low shear viscosity of F-actin solutions nor was bundling of F-actin observed by electron microscopy. The 110-kD dimer protein inhibited the actin-activated Mg2+-ATPase activities of Acanthamoeba myosin I and myosin II in a concentration-dependent manner. By indirect immunofluorescence, the 110-kD protein was found to be localized in the peripheral cytoplasm near the plasma membrane which is also enriched in F-actin filaments and myosin I.


Paranoia, self-deception and overconfidence.
Self-deception, paranoia, and overconfidence involve misbeliefs about the self, others, and world. They are often considered mistaken. Here we explore whether they might be adaptive, and further, whether they might be explicable in Bayesian terms. We administered a difficult perceptual judgment task with and without social influence (suggestions from a cooperating or competing partner). Crucially, the social influence was uninformative. We found that participants heeded the suggestions most under the most uncertain conditions and that they did so with high confidence, particularly if they were more paranoid. Model fitting to participant behavior revealed that their prior beliefs changed depending on whether the partner was a collaborator or competitor, however, those beliefs did not differ as a function of paranoia. Instead, paranoia, self-deception, and overconfidence were associated with participants' perceived instability of their own performance. These data are consistent with the idea that self-deception, paranoia, and overconfidence flourish under uncertainty, and have their roots in low self-esteem, rather than excessive social concern. The model suggests that spurious beliefs can have value-self-deception is irrational yet can facilitate optimal behavior. This occurs even at the expense of monetary rewards, perhaps explaining why self-deception and paranoia contribute to costly decisions which can spark financial crashes and devastating wars.


Blood pressure response to isometric handgrip testing and aerobic capacity and associations with sprint performance in middle-aged men following high-intensity interval training.
The aim of this study was to determine if a 6-week high-intensity interval training (HIIT) protocol with increasing exercise volume affects aerobic capacity and arterial blood pressure (BP) measured during a fatiguing isometric handgrip test (IHT) in middle-aged, normotensive men and to assess whether sprint performance is associated with training-induced changes in BP. Fourteen recreationally active men (age 43.0±0.9 years, body mass 74.4±2.1 kg, body height 1.74±0.02 m, BMI 24.7±0.5 kg/m2 [mean±SE]) participated in 12 HIIT sessions at an intensity of 85% of heart rate reserve. Sprinting ability was estimated at baseline using the 50-yard dash test (50YDT). Pre- and post-HIIT aerobic capacity and pressor response was assessed by the 12-min Cooper run test (12CT) and the IHT at 30% of maximal voluntary contraction, respectively. Following the intervention, the distance covered in the 12CT and handgrip time to fatigue (HTF) significantly increased (P<0.05) but only diastolic BP significantly decreased (P<0.05) compared with pre-HIIT values. Changes (Δ - post- vs. pre-HIIT values) in HTF correlated positively with 50YDT performance (P<0.05). However, significant negative relationships were found between 50YDT and Δ in systolic, diastolic, and mean BP during the IHT except for resting values. The findings suggest that a HIIT intervention in middle-aged, normotensive men enhances aerobic capacity with a concomitant decrease in DBP and that training-induced ΔBP during IHT are dependent on sprinting ability.


Child protection involvement and victims of intimate partner violence: is there a bias?
Several studies have explored the disproportionate number of children of color involved in child protective services, raising concerns that racial bias in the system results in more women of color being referred to child protection. The authors conducted a case series to analyze whether a woman's race and ethnicity influenced referrals to child protective services in a domestic violence context. Data were obtained through interview records of 263 women (38% women of color) at a Minneapolis-based advocacy and therapy organization. The findings suggest that women who face multiple forms of oppressions may have greater risk of being involved with child protection services.


A clinical study of brucellosis in adults in the Asir region of southern Saudi Arabia.
One hundred four Saudi patients with brucellosis who were admitted to Abha General Hospital in the Asir region of southern Saudi Arabia were studied. All the patients had Brucella melitensis infection; 61.5% were females while 38.5% were males. Their mean age was 32 years. Most of the patients (61.5%) lived in the lowland (Tihama) and the majority were shepherds (84.6%). The most common presenting symptoms were fever (100%), sweating (96.2%), headache (76.9%), joint pains (76.9%), and backache (73.1%). Physical findings included fever (96.2%), hepatomegaly (46.2%), splenomegaly (42.3%), tenderness over the spine (30.8%), arthritis (26.9%), and lymphadenopathy (19.2%). Mild anemia, leukopenia, and relative lymphocytosis were common. A history of raw milk ingestion was an important factor in disease transmission (84.6%), followed by close animal contact (73%) and raw liver consumption (63.3%). The study shows the effectiveness of several drug combinations in the treatment of brucellosis and the low relapse rate if the treatment is prolonged for not less than six weeks.


Iontophoresis of dexamethosone-phosphate into the equine tibiotarsal joint.
In human rehabilitation medicine, dexamethasone-phosphate is theoretically iontophoresed to localized subcutaneous tissue where conversion to dexamethasone occurs. This delivery system has recently been introduced into veterinary medicine for the same purpose. However, the pharmacokinetic justification for parenteral delivery of this prodrug remains undocumented. Utilizing iontophoretic methods that are relevant to both human and veterinary clinical practice, the present investigation compared injection and iontophoresis of dexamethasone-phosphate into the equine tibiotarsal joint, also known as the tarsocrual joint. The tibiotarsal joints of seven horses were injected with 4 mL of 6 mg/mL dexamethasone-phosphate. With a similar drug concentration and over the same application site, six different horses underwent simultaneous cathodic iontophoresis (4 mA, 40 min) or passive application (0 mA, 40 min) on contralateral limbs. Following all applications, tibiotarsal joint synovium was collected. Local venous blood samples were also collected from the iontophoretic and passive application sites for analysis of plasma drug concentrations. Because of the potential for conversion of dexamethasone-phosphate to dexamethasone, an extraction and analysis protocol was developed for both chemicals. The technique demonstrated a linear range of detection (0.39-12 microg/mL) and a capability for measuring both chemicals in plasma and synovium. Conversion of dexamethasone-phosphate to dexamethasone occurred during synovial incubation (37 degrees C) and following freeze-thaw cycles. In contrast to the measurable synovial concentrations of dexamethasone-phosphate (2.3 +/- 0.96 mg/mL) and dexamethasone (0.27 +/- 0.07 mg/mL) following injection, neither drug was detected in the synovium or the local venous blood following iontophoretic or passive applications. In conclusion, these results do not confirm iontophoretic or passive delivery of measurable dexamethasone-phosphate into the tibiotarsal joint using current clinical methods.


Beyond low-density lipoprotein cholesterol: defining the role of low-density lipoprotein heterogeneity in coronary artery disease.
Recent clinical trials in patients with coronary artery disease (CAD) provide evidence that low-density lipoprotein cholesterol (LDL-C) levels should be lowered even further to prevent recurrent CAD. However, despite more aggressive interventions for lowering LDL-C levels, the majority of CAD events go undeterred, perhaps related to the fact that intervention was not started earlier in life or that LDL-C levels represent an incomplete picture of atherogenic potential. Nevertheless, LDL-C remains the contemporary standard as the primary goal for aggressive LDL reduction. If triglycerides are >200 mg/dl, the measurement of non-high-density lipoprotein cholesterol (HDL-C) is recommended. Measurement of apolipoprotein (apo)B has been shown in nearly all studies to outperform LDL-C and non-HDL-C as a predictor of CAD events and as an index of residual CAD risk. This is because apoB reflects the total number of atherogenic apoB-containing lipoproteins and is a superior predictor of the number of low-density lipoprotein particles (LDL-P). Estimates of LDL-P and size can also be made by nuclear magnetic resonance spectroscopy, density gradient ultracentrifugation, and gradient gel electrophoresis. Although a number of studies show that such estimates predict CAD, LDL-P, and size often accompany low HDL-C and high triglyceride levels, and therefore such additional lipoprotein testing has not been recommended for routine screening and follow-up. Because apoB is a superior predictor of LDL-P, we recommend that apoB and the apoB/apoA-I ratio be determined after measurement of LDL-C, non-HDL-C, and the ratio of total cholesterol/HDL-C to better predict CAD and assess efficacy of treatment.


Anomalous magnetic properties of mechanically milled cobalt oxide nanoparticles.
Defect induced magnetic properties of CoO nanoparticles produced via mechanical ball milling have been assessed by detailed magnetic measurements. A progressive decrease in the particle size and a concomitant increase in the induced strain have been observed with the milling times. The mechanically milled nanoparticles of CoO exhibit anomalous magnetic properties such as FM hysteresis when compared with the unmilled CoO sample. The presence of weak ferromagnetism, with a highest value of magnetization of 0.532 emu/g at 10 K in the 100 h milled sample, is attributed to the uncompensated surface spins resulting from induced surface defects via mechanical milling. The ZFC coercive force, measured at 10 K, increases with milling time reaching a maximum value of 1066 Oe for the 100 h milled sample. The temperature dependent field-cooled (FC) and zero-field-cooled (ZFC) magnetic measurements indicate a presence of an exchange bias field arising from uncompensated moments generated by mechanical strain and the antiferromagnetic (AFM) core. The exchange bias field measured at 10 K reaches a value 210 Oe for the 50 h milled sample and decreases upon prolonged milling. The exchange bias field vanishes at a temperature approximately 200 K, a temperature much lower than the Neel temperature of CoO (TN approximately 291 K). The observed anomalous magnetic behavior of CoO could be interpreted in terms of the exchanged bias FM-AFM model.


Anti-HIV-1 activity of eight monofloral Iranian honey types.
Monofloral Iranian honeys from eight floral sources were analyzed to determine their anti-HIV-1 activities as well as their effects on lymphocyte proliferation. The Peripheral Blood Mononuclear Cells (PBMCs) used in this study were prepared from five healthy volunteers who were seronegative for HIV, HCV, HBV and TB. The anti-HIV-1 activity of eight different honeys was performed by quantitative polymerase chain reaction (PCR) assay and high pure viral nucleic acid kit. The results demonstrated that monofloral honeys from Petro selinum sativum, Nigella sativa, Citrus sinensis, Zataria multiflora, Citrus aurantium and Zizyphus mauritiana flowers had potent anti-HIV-1 activity with half maximal effective concentration (EC50) values of 37.5, 88, 70, 88, 105 and 5 µg/ml respectively. However, monofloral Iranian honeys from Astragalus gummifer and Chamaemelum nobile flowers had weak anti-HIV-1 activity. The frequency and intensity of CD4 expression on PBMCs increased in the presence of all honey types. CD19 marker were also increased after the treatment with monofloral honeys from Z. multiflora and N. sativa. The anti-HIV-1 agent in monofloral honeys from P. sativum, N. sativa, Z. multiflora and Z. mauritiana flowers was detected by spectroscopic analysis as methylglyoxal. Time of drug addition studies demonstrated that the inhibitory effect of methylglyoxal is higher on the late stage of HIV-1 infection. The result demonstrated that methylglyoxal isolated from monofloral honey types is a good candidate for preclinical evaluation of anti-HIV-1 therapies.


Diversity of benzyl- and alkylsuccinate synthase genes in hydrocarbon-impacted environments and enrichment cultures.
Hydrocarbon-degrading microorganisms play an important role in the natural attenuation of spilled petroleum in a variety of anoxic environments. The role of benzylsuccinate synthase (BSS) in aromatic hydrocarbon degradation and its use as a biomarker for field investigations are well documented. The recent discovery of alkylsuccinate synthase (ASS) allows the opportunity to test whether its encoding gene, assA, can serve as a comparable biomarker of anaerobic alkane degradation. Degenerate assA- and bssA-targeted PCR primers were designed in order to survey the diversity of genes associated with aromatic and aliphatic hydrocarbon biodegradation in petroleum-impacted environments and enrichment cultures. DNA was extracted from an anaerobic alkane-degrading isolate (Desulfoglaeba alkenexedens ALDC), hydrocarbon-contaminated river and aquifer sediments, a paraffin-degrading enrichment, and a propane-utilizing mixed culture. Partial assA and bssA genes were PCR amplified, cloned, and sequenced, yielding several novel clades of assA genes. These data expand the range of alkane-degrading conditions for which relevant gene sequences are available and indicate that considerable diversity of assA genes can be found in hydrocarbon-impacted environments. The detection of genes associated with anaerobic alkane degradation in conjunction with the in situ detection of alkylsuccinate metabolites was also demonstrated. Comparable molecular signals of assA/bssA were not found when environmental metagenome databases of uncontaminated sites were searched. These data confirm that the assA gene is a useful biomarker for anaerobic alkane metabolism.


Risks in situations that are experienced as unfamiliar and confusing - the perspective of persons with dementia.
An increasing number of people with dementia are ageing at home in Sweden and in other countries. In order to meet the safety requirements, knowledge about how persons with dementia experience risks is required. The aim of the study was to explore and better understand how persons with dementia, living at home, experience risks in their daily life and how they handle these situations. Twelve persons with dementia were interviewed using open-ended questions, and the data were analyzed using a content analysis approach. Findings showed that participants experienced situations fraught with risks in their daily life as unfamiliar and confusing. Previously familiar places became unfamiliar to them, and details did not come together. They were uncertain about what actually had happened. How the participants handled these situations in order to reduce the risks are described.


Improved efficacy by using the pTnT-rhtTG plasmid for the detection of celiac disease specific tissue transglutaminase autoantibodies in radioligand binding assays.
Tissue transglutaminase (tTG) autoantibodies are serological markers for celiac disease. The aim was to study the efficacy of the pTnT-rhtTG plasmid and subsequent diagnostic accuracy of tTG autoantibodies for childhood celiac disease using radioligand binding assays. Coupled in vitro transcription and translation of tTG were performed by pTnT-rhtTG as well as by the pGEMt Easy-rhtTG vectors using the TNT SP6 Coupled Reticulocyte Lysate System in the presence of [³⁵S] methionine. Sera from 190 celiac disease children and 74 controls were measured for tTG autoantibodies in two separate radioligand binding assays using anti-human IgA agarose and protein A sepharose beads for the detection of IgA-tTG and IgG-tTG, respectively. Median incorporation of [³⁵S] methionine into the pTnT-rhtTG was 26% compared to 16% for the pGEMt Easy-rhtTG plasmid (p = 0.0016). Using pTnT-rhtTG (as compared to pGEMt Easy-rhtTG), sensitivities were IgA-tTG = 96.3% (95.7%) and IgG-tTG = 95.8% (97.3%) and specificities were IgA-tTG = 91.9% (90.5%) and IgG-tTG = 94.6% (98.4%). According to receiver operator characteristics for the pTnT (pGEMt Easy) assays, area under the curves were IgA-tTG = 98.4% (98.4%) and IgG-tTG = 97.7% (97.2%), respectively. The pTnT-rhtTG plasmid increased the efficacy of tTG antigen usage without reducing the diagnostic accuracy of IgA-tTG and IgG-tTG for childhood celiac disease. The pTnT-rhtTG plasmid is therefore recommended over the pGEMt Easy-rhtTG for the assessment of IgA-tTG and IgG-tTG using radioligand binding assays.


Monitoring Allergen Immunotherapy Effects by Microarray.
Allergen-specific immunotherapy (AIT) is the only treatment of IgE-mediated allergies so far that has a sustained effect on clinical symptoms and can modify the course of the disease. It is an allergen-specific treatment and therefore requires the correct identification of the disease-causing allergens. Furthermore, AIT is a time-consuming treatment for which the efficacy is dependent on several factors. Therefore, diagnostic tests and biomarkers are needed that facilitate (1) selection of the correct allergens according to the patient's individual sensitization profile and (2) to monitor the effects of AIT. This can provide support for the decision to continue, modify, or discontinue vaccination. One significant mechanism of action of AIT is the induction of allergen-specific antibodies that compete with IgE for the binding to allergen molecules, hence referred to as blocking antibodies. It was shown in several studies that the induction of blocking antibodies by AIT, and their specificity can be measured by allergen microarrays. Inhibition of allergen-specific IgE binding by blocking antibodies can also be determined by microarrays and is associated with changes in clinical parameters or other in vivo and in vitro assays demonstrating efficacy of AIT. Furthermore, allergen microarrays allow determination of IgE sensitizations towards a comprehensive set of allergen molecules and therefore are well suited for identifying the disease-causing allergens for correct prescription of AIT. Thus, diagnostic tests based on microarrayed allergens can be useful in determining the correct prescription of AIT and can be used to monitor efficacy of AIT.


Post-translational protein modifications in malaria parasites.
Post-translational modifications play crucial parts in regulating protein function and thereby control several fundamental aspects of eukaryotic biology, including cell signalling, protein trafficking, epigenetic control of gene expression, cell-cell interactions, and cell proliferation and differentiation. In this Review, we discuss protein modifications that have been shown to have a key role in malaria parasite biology and pathogenesis. We focus on phosphorylation, acetylation, methylation and lipidation. We provide an overview of the biological significance of these modifications and discuss prospects and progress in antimalarial drug discovery based on the inhibition of the enzymes that mediate these modifications.


Dipyridamole thallium-201 scintigraphy for improved detection of left anterior descending coronary artery stenosis in patients with left bundle branch block.
In patients with left bundle branch block (LBBB) undergoing thallium-201 exercise scintigraphy septal perfusion defects are frequently observed irrespective of the presence or absence of coronary artery disease. Consequently, in these patients the specificity for detecting stenoses in the left anterior descending coronary artery (LAD) by thallium-201 scintigraphy is relatively low. It has been hypothesized that pharmacological vasodilation would result in higher specificity than conventional exercise thallium-201 scintigraphy because of a more uniform exploitation of coronary flow reserve. Twenty-five consecutive patients with LBBB underwent dipyridamole thallium-201 imaging and coronary arteriography within 3 months. The prevalence of coronary artery disease was estimated at 48 +/- 19%. Sensitivity for detection of LAD lesions was 100% and specificity was 88%. The positive predictive accuracy and the negative predictive accuracy were 80% and 100% respectively. The 'false positive' septal defects were of the non-reversible type. The appearance of septal perfusion defects was not dependent on changes in heart rate during dipyridamole infusion. This study indicates the value of dipyridamole thallium-201 imaging for non-invasive detection of LAD stenosis in patients with LBBB.


Preclinical characterization of OSI-027, a potent and selective inhibitor of mTORC1 and mTORC2: distinct from rapamycin.
The phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway is frequently activated in human cancers, and mTOR is a clinically validated target. mTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, metabolism, proliferation, and survival. Rapamycin and its analogues partially inhibit mTOR through allosteric binding to mTORC1, but not mTORC2, and have shown clinical utility in certain cancers. Here, we report the preclinical characterization of OSI-027, a selective and potent dual inhibitor of mTORC1 and mTORC2 with biochemical IC(50) values of 22 nmol/L and 65 nmol/L, respectively. OSI-027 shows more than 100-fold selectivity for mTOR relative to PI3Kα, PI3Kβ, PI3Kγ, and DNA-PK. OSI-027 inhibits phosphorylation of the mTORC1 substrates 4E-BP1 and S6K1 as well as the mTORC2 substrate AKT in diverse cancer models in vitro and in vivo. OSI-027 and OXA-01 (close analogue of OSI-027) potently inhibit proliferation of several rapamycin-sensitive and -insensitive nonengineered and engineered cancer cell lines and also, induce cell death in tumor cell lines with activated PI3K-AKT signaling. OSI-027 shows concentration-dependent pharmacodynamic effects on phosphorylation of 4E-BP1 and AKT in tumor tissue with resulting tumor growth inhibition. OSI-027 shows robust antitumor activity in several different human xenograft models representing various histologies. Furthermore, in COLO 205 and GEO colon cancer xenograft models, OSI-027 shows superior efficacy compared with rapamycin. Our results further support the important role of mTOR as a driver of tumor growth and establish OSI-027 as a potent anticancer agent. OSI-027 is currently in phase I clinical trials in cancer patients.


Disappearing in the Night: An Overview on Trade and Legislation of Night Monkeys in South and Central America.
The international trade in night monkeys (Aotus spp.), found throughout Central and South America, has been regulated by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) since 1975. We present a quantitative analysis of this trade from all 9 range countries, over 4 decades, and compare domestic legislation to CITES regulations. Night monkeys were exported from 8 of the 9 habitat countries, totalling 5,968 live individuals and 7,098 specimens, with trade of live individuals declining over time. In terms of species, the most commonly traded was Aotus nancymaae (present in Brazil, Colombia, Peru) followed by A. vociferans (Brazil, Colombia, Ecuador, Peru) and A. zonalis (Colombia, Panama). There was no significant correlation between levels of trade and species' geographic range size or the number of countries in which a species occurs. Five countries have legislation that meets CITES requirements for implementation, whereas the other 4 countries' legislation showed deficiencies. Research conducted in Colombia, Peru, and Brazil suggests significant cross-border trade not captured in official international trade registers. Although international trade has diminished, current trends suggest that populations of rarer species may be under unsustainable pressure. Further research is needed to quantify real trade numbers occurring between habitat countries.


Off-label long acting injectable antipsychotics in real-world clinical practice: a cross-sectional analysis of prescriptive patterns from the STAR Network DEPOT study.
Information on the off-label use of Long-Acting Injectable (LAI) antipsychotics in the real world is lacking. In this study, we aimed to identify the sociodemographic and clinical features of patients treated with on- vs off-label LAIs and predictors of off-label First- or Second-Generation Antipsychotic (FGA vs. SGA) LAI choice in everyday clinical practice. In a naturalistic national cohort of 449 patients who initiated LAI treatment in the STAR Network Depot Study, two groups were identified based on off- or on-label prescriptions. A multivariate logistic regression analysis was used to test several clinically relevant variables and identify those associated with the choice of FGA vs SGA prescription in the off-label group. SGA LAIs were more commonly prescribed in everyday practice, without significant differences in their on- and off-label use. Approximately 1 in 4 patients received an off-label prescription. In the off-label group, the most frequent diagnoses were bipolar disorder (67.5%) or any personality disorder (23.7%). FGA vs SGA LAI choice was significantly associated with BPRS thought disorder (OR = 1.22, CI95% 1.04 to 1.43, p = 0.015) and hostility/suspiciousness (OR = 0.83, CI95% 0.71 to 0.97, p = 0.017) dimensions. The likelihood of receiving an SGA LAI grew steadily with the increase of the BPRS thought disturbance score. Conversely, a preference towards prescribing an FGA was observed with higher scores at the BPRS hostility/suspiciousness subscale. Our study is the first to identify predictors of FGA vs SGA choice in patients treated with off-label LAI antipsychotics. Demographic characteristics, i.e. age, sex, and substance/alcohol use co-morbidities did not appear to influence the choice towards FGAs or SGAs. Despite a lack of evidence, clinicians tend to favour FGA over SGA LAIs in bipolar or personality disorder patients with relevant hostility. Further research is needed to evaluate treatment adherence and clinical effectiveness of these prescriptive patterns.


Drosophila Spd-2 recruits PCM to the sperm centriole, but is dispensable for centriole duplication.
In C. elegans, genome-wide screens have identified just five essential centriole-duplication factors: SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4 [1-8]. These proteins are widely believed to comprise a conserved core duplication module [3, 9-14]. In worm embryos, SPD-2 is the most upstream component of this module, and it is also essential for pericentriolar material (PCM) recruitment to the centrioles [1, 4, 15, 16]. Here, we show that Drosophila Spd-2 (DSpd-2) is a component of both the centrioles and the PCM and has a role in recruiting PCM to the centrioles. DSpd-2 appears not, however, to be essential for centriole duplication in somatic cells. Moreover, PCM recruitment in DSpd-2 mutant somatic cells is only partially compromised, and mitosis appears unperturbed. In contrast, DSpd-2 is essential for proper PCM recruitment to the fertilizing sperm centriole, and hence for microtubule nucleation and pronuclear fusion. DSpd-2 therefore appears to have a particularly important role in recruiting PCM to the sperm centriole. We speculate that the SPD-2 family of proteins might only be absolutely essential for the recruitment of centriole duplication factors and PCM to the centriole(s) that enter the egg with the fertilizing sperm.


Advances in Toll-like receptor biology: Modes of activation by diverse stimuli.
As we learn more about the biology of the Toll-like receptors (TLRs), a wide range of molecules that can activate this fascinating family of pattern recognition receptors emerges. In addition to conserved pathogenic components, endogenous danger signals created upon tissue damage are also sensed by TLRs. Detection of these types of stimuli results in TLR mediated inflammation that is vital to fight pathogenic invasion and drive tissue repair. Aberrant activation of TLRs by pathogenic and endogenous ligands has also been linked with the pathogenesis of an increasing number of infectious and autoimmune diseases, respectively. Most recently, allergen activation of TLRs has also been described, creating a third broad class of TLR stimulus that has helped to shed light on the pathogenesis of allergic disease. To date, microbial activation of TLRs remains best characterized. Each member of the TLR family senses a specific subset of pathogenic ligands, pathogen associated molecular patterns (PAMPS), and a wealth of structural and biochemical data continues to reveal the molecular mechanisms of TLR activation by PAMPs, and to demonstrate how receptor specificity is achieved. In contrast, the mechanisms by which endogenous molecules and allergens activate TLRs remain much more mysterious. Here, we provide an overview of our current knowledge of how very diverse stimuli activate the same TLRs and the structural basis of these modes of immunity.


Regulation of polyethylene glycol 400 intestinal permeability by endogenous and exogenous prostanoids. Influence of non-steroidal anti-inflammatory drugs.
Polyethylene glycol 400 (PEG 400) is a clinically useful intestinal permeability probe whose rate of intestinal permeation is influenced in part by solvent drag. As mucosal prostanoids are increased in inflammatory bowel disease and affect water transport we examined the possible relationship between prostaglandin E2 (PGE2) and the inhibitors of endogenous prostaglandins--the non-steroidal anti-inflammatory drugs (NSAIDS)--on PEG 400 absorption in vivo using segmental perfusion of rat small intestine. We found that the addition of exogenous PGE2 in concentrations of 0.5, 1.0, and 1.5 micrograms/ml significantly (p less than 0.01) decreased PEG 400 and water absorption. Addition of 5 mmol/l of the cyclooxygenase inhibitors acetylsalicylic acid (ASA) or indomethacin in concentrations 2.5 or 5.0 mmol/l to the perfusate significantly (p less than 0.01) increased PEG 400 and water absorption. The simultaneous addition of 1.0 micrograms/ml of exogenous PGE2 to the perfusate with 5 mmol/l of ASA or with 2.5 mmol/l of indomethacin reversed the increase of PEG 400 and water transport (p less than 0.01). There were no differences in PEG 400 and water absorption when PGE2 was given alone or in combination with ASA or indomethacin. This study suggests that endogenous or exogenous prostanoids play an important role in the regulation of PEG 400 permeation. PGE2 and NSAIDS modify PEG 400 permeation in parallel with changes in water transport indicating that their effect on permeability is through changes in solvent drag. These findings provide a mechanism which might explain the increase in PEG 400 intestinal permeability in Crohn's disease patients and the increase in intestinal permeability found in patients receiving NSAIDS.


Alcohol intake, hypertension development and mortality in black South Africans.
Excessive alcohol intake is a risk factor for cardiovascular disease (CVD) and predicts cardiovascular and all-cause mortality. We determined which alcohol marker (self-reported alcohol intake, gamma-glutamyltransferase (GGT) or percentage carbohydrate deficient transferrin (%CDT)) relates best with mortality and predicts hypertension development over five years in black South Africans. This was a longitudinal study as part of the PURE (Prospective Urban and Rural Epidemiology) study in the North West Province, South Africa. We included 2010 participants and followed 1471 participants. Over five years, 230 deaths occurred, of which 66 were cardiovascular-related. At enrolment, participants completed questionnaires on alcohol intake (yes, for former and current use; no, for alcohol never used). We measured blood pressure, collected blood samples and measured GGT and %CDT. When comparing hazard ratios (HRs) of self-report, GGT and %CDT, we found that only GGT predicted cardiovascular (HR = 2.76 (1.49-5.12)) and all-cause mortality (HR = 2.47 (1.75-3.47)) and hypertension development ((HR = 1.31 (1.06-1.62)). Participants self-reporting yes for alcohol intake had a 30% increased risk of developing hypertension (HR = 1.30 (1.07-1.60)) but not an increased risk for mortality. When adding both GGT and self-report in the prediction model for hypertension, only self-reporting of alcohol was significant (HR = 1.24 (1.01-1.53)). The alcohol marker, %CDT, did not show any significant association with mortality or hypertension development. GGT independently predicted cardiovascular and all-cause mortality, as well as hypertension development in black South Africans. Despite non-specificity to excessive alcohol consumption, GGT may be a useful general marker for hypertension development and mortality, also due to its significant association with self-reported alcohol intake.


Development of a nomogram for prediction of vaginal birth after cesarean delivery.
To develop a model based on factors available at the first prenatal visit that predicts chance of successful vaginal birth after cesarean delivery (VBAC) for individual patients who undergo a trial of labor. All women with one prior low transverse cesarean who underwent a trial of labor at term with a vertex singleton gestation were identified from a concurrently collected database of deliveries at 19 academic centers during a 4-year period. Using factors identifiable at the first prenatal visit, we analyzed different classification techniques in an effort to develop a meaningful prediction model for VBAC success. After development and cross-validation, this model was represented by a graphic nomogram. Seven-thousand six hundred sixty women were available for analysis. The prediction model is based on a multivariable logistic regression, including the variables of maternal age, body mass index, ethnicity, prior vaginal delivery, the occurrence of a VBAC, and a potentially recurrent indication for the cesarean delivery. After analyzing the model with cross-validation techniques, it was found to be both accurate and discriminating. A predictive nomogram, which incorporates six variables easily ascertainable at the first prenatal visit, has been developed that allows the determination of a patient-specific chance for successful VBAC for those women who undertake trial of labor. II.


Effects of magnitude and frequency of variations in external power output on simulated cycling time-trial performance.
Mechanical models of cycling time-trial performance have indicated adverse effects of variations in external power output on overall performance times. Nevertheless, the precise influences of the magnitude and number of these variations over different distances of time trial are unclear. A hypothetical cyclist (body mass 70 kg, bicycle mass 10 kg) was studied using a mathematical model of cycling, which included the effects of acceleration. Performance times were modelled over distances of 4-40 km, mean power outputs of 200-600 W, power variation amplitudes of 5-15% and variation frequencies of 2-32 per time-trial. Effects of a "fast-start" strategy were compared with those of a constant-power strategy. Varying power improved 4-km performance at all power outputs, with the greatest improvement being 0.90 s for ± 15% power variation. For distances of 16.1, 20 and 40 km, varying power by ± 15% increased times by 3.29, 4.46 and 10.43 s respectively, suggesting that in long-duration cycling in constant environmental conditions, cyclists should strive to reduce power variation to maximise performance. The novel finding of the present study is that these effects are augmented with increasing event distance, amplitude and period of variation. These two latter factors reflect a poor adherence to a constant speed.


Prospective audit of parastomal hernia: prevalence and associated comorbidities.
There is little evidence regarding the prevalence or incidence of parastomal hernia, but it is thought to be common. Repair of parastomal hernia can be troublesome, and methods of repair need to be validated based on reduced incidence following surgery. The true rate of parastomal herniation needs to be determined prospectively, and risk factors for developing such hernias need to be more clearly defined. To determine prevalence and associated risk factors, prospective data were collected regarding initial stoma surgery, presence of parastomal hernia, and comorbidities. Ninety patients were prospectively audited. For stomas formed at emergency or elective surgery, regardless of surgical indication, the overall rate of parastomal hernia was 33%. Aperture size and patient age were independently predictive of parastomal hernia in multivariate analysis. For every millimeter increase in aperture size, the risk of developing a hernia increased by 10% (odds ratio, 1.10 (CI, 1.03-1.18); P = .005). For every additional year of patient age, the risk of developing a hernia increased by 4% (odds ratio, 1.04 (CI, 1.00-1.08); P = .04). There was a significantly higher prevalence of hernia following sigmoid colostomy than following ileostomy (45.9% vs. 22%; P < .05). The hernia rate was higher but did not reach statistical significance in patients with disseminated malignancy, body mass index >35 kg/m2, diabetes, prostate hypertrophy, ascites, or chronic constipation. This study of carefully and prospectively collected data shows the prevalence of parastomal herniation to be 33%. This rate was higher with larger aperture size and increased age in multivariate analysis.


Standardised surveillance of Clostridium difficile infection in European acute care hospitals: a pilot study, 2013.
Clostridium difficile infection (CDI) remains poorly controlled in many European countries, of which several have not yet implemented national CDI surveillance. In 2013, experts from the European CDI Surveillance Network project and from the European Centre for Disease Prevention and Control developed a protocol with three options of CDI surveillance for acute care hospitals: a 'minimal' option (aggregated hospital data), a 'light' option (including patient data for CDI cases) and an 'enhanced' option (including microbiological data on the first 10 CDI episodes per hospital). A total of 37 hospitals in 14 European countries tested these options for a three-month period (between 13 May and 1 November 2013). All 37 hospitals successfully completed the minimal surveillance option (for 1,152 patients). Clinical data were submitted for 94% (1,078/1,152) of the patients in the light option; information on CDI origin and outcome was complete for 94% (1,016/1,078) and 98% (294/300) of the patients in the light and enhanced options, respectively. The workload of the options was 1.1, 2.0 and 3.0 person-days per 10,000 hospital discharges, respectively. Enhanced surveillance was tested and was successful in 32 of the hospitals, showing that C. difficile PCR ribotype 027 was predominant (30% (79/267)). This study showed that standardised multicountry surveillance, with the option of integrating clinical and molecular data, is a feasible strategy for monitoring CDI in Europe.


Silicon nitride based plasmonic components for CMOS back-end-of-line integration.
Silicon nitride waveguides provide low propagation loss but weak mode confinement due to the relatively small refractive index contrast between the Si₃N₄ core and the SiO2 cladding. On the other hand, metal-insulator-metal (MIM) plasmonic waveguides offer strong mode confinement but large propagation loss. In this work, MIM-like plasmonic waveguides and passive devices based on horizontal Cu-Si₃N₄-Cu or Cu-SiO₂-Si₃N₄-SiO₂-Cu structures are integrated in the conventional Si₃N₄ waveguide circuits using standard CMOS backend processes, and are characterized around 1550-nm telecom wavelengths using the conventional fiber-waveguide-fiber method. The Cu-Si₃N₄(~100 nm)-Cu devices exhibit ~0.78-dB/μm propagation loss for straight waveguides, ~38% coupling efficiency with the conventional 1-μm-wide Si₃N₄ waveguide through a 2-μm-long taper coupler, ~0.2-dB bending loss for sharp 90° bends, and ~0.1-dB excess loss for ultracompact 1 × 2 and 1 × 4 power splitters. Inserting a ~10-nm SiO₂ layer between the Si3N4 core and the Cu cover (i.e., the Cu-SiO2(~10 nm)-Si₃N₄(~100 nm)-SiO2(~10 nm)-Cu devices), the propagation loss and the coupling efficiency are improved to ~0.37 dB/μm and ~52% while the bending loss and the excess loss are degraded to ~3.2 dB and ~2.1 dB, respectively. These experimental results are roughly consistent with the numerical simulation results after taking the influence of possible imperfect fabrication into account. Ultracompact plasmonic ring resonators with 1-μm radius are demonstrated with an extinction ratio of ~18 dB and a quality factor of ~84, close to the theoretical prediction.


Modeling hemoglobin and hemoglobin:haptoglobin complex clearance in a non-rodent species-pharmacokinetic and therapeutic implications.
Haptoglobin (Hp) prevents hemoglobin (Hb) extravasation and attenuates Hb induced tissue oxidation and vasoconstriction. Small animal models such as mouse, rat and guinea pig appear to demonstrate proof-of-concept for Hb neutralization by Hp in diverse pre-clinical conditions. However, these species differ significantly from humans in the clearance of Hb:Hp and demonstrate long persistence of circulating Hb:Hp complexes. The focus of this study is to understand Hb:Hp clearance in a non-rodent species. In contrast to rodents, dogs maintain high plasma Hp concentrations comparable to humans and demonstrate more rapid clearance of Hb:Hp when compared to rodent species, therefore dogs may represent a relevant species to evaluate Hb:Hp pharmacokinetics and cellular clearance. In this study we show, that like human macrophages, dog peripheral blood monocyte derived macrophages express a glucocorticoid inducible endocytic clearance pathways with a high specificity for the Hb:Hp complex. Evaluating the Beagle dog as a non-rodent model species we provide the first pharmacokinetic parameter estimates of free Hb and Hb:Hp complexes. The data demonstrate a significantly reduced volume of distribution (Vc) for Hb:Hp compared to free Hb, increased maximum plasma concentrations and areas under plasma concentration time curves (Cmax and AUC). Significantly reduced total body clearance (CL) and a longer terminal half-life (t1/2) of approximately 12 h were also observed for the Hb:Hp complex. Distribution and clearance were identical for dimeric and multimeric Hb:Hp complexes. We found no significant effect of a high-dose glucocorticoid treatment protocol on Hb:Hp pharmacokinetic parameter estimates. Collectively, our study supports the dog as a non-rodent animal model to study pharmacological and pharmacokinetic aspects of Hb clearance systems and apply the model to studying Hp as a therapeutic in diseases of hemolysis.


Galanin-expression and galanin-dependent sensory neurons are not required for itch.
Galanin is a key modulator of nociception, and it is also required for the developmental survival of a subset of C-fibre sensory neurons which are critical to the mediation of neuropathic and inflammatory pain. However, the potential modulatory roles played by galanin, or the galanin-dependent neurons, in pruritoceptive mechanisms underlying the sensation of itch have not been investigated. Here we report that mice carrying a loss-of-function mutation in the galanin gene (Gal-KO) show no differences in spontaneous behavioural itch responses compared to wild-type (WT) controls. Similarly, the responses to a range of pruritogens are not significantly different between the two genotypes. These results suggest that neither galanin expression, nor the galanin-dependent subpopulation of sensory neurons is required for itch-related behaviours.


Manifestation of incompleteness in obsessive-compulsive disorder (OCD) as reduced functionality and extended activity beyond task completion.
This study focused on hypotheses regarding the source of incompleteness in obsessive-compulsive disorder (OCD). For this, we had to document the behavioral manifestation of incompleteness in compulsive rituals, predicting that an exaggerated focus on acts that are appropriate for the task will support the hypothesis on heightened responsibility/perfectionism. In contrast, activity past the expected terminal act for the motor task would support the "stop signal deficiency" hypothesis. We employed video-telemetry to analyze 39 motor OCD rituals and compared each with a similar task performed by a non-OCD individual, in order to objectively and explicitly determine the functional end of the activity. We found that 75% of OCD rituals comprised a "tail," which is a section that follows the functional end of the task that the patients ascribed to their activity. The other 25% tailless rituals comprised a relatively high number and higher rate of repetition of non-functional acts. Thus, in rituals with tail, incompleteness was manifested by the mere presence of the tail whereas in tailless rituals, incompleteness was manifested by the reduced functionality of the task due to an inflated execution and repetition of non-functional acts. The prevalence of activity after the functional end ("tail") and the elevated non-functionality in OCD motor rituals support the "lack of stop signal" theories as the underlying mechanism in OCD. Furthermore, the presence and content of the tail might have a therapeutic potential in cognitive-behavior therapy.


Hepatic uptake of cholecystokinin octapeptide by organic anion-transporting polypeptides OATP4 and OATP8 of rat and human liver.
Cholecystokinin (CCK) is a major gastrointestinal peptide hormone that is released postprandially from the small intestine and exerts marked effects on gallbladder and gastrointestinal motility. The smaller isoforms CCK-8 and CCK-4 are rapidly taken up into hepatocytes, metabolized, and excreted into bile. Our aim was to identify and characterize the hepatocellular CCK-8 uptake system. CCK-8 uptake was measured in Xenopus laevis oocytes expressing the organic anion-transporting polypeptides of rat liver (Oatp1, Oatp2, Oatp3, or Oatp4) and of human liver (OATP-A, OATP-B, OATP-C, or OATP8) and in primary cultured rat hepatocytes. Rat Oatp4 and human OATP8 efficiently mediated CCK-8 uptake in oocytes, with Michaelis constant (Km) values of 14.9 +/- 2.9 micromol/L and 11.1 +/- 2.9 micromol/L, respectively. CCK-8 uptake by hepatocytes was also saturable, with a Km of 6.7 +/- 2.1 micromol/L. The Km value in rat hepatocytes is consistent with Oatp4-mediated transport. CCK-8 is selectively transported by rat Oatp4 and human OATP8, both of which are exclusively expressed at the basolateral membrane of hepatocytes. These 2 transporters are the first and probably the predominant hepatic uptake systems for CCK-8 and may be critical for the rapid clearance of this hormone from the circulation.


Neural systems underlying the reappraisal of personally craved foods.
Craving of unhealthy food is a common target of self-regulation, but the neural systems underlying this process are understudied. In this study, participants used cognitive reappraisal to regulate their desire to consume idiosyncratically craved or not craved energy-dense foods, and neural activity during regulation was compared with each other and with the activity during passive viewing of energy-dense foods. Regulation of both food types elicited activation in classic top-down self-regulation regions including the dorsolateral prefrontal, inferior frontal, and dorsal anterior cingulate cortices. This main effect of regulation was qualified by an interaction, such that activation in these regions was significantly greater during reappraisal of craved (versus not craved) foods and several regions, including the dorsolateral prefrontal, inferior frontal, medial frontal, and dorsal anterior cingulate cortices, were uniquely active during regulation of personally craved foods. Body mass index significantly negatively correlated with regulation-related activation in the right dorsolateral PFC, thalamus, and bilateral dorsal ACC and with activity in nucleus accumbens during passive viewing of craved (vs. neutral, low-energy density) foods. These results suggest that several of the brain regions involved in the self-regulation of food craving are similar to other kinds of affective self-regulation and that others are sensitive to the self-relevance of the regulation target.


Cardiorespiratory fitness levels and associations with physical activity and body composition in young South African adults from Soweto.
This observational study aims to describe fitness, and objectively measured physical activity levels and patterns in 409 young black South African adults (aged 19-20 years) from Soweto, as well as to examine associations between physical activity, fitness and BMI. A sub-maximal ramped step test was used to obtain an estimate of maximal oxygen uptake (VO2max). Physical activity was measured using ActiGraph (GT1M) for 7 days in 256 participants. Time spent in sedentary (<100 counts per minute (cpm)), moderate (2020-5998 cpm) and vigorous (≥5999 cpm) intensity activity was calculated, and 90% of participants were considered active. Data are presented as mean(CI) or median(CI). Overweight and obesity was more prevalent in females than males (35% vs 8%, p < 0.001). Males had a higher VO2max than females (41.9(41, 43) vs 32.6(32, 33)mlO2/kg/min, p < 0.001); spent more time in moderate to vigorous intensity physical activity (MVPA) (83(80, 94) vs 43(38, 45)min/day, p < 0.001), and less time in sedentary behaviours (541(541, 567) vs 575(568, 597)min/day, p < 0.01). Sedentary time was not associated with VO2max, however BMI was inversely associated, and MVPA was positively associated, with VO2max (both p < 0.001). The majority of young South African adults in this study were sufficiently active, and higher MVPA was associated with fitness. However, the high level of sedentary behaviour in this population is of concern and may be contributing to the increasing prevalence of overweight and obesity in this population. Young South African females are at greatest risk for decreased cardiovascular fitness and should be the focus for future interventions.


HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia.
Adaptation to hypoxia is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-regulated alpha subunit and a constitutively expressed beta subunit. Although HIF-1 is regulated mainly by oxygen tension through the oxygen-dependent degradation of its alpha subunit, in vitro it can also be modulated by cytokines, hormones and genetic alterations. To investigate HIF-1 activation in vivo, we determined the spatial and temporal distribution of HIF-1 in healthy mice subjected to varying fractions of inspiratory oxygen. Immunohistochemical examination of brain, kidney, liver, heart, and skeletal muscle revealed that HIF-1alpha is present in mice kept under normoxic conditions and is further increased in response to systemic hypoxia. Moreover, immunoblot analysis showed that the kinetics of HIF-1alpha expression varies among different organs. In liver and kidney, HIF-1alpha reaches maximal levels after 1 h and gradually decreases to baseline levels after 4 h of continuous hypoxia. In the brain, however, HIF-1alpha is maximally expressed after 5 h and declines to basal levels by 12 h. Whereas HIF-1beta is constitutively expressed in brain and kidney nuclear extracts, its hepatic expression increases concomitantly with HIF-1alpha. Overall, HIF-1alpha expression in normoxic mice suggests that HIF-1 has an important role in tissue homeostasis.


A Brief, 6-Item Scale for Caregiver Grief in Dementia Caregiving.
Caregivers of persons with dementia (PWD) can experience loss and grief long before the death of the person. Although such experience of caregiver grief is measurable, available scales (such as the Marwit-Meuser Caregiver Grief Inventory, MM-CGI) are lengthy and have overlaps with other caregiving constructs. We developed a briefer scale that captures the essence of caregiver grief-with comparable psychometric properties and total score to MM-CGI, as well as less overlap with other caregiving constructs. Family caregivers of community-dwelling PWD (N = 394) completed questionnaires containing MM-CGI and other caregiving scales. Initially, we split the study samples into two -the derivation sample (n = 179) was used to develop a brief scale that best predicts MM-CGI (using the best-subset approach with tenfold cross-validation), whereas the validation sample (n = 215) verified its actual performance in predicting MM-CGI. Thereafter, we evaluated the derived scale in its reliability and validity, and mapped its scores to MM-CGI using the equipercentile equating method. We derived a 6-item scale, which explained 84.1% of the variability in MM-CGI and had area under the receiver operating characteristic curve of .96 in discriminating high caregiver grief (95% CI: .94-.99). It had single dimension in confirmatory factor analysis (comparative fit index = .98; Tucker-Lewis index = .97) and maintained good psychometric properties similar to those of MM-CGI, while showing lower correlation with caregiver burden and depression. It also had scores that could be mapped to MM-CGI with reasonable precision. We developed the first brief scale with less than 10 items that can conveniently and accurately measure caregiver grief, which opens the way for grief-related interventions in clinical care. Notably, this 6-item scale was developed using rigorous methods and demonstrated consistent evidence of capturing the essence of caregiver grief.


Visualization of Active Glucocerebrosidase in Rodent Brain with High Spatial Resolution following In Situ Labeling with Fluorescent Activity Based Probes.
Gaucher disease is characterized by lysosomal accumulation of glucosylceramide due to deficient activity of lysosomal glucocerebrosidase (GBA). In cells, glucosylceramide is also degraded outside lysosomes by the enzyme glucosylceramidase 2 (GBA2) of which inherited deficiency is associated with ataxias. The interest in GBA and glucosylceramide metabolism in the brain has grown following the notion that mutations in the GBA gene impose a risk factor for motor disorders such as α-synucleinopathies. We earlier developed a β-glucopyranosyl-configured cyclophellitol-epoxide type activity based probe (ABP) allowing in vivo and in vitro visualization of active molecules of GBA with high spatial resolution. Labeling occurs through covalent linkage of the ABP to the catalytic nucleophile residue in the enzyme pocket. Here, we describe a method to visualize active GBA molecules in rat brain slices using in vivo labeling. Brain areas related to motor control, like the basal ganglia and motor related structures in the brainstem, show a high content of active GBA. We also developed a β-glucopyranosyl cyclophellitol-aziridine ABP allowing in situ labeling of GBA2. Labeled GBA2 in brain areas can be identified and quantified upon gel electrophoresis. The distribution of active GBA2 markedly differs from that of GBA, being highest in the cerebellar cortex. The histological findings with ABP labeling were confirmed by biochemical analysis of isolated brain areas. In conclusion, ABPs offer sensitive tools to visualize active GBA and to study the distribution of GBA2 in the brain and thus may find application to establish the role of these enzymes in neurodegenerative disease conditions such as α-synucleinopathies and cerebellar ataxia.


Induction of phenolic glycosides by quaking aspen (Populus tremuloides) leaves in relation to extrafloral nectaries and epidermal leaf mining.
We studied the effect of epidermal leaf mining on the leaf chemistry of quaking aspen, Populus tremuloides, during an outbreak of the aspen leaf miner, Phyllocnistis populiella, in the boreal forest of interior Alaska. Phyllocnistis populiella feeds on the epidermal cells of P. tremuloides leaves. Eleven days after the onset of leaf mining, concentrations of the phenolic glycosides tremulacin and salicortin were significantly higher in aspen leaves that had received natural levels of leaf mining than in leaves sprayed with insecticide to reduce mining damage. In a second experiment, we examined the time course of induction in more detail. The levels of foliar phenolic glycosides in naturally mined ramets increased relative to the levels in insecticide-treated ramets on the ninth day following the onset of leaf mining. Induction occurred while some leaf miner larvae were still feeding and when leaves had sustained mining over 5% of the leaf surface. Leaves with extrafloral nectaries (EFNs) had significantly higher constitutive and induced levels of phenolic glycosides than leaves lacking EFNs, but there was no difference in the ability of leaves with and without EFNs to induce phenolic glycosides in response to mining. Previous work showed that the extent of leaf mining damage was negatively related to the total foliar phenolic glycoside concentration, suggesting that phenolic glycosides deter or reduce mining damage. The results presented here demonstrate that induction of phenolic glycosides can be triggered by relatively small amounts of mining damage confined to the epidermal tissue, and that these changes in leaf chemistry occur while a subset of leaf miners are still feeding within the leaf.


The CIN4 chromosomal instability qPCR classifier defines tumor aneuploidy and stratifies outcome in grade 2 breast cancer.
Quantifying chromosomal instability (CIN) has both prognostic and predictive clinical utility in breast cancer. In order to establish a robust and clinically applicable gene expression-based measure of CIN, we assessed the ability of four qPCR quantified genes selected from the 70-gene Chromosomal Instability (CIN70) expression signature to stratify outcome in patients with grade 2 breast cancer. AURKA, FOXM1, TOP2A and TPX2 (CIN4), were selected from the CIN70 signature due to their high level of correlation with histological grade and mean CIN70 signature expression in silico. We assessed the ability of CIN4 to stratify outcome in an independent cohort of patients diagnosed between 1999 and 2002. 185 formalin-fixed, paraffin-embedded (FFPE) samples were included in the qPCR measurement of CIN4 expression. In parallel, ploidy status of tumors was assessed by flow cytometry. We investigated whether the categorical CIN4 score derived from the CIN4 signature was correlated with recurrence-free survival (RFS) and ploidy status in this cohort. We observed a significant association of tumor proliferation, defined by Ki67 and mitotic index (MI), with both CIN4 expression and aneuploidy. The CIN4 score stratified grade 2 carcinomas into good and poor prognostic cohorts (mean RFS: 83.8±4.9 and 69.4±8.2 months, respectively, p = 0.016) and its predictive power was confirmed by multivariate analysis outperforming MI and Ki67 expression. The first clinically applicable qPCR derived measure of tumor aneuploidy from FFPE tissue, stratifies grade 2 tumors into good and poor prognosis groups.


Goal neglect and working memory capacity in 4- to 6-year-old children.
Goal neglect is the phenomenon of failing to execute the momentary demands of a task despite understanding and being able to recall the task instructions. Successful goal maintenance is more likely to occur in adults with high working memory capacity (WMC) who can keep rules mentally accessible while performing the task. The current study predicted that goal neglect would also be related to WMC in children. It assessed thirty-seven 4-year-old and twenty-eight 6-year-old children on the goal neglect version of the Dimensional Change Card Sort, and 3 tasks that measure WMC. As predicted, children with higher WMC scores were more likely to maintain goals adequately for task performance. The findings are consistent with a 2-factor model of working memory and its development.


White Matter Integrity Declined Over 6-Months, but Dance Intervention Improved Integrity of the Fornix of Older Adults.
Degeneration of cerebral white matter (WM), or structural disconnection, is one of the major neural mechanisms driving age-related decline in cognitive functions, such as processing speed. Past cross-sectional studies have demonstrated beneficial effects of greater cardiorespiratory fitness, physical activity, cognitive training, social engagement, and nutrition on cognitive functioning and brain health in aging. Here, we collected diffusion magnetic resonance (MRI) imaging data from 174 older (age 60-79) adults to study the effects of 6-months lifestyle interventions on WM integrity. Healthy but low-active participants were randomized into Dance, Walking, Walking + Nutrition, and Active Control (stretching and toning) intervention groups (NCT01472744 on ClinicalTrials.gov). Only in the fornix there was a time × intervention group interaction of change in WM integrity: integrity declined over 6 months in all groups but increased in the Dance group. Integrity in the fornix at baseline was associated with better processing speed, however, change in fornix integrity did not correlate with change in processing speed. Next, we observed a decline in WM integrity across the majority of brain regions in all participants, regardless of the intervention group. This suggests that the aging of the brain is detectable on the scale of 6-months, which highlights the urgency of finding effective interventions to slow down this process. Magnitude of WM decline increased with age and decline in prefrontal WM was of lesser magnitude in older adults spending less time sedentary and more engaging in moderate-to-vigorous physical activity. In addition, our findings support the anterior-to-posterior gradient of greater-to-lesser decline, but only in the in the corpus callosum. Together, our findings suggest that combining physical, cognitive, and social engagement (dance) may help maintain or improve WM health and more physically active lifestyle is associated with slower WM decline. This study emphasizes the importance of a physically active and socially engaging lifestyle among aging adults.


Comparative studies on the ultrastructure of malignant melanoma in fish and human by freeze-etching and transmission electron microscopy.
Malignant melanomas (MM) in the fish Xiphophorus and in humans were studied both by transmission electron microscopy (TEM) and freeze-etching (FE). In both fish and human melanomas the cells show interdigitations of the plasma membranes. The nuclei are large and lobulated and have many nuclear pores. Melanosomes are abundant and melanosome complexes ("compound melanosomes") occur regularly. Pinocytotic vesicles could be demonstrated in fish and human melanomas showing local differences in frequency and distribution patterns in the tumor. Intercellular junctions are lacking in MM cells from fish and humans. The FE technique showed considerable advantages in demonstrating membrane-surface peculiarities such as nuclear pores or pinocytotic vesicles. The FE replicas of fish melanomas are like those of humans. These findings may support the hypothesis that melanoma in fish and humans reflect the same biological phenomenon.


Study of singular radius and surface boundary constraints in refractive beam shaper design.
This paper presents analysis of important issues associated with the design of refractive laser beam shaping systems. The concept of "singular radius" is introduced along with solutions to minimize its adverse effect on shaper performance. In addition, the surface boundary constraint is discussed in detail. This study provides useful guidelines to circumvent possible design errors that would degrade the shaper quality or add undesired complication to the system.


Chimpanzee Adenovirus Vector Ebola Vaccine.
The unprecedented 2014 epidemic of Ebola virus disease (EVD) prompted an international response to accelerate the availability of a preventive vaccine. A replication-defective recombinant chimpanzee adenovirus type 3-vectored ebolavirus vaccine (cAd3-EBO), encoding the glycoprotein from Zaire and Sudan species, that offers protection in the nonhuman primate model, was rapidly advanced into phase 1 clinical evaluation. We conducted a phase 1, dose-escalation, open-label trial of cAd3-EBO. Twenty healthy adults, in sequentially enrolled groups of 10 each, received vaccination intramuscularly in doses of 2×1010 particle units or 2×1011 particle units. Primary and secondary end points related to safety and immunogenicity were assessed throughout the first 8 weeks after vaccination; in addition, longer-term vaccine durability was assessed at 48 weeks after vaccination. In this small study, no safety concerns were identified; however, transient fever developed within 1 day after vaccination in two participants who had received the 2×1011 particle-unit dose. Glycoprotein-specific antibodies were induced in all 20 participants; the titers were of greater magnitude in the group that received the 2×1011 particle-unit dose than in the group that received the 2×1010 particle-unit dose (geometric mean titer against the Zaire antigen at week 4, 2037 vs. 331; P=0.001). Glycoprotein-specific T-cell responses were more frequent among those who received the 2×1011 particle-unit dose than among those who received the 2×1010 particle-unit dose, with a CD4 response in 10 of 10 participants versus 3 of 10 participants (P=0.004) and a CD8 response in 7 of 10 participants versus 2 of 10 participants (P=0.07) at week 4. Assessment of the durability of the antibody response showed that titers remained high at week 48, with the highest titers in those who received the 2×1011 particle-unit dose. Reactogenicity and immune responses to cAd3-EBO vaccine were dose-dependent. At the 2×1011 particle-unit dose, glycoprotein Zaire-specific antibody responses were in the range reported to be associated with vaccine-induced protective immunity in challenge studies involving nonhuman primates, and responses were sustained to week 48. Phase 2 studies and efficacy trials assessing cAd3-EBO are in progress. (Funded by the Intramural Research Program of the National Institutes of Health; VRC 207 ClinicalTrials.gov number, NCT02231866 .).


Speckle imaging and hidden phase.
The Knox-Thompson, or cross-spectrum, method provides two two-dimensional difference equations for the phase of the object spectrum. We demonstrate that, in general, the object spectrum phase can be decomposed into a regular, single-valued function determined by the divergence of the phase gradient, as well as a multivalued function determined by the circulation of the phase gradient; this second function has been called the hidden phase. The standard least-squares solution to the two-dimensional difference equations will always miss this hidden phase. We present a solution method that gives both the regular and the hidden parts of the object spectrum phase. Finally, we illustrate several examples of imaging through turbulence and postprocessing with the Knox-Thompson method, including the hidden phase.


Anodic stripping voltammetry of gold nanoparticles at boron-doped diamond electrodes and its application in immunochromatographic strip tests.
Anodic stripping voltammetry (ASV) of colloidal gold-nanoparticles (AuNPs) was investigated at boron-doped diamond (BDD) electrodes in 50 mM HClO4. A deposition time of 300 s at-0.2 V (vs. Ag/AgCl) was fixed as the condition for the ASV. The voltammograms showed oxidation peaks that could be attributed to the oxidation of gold. These oxidation peaks were then investigated for potential application in immunochromatographic strip tests for the selective and quantitative detection of melamine, in which AuNPs were used as the label for the antibody of melamine. Linear regression of the oxidation peak currents appeared in the concentration range from 0.05-0.6 μg/mL melamine standard, with an estimated LOD of 0.069 μg/mL and an average relative standard deviation of 8.0%. This indicated that the method could be considered as an alternative method for selective and quantitative immunochromatographic applications. The validity was examined by the measurements of melamine injected into milk samples, which showed good recovery percentages during the measurements.


Television Viewing Time and Inflammatory-Related Mortality.
Television (TV) viewing time is associated with increased risk of all-cause, cardiovascular and cancer mortality. Although TV time is detrimentally associated with key inflammatory markers, the associations of TV time with other inflammatory-related mortality (with a predominant inflammatory, oxidative or infectious component, but not attributable to cancer or cardiovascular causes), are unknown. Among 8933 Australian adults (4593 never-smokers) from the baseline (1999-2000) Australian Diabetes, Obesity and Lifestyle Study (median follow-up, 13.6 yr), we examined TV time in relation to noninflammatory and inflammatory-related mortality (not attributable to cancer or cardiovascular causes, hereafter "inflammatory-related" mortality). Because smoking has a significant inflammatory component, we also examined this relationship in never-smokers. Of 896 deaths, 248 were attributable to cardiovascular disease, 346 to cancer, 130 to other inflammatory-related causes (71 for never-smokers), and 172 to noninflammatory-related causes (87 for never-smokers). After multivariate adjustment for age, sex, education, household income, smoking status, alcohol intake, energy intake, diet, and cardiometabolic risk biomarkers (model 3), every additional hours per day of TV time was associated with increased risk of inflammatory-related mortality in the overall population (hazard ratio, 1.12; 95% confidence interval, 1.00-1.25) and in never-smokers (1.18; 1.00, 1.40). These results were attenuated after additional adjustment for leisure-time physical activity. After multivariate adjustment (model 3), no association was observed for noninflammatory mortality in the overall population (0.95; 0.85, 1.07), but risk tended to decrease for never-smokers (0.85; 0.75, 1.02). In summary, before adjustment for leisure-time physical activity, TV time was associated with increased risk of inflammatory-related mortality. This is consistent with the hypothesis that high TV viewing may be associated with a chronic inflammatory state.


Age-associated changes in histology and gene-expression profile in the rat ventral prostate.
The incidence of prostate diseases rises dramatically with age in men, yet little is understood of the mechanisms underlying prostatic senescence and its contribution to disease development in the gland. In Noble rats, aging of the ventral prostate (VP) is characterized morphologically by widespread atrophy of acini, increased accumulation of concretions in glandular lumen, infiltration of inflammatory cells, and focal epithelial atypia. We used a cDNA microarray containing 2388 known transcripts, together with the Tyramide Amplification System and t statistics, to identify differentially expressed genes in the VPs of young (3 months old) and old (16 months old) rats. A total of 78 VP genes were found to be differentially expressed by the two groups; in old rats, 65 VP genes (83%) showed reduced expression and 13 genes (17%) showed increased expression compared with young animals. The age-dependent underexpressed genes fell into several functional clusters: those involved in amino-acid metabolism, protein synthesis, protein secretion and degradation, vesicle/membrane trafficking, energy metabolism, signal transduction, spermidine and spermine syntheses, and cellular defense against stress. The overexpressed genes included iduronate 2-sulfatase, HLA class I locus C heavy chain, membrane cofactor protein of the complement system, TRPM-2, cadherin-associated protein-related, and X-CGD. Post hoc analyses confirmed a progressive decline in the expression of ribophorin II and BiP and a gradual increase in the expression of TRPM-2 in rat VPs as animals aged from 3 to 19 months old. In conclusion, the observed widespread declines in expression of genes involved in protein synthesis, protein fidelity maintenance, anabolism, growth inhibition, and energy metabolism, together with increased expression of genes implicated in cell survival in the VPs of senescent rats, may help explain the susceptibility of the prostates of elderly men to development of disease.


Selective immunocytochemical staining of mitral cells in rat olfactory bulb with affinity purified antibodies against N-acetyl-aspartyl-glutamate.
Polyclonal antibodies were raised in rabbits against N-acetyl-aspartyl-glutamate (NAAG) coupled to bovine serum albumin (BSA) with carbodiimide and were purified by affinity chromatography sequentially over BSA-agarose and NAAG-agarose resins. Solid-phase RIA revealed a distinct pattern of specificity of the antibodies for N-acetylated acidic peptides, with highest signal obtained for NAAG, and essentially no immunoreactivity demonstrable for aspartate or glutamate. Coronal sections through carbodiimide-fixed rat olfactory bulb were incubated with the purified antiserum and antigen localization visualized by the avidin-biotin peroxidase techniques. Immunoreactivity was restricted to the mitral cells, the major excitatory projection neurons of the lateral olfactory tract, a putative glutamatergic pathway. Immunoreactivity was selectively blocked by preincubation of the antibody with 1 microgram/ml of NAAG-BSA. These results demonstrate a specific neuronal localization of NAAG-like immunoreactivity and support the candidacy of NAAG as a neurotransmitter of the lateral olfactory tract.


Correlations between carbene and carbenium stability: ab initio calculations on substituted phenylcarbenes, nonbenzenoid arylcarbenes, heteroatom-substituted carbenes, and the corresponding carbocations and hydrogenation products.
Calculations were completed at the G3MP2 level on a large group of carbon- and heteroatom-substituted carbenes (X-CH, singlets and triplets), carbenium ions (X-CH2(+)), and their hydrogen addition products (X-CH3). One series includes 11 meta- and 12 para-substituted phenylcarbenes, X = Ar. Gas-phase enthalpies of reaction were calculated for four processes: singlet-triplet enthalpy gaps of the carbenes, DeltaH(ST); enthalpies for deprotonation of the cations yielding singlet carbenes, DeltaH(ACID); hydride ion affinities of the carbenium ions, HIA; and enthalpies of hydrogenation of the singlet carbenes, DeltaH(HYDROG). A plot of HIA vs DeltaH(HYDROG) values provides a direct comparison of substituent effects on the stabilities of the singlet carbenes and the corresponding benzylic cations. These effects are larger for the cations but are remarkably consistent over a wide range of reactivity: 166 kcal/mol in HIA. All four processes were analyzed according to the relative importance of polarizability, polar, and resonance effects. Polar and resonance effects are large and of similar magnitude for meta compounds. For the para compounds resonance effects are more dominant. Calculations were made on three nonbenzenoid arylcarbenes: Ar = cycloheptatrienyl(+), cyclopentadienyl(-), and cyclopropenyl(+). The cyclopentadienyl(-)-substituted system fits the HIA vs DeltaH(HYDROG) correlation, but the other two fall well off the line, suggesting markedly different interactions are at play. A set of heteroatom-substituted carbenes and carbocations was also examined. Points for these groups lie well above the correlation line for the HIA vs DeltaH(HYDROG) plot defined by the aryl compounds, confirming that heteroatoms stabilize the singlet carbene proportionally more than the carbocation.


Expression of phosphofructokinase in skeletal muscle is influenced by genetic variation and associated with insulin sensitivity.
Using an integrative approach in which genetic variation, gene expression, and clinical phenotypes are assessed in relevant tissues may help functionally characterize the contribution of genetics to disease susceptibility. We sought to identify genetic variation influencing skeletal muscle gene expression (expression quantitative trait loci [eQTLs]) as well as expression associated with measures of insulin sensitivity. We investigated associations of 3,799,401 genetic variants in expression of >7,000 genes from three cohorts (n = 104). We identified 287 genes with cis-acting eQTLs (false discovery rate [FDR] <5%; P < 1.96 × 10(-5)) and 49 expression-insulin sensitivity phenotype associations (i.e., fasting insulin, homeostasis model assessment-insulin resistance, and BMI) (FDR <5%; P = 1.34 × 10(-4)). One of these associations, fasting insulin/phosphofructokinase (PFKM), overlaps with an eQTL. Furthermore, the expression of PFKM, a rate-limiting enzyme in glycolysis, was nominally associated with glucose uptake in skeletal muscle (P = 0.026; n = 42) and overexpressed (Bonferroni-corrected P = 0.03) in skeletal muscle of patients with T2D (n = 102) compared with normoglycemic controls (n = 87). The PFKM eQTL (rs4547172; P = 7.69 × 10(-6)) was nominally associated with glucose uptake, glucose oxidation rate, intramuscular triglyceride content, and metabolic flexibility (P = 0.016-0.048; n = 178). We explored eQTL results using published data from genome-wide association studies (DIAGRAM and MAGIC), and a proxy for the PFKM eQTL (rs11168327; r(2) = 0.75) was nominally associated with T2D (DIAGRAM P = 2.7 × 10(-3)). Taken together, our analysis highlights PFKM as a potential regulator of skeletal muscle insulin sensitivity.


A Novel Risk-based Approach Simulating Oncological Surveillance After Radical Nephroureterectomy in Patients with Upper Tract Urothelial Carcinoma.
The current guideline lacks evidence for creating individualized surveillance strategies for upper tract urothelial carcinoma (UTUC) patients after radical nephroureterectomy (RNU). To create a novel risk model and to simulate individualized surveillance duration that dynamically illustrates the changing risk relationship of UTUC-related death and non-UTUC death, considering the impact of cigarette smoking. This multicenter cohort study comprised 714 pTa-T4N0M0 UTUC patients, with a median follow-up duration of 65mo. There were 279 (39.1%) nonsmokers, 260 (36.4%) current smokers, and 175 (24.5%) ex-smokers. All patients underwent RNU. The risks of UTUC death and non-UTUC death over time were estimated using parametric models for time to failure with Weibull distributions. Age-specific, stage-specific, and smoking status-specific surveillance durations were simulated based upon Weibull estimates. The hazard rate (HR) of non-UTUC death gradually increased over time in all age groups regardless of the smoking status, whereas that of UTUC-related death decreased markedly according to the pathological T (pT) stage and was affected by the smoking status. Among current smokers, the baseline HR of UTUC-related death in pT3/4 was higher than that of pT ≤2 and remained high even 10yr after RNU. Among heavy smokers, the HR of UTUC-related death in all pT stages was highest at baseline and remained high after RNU, compared with nonsmokers, current smokers, or ex-smokers. We simulated specific time points when the risk of non-UTUC death was greater than that of UTUC-related death. Among patients ≥80yr of with pT3N0M0, the risk of non-UTUC death was greater than that of UTUC-related death 1yr after RNU in nonsmokers, but 7yr for heavy smokers. Our result revealed that smokers bear a long-term risk burden of UTUC-related death more than the risk of non-UTUC death. For UTUC smokers, longer-term surveillance duration is recommended even in elderly stage. In the present study, we evaluated the risk transition of upper tract urothelial carcinoma (UTUC)-related death and non-cancer-related death over time. We found that smoking weighed a huge impact upon UTUC-related death compared with death from other cause, and therefore, we created a more individualized surveillance duration model.