Detection of light and flicker at low luminance levels in the human peripheral visual system. I. Psychophysical experiments.
The interaction between summation and adaptation mechanisms near the absolute threshold of vision is studied. In this paper, results are presented of measurements of both light detection and flicker detection for circular flashes with a diameter of 5.7-480 min of arc, a flash duration of 5-1000 msec, a period of 0-8000 msec, and at eccentricities of 7 and 40 deg in the temporal retina. It is confirmed that the estimates of the summation times obtained from the light-detection-threshold energy as a function of the period and from the light-detection threshold as a function of the flash duration are similar; these estimates depend on the stimulus size and eccentricity. It is suggested that two summation mechanisms can be distinguished, one for the preprocessing and one for the detection mechanism. The summation time of the first mechanism is estimated to be about 100 msec; that of the latter, about 500 msec. At 40-deg eccentricity, decreasing the period of presentation leads initially to a rise in the light-detection threshold, pointing to adaptational phenomena. The flicker threshold is determined by the energy per flash (for brief flashes); when the flash duration exceeds 100 msec, the intensity of the flash is the important parameter. The flicker threshold depends strongly and in an irregular way on the stimulus size because of lateral inhibition effects.


Individual- and school-level sociodemographic predictors of obesity among New York City public school children.
To identify student- and school-level sociodemographic characteristics associated with overweight and obesity, the authors conducted cross-sectional analyses of data from 624,204 public school children (kindergarten through 12th grade) who took part in the 2007-2008 New York City Fitnessgram Program. The overall prevalence of obesity was 20.3%, and the prevalence of overweight was 17.6%. In multivariate models, the odds of being obese as compared with normal weight were higher for boys versus girls (odds ratio (OR) = 1.39, 95% confidence interval (CI): 1.36, 1.42), for black (OR = 1.11, 95% CI: 1.07, 1.15) and Hispanic (OR = 1.48, 95% CI: 1.43, 1.53) children as compared with white children, for children receiving reduced-price (OR = 1.17, 95% CI: 1.13, 1.21) or free (OR = 1.12, 95% CI: 1.09, 1.15) school lunches as compared with those paying full price, and for US-born students (OR = 1.54, 95% CI: 1.50, 1.58) as compared with foreign-born students. After adjustment for individual-level factors, obesity was associated with the percentage of students who were US-born (across interquartile range (75th percentile vs. 25th), OR = 1.10, 95% CI: 1.07, 1.14) and the percentage of students who received free or reduced-price lunches (across interquartile range, OR = 1.13, 95% CI: 1.10, 1.18). The authors conclude that individual sociodemographic characteristics and school-level sociodemographic composition are associated with obesity among New York City public school students.


Experiences of nurses caring for mental health care users in an acute admission unit at a psychiatric hospital in the Western Cape Province.
Caring for mental health care users (MHCUs) with mental illnesses is a major task that confronts nurses globally. It has been argued that caring for this group of patients is accompanied by unique challenges. Despite the available abundance of data about nursing patients suffering from mental illnesses, little is known about the lived experiences of nurses who care for MHCUs in acute admission units in the Western Cape province. This study's aim is to explore and describe the lived experiences of nurses who care for MHCUs in an acute admission unit at a psychiatric hospital in the Western Capeprovince. A qualitative, descriptive, phenomenological study was conducted. A purposive sampling procedure was applied which resulted in a sample that comprised eight nurses. Indepth, individual, semi-structured interviews were conducted with these eight participants. Interviews were audio-recorded and transcribed verbatim and the researcher utilised Collaizzi's method to analyse collected data. Both positive and negative experiences were reported. Positive experiences werethe recovery of patients, teamwork, and passion for caring. Negative experiences were thefeelings of being unappreciated and unsupported by authorities. Physical assault by MHCUs,shortage of staff, increased workload and burnout was also reported. In-service training about management of aggression needs to be provided,debriefing sessions to deal with burnout needs to be arranged, and research to quantify levelsof burnout should be conducted.


Clonally expanded γδ T cells protect against Staphylococcus aureus skin reinfection.
The mechanisms that mediate durable protection against Staphylococcus aureus skin reinfections are unclear, as recurrences are common despite high antibody titers and memory T cells. Here, we developed a mouse model of S. aureus skin reinfection to investigate protective memory responses. In contrast with WT mice, IL-1β-deficient mice exhibited poor neutrophil recruitment and bacterial clearance during primary infection that was rescued during secondary S. aureus challenge. The γδ T cells from skin-draining LNs utilized compensatory T cell-intrinsic TLR2/MyD88 signaling to mediate rescue by trafficking and producing TNF and IFN-γ, which restored neutrophil recruitment and promoted bacterial clearance. RNA-sequencing (RNA-seq) of the LNs revealed a clonotypic S. aureus-induced γδ T cell expansion with a complementarity-determining region 3 (CDR3) aa sequence identical to that of invariant Vγ5+ dendritic epidermal T cells. However, this T cell receptor γ (TRG) aa sequence of the dominant CDR3 sequence was generated from multiple gene rearrangements of TRGV5 and TRGV6, indicating clonotypic expansion. TNF- and IFN-γ-producing γδ T cells were also expanded in peripheral blood of IRAK4-deficient humans no longer predisposed to S. aureus skin infections. Thus, clonally expanded γδ T cells represent a mechanism for long-lasting immunity against recurrent S. aureus skin infections.


A comparative study for performance evaluation of sit-to-stand task with body worn sensor and existing laboratory methods.
uman movement such as sit-to-stand (STS) is one of the most fundamental and essential movement among daily living activities. Elderly who are unable to stand are severely limited in daily activities and rely on others for their care. In the coming next two decades about 20% of US population will be above the age of 65 years, and considering ever-increasing elderly population and health care expenses, the rehabilitation of functionally limited elderly is becoming a topic of greater concern. Clinically STS movement is looked upon with great interest, such as its analysis is meaningful in order to evaluate motor control and stability in elderly patients with functional limitations and may be more helpful than traditional medical evaluations in diagnosing and treating mobility problems in elderly. A few previous research studies describe controlled STS movements with constrains in laboratory environments and have established STS standardized events. A comparative study is presented in this work using and inertial measurement unit (IMU) and existing laboratory based sit-to-stand phases and events. The aims of this study are (1) to define phases and identify events of STS movement based on those previously described, using wireless IMU (2) to test the consistency of these events and phases in total of seven young healthy subjects (ages 25-35 years). We found significant differences in peak flexion angular velocities (p<0.001), peak extension angular velocities (p<0.01) and accelerations at seat-off (p<0.01) between participants when using chair arm rest versus knee as support during rising. Also, we have found significant differences in time events for pre seat off flexion decelerations and post seat off extension angular accelerations with the two different rising supports. Thus these distinct and consistent phases of STS movement using IMU can help identify individuals at fall risk in non-laboratory environments.


Links between personality, time perspective, and intention to practice physical activity during cancer treatment: an exploratory study.
The purpose of the study was to analyze links between personality, time perspective, and intention to practice physical activity during cancer treatment. One hundred forty-three patients participated in survey by questionnaire. Intention to practice physical activity, time perspective using Zimbardo Time Perspective Inventory, and personality with the Big Five Inventory were measured. Structural equation models using Lisrel were developed to examine hypothetical links between the variables. The adjusted model evidenced an excellent fit (comparative fit index = 0.92; root-mean-square error of approximation = 0.076; P = .014). Results showed that intention to practice exercise was positively linked with openness to experience and negatively with present fatalist time perspective. Moreover, conscientiousness and neuroticism were found to be linked with future time perspective, which was positively related with intention to practice physical activity. The present exploratory study with patients suffering from cancer underlined the importance of considering jointly time perspective dimensions and personality factors for health behavior recommendations. Based on our results, we propose some reflections on practice to help nurses and physicians increase patient's motivation to be physically active. Taking into account patients' personality and time perspective, we would be able to propose specific awareness messages and offer short interventions to have an impact on patients' motivation to practice.


Fast 18F labeling of a near-infrared fluorophore enables positron emission tomography and optical imaging of sentinel lymph nodes.
We combine a novel boronate trap for F(-) with a near-infrared fluorophore into a single molecule. Attachment to targeting ligands enables localization by positron emission tomography (PET) and near-infrared fluorescence (NIRF). Our first application of this generic tag is to label Lymphoseek (tilmanocept), an agent designed for receptor-specific sentinel lymph node (SLN) mapping. The new conjugate incorporates (18)F(-) in a single, aqueous step, targets mouse SLN rapidly (1 h) with reduced distal lymph node accumulation, permits PET or scintigraphic imaging of SLN, and enables NIRF-guided excision and histological verification even after (18)F decay. This embodiment is superior to current SLN mapping agents such as nontargeted [(99m)Tc]sulfur colloids and Isosulfan Blue, as well as the phase III targeted ligand [(99m)Tc]SPECT Lymphoseek counterpart, species that are visible by SPECT or visible absorbance separately. Facile incorporation of (18)F into a NIRF probe should promote many synergistic PET and NIRF combinations.


Time estimation and reproduction is abnormal in Parkinson's disease.
We compared the performance of 44 patients with Parkinson's disease (PD), tested 12-24 h after withdrawal of dopaminergic medication, with 20 age-matched controls, on a verbal test for time estimation and in several time reproduction tasks. Patients with PD underestimated the duration of a time interval in the verbal time estimation task and showed overproduction of time intervals when required to reproduce a short time sample. Absolute errors were greater in the reproduction of longer time intervals in both control and PD patients, but especially in the latter. The presentation of time markers at faster rates had a detrimental effect on the performance of the patients but not of the controls. Patients with more severe PD performed worse on the time estimation and reproduction tasks compared with those with milder disease. Administration of levodopa-carbidopa (205/25 mg, p.o.) significantly reduced absolute errors in time estimation and reproduction in conditions where time markers were presented at the two faster rates of 5 Hz and 3.3 Hz. Performance in these two latter tests best discriminated patients and controls and had a positive significant association with simple reaction time and movement time. These results lead us to suggest that time estimation, i.e. the 'internal clock', is abnormally slow in PD.


Basic autonomy as a fundamental step in the synthesis of life.
In the search for the primary roots of autonomy (a pivotal concept in Varela's comprehensive understanding of living beings), the theory of autopoiesis provided an explicit criterion to define minimal life in universal terms, and was taken as a guideline in the research program for the artificial synthesis of biological systems. Acknowledging the invaluable contribution of the autopoietic school to present biological thinking, we offer an alternative way of conceiving the most basic forms of autonomy. We give a bottom-up account of the origins of "self-production" (or self-construction, as we propose to call it), pointing out which are the minimal material and energetic requirements for the constitution of basic autonomous systems. This account is, indeed, committed to the project of developing a general theory of biology, but well grounded in the universal laws of physics and chemistry. We consider that the autopoietic theory was formulated in highly abstract terms and, in order to advance in the implementation of minimal autonomous systems (and, at the same time, make major progress in exploring the origins of life), a more specific characterization of minimal autonomous systems is required. Such a characterization will not be drawn from a review of the autopoietic criteria and terminology (à la Fleischaker) but demands a whole reformulation of the question: a proper naturalization of the concept of autonomy. Finally, we also discuss why basic autonomy, according to our account, is necessary but not sufficient for life, in contrast with Varela's idea that autopoiesis was a necessary and sufficient condition for it.


A Double-Blind, Randomized, Placebo-Controlled Trial of Panax Ginseng for Cancer-Related Fatigue in Patients With Advanced Cancer.
Background: Despite the high frequency, severity, and effects of cancer-related fatigue (CRF) on the quality of life (QoL) of patients with cancer, limited treatment options are available. The primary objective of this study was to compare the effects of oral Panax ginseng extract (PG) and placebo on CRF. Secondary objectives were to determine the effects of PG on QoL, mood, and function. Methods: In this randomized, double-blind, placebo-controlled study, patients with CRF ≥4/10 on the Edmonton Symptom Assessment System (ESAS) were eligible. Based on a pilot study, we randomized patients to receive either 400 mg of standardized PG twice daily or a matching placebo for 28 days. The primary end point was change in the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F) subscale from baseline to day 29. Results: Of 127 patients, 112 (88.2%) were evaluable. The mean (SD) FACIT-F subscale scores at baseline, day 15, and day 29 were 22.4 (10.1), 29.9 (10.6), and 30.1 (11.6) for PG (P<.001), and 24.0 (9.4), 30.0 (10.1), and 30.4 (11.5) for placebo (P<.001). Mean (SD) improvement in the FACIT-F subscale at day 29 was not significantly different in the PG than in the placebo group (7.5 [12.7] vs 6.5 [9.9]; P=.67). QoL, anxiety, depression, symptoms, and functional scores were not significantly different between the PG and placebo groups. Improvement in the FACIT-F subscale correlated with baseline scores (P=.0005), Hospital Anxiety and Depression Scale results (P=.032), and sex (P=.023). There were fewer any-grade toxicities in the PG versus placebo group (28/63 vs 33/64; P=.024). Conclusions: Both PG and placebo result in significant improvement in CRF. PG was not significantly superior to placebo after 4 weeks of treatment. There is no justification to recommend the use of PG for CRF. Further studies are needed. Trial Registration: ClinicalTrials.gov identifier: NCT01375114.


Effect of germination and thermal treatments on folates in rye.
Effects of germination conditions and thermal processes on folate contents of rye were investigated. Total folate contents were determined microbiologically with Lactobacillus rhamnosus (ATCC 7469) as the growth indicator organism, and individual folates were determined by high-performance liquid chromatography after affinity chromatographic purification. Germination increased the folate content by 1.7-3.8-fold, depending on germination temperature, with a maximum content of 250 micro g/100 g dry matter. Hypocotylar roots with their notably high folate concentrations (600-1180 micro g/100 g dry matter) contributed 30-50% of the folate contents of germinated grains. Germination altered the proportions of folates, increasing the proportion of 5-methyltetrahydrofolate and decreasing the proportion of formylated folate compounds. Thermal treatments (extrusion, autoclaving and puffing, and IR and toasting) resulted in significant folate losses. However, folate levels in grains that were germinated and then were heat processed were higher than for native (nongerminated) grains. Opportunities to optimize rye processing to enhance folate levels in rye-based foods are discussed.


Thermal dependence of maximum Ca2+-activated force in skinned muscle fibres of the toad Bufo marinus acclimated at different temperatures.
Mechanically skinned muscle fibres from the twitch region of the iliofibularis muscle of cool- (16 +/- 1 degree C) and warm- (32 +/- 1 degree C) acclimated cane toads (Bufo marinus) were activated maximally by Ca2+ in solutions of different pH and at different temperatures (approx. 1-35 degrees C). Acclimation of up to 12 weeks at 16 degrees C and up to 8 weeks at 32 degrees C did not modify the marked thermal dependence of isometric force in the skeletal muscle fibres of the cane toad. The prominent decline of maximum Ca2+-activated force at lower temperatures, a property which is not characteristic of muscles from other anurans, was associated with an obvious decline in fibre stiffness at temperatures below about 20 degrees C, regardless of the temperatures at which the toads were kept prior to experimentation. The results suggest that the decline of isometric force at lower temperatures is due both to a reduction in the number of cross-bridges and to a decrease in the force output per cross-bridge. The maximum Ca2+-activated force response increased when fibres were activated in solutions of increasing pH at all temperatures investigated. This trend is expected to have a compensatory effect on the thermal dependence of the maximum Ca2+-activated force under physiological conditions, because of the elevation of intracellular pH as temperature declines. The isometric force did not depend on the concentration of the zwitterionic species of the pH buffer in solutions. The skinned fibre preparation developed a Ca2+-insensitive residual force following maximal activation. The increment in residual force followed a linear relationship with the duration of activation at a given temperature and a power relationship of activation temperature for a given duration of activation. Fibres from warm-acclimated animals developed less residual force following activations at 15 degrees C than did fibres from cool-acclimated animals, suggesting that thermal acclimation may substantially reduce the magnitude of this phenomenon at temperatures below 20 degrees C.


The effect of dominance on food hoarding: a game theoretical model.
Many food hoarding animals live in small groups structured by rank. The presence of conspecifics in the hoarding area increases the risk of losing stored supplies. The possibility of stealing from others depends on a forager's rank in the group. Highly ranked individuals can steal from subordinates and also protect their own caches. Since storing incurs both costs and benefits, the optimal hoarding investment will differ between individuals of different rank. In a game theoretical model, we investigate how dominant and subordinate individuals should optimize their hoarding effort. Our model imagines animals that are large-scale hoarders in autumn and dependent on stored supplies for winter survival. Many examples can be found in the bird families Paridae and Corvidae, but the model can be used for any hoarding species that forage in groups. Predictions from the model are as follows: First, subordinates should store more than dominants, but in a predictable environment, this difference will decrease as the environment gets harsher. Under harsh conditions, dominants should store almost as much as subordinates and, later, spend almost as much time retrieving their own caches as subordinates. Second, if on the other hand, bad winter conditions were not expected when storing, dominants should spend more time pilfering caches from subordinates. Third, in populations that are highly dependent on stored supplies, dominants should store relatively more than in populations that are less dependent on stored supplies. Fourth, harsher environments will favor hoarding. And finally, if dominant individuals store, it implies that hoarders have a selfish recovery advantage over conspecific pilferers.


The technological and economic prospects for CO2 utilization and removal.
The capture and use of carbon dioxide to create valuable products might lower the net costs of reducing emissions or removing carbon dioxide from the atmosphere. Here we review ten pathways for the utilization of carbon dioxide. Pathways that involve chemicals, fuels and microalgae might reduce emissions of carbon dioxide but have limited potential for its removal, whereas pathways that involve construction materials can both utilize and remove carbon dioxide. Land-based pathways can increase agricultural output and remove carbon dioxide. Our assessment suggests that each pathway could scale to over 0.5 gigatonnes of carbon dioxide utilization annually. However, barriers to implementation remain substantial and resource constraints prevent the simultaneous deployment of all pathways.


Next-generation sequencing approaches for improvement of lactic acid bacteria-fermented plant-based beverages.
Plant-based beverages and milk alternatives produced from cereals and legumes have grown in popularity in recent years due to a range of consumer concerns over dairy products. These plant-based products can often have undesirable physiochemical properties related to flavour, texture, and nutrient availability and/or deficiencies. Lactic acid bacteria (LAB) fermentation offers potential remediation for many of these issues, and allows consumers to retain their perception of the resultant products as natural and additive-free. Using next-generation sequencing (NGS) or omics approaches to characterize LAB isolates to find those that will improve properties of plant-based beverages is the most direct way to product improvement. Although NGS/omics approaches have been extensively used for selection of LAB for use in the dairy industry, a comparable effort has not occurred for selecting LAB for fermenting plant raw substrates, save those used in producing wine and certain types of beer. Here we review the few and recent applications of NGS/omics to profile and improve LAB fermentation of various plant-based substrates for beverage production. We also identify specific issues in the production of various LAB fermented plant-based beverages that such NGS/omics applications have the power to resolve.


Pediatric pacemakers and ICDs: how to optimize perioperative care.
An increasing number of pediatric patients with permanent pacemakers and implantable cardioverter defibrillators (ICDs) require cardiac and noncardiac surgery. It is critical that the anesthesiologist caring for these patients understands the management of the device and the underlying heart disease. Children with these devices are more vulnerable to lead failure and inappropriate shocks compared with the adult population. Preoperative assessment and appropriate reprogramming of the device, in addition to minimizing sources of electromagnetic interference, are keystones in the perioperative care of these patients. Prior consultation with qualified programmers is recommended to enable timely optimization of the device. Magnets may be used in emergency situations but it is important to appreciate the limitations of magnet use on different models of pacemakers and ICDs. Safe and successful perioperative care is dependent upon a well-organized and coordinated multidisciplinary team approach.


Proteasome inhibitor MG132 modulates inflammatory pain by central mechanisms in adjuvant arthritis.
In rheumatoid arthritis (RA), pain and inflammation are initial symptoms followed by various degrees of bone and cartilage destruction. Previously, we have shown that reversible proteasome inhibitor MG132 attenuates pain and joint inflammation in a rat model of adjuvant-arthritis. Our present study aims to study the effects of MG132 on molecular changes in the dorsal root ganglia (DRG) and in the spinal cord (SC) using the same animal model. Arthritis was induced by heat-killed Mycobacterium butyricum in rats. The expression of substance P (SP) was analyzed by quantitative reverse transcription polymerase chain reaction and immunohistochemistry in DRG and in the SC. The nuclear factor-κB (NF-κB) DNA-binding activity in the SC was analyzed by electromobility shift assay. Arthritic rats treated daily with MG132 demonstrated a marked reduction of SP gene expression in the DRG and number of SP-positive cells was reduced. In the spinal cord of arthritic rats elevated SP messenger RNA levels were normalized and NF-κB-DNA-binding activity was down-regulated in arthritic rats treated with MG132. Our results indicate that proteasome inhibitor MG132 attenuates pain in adjuvant arthritis by targeting the sensory neuropeptide substance P in the peripheral and central nervous systems. These effects may be mediated through the inhibition of NF-κB activation.


Genomic Characterization of Dairy Associated Leuconostoc Species and Diversity of Leuconostocs in Undefined Mixed Mesophilic Starter Cultures.
Undefined mesophilic mixed (DL-type) starter cultures are composed of predominantly Lactococcus lactis subspecies and 1-10% Leuconostoc spp. The composition of the Leuconostoc population in the starter culture ultimately affects the characteristics and the quality of the final product. The scientific basis for the taxonomy of dairy relevant leuconostocs can be traced back 50 years, and no documentation on the genomic diversity of leuconostocs in starter cultures exists. We present data on the Leuconostoc population in five DL-type starter cultures commonly used by the dairy industry. The analyses were performed using traditional cultivation methods, and further augmented by next-generation DNA sequencing methods. Bacterial counts for starter cultures cultivated on two different media, MRS and MPCA, revealed large differences in the relative abundance of leuconostocs. Most of the leuconostocs in two of the starter cultures were unable to grow on MRS, emphasizing the limitations of culture-based methods and the importance of careful media selection or use of culture independent methods. Pan-genomic analysis of 59 Leuconostoc genomes enabled differentiation into twelve robust lineages. The genomic analyses show that the dairy-associated leuconostocs are highly adapted to their environment, characterized by the acquisition of genotype traits, such as the ability to metabolize citrate. In particular, Leuconostoc mesenteroides subsp. cremoris display telltale signs of a degenerative evolution, likely resulting from a long period of growth in milk in association with lactococci. Great differences in the metabolic potential between Leuconostoc species and subspecies were revealed. Using targeted amplicon sequencing, the composition of the Leuconostoc population in the five commercial starter cultures was shown to be significantly different. Three of the cultures were dominated by Ln. mesenteroides subspecies cremoris. Leuconostoc pseudomesenteroides dominated in two of the cultures while Leuconostoc lactis, reported to be a major constituent in fermented dairy products, was only present in low amounts in one of the cultures. This is the first in-depth study of Leuconostoc genomics and diversity in dairy starter cultures. The results and the techniques presented may be of great value for the dairy industry.


Synthetic analogs of stryphnusin isolated from the marine sponge Stryphnus fortis inhibit acetylcholinesterase with no effect on muscle function or neuromuscular transmission.
The marine secondary metabolite stryphnusin (1) was isolated from the boreal sponge Stryphnus fortis, collected off the Norwegian coast. Given its resemblance to other natural acetylcholinesterase antagonists, it was evaluated against electric eel acetylcholinesterase and displayed inhibitory activity. A library of twelve synthetic phenethylamine analogs, 2a-7a and 2b-7b, containing tertiary and quaternary amines respectively were synthesized to investigate the individual structural contributions to the activity. Compound 7b was the strongest competitive inhibitor of both acetylcholinesterase and butyrylcholinesterase with IC50 values of 57 and 20 μM, respectively. This inhibitory activity is one order of magnitude higher than the positive control physostigmine, and is comparable with several other marine acetylcholinesterase inhibitors. The physiological effect of compound 7b on muscle function and neuromuscular transmission was studied and revealed a selective mode of action at the investigated concentration. This data is of importance as the interference of therapeutic acetylcholinesterase inhibitors with neuromuscular transmission can be problematic and lead to unwanted side effects. The current findings also provide additional insights into the structure-activity relationship of both natural and synthetic acetylcholinesterase inhibitors.


Tumour genomic and microenvironmental heterogeneity for integrated prediction of 5-year biochemical recurrence of prostate cancer: a retrospective cohort study.
Clinical prognostic groupings for localised prostate cancers are imprecise, with 30-50% of patients recurring after image-guided radiotherapy or radical prostatectomy. We aimed to test combined genomic and microenvironmental indices in prostate cancer to improve risk stratification and complement clinical prognostic factors. We used DNA-based indices alone or in combination with intra-prostatic hypoxia measurements to develop four prognostic indices in 126 low-risk to intermediate-risk patients (Toronto cohort) who will receive image-guided radiotherapy. We validated these indices in two independent cohorts of 154 (Memorial Sloan Kettering Cancer Center cohort [MSKCC] cohort) and 117 (Cambridge cohort) radical prostatectomy specimens from low-risk to high-risk patients. We applied unsupervised and supervised machine learning techniques to the copy-number profiles of 126 pre-image-guided radiotherapy diagnostic biopsies to develop prognostic signatures. Our primary endpoint was the development of a set of prognostic measures capable of stratifying patients for risk of biochemical relapse 5 years after primary treatment. Biochemical relapse was associated with indices of tumour hypoxia, genomic instability, and genomic subtypes based on multivariate analyses. We identified four genomic subtypes for prostate cancer, which had different 5-year biochemical relapse-free survival. Genomic instability is prognostic for relapse in both image-guided radiotherapy (multivariate analysis hazard ratio [HR] 4·5 [95% CI 2·1-9·8]; p=0·00013; area under the receiver operator curve [AUC] 0·70 [95% CI 0·65-0·76]) and radical prostatectomy (4·0 [1·6-9·7]; p=0·0024; AUC 0·57 [0·52-0·61]) patients with prostate cancer, and its effect is magnified by intratumoral hypoxia (3·8 [1·2-12]; p=0·019; AUC 0·67 [0·61-0·73]). A novel 100-loci DNA signature accurately classified treatment outcome in the MSKCC low-risk to intermediate-risk cohort (multivariate analysis HR 6·1 [95% CI 2·0-19]; p=0·0015; AUC 0·74 [95% CI 0·65-0·83]). In the independent MSKCC and Cambridge cohorts, this signature identified low-risk to high-risk patients who were most likely to fail treatment within 18 months (combined cohorts multivariate analysis HR 2·9 [95% CI 1·4-6·0]; p=0·0039; AUC 0·68 [95% CI 0·63-0·73]), and was better at predicting biochemical relapse than 23 previously published RNA signatures. This is the first study of cancer outcome to integrate DNA-based and microenvironment-based failure indices to predict patient outcome. Patients exhibiting these aggressive features after biopsy should be entered into treatment intensification trials. Movember Foundation, Prostate Cancer Canada, Ontario Institute for Cancer Research, Canadian Institute for Health Research, NIHR Cambridge Biomedical Research Centre, The University of Cambridge, Cancer Research UK, Cambridge Cancer Charity, Prostate Cancer UK, Hutchison Whampoa Limited, Terry Fox Research Institute, Princess Margaret Cancer Centre Foundation, PMH-Radiation Medicine Program Academic Enrichment Fund, Motorcycle Ride for Dad (Durham), Canadian Cancer Society.


Systemic inflammation in decompensated cirrhosis: Characterization and role in acute-on-chronic liver failure.
Acute-on-chronic liver failure (ACLF) in cirrhosis is characterized by acute decompensation (AD), organ failure(s), and high short-term mortality. Recently, we have proposed (systemic inflammation [SI] hypothesis) that ACLF is the expression of an acute exacerbation of the SI already present in decompensated cirrhosis. This study was aimed at testing this hypothesis and included 522 patients with decompensated cirrhosis (237 with ACLF) and 40 healthy subjects. SI was assessed by measuring 29 cytokines and the redox state of circulating albumin (HNA2), a marker of systemic oxidative stress. Systemic circulatory dysfunction (SCD) was estimated by plasma renin (PRC) and copeptin (PCC) concentrations. Measurements were performed at enrollment (baseline) in all patients and sequentially during hospitalization in 255. The main findings of this study were: (1) Patients with AD without ACLF showed very high baseline levels of inflammatory cytokines, HNA2, PRC, and PCC. Patients with ACLF showed significantly higher levels of these markers than those without ACLF; (2) different cytokine profiles were identified according to the type of ACLF precipitating event (active alcoholism/acute alcoholic hepatitis, bacterial infection, and others); (3) severity of SI and frequency and severity of ACLF at enrollment were strongly associated. The course of SI and the course of ACLF (improvement, no change, or worsening) during hospitalization and short-term mortality were also strongly associated; and (4) the strength of association of ACLF with SI was higher than with SCD. These data support SI as the primary driver of ACLF in cirrhosis. (Hepatology 2016;64:1249-1264).


High thermal stability of core-shell structures dominated by negative interface energy.
Nanoscale core/shell structures are of interest in catalysis due to their superior catalytic properties. Here we investigated the thermal stability of the coherent core-shell structures in a thermodynamic way by considering the impact from the core with the bulk melting point Tm(∞) lower or higher than the shell. When a low-Tm(∞) core is adopted, core-shell melting induced by the melting depression of the core does not occur upon heating because of the superheating, although the melting depression of the core can be triggered ultimately by the preferential melting of the high-Tm(∞) shell for small cores. The superheating of the core is contributed by the negative solid-solid interface energy, while the depression is originated from the positive solid-liquid interface energy. Owing to the presence of the negative interface energy, moreover, the low-Tm(∞)-core structure possesses a low difference in thermal expansion between the core and the shell, high activation energy of outward atomic diffusion from the core to shell, and low heat capacity. This result is beneficial for the core-shell structure design for its application in catalysis.


Anti-Inflammatory Effects of Four Psilocybin-Containing Magic Mushroom Water Extracts in vitro on 15-Lipoxygenase Activity and on Lipopolysaccharide-Induced Cyclooxygenase-2 and Inflammatory Cytokines in Human U937 Macrophage Cells.
During a pathological inflammation, macrophages are activated to produce accumulation of inflammatory mediators such as induced-cyclooxygenase-2 (COX-2), 15-lipoxygenase (15-LOX) and pro-inflammatory cytokines. Pathological inflammation is a significant problem in many chronic diseases. As a result, more research into natural remedies with anti-inflammatory potential is crucial. Since ancient times, psilocybin-containing mushrooms, also known as magic mushrooms, were used for mind healing and also to advance the quality of life. However, not much is known about their anti-inflammatory potential. This study aimed at investigating the anti-inflammatory effects of four psilocybin-containing mushrooms (Panaeolus cyanescens, Psilocybe natalensis, Psilocybe cubensis and Psilocybe cubensis leucistic A+ strain) from genus Panaeolus and Psilocybe for the first time in vitro on 15-LOX activity and also on lipopolysaccharide (LPS)-induced inflammation in human U937 macrophage cells. Mushrooms were grown and extracted with boiling hot water. Effects of the four water extracts on 15-LOX activity were determined. Confluent human U937 cells were differentiated with phorbol 12-myristate 13-acetate and treated with the hot-water extracts (25 and 50 µg/mL) 2 hours before being stimulated with 1 µg/mL LPS over 24 hours. Quercetin was used as a positive control. Control cells were differentiated but not LPS-induced nor treated. Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10 concentrations were measured. Levels of COX-2 and mitochondrial activity were also determined. The four water extracts had poor 15-LOX inhibition activity with IC50 > 250 µg/mL. Extracts were safe at the concentration studied and inhibited the LPS-induced production of pro-inflammatory mediators, TNF-α and IL-1β significantly and lowered IL-6 and COX-2 concentrations in treated human U937 macrophage cells. Water extracts also increased percentage viability of treated cells and levels of anti-inflammatory IL-10 non-significantly. The study suggested that the hot-water extracts of the four psilocybin-containing magic mushrooms have potential anti-inflammatory effects executed by down-regulating pro-inflammatory mediators.


Barriers to Trace-back in a Salad-associated EHEC Outbreak, Sweden, June 2013.
In June-July 2013, six counties notified the Swedish Institute for Communicable Disease Control of enterohaemorrhagic E.coli (EHEC) infections among attendees at a hotel in Dalarna, Sweden. An outbreak control team investigated to identify the source and implement control measures. We included individuals who attended the hotel between June 19th-25th in a cohort. We asked them about animal contact, swimming, and consumption of food items during this time using a questionnaire. A confirmed case was an EHEC O157:H7 outbreak strain positive individual who developed abdominal pain or diarrhoea between June 20th-July 2nd. We described the outbreak in time, place and person, calculated risk ratios (RR) and 95% confidence intervals (CI). We investigated the kitchen, tested and traced back implicated food items. 172 individuals responded. We identified 19 confirmed cases (Median age: 17 years, 64% female) with symptom onset between June 22nd-27th. Eating green salad on June 20th was associated with illness (RR:3.7;CI:1.3-11). The kitchen mixed green salads without records and destroyed leftovers immediately. Hence we could not conduct trace-back or obtain microbiological confirmation. Green salad contaminated before entering the kitchen was the likely outbreak source. We recommended early collaboration with food agencies and better restaurant records to facilitate future investigations.


Effect of etching time and light source on the bond strength of metallic brackets to ceramic.
This study evaluated the bond strength of brackets to ceramic testing different etching times and light sources for photo-activation of the bonding agent. Cylinders of feldspathic ceramic were etched with 10% hydrofluoric acid for 20 or 60 s. After application of silane on the ceramic surface, metallic brackets were bonded to the cylinders using Transbond XT (3M Unitek). The specimens for each etching time were assigned to 4 groups (n=15), according to the light source: XL2500 halogen light, UltraLume 5 LED, AccuCure 3000 argon laser, and Apollo 95E plasma arc. Light-activation was carried out with total exposure times of 40, 40, 20 and 12 s, respectively. Shear strength testing was carried out after 24 h. The adhesive remnant index (ARI) was evaluated under magnification. Data were subjected to two-way ANOVA and Tukey's test (α=0.05). Specimens etched for 20 s presented significantly lower bond strength (p<0.05) compared with those etched for 60 s. No significant differences (p>0.05) were detected among the light sources. The ARI showed a predominance of scores 0 in all groups, with an increase in scores 1, 2 and 3 for the 60 s time. In conclusion, only the etching time had significant influence on the bond strength of brackets to ceramic.


Real-time characterization of risks of death associated with the Middle East respiratory syndrome (MERS) in the Republic of Korea, 2015.
An outbreak of the Middle East respiratory syndrome (MERS), comprising 185 cases linked to healthcare facilities, occurred in the Republic of Korea from May to July 2015. Owing to the nosocomial nature of the outbreak, it is particularly important to gain a better understanding of the epidemiological determinants characterizing the risk of MERS death in order to predict the heterogeneous risk of death in medical settings. We have devised a novel statistical model that identifies the risk of MERS death during the outbreak in real time. While accounting for the time delay from illness onset to death, risk factors for death were identified using a linear predictor tied to a logit model. We employ this approach to (1) quantify the risks of death and (2) characterize the temporal evolution of the case fatality ratio (CFR) as case ascertainment greatly improved during the course of the outbreak. Senior persons aged 60 years or over were found to be 9.3 times (95% confidence interval (CI), 5.3-16.9) more likely to die compared to younger MERS cases. Patients under treatment were at a 7.8-fold (95% CI, 4.0-16.7) significantly higher risk of death compared to other MERS cases. The CFR among patients aged 60 years or older under treatment was estimated at 48.2% (95% CI, 35.2-61.3) as of July 31, 2015, while the CFR among other cases was estimated to lie below 15%. From June 6, 2015, onwards, the CFR declined 0.3-fold (95% CI, 0.1-1.1) compared to the earlier epidemic period, which may perhaps reflect enhanced case ascertainment following major contact tracing efforts. The risk of MERS death was significantly associated with older age as well as treatment for underlying diseases after explicitly adjusting for the delay between illness onset and death. Because MERS outbreaks are greatly amplified in the healthcare setting, enhanced infection control practices in medical facilities should strive to shield risk groups from MERS exposure.


Integrated Delivery of Antiretroviral Treatment and Pre-exposure Prophylaxis to HIV-1-Serodiscordant Couples: A Prospective Implementation Study in Kenya and Uganda.
Antiretroviral-based interventions for HIV-1 prevention, including antiretroviral therapy (ART) to reduce the infectiousness of HIV-1 infected persons and pre-exposure prophylaxis (PrEP) to reduce the susceptibility of HIV-1 uninfected persons, showed high efficacy for HIV-1 protection in randomized clinical trials. We conducted a prospective implementation study to understand the feasibility and effectiveness of these interventions in delivery settings. Between November 5, 2012, and January 5, 2015, we enrolled and followed 1,013 heterosexual HIV-1-serodiscordant couples in Kenya and Uganda in a prospective implementation study. ART and PrEP were offered through a pragmatic strategy, with ART promoted for all couples and PrEP offered until 6 mo after ART initiation by the HIV-1 infected partner, permitting time to achieve virologic suppression. One thousand thirteen couples were enrolled, 78% of partnerships initiated ART, and 97% used PrEP, during a median follow-up of 0.9 years. Objective measures of adherence to both prevention strategies demonstrated high use (≥85%). Given the low HIV-1 incidence observed in the study, an additional analysis was added to compare observed incidence to incidence estimated under a simulated counterfactual model constructed using data from a prior prospective study of HIV-1-serodiscordant couples. Counterfactual simulations predicted 39.7 HIV-1 infections would be expected in the population at an incidence of 5.2 per 100 person-years (95% CI 3.7-6.9). However, only two incident HIV-1 infections were observed, at an incidence of 0.2 per 100 person-years (95% CI 0.0-0.9, p < 0.0001 versus predicted). The use of a non-concurrent comparison of HIV-1 incidence is a potential limitation of this approach; however, it would not have been ethical to enroll a contemporaneous population not provided access to ART and PrEP. Integrated delivery of time-limited PrEP until sustained ART use in African HIV-1-serodiscordant couples was feasible, demonstrated high uptake and adherence, and resulted in near elimination of HIV-1 transmission, with an observed HIV incidence of <0.5% per year compared to an expected incidence of >5% per year.


Incomplete normalization of dog gracilis muscle grafts with neurovascular repair despite long-term recovery.
Skeletal muscle grafts performed with neurovascular repair are used extensively in clinical situations. However, most controlled experimental studies on the efficacy of such grafts have been conducted on muscles with a relatively small mass and over a limited recovery period. Therefore, selected cellular and matrix component properties of the comparatively large dog gracilis muscle (75 g) were studied 9-12 mo after orthotopic neurovascular grafting. The grafted muscle wet weights were 71% of the contralateral control (sham-operated) muscles. In addition, the concentrations of noncollagenous protein (13%), DNA (28%), and RNA (34%) were significantly reduced in the grafts. However, the concentration of collagen was significantly higher (41%) in the grafts. In this regard, the type III collagen phenotype showed the greatest relative increase. There was no difference between the grafted and control proteoglycan concentration. The metabolic profiles of the grafted muscles were significantly different from control. The activities of myofibrillar adenosinetriphosphatase (34%) and alpha-glycerophosphate dehydrogenase (25%) were reduced, whereas citrate synthase remained unchanged. These data suggest that recovery of up to 1 yr was insufficient for the normalization of several connective tissue matrix components and biochemical properties of the grafts.


Possible roles of exceptionally conserved residues around the selectivity filters of sodium and calcium channels.
In the absence of x-ray structures of sodium and calcium channels their homology models are used to rationalize experimental data and design new experiments. A challenge is to model the outer-pore region that folds differently from potassium channels. Here we report a new model of the outer-pore region of the NaV1.4 channel, which suggests roles of highly conserved residues around the selectivity filter. The model takes from our previous study (Tikhonov, D. B., and Zhorov, B. S. (2005) Biophys. J. 88, 184-197) the general disposition of the P-helices, selectivity filter residues, and the outer carboxylates, but proposes new intra- and inter-domain contacts that support structural stability of the outer pore. Glycine residues downstream from the selectivity filter are proposed to participate in knob-into-hole contacts with the P-helices and S6s. These contacts explain the adapted tetrodotoxin resistance of snakes that feed on toxic prey through valine substitution of isoleucine in the P-helix of repeat IV. Polar residues five positions upstream from the selectivity filter residues form H-bonds with the ascending-limb backbones. Exceptionally conserved tryptophans are engaged in inter-repeat H-bonds to form a ring whose π-electrons would facilitate passage of ions from the outer carboxylates to the selectivity filter. The outer-pore model of CaV1.2 derived from the NaV1.4 model is also stabilized by the ring of exceptionally conservative tryptophans and H-bonds between the P-helices and ascending limbs. In this model, the exceptionally conserved aspartate downstream from the selectivity-filter glutamate in repeat II facilitates passage of calcium ions to the selectivity-filter ring through the tryptophan ring. Available experimental data are discussed in view of the models.


Current diagnostic and therapeutic approaches for colorectal cancer liver metastasis.
Colorectal cancer is the second leading cause of cancer death in the developed world, due to formation of distant metastases. The liver is a primary target organ of metastatic lesions, which substantially influence the morbidity of the disease. Prompt diagnosis of colorectal liver metastases leads to early treatment, which favours a better prognosis. Consequently, the diagnostic process has shifted from traditional clinical and biochemical procedures to technologically advanced imaging modalities, such as CT, MRI, FDG-PET and PET-CT. However, the only current curative therapeutic approach is the surgical resection of metastases, using the new methods of tissue excision and haemostasis. New therapeutic modalities like cryo- or radiofrequency ablation and portal vein embolisation as well as pharmaceutical innovations such as hepatic arterial infusion chemotherapy, isolated hepatic perfusion and contemporary chemotherapeutic regimens have emerged. While still under evaluation, they present promise for the future treatment of unresectable liver metastases.


Neuronal excitability and calcium/calmodulin-dependent protein kinase type II: location, location, location.
Calcium/calmodulin-dependent protein kinase type II (CaMKII) is a highly abundant serine/threonine kinase comprising a significant fraction of total protein in mammalian forebrain and forming a major component of the postsynaptic density. CaMKII is essential for certain forms of synaptic plasticity and memory consolidation and this is mediated through substrate binding and intramolecular phosphorylation of holoenzyme subunits. CaMKII is multifunctional; it targets a variety of cellular substrates, and this diversity depends on holoenzyme subunit composition. CaMKII comprises homooligomeric and heterooligomeric complexes generated from four subunits (α, β, δ, and γ) encoded by separate genes that are further expanded by extensive alternative splicing to more than 30 different isoforms. Much attention has been paid to understanding the regulation of CaMKII function through its structural diversity and/or substrate specificity. However, given the importance of subunit composition to holoenzyme activity, it is likely that specificity of cellular expression of CaMKII isoforms also plays a major role in regulation of enzyme function. Herein we review the cellular colocalization of CaMKII isoforms with special regard to the cell-type specificity of isoform expression in brain. In addition, we highlight the remarkable specificity of subcellular localization by the CaMKIIα isoform. In addition, we discuss the role that this cellular specificity of expression might play in propagating the type of recurrent neuronal activity associated with disorders such as temporal lobe epilepsy.


Assessment of the trends in medical use and misuse of opioid analgesics from 2004 to 2011.
The epidemic of medical use and abuse of opioid analgesics is linked to the economic burden of opioid-related abuse and fatalities in the United States. Multiple studies have estimated the extent to which prescription opioid analgesics contribute to the national drug abuse problem; studies also assessing the trends in medical use and abuse of opioid analgesics have confirmed the relationship between increasing medical use of opioids and increasing fatalities.The available data is limited until 2002. Retrospective analysis of data from 2004 to 2011 from 2 databases: Automation of Reports and Consolidated Orders System (ARCOS) for opioid use data and Drug Abuse Warning Network (DAWN) for drug misuse data. To determine the proportion of drug abuse related to opioid analgesics and the various trends in the medical use and abuse of 8 opioid analgesics commonly used to treat pain: buprenorphine, codeine, fentanyl, hydrocodone, hydromorphone, methadone, morphine, and oxycodone. The data obtained from DAWN is a nationally representative sample of hospital emergency department admissions resulting from drug abuse. Main outcome measure was the identification of trends in the medical use and misuse of opioid analgesics from 2004 to 2011. From 2004 to 2011, there was an increase in the medical use of all opioids except for a 20% decrease in codeine. The abuse of all opioids including codeine increased during this period. Increases in medical use ranged from 2,318% for buprenorphine to 35% for fentanyl, including 140% for hydromorphone, 117% for oxycodone, 73% for hydrocodone, 64% for morphine, and 37% for methadone. The misuse increased 384% for buprenorphine with available data from 2006 to 2011, whereas from 2004 to 2011, it increased 438% for hydromorphone, 263% for oxycodone, 146% for morphine, 107% for hydrocodone, 104% for fentanyl, 82% for methadone, and 39% for codeine. Comparison of opioid use showed an overall increase of 1,448% from 1996 to 2011, with increases of 690% from 1996 to 2004 and 100% from 2004 to 2011. In contrast, misuse increased more dramatically: 4,680% from 1996 to 2011, with increases of 1,372% from 1996 through 2004 and 245% from 2004 to 2011. The number of patients seeking rehabilitation for substance abuse also increased 187% for opioids, whereas it increased 87% for heroin, 40% for marijuana, and decreased 7% for cocaine. Limitations of this assessment include the lack of data from 2003, lack of data available on meperidine, and that the aggregate data systems used in the study did not identify specific formulations or commercial products. The present trend of continued increase in the medical use of opioid analgesics appears to contribute to increases in misuse, resulting in multiple health consequences.


Posterior expression of a homeobox gene in early Xenopus embryos.
The homeobox containing transcript Xhox-36 is expressed exclusively in the posterior mesoderm and ectoderm of early Xenopus embryos. Therefore, the transcript shows region-specific rather than tissue-specific expression in the gastrula and neurula, a time when cells are becoming committed to defined fates. Exposure of early embryos to LiCl, which shifts posterior cells to more anterior fates, reduces the abundance of this posterior-specific transcript. In contrast, embryos ventralized by u.v. treatment express normal levels of the transcript, implying that expression of the gene is not absolutely linked to dorsal cell identity. The sequence of a full-length cDNA corresponding to this transcript predicts a homeodomain-containing protein of 209 amino acids.


Prognostic factors in inoperable adenocarcinoma of the lung: a multivariate regression analysis of 259 patients.
The prognostic factors for survival in advanced adenocarcinoma of the lung were investigated in a consecutive series of 259 patients treated with chemotherapy. Twenty-eight pretreatment variables were investigated by use of Cox's multivariate regression model, including histological subtypes and degree of differentiation, the new international staging system for lung cancer, and seven laboratory parameters. Staging of the patients included bone marrow examination but were otherwise nonextensive without routine bone, liver, and brain scans. Factors predicting poor survival were low performance status, stage IV disease, no prior nonradical resection, liver metastases, high values of white blood cell count, and lactate dehydrogenase, and low values of asparatate aminotransaminase. The nonradical resection may not be a prognostic factor because of the resection itself but may rather serve as an indicator for patients having minimal disease spread. Liver metastases were of limited clinical value as a prognostic factor because they were detected in only seven cases in this patient population. A new Cox analysis ignoring the influence of this variable revealed no other variables than those occurring in the former Cox model to be of importance (performance status, stage, surgical resection, WBC, aspartate aminotransaminase, and lactate dehydrogenase). This simplified model appears to be a feasible clinical tool, allowing for prognostic stratification of patients when first the inoperability of the patient is known.


Instrumental variable estimation in a survival context.
Bias due to unobserved confounding can seldom be ruled out with certainty when estimating the causal effect of a nonrandomized treatment. The instrumental variable (IV) design offers, under certain assumptions, the opportunity to tame confounding bias, without directly observing all confounders. The IV approach is very well developed in the context of linear regression and also for certain generalized linear models with a nonlinear link function. However, IV methods are not as well developed for regression analysis with a censored survival outcome. In this article, we develop the IV approach for regression analysis in a survival context, primarily under an additive hazards model, for which we describe 2 simple methods for estimating causal effects. The first method is a straightforward 2-stage regression approach analogous to 2-stage least squares commonly used for IV analysis in linear regression. In this approach, the fitted value from a first-stage regression of the exposure on the IV is entered in place of the exposure in the second-stage hazard model to recover a valid estimate of the treatment effect of interest. The second method is a so-called control function approach, which entails adding to the additive hazards outcome model, the residual from a first-stage regression of the exposure on the IV. Formal conditions are given justifying each strategy, and the methods are illustrated in a novel application to a Mendelian randomization study to evaluate the effect of diabetes on mortality using data from the Health and Retirement Study. We also establish that analogous strategies can also be used under a proportional hazards model specification, provided the outcome is rare over the entire follow-up.


Comparative genomic identification and validation of β-defensin genes in the Ovis aries genome.
β-defensins are small, cationic, antimicrobial peptides found in species across the plant and animal kingdoms. In addition to microbiocidal activity, roles in immunity as well as reproduction have more recently been documented. β-defensin genes in Ovis aries (domestic sheep) have been poorly annotated, having been identified only by automatic gene prediction algorithms. The objective of this study was to use a comparative genomics approach to identify and characterise the β-defensin gene repertoire in sheep using the bovine genome as the primary reference. All 57 currently predicted bovine β-defensin genes were used to find orthologous sequences in the most recent version of the sheep genome (OAR v4.0). Forty three genes were found to have close genomic matches (>70% similarity) between sheep and cattle. The orthologous genes were located in four clusters across the genome, with 4 genes on chromosome 2, 19 genes on chromosome 13, 5 genes on chromosome 20 and 15 genes on chromosome 26. Conserved gene order for the β-defensin genes was apparent in the two smaller clusters, although gene order was reversed on chromosome 2, suggesting an inversion between sheep and cattle. Complete conservation of gene order was also observed for chromosome 13 β-defensin orthologs. More structural differences were apparent between chromosome 26 genes and the orthologous region in the bovine reference genome, which is known to be copy-number variable. In this cluster, the Defensin-beta 1 (DEFB1) gene matched to eleven Bovine Neutrophil beta-Defensin (BNBD) genes on chromosome 27 with almost uniform similarity, as well as to tracheal, enteric and lingual anti-microbial peptides (TAP, EAP and LAP), suggesting that annotation of the bovine reference sequence is still incomplete. qPCR was used to profile the expression of 34 β-defensin genes, representing each of the four clusters, in the ram reproductive tract. Distinct site-specific and differential expression profiles were detected across the reproductive tract of mature rams with preferential β-defensin gene expression in the epididymis, recapitulating observations for orthologous genes in other species. This is the first comprehensive analysis of β-defensin genes encoded by the ovine reference sequence, and the first report of an expanded repertoire of β-defensin genes in this species. The preferential expression of these genes in the epididymis suggests a role in fertility, possibly providing immunoprotection for sperm within the female reproductive tract.


Functional imaging of legumain in cancer using a new quenched activity-based probe.
Legumain is a lysosomal cysteine protease whose biological function remains poorly defined. Legumain activity is up-regulated in most human cancers and inflammatory diseases most likely as the result of high expression in populations of activated macrophages. Within the tumor microenvironment, legumain activity is thought to promote tumorigenesis. To obtain a greater understanding of the role of legumain activity during cancer progression and inflammation, we developed an activity-based probe that becomes fluorescent only upon binding active legumain. This probe is highly selective for legumain, even in the context of whole cells and tissues, and is also a more effective label of legumain than previously reported probes. Here we present the synthesis and application of our probe to the analysis of legumain activity in primary macrophages and in two mouse models of cancer. We find that legumain activity is highly correlated with macrophage activation and furthermore that it is an ideal marker for primary tumor inflammation and early stage metastatic lesions.


Dissection of exopolysaccharide biosynthesis in Kozakia baliensis.
Acetic acid bacteria (AAB) are well known producers of commercially used exopolysaccharides, such as cellulose and levan. Kozakia (K.) baliensis is a relatively new member of AAB, which produces ultra-high molecular weight levan from sucrose. Throughout cultivation of two K. baliensis strains (DSM 14400, NBRC 16680) on sucrose-deficient media, we found that both strains still produce high amounts of mucous, water-soluble substances from mannitol and glycerol as (main) carbon sources. This indicated that both Kozakia strains additionally produce new classes of so far not characterized EPS. By whole genome sequencing of both strains, circularized genomes could be established and typical EPS forming clusters were identified. As expected, complete ORFs coding for levansucrases could be detected in both Kozakia strains. In K. baliensis DSM 14400 plasmid encoded cellulose synthase genes and fragments of truncated levansucrase operons could be assigned in contrast to K. baliensis NBRC 16680. Additionally, both K. baliensis strains harbor identical gum-like clusters, which are related to the well characterized gum cluster coding for xanthan synthesis in Xanthomanas campestris and show highest similarity with gum-like heteropolysaccharide (HePS) clusters from other acetic acid bacteria such as Gluconacetobacter diazotrophicus and Komagataeibacter xylinus. A mutant strain of K. baliensis NBRC 16680 lacking EPS production on sucrose-deficient media exhibited a transposon insertion in front of the gumD gene of its gum-like cluster in contrast to the wildtype strain, which indicated the essential role of gumD and of the associated gum genes for production of these new EPS. The EPS secreted by K. baliensis are composed of glucose, galactose and mannose, respectively, which is in agreement with the predicted sugar monomer composition derived from in silico genome analysis of the respective gum-like clusters. By comparative sugar monomer and genome analysis, the polymeric substances secreted by K. baliensis can be considered as unique HePS. Via genome sequencing of K. baliensis DSM 14400 + NBRC 16680 we got first insights into the biosynthesis of these novel HePS, which is related to xanthan and acetan biosynthesis. Consequently, the present study provides the basis for establishment of K. baliensis strains as novel microbial cell factories for biotechnologically relevant, unique polysaccharides.


Rab25 regulates integrin expression in polarized colonic epithelial cells.
Rab25 is a tumor suppressor for colon cancer in humans and mice. To identify elements of intestinal polarity regulated by Rab25, we developed Caco2-BBE cell lines stably expressing short hairpin RNA for Rab25 and lines rescuing Rab25 knockdown with reexpression of rabbit Rab25. Rab25 knockdown decreased α2-, α5-, and β1-integrin expression. We observed colocalization and direct association of Rab25 with α5β1-integrins. Rab25 knockdown also up-regulated claudin-1 expression, increased transepithelial resistance, and increased invasive behavior. Rab25-knockdown cells showed disorganized brush border microvilli with decreases in villin expression. All of these changes were reversed by reintroduction of rabbit Rab25. Rab25 knockdown altered the expression of 29 gene transcripts, including the loss of α5-integrin transcripts. Rab25 loss decreased expression of one transcription factor, ETV4, and overexpression of ETV4 in Rab25-knockdown cells reversed losses of α5β1-integrin. The results suggest that Rab25 controls intestinal cell polarity through the regulation of gene expression.


Evaluation of a protocol for metabolic profiling studies on human blood plasma by combined ultra-performance liquid chromatography/mass spectrometry: From extraction to data analysis.
The investigation presented here describes a protocol designed to perform high-throughput metabolic profiling analysis on human blood plasma by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS). To address whether a previous extraction protocol for gas chromatography (GC)/MS-based metabolic profiling of plasma could be used for UPLC/MS-based analysis, the original protocol was compared with similar methods for extraction of low-molecular-weight compounds from plasma via protein precipitation. Differences between extraction methods could be observed, but the previously published extraction method was considered the best. UPLC columns with three different stationary phases (C8, C18, and phenyl) were used in identical experimental runs consisting of a total of 60 injections of extracted male and female plasma samples. The C8 column was determined to be the best for metabolic profiling analysis on plasma. The acquired UPLC/MS data of extracted male and female plasma samples was subjected to principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA). Furthermore, a strategy for compound identification was applied here, demonstrating the strength of high-mass-accuracy time-of-flight (TOF)/MS analysis in metabolic profiling.


Characterization of the cytochrome P-450 gene family responsible for the N-dealkylation of the ergot alkaloid CQA 206-291 in humans.
The ergot alkaloid CQA 206-291 (CQA) was converted by human liver microsomes (n = 16) almost exclusively to the N-deethylated metabolite (I), as identified by the on-line coupling of liquid chromatography and mass spectroscopy. Metabolite I formation exhibited monophasic and linear enzyme kinetics (2.9-300 microM), and a 5.6-fold interindividual variability (7.2-40.2 nmol/mg/hr). Chemical inhibition experiments revealed that imidazole antimycotic agents (ketoconazole, miconazole, and clotrimazole) were potent inhibitors of this N-deethylation. Polymorphically metabolized substrates (sparteine and phenytoin), well-established cytochrome P-450 probe substrates (antipyrine and tolbutamide), and steroid hormones (estradiol and testosterone) were noninhibitory, indicating that their metabolism is catalyzed by forms of cytochrome P-450 that do not catalyze this route of CQA biotransformation. The ergot alkaloids--dihydroergotamine, bromocriptine, and SDZ 208-911--were competitive inhibitors of metabolite I formation, suggesting that these compounds are metabolized by similar enzymes. Cyclosporine A was a potent competitive inhibitor of CQA metabolism, providing initial evidence that formation of metabolite I was catalyzed by proteins of the CYP3 gene family. This was substantiated by the finding that CQA metabolism was completely inhibited by a polyclonal antibody directed against a pregnenolone 16 alpha-carbonitrile-inducible cytochrome P-450 of rat liver. The rate of CQA metabolism correlated significantly to the level of CYP3A4 expression, the rate of cyclosporine A metabolism to each of the primary metabolites (M-1, M-17, and M-21), and the rate of midazolam 4-hydroxylation. COS 1 cells transfected with human CYP3A4 and CYP3A5 provided direct evidence that these enzymes catalyze the metabolism of CQA.(ABSTRACT TRUNCATED AT 250 WORDS)


Materials used for indirect pulp treatment in primary teeth: a mixed treatment comparisons meta-analysis.
This study aimed to systematically review the literature to address the question regarding the influence of different materials in the clinical and radiographic success of indirect pulp treatment in primary teeth. A literature search was carried out for articles published prior to January 2017 in PubMed/MEDLINE, CENTRAL, Scopus, TRIP and ClinicalTrials databases; relevant articles included randomized clinical trials that compared materials used for indirect pulp treatment in primary teeth. Two reviewers independently selected the studies and extracted the data. The effects of each material on the outcome (clinical and radiographic failures) were analyzed using a mixed treatment comparisons meta-analysis. The ranking of treatments according to their probability of being the best choice was also calculated. From 1,088 potentially eligible studies, 11 were selected for full-text analysis, and 4 were included in the meta-analysis. In all papers, calcium hydroxide liner was used as the control group versus an adhesive system, resin-modified glass ionomer cement or placebo. The follow-up period ranged from 24 to 48 months, with dropout rates of 0-25.7%. The material type did not significantly affect the risk of failure of the indirect pulp treatment. However, calcium hydroxide presented a higher probability of failure. In conclusion, there is no scientific evidence showing the superiority of any material used for indirect pulp treatment in primary teeth.


High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome.
African trypanosomes are major pathogens of humans and livestock and represent a model for studies of unusual protozoal biology. We describe a high-throughput phenotyping approach termed RNA interference (RNAi) target sequencing, or RIT-seq that, using Illumina sequencing, maps fitness-costs associated with RNAi. We scored the abundance of >90,000 integrated RNAi targets recovered from trypanosome libraries before and after induction of RNAi. Data are presented for 7435 protein coding sequences, >99% of a non-redundant set in the Trypanosoma brucei genome. Analysis of bloodstream and insect life-cycle stages and differentiated libraries revealed genome-scale knockdown profiles of growth and development, linking thousands of previously uncharacterized and "hypothetical" genes to essential functions. Genes underlying prominent features of trypanosome biology are highlighted, including the constitutive emphasis on post-transcriptional gene expression control, the importance of flagellar motility and glycolysis in the bloodstream, and of carboxylic acid metabolism and phosphorylation during differentiation from the bloodstream to the insect stage. The current data set also provides much needed genetic validation to identify new drug targets. RIT-seq represents a versatile new tool for genome-scale functional analyses and for the exploitation of genome sequence data.


Relationships between serum adiponectin and bone density, adiposity and calcified atherosclerotic plaque in the African American-Diabetes Heart Study.
Adiposity, bone mineral density (BMD), and calcified atherosclerotic plaque (CP) exhibit complex interrelationships that are not well understood. Adipokines vary in relation to changes in body composition and may play roles in regulation of BMD and risk of cardiovascular disease. Our objective was to examine the relationship between serum adiponectin and quantitative computed tomography-derived measures of volumetric BMD (vBMD) in thoracic and lumbar vertebrae, adipose tissue volumes, and CP in coronary, carotid, and infrarenal aortoiliac arteries. Generalized linear models were fitted to test for associations between adiponectin and measured phenotypes. A total of 479 unrelated African Americans with type 2 diabetes, 57% female with a mean ± SD (median) age of 55.6 ± 9.5 (55.0) years and diabetes duration of 10.3 ± 8.2 (8.0) years. Serum adiponectin was 8.26 ± 7.41 (6.10) μg/mL, coronary artery CP mass score was 280 ± 634 (14), carotid artery CP was 47 ± 133 (0), and aortoiliac CP was 1616 ± 2864 (319). Women had significantly higher body mass index and serum adiponectin and lower coronary and carotid artery calcium than males (all P < .05). Before and after adjusting for age, sex, body mass index, mean arterial pressure, smoking status, hemoglobin A1c, thiazolidinedione use, and low-density lipoprotein-cholesterol, adiponectin was inversely associated with thoracic and lumbar vertebral vBMD [parameter estimates (PEs) of -0.06 and -0.021, respectively; both P < .0005], visceral adipose tissue (PE -0.02; P < 0.0001), and C-reactive protein (PE -0.07; P < .0001) and positively associated with intermuscular adipose tissue (PE 0.01; P = .03). After covariate adjustment, significant associations were not observed between adiponectin and CP in any vascular bed (P > .1). Serum adiponectin levels were inversely associated with cross-sectional measures of thoracic and lumbar vertebral vBMD, inflammation, and visceral adiposity in African Americans but not with vascular CP after adjustment for covariates. The data support a regulatory/signaling role for adiponectin in the modulation of bone density.


Neurotoxicity, general anesthesia in young children, and a survey of current pediatric anesthesia practice at US teaching institutions.
Recent articles in both scholarly journals and the lay press about the topic of anesthetic related neurotoxicity have increased the awareness and discussion of this topic with parents and other pediatric medical specialties (i.e., surgeons, radiologists, and pediatricians). The purpose of the present study was to survey how a subset of pediatric anesthesia departments in the US have responded to the issue of anesthetic related neurotoxicity in terms of clinical practice, training and communication with other medical specialties, and the frequency and timing of discussions with families. A survey consisting of 22 questions was sent to PALC (Pediatric Anesthesia Leadership Council) & PAPDA (Pediatric Anesthesia Program Directors Association) via SurveyMonkey. The survey was divided into sections on Anesthesia Faculty/Trainees, Parents and Non-Anesthesia Providers. Responses to the survey were solicited via email to PALC and PAPDA, and then followed up with reminders to individual emails using the mailing lists of both organizations. The results of this survey demonstrate that pediatric anesthesia programs around the US do not have a consistent approach in managing the topic of anesthesia-related neurotoxicity with pediatric anesthesiologists, anesthesiology residents, pediatric anesthesiology fellows and their non-anesthesia medical and surgical colleagues, as well as the discussion of this topic with parents. A significant need exists to provide information to other pediatric professionals and parents. A consistent message from all providers that includes what is known, and indeed more importantly what is not known may be a useful approach.


Design of a specific colonic mucus marker using a human commensal bacterium cell surface domain.
Imaging living cells and organs requires innovative, specific, efficient, and well tolerated fluorescent markers targeting cellular components. Such tools will allow proceeding to the dynamic analysis of cells and the adaptation of tissues to environmental cues. In this study, we have identified and synthesized a novel non-toxic fluorescent marker allowing a specific fluorescent staining of the human colonic mucus. Our strategy to identify a molecule able to specifically bind to the human colonic mucus was on the basis of the mucus adhesion properties of commensal bacteria. We identified and characterized the mucus-binding property of a 70-amino acid domain (MUB(70)) expressed on the surface of Lactobacillus strains. The chemical synthesis of MUB(70) was achieved using the human commensal bacterium Lactobacillus reuteri AF120104 protein as a template. The synthesized Cy5-conjugated MUB(70) marker specifically stained the colonic mucus on fixed human, rabbit, and guinea pig tissues. Interestingly, murine tissue was not stained, suggesting significant differences in the composition of the murine colonic mucus. In addition, this marker stained the mucus of living cultured human colonic cells (HT29-MTX) and human colonic tissue explants. Using a biotinylated derivative of MUB(70), we demonstrated that this peptide binds specifically to Muc2, the most abundant secreted mucin, through its glycosylated moieties. Hence, Cy5-MUB(70) is a novel and specific fluorescent marker for mammalian colonic mucus. It may be used for live imaging analysis but also, as demonstrated in this study, as a marker for the diagnosis and the prognosis of colonic mucinous carcinomas.


Phase I study of neoadjuvant chemoradiotherapy with S-1 plus biweekly cisplatin for advanced gastric cancer patients with lymph node metastasis: -KOGC04-.
In patients with highly advanced gastric cancer, the recurrence rate remains high and the prognosis disappointing. We previously reported a phase I study of a neoadjuvant chemoradiotherapy of S-1 plus weekly cisplatin. Although adequate safety and efficacy were reported, myelosuppression was frequently observed, leading to treatment delay in several cases. To decrease toxicity and improve efficacy, we planned a phase I study with a modified chemotherapy regimen with biweekly cisplatin. Patients with advanced gastric cancer and lymph node metastasis who were treated by our institution between 2011 and 2012 were eligible for inclusion. The initial chemoradiotherapy schedule consisted of 6 weeks of S-1 orally administered on days 1-15 with an escalating dose of cisplatin administered on days 1 and 15. The starting dose (level 1) of cisplatin was 15 mg/m(2), the second dose (level 2) was 20 mg/m(2), and the third dose (level 3) was 25 mg/m(2). Radiation of 40 Gy was administered in 20 fractions. After initial chemoradiotherapy, one cycle of combination chemotherapy with S-1 plus cisplatin was delivered. The second cycle was 42 days in duration and included S-1 administered on days 1-29 plus biweekly cisplatin administered on days 1, 15, and 29. After neoadjuvant treatment, a curative gastrectomy with extended (D2) lymph node dissection was planned. Nine patients were enrolled. At level 3, one patient had dose-limiting grade 3 diarrhea. Another patient experienced grade 3 nausea and intended to discontinue the treatment. Overall, because 2 of 3 patients experienced dose-limiting toxicity at level 3, we confirmed level 3 (Cisplatin 25 mg/m(2)) as the maximum tolerated dose and level 2 (Cisplatin 20 mg/m(2)) as the recommended dose (RD). The response rate was 78%, and 8 patients underwent curative gastrectomy. Resected specimens showed a histological response in 6 patients (75%), including one with a pathological complete response. In this phase I trial, RD of cisplatin was identified as 20 mg/m(2). Generally, S-1 plus biweekly cisplatin can be given safely with concurrent radiation. We have initiated a multicenter phase II trial to further confirm the efficacy and safety of this approach. UMIN000008941.


Frequency effects in compound production.
Four experiments investigated the role of frequency information in compound production by independently varying the frequencies of the first and second constituent as well as the frequency of the compound itself. Pairs of Dutch noun-noun compounds were selected such that there was a maximal contrast for one frequency while matching the other two frequencies. In a position-response association task, participants first learned to associate a compound with a visually marked position on a computer screen. In the test phase, participants had to produce the associated compound in response to the appearance of the position mark, and we measured speech onset latencies. The compound production latencies varied significantly according to factorial contrasts in the frequencies of both constituting morphemes but not according to a factorial contrast in compound frequency, providing further evidence for decompositional models of speech production. In a stepwise regression analysis of the joint data of Experiments 1-4, however, compound frequency was a significant nonlinear predictor, with facilitation in the low-frequency range and a trend toward inhibition in the high-frequency range. Furthermore, a combination of structural measures of constituent frequencies and entropies explained significantly more variance than a strict decompositional model, including cumulative root frequency as the only measure of constituent frequency, suggesting a role for paradigmatic relations in the mental lexicon.


Use of Nanotrap particles for the capture and enrichment of Zika, chikungunya and dengue viruses in urine.
Nanotrap® (NT) particles are hydrogel microspheres developed for target analyte separation and discovery applications. NT particles consist of cross-linked N-isopropylacrylamide (NIPAm) copolymers that are functionalized with a variety of chemical affinity baits to enable broad-spectrum collection and retention of target proteins, nucleic acids, and pathogens. NT particles have been previously shown to capture and enrich arboviruses including Rift Valley fever and Venezuelan equine encephalitis viruses. Yet, there is still a need to enhance the detection ability for other re-emerging viruses such as Zika (ZIKV), chikungunya (CHIKV), and dengue (DENV) viruses. In this study, we exploited NT particles with different affinity baits, including cibacron blue, acrylic acid, and reactive red 120, to evaluate their capturing and enrichment capability for ZIKV, DENV and CHIKV in human fluids. Our results demonstrate that CN1030, a NT particle conjugated with reactive red 120, can recover between 8-16-fold greater genomic copies of ZIKV, CHIKV and DENV in virus spiked urine samples via RT-qPCR, superior to the other chemical baits. Also, we observed that CN1030 simultaneously enriched ZIKV, CHIKV and DENV in co-infection-based settings and could stabilize ZIKV, but not CHIKV infectivity in saliva spiked samples. CN1030 enriched viral detection at various viral concentrations, with significant enhancement observed at viral titers as low as 100 PFU/mL for ZIKV and 10 PFU/mL for CHIKV. The detection of ZIKV was further enhanced with NT particles by processing of larger volume urine samples. Furthermore, we developed a magnetic NT particle, CN3080, based on the same backbone of CN1030, and demonstrated that CN3080 could also capture and enrich ZIKV and CHIKV in a dose-dependent manner. Finally, in silico docking predictions support that the affinity between reactive red 120 and ZIKV or CHIKV envelope proteins appeared to be greater than acrylic acid. Overall, our data show that NT particles along with reactive red 120 can be utilized as a pre-processing technology for enhancement of detecting febrile-illness causing viruses.


Effect of transforming growth factor-β3 on mono and multilayer chondrocytes.
Articular cartilage is an avascular and flexible connective tissue found in joints. It produces a cushioning effect at the joints and provides low friction to protect the ends of the bones from wear and tear/damage. It has poor repair capacity and any injury can result pain and loss of mobility. Transforming growth factor-beta (TGF-β), a cytokine superfamily, regulates cell function, including differentiation and proliferation. Although the function of the TGF-βs in various cell types has been investigated, their function in cartilage repair is as yet not fully understood. The effect of TGF-β3 in biological regulation of primary chondrocyte was investigated in this work. TGF-β3 provided fibroblastic morphology to chondrocytes and therefore overall reduction in cell proliferation was observed. The length of the cells supplemented with TGF-β3 were larger than the cells without TGF-β3 treatment. This was caused by the fibroblast like cells (dedifferentiated chondrocytes) which occupied larger areas compared to cells without TGF-β3 addition. The healing process of the model wound closure assay of chondrocyte multilayer was slowed down by TGF-β3, and this cytokine negatively affected the strength of chondrocyte adhesion to the cell culture surface.


Escherichia coli infections in newborn puppies--clinical and epidemiological investigations.
Epidemiological analyses were performed in five breeding kennels with Escherichia coli infections in newborn pups using pulsed field gel electrophoresis (PFGE). Previous reports demonstrated the high discriminatory power of this method and its usefulness for detecting epidemiologically related isolates. A total of 113 E. coli strains were isolated from vagina, faeces, oral cavity, milk and organs from 19 adult dogs, and 57 diseased or dead pups from 12 litters. Restriction enzyme analyses were performed using XbaI and BlnI digests and the resulting 91 DNA patterns were aligned for comparison. The results showed that a total of 60% of E. coli strains from progeny were also found in vaginal samples of the mothers. Another bacterial source was the faeces found within the kennels. One instance of milk and oral cavity isolates of the mother was found to be identical with strains isolated from the pups. The results indicate that for repeated cases of E. coli infections in neonates, diagnostic procedures of vaginal and faecal swabs from dams result in isolation of the responsible bacteria with high probability and further suggest that preterm treatment could help to control bacterial diseases and losses in pups. In addition, the observation that two canine strains were found to be identical with an E. coli strain isolated from a human case of diarrhoea strongly supports the canine reservoir hypothesis.


Estimation of direct costs of melanoma in the Veneto Region: a budget assessment and cost-consequence analysis.
While many evidence-based pathways have been introduced to drive quality improvements in cancer care, most of these do not include evidence about their affordability. The main aim of this study was to provide an estimation of the overall budget to cover all the needs of melanoma patients in Veneto Region, managed according to the clinical pathway defined by the Rete Oncologica Veneta. A second objective is to conduct a cost-consequence analysis, comparing two different treatments. A very detailed whole-disease model was developed describing the patient's pathway from diagnosis through the first year of follow-up. Each procedure involved in the model was associated with a likelihood measure and a cost. The model can be used to estimate the expected direct costs associated with melanoma. We can observe that 0 and I stage, despite accounting for a huge percentage of new melanoma cases are characterized by a small percentage of the total costs. Stage III can be considered as the most expensive stage accounting for 54% of the total costs with a 12% of patients. Finally, the stage IV patients, although very few accounts for almost the 7% of the total costs. Regarding the cost-consequence analysis, it was estimated that the therapies introduced in 2016 led to an approximately 14% increase in the total costs. Modeling a clinical pathway with a high level of detail enables to identify the main sources of spending. The consequent analysis can thus help policymakers to plan the future resources allocation.


Effects of Changes in Arterial Carbon Dioxide and Oxygen Partial Pressures on Cerebral Oximeter Performance.
Cerebral oximetry (cerebral oxygen saturation; ScO2) is used to noninvasively monitor cerebral oxygenation. ScO2 readings are based on the fraction of reduced and oxidized hemoglobin as an indirect estimate of brain tissue oxygenation and assume a static ratio of arterial to venous intracranial blood. Conditions that alter cerebral blood flow, such as acute changes in PaCO2, may decrease accuracy. We assessed the performance of two commercial cerebral oximeters across a range of oxygen concentrations during normocapnia and hypocapnia. Casmed FORE-SIGHT Elite (CAS Medical Systems, Inc., USA) and Covidien INVOS 5100C (Covidien, USA) oximeter sensors were placed on 12 healthy volunteers. The fractional inspired oxygen tension was varied to achieve seven steady-state levels including hypoxic and hyperoxic PaO2 values. ScO2 and simultaneous arterial and jugular venous blood gas measurements were obtained with both normocapnia and hypocapnia. Oximeter bias was calculated as the difference between the ScO2 and reference saturation using manufacturer-specified weighting ratios from the arterial and venous samples. FORE-SIGHT Elite bias was greater during hypocapnia as compared with normocapnia (4 ± 9% vs. 0 ± 6%; P < 0.001). The INVOS 5100C bias was also lower during normocapnia (5 ± 15% vs. 3 ± 12%; P = 0.01). Hypocapnia resulted in a significant decrease in mixed venous oxygen saturation and mixed venous oxygen tension, as well as increased oxygen extraction across fractional inspired oxygen tension levels (P < 0.0001). Bias increased significantly with increasing oxygen extraction (P < 0.0001). Changes in PaCO2 affect cerebral oximeter accuracy, and increased bias occurs with hypocapnia. Decreased accuracy may represent an incorrect assumption of a static arterial-venous blood fraction. Understanding cerebral oximetry limitations is especially important in patients at risk for hypoxia-induced brain injury, where PaCO2 may be purposefully altered.


Probing changes in tilt angle with 20 nanoradian resolution using an extrinsic Fabry-Perot interferometer-based optical fiber inclinometer.
In this paper, we introduce and demonstrate a novel optical fiber extrinsic Fabry-Perot interferometer (EFPI) for tilt measurements with 20 nrad resolution. Compared with in-line optical fiber inclinometers, an extrinsic sensing structure is used in the inclinometer reported herein. Our design greatly improves on the tilt angle resolution, the temperature stability, and the mechanical robustness of inclinometers with advanced designs. An EFPI cavity, which is formed between endfaces of a suspended rectangular mass block and a fixed optical fiber, is packaged inside a rectangular container box with an oscillation dampening mechanism. Importantly, the two reflectors of the EFPI sensor remain parallel while the cavity length of the EFPI sensor meters a change in tilt. According to the Fabry-Perot principle, the change in the cavity length can be determined, and the tilt angle of the inclinometer can be calculated. The sensor design and the measurement principle are discussed. An experiment based on measuring the tilt angle of a simply-supported 70-cm beam induced by a small load is presented to verify the resolution of our prototype inclinometer. The experimental results demonstrate significantly higher resolution (ca. 20 nrad) compared to commercial devices. The temperature cross-talk of the inclinometer was also investigated in a separate experiment and found to be 0.0041 μrad /°C. Our inclinometer was also employed for monitoring the daily periodic variations in the tilt angle of a windowsill in a cement building caused by local temperature changes during a five-day period. The multi-day study demonstrated excellent stability and practicability for the novel device. The significant inclinometer improvements in differential tilt angle resolution, temperature compensation, and mechanical robustness also provide unique opportunities for investigating spatial-temporal modulations of gravitational fields.


Proton release on binding of glutathione to alpha, Mu and Delta class glutathione transferases.
Potentiometric, spectroscopic and stopped-flow experiments have been performed to dissect the binding mechanism of GSH to selected glutathione S-transferases (GSTs), A1-1, M2-2 and Lucilia cuprina GST, belonging to Alpha, Mu and Delta classes respectively. Both Alpha and Mu isoenzymes quantitatively release the thiol proton of the substrate when the binary complex is formed. Proton extrusion, quenching of intrinsic fluorescence and thiolate formation, diagnostic of different steps along the binding pathway, have been monitored by stopped-flow analysis. Kinetic data are consistent with a multi-step binding mechanism: the substrate is initially bound to form an un-ionized pre-complex [k(1)>/=(2-5)x10(6) M(-1).s(-1)], which is slowly converted into the final Michaelis complex (k(2)=1100-1200 s(-1)). Ionization of GSH, fluorescence quenching and proton extrusion are fast events that occur either synchronously or rapidly after the final complex formation. The Delta isoenzyme shows an interesting difference: proton extrusion is almost stoichiometric with thiolate formed at the active site only up to pH 7.0. Above this pH, at least one protein residue acts as internal base to neutralize the thiol proton. These results suggest that the Alpha and Mu enzymes retain not only a similar catalytic outcome and overall three-dimensional structure but also share a similar kinetic mechanism for GSH binding. The Delta GST, which is closely related to the mammalian Theta class enzymes and is distantly related to Alpha and Mu GSTs in the evolutionary pathway, might display a different activation mechanism for GSH.


Advanced microscopy techniques used for comparison of UVA- and γ-irradiation-induced DNA damage in the cell nucleus and nucleolus.
Every day, genomes are affected by genotoxic factors that create multiple DNA lesions. Several DNA repair systems have evolved to counteract the deleterious effects of DNA damage. These systems include a set of DNA repair mechanisms, damage tolerance processes, and activation of cell-cycle checkpoints. This study describes selected confocal microscopy techniques that investigate DNA damage-related nuclear events after UVA- and γ-irradiation and compare the DNA damage response (DDR) induced by the two experimental approaches. In both cases, we observed induction of the nucleotide excision repair (NER) pathway and formation of localized double-strand breaks (DSBs). This was confirmed by analysis of cyclobutane pyrimidine dimers (CPDs) in the DNA lesions and by increased levels of γH2AX and 53BP1 proteins in the irradiated genome. DNA damage by UVA-lasers was potentiated by either BrdU or Hoechst 33342 pre-sensitization and compared to non-photosensitized cells. DSBs were also induced without BrdU or Hoechst 33342 pre-treatment. Interestingly, no cyclobutane pyrimidine dimers (CPDs) were detected after 405 nm UVA laser micro-irradiation in non-photosensitized cells. The effects of UVA and γ-irradiation were also studied by silver staining of nucleolar organizer regions (AgNORs). This experimental approach revealed changes in the morphology of nucleoli after genome injury. Additionally, to precisely characterize DDR in locally induced DNA lesions, we analysed the kinetics of the 53BP1 protein involved in DDR by fluorescence recovery after photobleaching (FRAP).


Mechanisms of nitric oxide-mediated neurotoxicity in primary brain cultures.
In addition to mediating several physiological functions, nitric oxide (NO) has been implicated in the cytotoxicities observed following activation of macrophages or excess stimulation of neurons by glutamate. We extend our previous observations of glutamate-stimulated, NO-mediated neurotoxicity in primary cultures of rat fetal cortical, striatal, and hippocampal neurons. Neurotoxicity elicited by either NMDA or sodium nitroprusside (SNP) exhibits a similar concentration-effect relationship and time course. The concentration-effect curve of NMDA-induced neurotoxicity is shifted to the right in the presence of nitro-L-arginine and farther to the right in arginine-free media. The rank order of potency of several NO synthase (NOS) inhibitors in preventing neurotoxicity is the same as the rank order of these compounds in inhibiting NOS, and this inhibition is stereospecific. NMDA neurotoxicity is also prevented by flavoprotein inhibitors and calmodulin inhibitors, fitting with the roles of flavoproteins and calmodulin as NOS regulators. 8-Bromo-cGMP and guanylyl cyclase inhibitors do not affect neurotoxicity, while superoxide dismutase attenuates neurotoxicity. NOS neurons appear to be the source of neurotoxic NO in culture, as lesions of these neurons with 20 microM quisqualate diminish subsequent NMDA neurotoxicity. Moreover, NMDA neurotoxicity develops over time in culture coincident with the expression of NOS. Immunohistochemical localization of NOS in cultures and intact brain demonstrates widespread distribution of the cell processes suggesting that NOS neurons contact the majority of cortical neurons and so could mediate widespread neurotoxicity.