Distribution patterns of postmortem damage in human mitochondrial DNA.
The distribution of postmortem damage in mitochondrial DNA retrieved from 37 ancient human DNA samples was analyzed by cloning and was compared with a selection of published animal data. A relative rate of damage (rho(v)) was calculated for nucleotide positions within the human hypervariable region 1 (HVR1) and cytochrome oxidase subunit III genes. A comparison of damaged sites within and between the regions reveals that damage hotspots exist and that, in the HVR1, these correlate with sites known to have high in vivo mutation rates. Conversely, HVR1 subregions with known structural function, such as MT5, have lower in vivo mutation rates and lower postmortem-damage rates. The postmortem data also identify a possible functional subregion of the HVR1, termed "low-diversity 1," through the lack of sequence damage. The amount of postmortem damage observed in mitochondrial coding regions was significantly lower than in the HVR1, and, although hotspots were noted, these did not correlate with codon position. Finally, a simple method for the identification of incorrect archaeological haplogroup designations is introduced, on the basis of the observed spectrum of postmortem damage.


An HER2 DNA vaccine with evolution-selected amino acid substitutions reveals a fundamental principle for cancer vaccine formulation in HER2 transgenic mice.
Enhancement of endogenous immunity to tumor-associated self-antigens and neoantigens is the goal of preventive vaccination. Toward this goal, we compared the efficacy of the following HER2 DNA vaccine constructs: vaccines encoding wild-type HER2, hybrid HER2 vaccines consisting of human HER2 and rat Neu, HER2 vaccines with single residue substitutions and a novel human HER2 DNA vaccine, ph(es)E2TM. ph(es)E2TM was designed to contain five evolution-selected substitutions: M198V, Q398R, F425L, H473R and A622T that occur frequently in 12 primate HER2 sequences. These ph(es)E2TM substitutions score 0 to 1 in blocks substitutions matrix (BLOSUM), indicating minimal biochemical alterations. h(es)E2TM recombinant protein is recognized by a panel of anti-HER2 mAbs, demonstrating the preservation of HER2 protein structure. Compared to native human HER2, electrovaccination of HER2 transgenic mice with ph(es)E2TM induced a threefold increase in HER2-binding antibody (Ab) and elevated levels of IFNγ-producing T cells. ph(es)E2TM, but not pE2TM immune serum, recognized HER2 peptide p95 355LPESFDGDPASNTAP369, suggesting a broadening of epitope recognition induced by the minimally modified HER2 vaccine. ph(es)E2TM vaccination reduced tumor growth more effectively than wild-type HER2 or HER2 vaccines with more extensive modifications. The elevation of tumor immunity by ph(es)E2TM vaccination would create a favorable tumor microenvironment for neoantigen priming, further enhancing the protective immunity. The fundamental principle of exploiting evolution-selected amino acid substitutions is novel, effective and applicable to vaccine development in general.


On the robust circuit design schemes of biochemical networks: steady-state approach.
Based on the steady-state analyses of the synergism and saturation system (S-system) model, a robust control method is proposed for biochemical networks via feedback and feedforward biochemical circuits. Two robust biochemical circuit design schemes are developed. One scheme is to improve the system's structural stability so as to tolerate larger kinetic parameter variations, whereas the other is to compensate for the kinetic parameter variations to eliminate their effects. In addition, a multi-objective biochemical circuit design scheme is introduced for both the robust design against kinetic parameter variations and a desired sensitivity design to eliminate the effect of external disturbance simultaneously. The proposed robust circuit design schemes will provide a systematic method with potential applications in synthetic circuit design for biotechnological purpose and drug design purpose. Recent advances in both metabolic and genetic engineering have made the robust biochemical circuit control approach feasible through the design and implementation of synthetic biological networks amenable to mathematical modeling and quantitative analysis. Finally, several examples including the robust circuit design of the tricarboxylic acid cycle are used in silico to illustrate the design procedure and to confirm the performance of the proposed design method.


Short pressure reactivity index versus long pressure reactivity index in the management of traumatic brain injury.
The pressure reactivity index (PRx) correlates with outcome after traumatic brain injury (TBI) and is used to calculate optimal cerebral perfusion pressure (CPPopt). The PRx is a correlation coefficient between slow, spontaneous changes (0.003-0.05 Hz) in intracranial pressure (ICP) and arterial blood pressure (ABP). A novel index-the so-called long PRx (L-PRx)-that considers ABP and ICP changes (0.0008-0.008 Hz) was proposed. The authors compared PRx and L-PRx for 6-month outcome prediction and CPPopt calculation in 307 patients with TBI. The PRx- and L-PRx-based CPPopt were determined and the predictive power and discriminant abilities were compared. The PRx and L-PRx correlation was good (R = 0.7, p < 0.00001; Spearman test). The PRx, age, CPP, and Glasgow Coma Scale score but not L-PRx were significant fatal outcome predictors (death and persistent vegetative state). There was a significant difference between the areas under the receiver operating characteristic curves calculated for PRx and L-PRx (0.61 ± 0.04 vs 0.51 ± 0.04; z-statistic = -3.26, p = 0.011), which indicates a better ability by PRx than L-PRx to predict fatal outcome. The CPPopt was higher for L-PRx than for PRx, without a statistical difference (median CPPopt for L-PRx: 76.9 mm Hg, interquartile range [IQR] ± 10.1 mm Hg; median CPPopt for PRx: 74.7 mm Hg, IQR ± 8.2 mm Hg). Death was associated with CPP below CPPopt for PRx (χ(2) = 30.6, p < 0.00001), and severe disability was associated with CPP above CPPopt for PRx (χ(2) = 7.8, p = 0.005). These relationships were not statistically significant for CPPopt for L-PRx. The PRx is superior to the L-PRx for TBI outcome prediction. Individual CPPopt for L-PRx and PRx are not statistically different. Deviations between CPP and CPPopt for PRx are relevant for outcome prediction; those between CPP and CPPopt for L-PRx are not. The PRx uses the entire B-wave spectrum for index calculation, whereas the L-PRX covers only one-third of it. This may explain the performance discrepancy.


The Dehydrogenase Hypothesis.
Circulating glucocorticoid (GC) levels are controlled by the Hypothalamo-Pituitary-Adrenal (HPA) axis, but within tissues, GC availability is controlled by the isoforms of 11β (Beta)-Hydroxysteroid Dehydrogenase 11β (Beta)-HSD that interconvert inactive cortisone and active cortisol. Two isoforms have been identified; in key metabolic target tissues (including liver and adipose), expression of 11β (Beta)-HSD1 predominates that in vivo converts cortisone to cortisol and thus amplifies local GC action. In contrast, in mineralocorticoid target tissues 11β (Beta)-HSD2 is the isoform that is most abundantly expressed. This inactivates cortisol to cortisone and offers protection for the mineralocorticoid receptor form occupation and activation by cortisol. Dysregulated 11β (Beta)-HSD1 activity has been implicated in many metabolic diseases such as obesity and diabetes and inhibition of 11β (Beta)-HSD1 represents a promising therapeutic target. Mutations within the gene encoding 11β (Beta)-HSD2 cause the Syndrome of Apparent Mineralocorticoid Excess and decreases in activity are linked to hypertension as well as impairment in placental function and neonatal growth. We will discuss the molecular biology and enzymology of 11β (Beta)-HSD and its role in normal physiology and discuss altered 11β (Beta)-HSD activity in pathological states and the potential for therapeutic targeting.


Early mortality following stroke: a prospective review.
Analysis of early deaths after stroke is important, since some deaths may be preventable. Previous studies have relied on retrospective and often incomplete clinical data, for comparison with pathological findings. The present study is based on 1073 consecutive stroke patients admitted to an intensive care stroke unit from a well-defined population. There were 212 deaths within the first 30 days, yielding a mortality rate of 20%. Clinical, radiological, and laboratory data were collected prospectively according to a standardized protocol. Autopsies were performed on 90 of the 212 patients, and CT scanning on a further 27. Early mortality after stroke exhibits a bimodal distribution. One peak occurs during the first week, and a second during the second and third weeks. The majority of deaths in the first week are due to transtentorial herniation. Of these, deaths due to hemorrhage usually occur within the first three days, whilst deaths due to infarction peak between the third and sixth day post ictus. After the first week, deaths due to relative immobility (pneumonia, pulmonary embolism and sepsis) predominate, peaking towards the end of the second week. Cardiac deaths occur throughout the first month, and unfortunately account for many deaths in patients with small functional deficits.


Full can test: Mechanisms of a positive test in patients with shoulder pain.
The full can test is theorized to produce compressive loads on the supraspinatus tendon within the subacromial space. Characterizing the width of the subacromial outlet, scapular orientation, and shoulder pain during the full can test will improve the mechanistic understanding of the positive full can test. Cross-sectional repeated measures design. Participants with subacromial pain syndrome (n=30) were compared to a matched control group (n=30) during 2 conditions: passive support, and the full can test. The full can test was performed with the arm elevated to 90° in the scapular plane. In both conditions, measurements were taken of acromiohumeral distance with ultrasonography, scapular position using electromagnetic tracking, shoulder strength using a dynamometer, and shoulder pain with the 11-point rating scale. During the full can test, both groups had a decreased acromial humeral distance, scapular upward rotation, posterior tilt, external rotation and clavicular protraction as compared to passive support (p<0.05). The subacromial pain group as compared to the control group reported greater shoulder pain (p<0.001), reduced strength (p=0.002) and greater scapular anterior tilt (p<0.05) during the full can test. This study indicates the mechanisms of a full can test are a reduction in the acromial humeral distance, accompanied by scapular kinematic changes. A positive test of increased pain and reduced strength in those with subacromial pain syndrome can be explained additionally by an increase in scapular anterior tilt. These mechanistic changes may lead to tendon compression, but this cannot be verified as direct tendon compression was not measured.


Sensitivity of Seven Diverse Species to Blue and Green Light: Interactions with Photon Flux.
Despite decades of research, the effects of spectral quality on plant growth, and development are not well understood. Much of our current understanding comes from studies with daily integrated light levels that are less than 10% of summer sunlight thus making it difficult to characterize interactions between light quality and quantity. Several studies have reported that growth is increased under fluorescent lamps compared to mixtures of wavelengths from LEDs. Conclusions regarding the effect of green light fraction range from detrimental to beneficial. Here we report the effects of eight blue and green light fractions at two photosynthetic photon fluxes (PPF; 200 and 500 μmol m-2 s-1; with a daily light integral of 11.5 and 29 mol m-2 d-1) on growth (dry mass), leaf expansion, stem and petiole elongation, and whole-plant net assimilation of seven diverse plant species. The treatments included cool, neutral, and warm white LEDs, and combinations of blue, green and/or red LEDs. At the higher PPF (500), increasing blue light in increments from 11 to 28% reduced growth in tomato, cucumber, and pepper by 22, 26, and 14% respectively, but there was no statistically significant effect on radish, soybean, lettuce or wheat. At the lower PPF (200), increasing blue light reduced growth only in tomato (41%). The effects of blue light on growth were mediated by changes in leaf area and radiation capture, with minimal effects on whole-plant net-assimilation. In contrast to the significant effects of blue light, increasing green light in increments from 0 to 30% had a relatively small effect on growth, leaf area and net assimilation at either low or high PPF. Surprisingly, growth of three of the seven species was not reduced by a treatment with 93% green light compared to the broad spectrum treatments. Collectively, these results are consistent with a shade avoidance response associated with either low blue or high green light fractions.


Characterization of In Vitro Engineered Human Adipose Tissues: Relevant Adipokine Secretion and Impact of TNF-α.
Representative modelling of human adipose tissue functions is central to metabolic research. Tridimensional models able to recreate human adipogenesis in a physiological tissue-like context in vitro are still scarce. We describe the engineering of white adipose tissues reconstructed from their cultured adipose-derived stromal precursor cells. We hypothesize that these reconstructed tissues can recapitulate key functions of AT under basal and pro-inflammatory conditions. These tissues, featuring human adipocytes surrounded by stroma, were stable and metabolically active in long-term cultures (at least 11 weeks). Secretion of major adipokines and growth factors by the reconstructed tissues was determined and compared to media conditioned by human native fat explants. Interestingly, the secretory profiles of the reconstructed adipose tissues indicated an abundant production of leptin, PAI-1 and angiopoietin-1 proteins, while higher HGF levels were detected for the human fat explants. We next demonstrated the responsiveness of the tissues to the pro-inflammatory stimulus TNF-α, as reflected by modulation of MCP-1, NGF and HGF secretion, while VEGF and leptin protein expression did not vary. TNF-α exposure induced changes in gene expression for adipocyte metabolism-associated mRNAs such as SLC2A4, FASN and LIPE, as well as for genes implicated in NF-κB activation. Finally, this model was customized to feature adipocytes representative of progressive stages of differentiation, thereby allowing investigations using newly differentiated or more mature adipocytes. In conclusion, we produced tridimensional tissues engineered in vitro that are able to recapitulate key characteristics of subcutaneous white adipose tissue. These tissues are produced from human cells and their neo-synthesized matrix elements without exogenous or synthetic biomaterials. Therefore, they represent unique tools to investigate the effects of pharmacologically active products on human stromal cells, extracellular matrix and differentiated adipocytes, in addition to compounds modulating adipogenesis from precursor cells.


Causes and management of thunderclap headache: a comprehensive review.
Thunderclap headache (or sudden severe headache) is an uncommon type of headache. Recognition and accurate diagnosis of this headache are important, because there is often a serious underlying brain disorder. In this article, causes and management of thunderclap headache are discussed. In the primary care setting, there is a serious cause in one third of patients, but in the hospital setting, up to two thirds of patients have a serious underlying brain disorder. Clues in history and physical examination can point to a possible serious underlying cause of thunderclap headache, such as subarachnoid hemorrhage, intracranial hematoma, or cerebral venous thrombosis. The remaining patients with thunderclap headache, however, have a primary headache disorder, such as migraine or (less frequently) tension headache with an unusual sudden onset, exertional headache, coital headache, cough headache, or cluster headache. The concept of thunderclap headache as a distinct clinical entity is discussed, with implications for its evaluation. Present radiological techniques are reviewed with regard to their diagnostic utility in detecting a serious brain disorder. Thunderclap headache is an uncommon type of headache, and a serious underlying cause should be excluded.


Noise-cancelling repeat finder: uncovering tandem repeats in error-prone long-read sequencing data.
Tandem DNA repeats can be sequenced with long-read technologies, but cannot be accurately deciphered due to the lack of computational tools taking high error rates of these technologies into account. Here we introduce Noise-Cancelling Repeat Finder (NCRF) to uncover putative tandem repeats of specified motifs in noisy long reads produced by Pacific Biosciences and Oxford Nanopore sequencers. Using simulations, we validated the use of NCRF to locate tandem repeats with motifs of various lengths and demonstrated its superior performance as compared to two alternative tools. Using real human whole-genome sequencing data, NCRF identified long arrays of the (AATGG)n repeat involved in heat shock stress response. NCRF is implemented in C, supported by several python scripts, and is available in bioconda and at https://github.com/makovalab-psu/NoiseCancellingRepeatFinder. Supplementary data are available at Bioinformatics online.


On the folding of a structurally complex protein to its metastable active state.
For successful protease inhibition, the reactive center loop (RCL) of the two-domain serine protease inhibitor, α1-antitrypsin (α1-AT), needs to remain exposed in a metastable active conformation. The α1-AT RCL is sequestered in a β-sheet in the stable latent conformation. Thus, to be functional, α1-AT must always fold to a metastable conformation while avoiding folding to a stable conformation. We explore the structural basis of this choice using folding simulations of coarse-grained structure-based models of the two α1-AT conformations. Our simulations capture the key features of folding experiments performed on both conformations. The simulations also show that the free energy barrier to fold to the latent conformation is much larger than the barrier to fold to the active conformation. An entropically stabilized on-pathway intermediate lowers the barrier for folding to the active conformation. In this intermediate, the RCL is in an exposed configuration, and only one of the two α1-AT domains is folded. In contrast, early conversion of the RCL into a β-strand increases the coupling between the two α1-AT domains in the transition state and creates a larger barrier for folding to the latent conformation. Thus, unlike what happens in several proteins, where separate regions promote folding and function, the structure of the RCL, formed early during folding, determines both the conformational and the functional fate of α1-AT. Further, the short 12-residue RCL modulates the free energy barrier and the folding cooperativity of the large 370-residue α1-AT. Finally, we suggest experiments to test the predicted folding mechanism for the latent state.


Infected Rathke cleft cysts: distinguishing factors and factors predicting recurrence.
Rathke cleft cysts (RCCs) are benign sellar lesions that are generally asymptomatic but sometimes warrant transsphenoidal drainage. Small case reports have described infected RCCs, but this phenomenon remains uncharacterized. We reviewed RCCs over 23 years at our institution to determine factors predicting infection and recurrence. We retrospectively reviewed the magnetic resonance images, laboratory results, microbiology, and pathology of 176 RCC patients (1985-2008) who underwent initial operation at our institution (n = 170) or at another institution followed by recurrence managed at our institution (n = 6). There were 3 RCC categories: cysts cultured intraoperatively during initial surgery (n = 21), cysts not cultured during initial surgery but cultured during subsequent surgery (n = 9), and cysts that were never cultured (n = 146). Cultured cysts were larger (1.6 vs 1.2 cm; P = .002) and had more frequent pituitary dysfunction (76% vs 30%; P < .001) than noncultured cysts. Restricted diffusion was also more common in cultured cysts (50% vs 0%; P = .02). Of cysts cultured at initial or subsequent surgery, 48% and 44%, respectively, had positive cultures (n = 14) and were treated with antibiotics. The most common organisms were Staphylococcus epidermidis (64%) and Propionibacterium acnes (57%). Kaplan-Meier recurrence rates were 13% (culture positive/antibiotic treated), 31% (culture negative/not antibiotic treated), and 9% (noncultured) (P = .002, cultured vs noncultured; P = .002, culture negative/not antibiotic treated vs non-cultured; P = .5 culture positive/antibiotic treated vs noncultured). Suspected RCC infection, regardless of culture results, is a strong predictor of recurrence and may warrant antibiotic treatment. With antibiotic treatment, the recurrence rate of infected RCC approaches that of noninfected cysts. The higher recurrence rates reported in other series may reflect underrecognition of occult infection.


Quantitative phosphoproteomics identifies substrates and functional modules of Aurora and Polo-like kinase activities in mitotic cells.
Mitosis is a process involving a complex series of events that require careful coordination. Protein phosphorylation by a small number of kinases, in particular Aurora A, Aurora B, the cyclin-dependent kinase-cyclin complex Cdk1/cyclinB, and Polo-like kinase 1 (Plk1), orchestrates almost every step of cell division, from entry into mitosis to cytokinesis. To discover more about the functions of Aurora A, Aurora B, and kinases of the Plk family, we mapped mitotic phosphorylation sites to these kinases through the combined use of quantitative phosphoproteomics and selective targeting of kinase activities by small-molecule inhibitors. Using this integrated approach, we connected 778 phosphorylation sites on 562 proteins with these enzymes in cells arrested in mitosis. By connecting the kinases to protein complexes, we associated these kinases with functional modules. In addition to predicting previously unknown functions, this work establishes additional substrate-recognition motifs for these kinases and provides an analytical template for further use in dissecting kinase signaling events in other areas of cellular signaling and systems biology.


Degradation of MDM2 by the interaction between berberine and DAXX leads to potent apoptosis in MDM2-overexpressing cancer cells.
Berberine, a natural product derived from a plant used in Chinese herbal medicine, is reported to exhibit anticancer effects; however, its mechanism of action is not clearly defined. Herein, we demonstrate that berberine induces apoptosis in acute lymphoblastic leukemia (ALL) cells by downregulating the MDM2 oncoprotein. The proapoptotic effects of berberine were closely associated with both the MDM2 expression levels and p53 status of a set of ALL cell lines. The most potent apoptosis was induced by berberine in ALL cells with both MDM2 overexpression and a wild-type (wt)-p53, whereas no proapoptotic effect was detected in ALL cells that were negative for MDM2 and wt-p53. In contrast to the conventional chemotherapeutic drug doxorubicin, which induces p53 activation and a subsequent upregulation of MDM2, berberine strongly induced persistent downregulation of MDM2 followed by a steady-state activation of p53. We discovered that downregulation of MDM2 in ALL cells by berberine occurred at a posttranslational level through modulation of death domain-associated protein (DAXX), which disrupted the MDM2-DAXX-HAUSP interactions and thereby promoted MDM2 self-ubiquitination and degradation. Given that MDM2-overexpressing cancer cells are commonly chemoresistant, our findings suggest that this naturally derived agent may have a highly useful role in the treatment of cancer patients with refractory disease.


Motor learning by observation: evidence from a serial reaction time task.
This study sought evidence of observational motor learning, a type of learning in which observation of the skilled performance of another person not only facilitates motor skill acquisition but does so by contributing to the formation of effector-specific motor representations. Previous research has indicated that observation of skilled performance engages cognitive processes similar to those occurring during action execution or physical practice, but has not demonstrated that these include processes involved in effector-specific representation. In two experiments, observer subjects watched the experimenter performing a serial reaction time (SRT) task with a six-item unique sequence before sequence knowledge was assessed by response time and/or free generation measures. The results suggest that: (1) subjects can acquire sequence information by watching another person performing the task (Experiments 1-2); (2) observation results in as much sequence learning as task practice when learning is measured by reaction times (RTs) and more than task practice when sequence learning is measured by free generation performance (Experiment 2, Part 1); and (3) sequence knowledge acquired by model observation can be encoded motorically--that is, in an effector-specific fashion (Experiment 2, Part 2).


Assessment of image quality in dental radiography, part 1: phantom validity.
The purpose of this study was to describe and validate an image-quality phantom to be used in dental radiography for comparison of film and digitally acquired images. An aluminum block of 12 steps, with 7 holes in each step, was covered by acrylic blocks. This phantom was radiographed with Kodak Ultra-speed and Ektaspeed Plus films at 70, 65, and 60 kVp with the whole exposure range available. All together, 50 dental films were randomly sequenced and presented to 7 observers. The average number of perceptible holes from all steps was plotted against exposure for each tube voltage and film type, generating a modified perceptibility curve. The tentative optimum exposure level was determined from perceptibility curves in each experimental condition and compared with that determined by means of the standard aluminum stepwedge and the preset time of the x-ray machine. The density range of this phantom at the optimum exposure was compared with that of clinical dental radiographs. Validity of the phantom was evaluated according to the optimum exposure level from the modified perceptibility curves and the overall density range. Finally, the average maximum numbers of perceptible holes at the tentative optimum exposure level were compared for each tube voltage and film type. The statistical test used was a 2-way factorial analysis of variance. The exposure at the perceptibility curve peak approximated that obtained by means of the standard aluminum step-wedge and the time preset by the manufacturer. The overall density range at the perceptibility curve peak covered the clinical density range for each tube voltage and film type. There were no statistically significant differences between film types or among tube voltages. The x-ray attenuation range for this phantom seemed to approximate clinical conditions. In addition, differences in image quality could be quantitatively evaluated by means of the number of the holes seen in the phantom.


Optimizing structure-function relationship by maximizing correspondence between glaucomatous visual fields and mathematical retinal nerve fiber models.
To introduce a method to optimize structural retinal nerve fiber layer (RNFL) models based on glaucomatous visual field data and to show how such an optimized model can be used to reduce noise in visual fields while probably preserving clinically important features. Correlation coefficients between age-adjusted deviation values of pairs of visual field test locations were calculated from 103 visual fields of eyes with moderate glaucomatous damage. Distances between those test locations were defined for various parameters of a mathematical RNFL model. Then, the correspondence between the structural and functional data was defined by the spread, or variance, of the correlation coefficients for all distances. The model parameters that minimized this spread constituted the optimized model. To reduce noise in visual fields, the optimized model was used to smooth visual field data according to the RNFL's structure. The resulting fields were compared with visual fields that were smoothed based on the regular testing grid. The optimal parameters for the RNFL model reduced the variance of the correlation coefficients by 78% and were well within the range of parameters previously determined from fundus photographs. Smoothing the visual fields based on the optimized RNFL model strongly reduced noise while keeping important features. Mathematic RNFL models can be optimized based on visual field data, resulting in a strong structure-function relationship. Taking the RNFL's shape, as defined by such an optimized model, into account when smoothing visual fields results in better noise reduction while preserving important details.


20-[18F]fluoroarachidonic acid: tissue biodistribution and incorporation into phospholipids.
The in vivo behavior of 20-[18F]fluoroarachidonic acid (18F-FAA) was investigated to evaluate its potential use as a radiotracer for studying the regional brain and heart lipid metabolism by positron emission tomography (PET). Tissue biodistribution studies in rats have revealed that 18F-FAA has a high uptake in the liver and lung, thus probably reflecting the metabolism, and is accompanied by both low in vivo defluorination and low blood levels. At 30 min postinjection, the uptake in the brain and heart reached values of 0.26 +/- 0.02 and 1.22 +/- 0.58% dose/g, respectively, with ratios to the blood radioactivity of 1.04 and 4.88, respectively. Lipid extraction at 30 min postinjection showed that 39% of the brain radioactivity was in the organic phase whereas the organic phase from heart tissue contained 73% of the total radioactivity. A TLC analysis demonstrated that 18F-FAA was mainly bound to phospholipids in the brain and heart tissue as expected. Based on the findings of this study, the utility of 18F-FAA as an in vivo tracer for cerebral phospholipid studies appears to be limited because of its relatively high radioactivity in the aqueous brain fraction. However, our findings do suggest that this agent might be useful as a tool for studies of cardiac phospholipid turnover, even though it demonstrated a poor heart-to-lung and heart-to-liver contrast.


Genome-wide miRNA profiling of mantle cell lymphoma reveals a distinct subgroup with poor prognosis.
miRNA deregulation has been implicated in the pathogenesis of mantle cell lymphoma (MCL). Using a high-throughput quantitative real-time PCR platform, we performed miRNA profiling on cyclin D1-positive MCL (n = 30) and cyclin D1-negative MCL (n = 7) and compared them with small lymphocytic leukemia/lymphoma (n = 12), aggressive B-cell lymphomas (n = 138), normal B-cell subsets, and stromal cells. We identified a 19-miRNA classifier that included 6 up-regulated miRNAs and 13 down regulated miRNA that was able to distinguish MCL from other aggressive lymphomas. Some of the up-regulated miRNAs are highly expressed in naive B cells. This miRNA classifier showed consistent results in formalin-fixed paraffin-embedded tissues and was able to distinguish cyclin D1-negative MCL from other lymphomas. A 26-miRNA classifier could distinguish MCL from small lymphocytic leukemia/lymphoma, dominated by 23 up-regulated miRNAs in MCL. Unsupervised hierarchical clustering of MCL patients demonstrated a cluster characterized by high expression of miRNAs from the polycistronic miR17-92 cluster and its paralogs, miR-106a-363 and miR-106b-25, and associated with high proliferation gene signature. The other clusters showed enrichment of stroma-associated miRNAs, and also had higher expression of stroma-associated genes. Our clinical outcome analysis in the present study suggested that miRNAs can serve as prognosticators.


Optimising the use of caesarean section: a generic formative research protocol for implementation preparation.
Caesarean section rates are rising across all geographical regions. Very high rates for some groups of women co-occur with very low rates for others. Both extremes are associated with short and longer term harms. This is a major public health concern. Making the most effective use of caesarean section is a critical component of good quality, sustainable maternity care. In 2018, the World Health Organization published evidence-based recommendations on non-clinical interventions to reduce unnecessary caesarean section. The guideline identified critical research gaps and called for formative research to be conducted ahead of any interventional research to define locally relevant determinants of caesarean birth and factors that may affect implementation of multifaceted optimisation strategies. This generic formative research protocol is designed as a guide for contextual assessment and understanding for anyone planning to take action to optimise the use of caesarean section. This formative protocol has three main components: (1) document review; (2) readiness assessment; and (3) primary qualitative research with women, healthcare providers and administrators. The document review and readiness assessment include tools for local mapping of policies, protocols, practices and organisation of care to describe and assess the service context ahead of implementation. The qualitative research is organized according to twelve identified interventions that may optimise use of caesarean section. Each intervention is designed as a "module" and includes a description of the intervention, supporting evidence, theory of change, and in-depth interview/focus group discussion guides. All study instruments are included in this protocol. This generic protocol is designed to underpin the formative stage of implementation research relating to optimal use of caesarean section. We encourage researchers, policy-makers and ministries of health to adapt and adopt this design to their context, and share their findings as a catalyst for rapid uptake of what works.


Insulin-like growth factor-II and heparin are anti-apoptotic survival factors in human villous cytotrophoblast.
This study aimed to determine the effects of insulin-like growth factors (IGF-I and IGF-II), heparin, aspirin and vitamin C on the proliferation and apoptosis of human villous cytotrophoblast from first trimester and term placentae. Villous cytotrophoblast cells were isolated from uncomplicated first trimester (n=12) and term placental tissues (n=12) using negative immunoselection with an antibody to HLA class I antigens. Cells were incubated with IGF-I, IGF-II, heparin, aspirin and vitamin C either alone, or in combination with either TNF-α/IFN-γ or staurosporine. Proliferation was determined by measurement of Ki67 expression using immunocytochemistry. Trophoblast apoptosis was determined by TUNEL staining. Finally RT-PCR was carried out to identify IGF-binding insulin receptor isoforms. Data were expressed as means±SEM. One way analysis of variance (ANOVA) with Bonferroni correction was used to determine if differences between groups were statistically significant. Following negative immunoselection >98% of cells were positively stained for cytokeratin 7, a marker for cytotrophoblasts, and <1% were vimentin positive. First trimester and term trophoblasts underwent spontaneous apoptosis which was inhibited by approximately 50% in the presence of IGF-II or heparin. Apoptosis was significantly increased following incubation with a combination of TNF-α and IFN-γ or staurosporine. Apoptosis was decreased to basal levels following coincubation with IGF-II or heparin. Incubation with IGFs or heparin resulted in a small, but significant increase in Ki67 expression. Insulin receptor isoform A, which binds IGF-II with high affinity, was present in all trophoblast samples tested. These results suggest that heparin and IGF-II, but not IGF-I are important regulators of villous cytotrophoblast survival in early and late pregnancy.


White matter correlates of psychopathic traits in a female community sample.
Psychopathy comprises interpersonal, affective, lifestyle and antisocial facets that vary dimensionally in the population and are associated with criminal offending and adverse psychosocial outcomes. Evidence associating these facets with white matter microstructure of the uncinate fasciculus and the cingulum tracts is inconsistent and derives principally from studies of male offenders. In a sample of 99 young women presenting a range of scores on the Psychopathy Checklist: Screening Version, we used Diffusion Tensor Imaging, tractography and Tract-Based Spatial Statistics to investigate microstructure across the brain and of the uncinate fasciculus and cingulum. Right uncinate fasciculus microstructure was negatively associated with the interpersonal facet, while cingulum integrity was not associated with any facet of psychopathy. Whole-brain analyses revealed that both affective and lifestyle facets were negatively correlated with white matter microstructure adjacent to the fusiform gyrus, and the interpersonal facet correlated negatively with the integrity of the fornix. Findings survived adjustment for the other facet scores, and age, verbal and performance IQ. A similar negative association between the interpersonal facet and uncinate fasciculus integrity was previously observed in male offenders. Thus, previous evidence showing that psychopathic traits are associated with functional and structural abnormalities within limbic networks may also apply to females.


LEM-4 promotes rapid dephosphorylation of BAF during mitotic exit.
The transitions between the successive cell cycle stages depend on reversible protein phosphorylation events. The phosphorylation state of every protein within a cell is strictly determined by spatiotemporally controlled kinase and phosphatase activities. Nuclear disassembly and reassembly during open mitosis in higher eukaryotic cells is one such process that is tightly regulated by the reversible phosphorylation of key proteins. However, little is known about the regulation of these mitotic events. In particular, although kinase function during entry into mitosis is better studied, very little is known about how proteins are dephosphorylated to allow nuclear reformation at the end of mitosis. We have identified LEM‑4, a conserved protein of the nuclear envelope, as an essential coordinator of kinase and phosphatase activities during mitotic exit. Inhibition of VRK‑1 kinase and promotion of a PP2A phosphatase complex by LEM‑4 tightly regulate the phosphorylation state of BAF, an essential player of nuclear reformation at the end of mitosis. Here I offer extended comments on the contribution of LEM‑4 in the regulation of protein phosphorylation and nuclear reformation.


Post-Translational Dosage Compensation Buffers Genetic Perturbations to Stoichiometry of Protein Complexes.
Understanding buffering mechanisms for various perturbations is essential for understanding robustness in cellular systems. Protein-level dosage compensation, which arises when changes in gene copy number do not translate linearly into protein level, is one mechanism for buffering against genetic perturbations. Here, we present an approach to identify genes with dosage compensation by increasing the copy number of individual genes using the genetic tug-of-war technique. Our screen of chromosome I suggests that dosage-compensated genes constitute approximately 10% of the genome and consist predominantly of subunits of multi-protein complexes. Importantly, because subunit levels are regulated in a stoichiometry-dependent manner, dosage compensation plays a crucial role in maintaining subunit stoichiometries. Indeed, we observed changes in the levels of a complex when its subunit stoichiometries were perturbed. We further analyzed compensation mechanisms using a proteasome-defective mutant as well as ribosome profiling, which provided strong evidence for compensation by ubiquitin-dependent degradation but not reduced translational efficiency. Thus, our study provides a systematic understanding of dosage compensation and highlights that this post-translational regulation is a critical aspect of robustness in cellular systems.


Expression of NF-kappaB in epidermis and the relationship between NF-kappaB activation and inhibition of keratinocyte growth.
Nuclear factor-kappaB (NF-kappaB) is a transcription factor involved in a number of signalling pathways in many cell types. NF-kappaB in mice has been implicated as an important regulator of keratinocyte proliferation and differentiation. To evaluate the role of NF-kappaB in keratinocyte growth in human beings, we examined its expression in keratinocytes both in culture and in situ, and studied the relationship between NF-kappaB activation and the inhibition of keratinocyte proliferation induced by known modulators of keratinocyte growth. The expression of subunits of the NF-kappaB family was examined in human skin, primary cultured keratinocytes and an immortalized keratinocyte line by immunohistochemistry and reverse transcriptase-polymerase chain reaction analysis. NF-kB activation was examined in keratinocytes treated with various modulating agents by electrophoretic mobility shift assay (for DNA-binding activity) and by immunocytochemistry (nuclear translocation). The proliferative capacity of treated keratinocytes was also examined by 3H-thymidine incorporation, cell cycle analysis, and expression of Ki-67, a nuclear marker for cell proliferation. The involvement of NF-kappaB was assessed using sodium salicylate, which inhibits NF-kappaB activation. The NF-kappaB subunits, p50, p65, RelB, and c-Rel (but not p52), were detected in keratinocytes and in normal epidermis at mRNA and protein levels. The four subunits were expressed in a cytoplasmic (rather than a nuclear) pattern in both basal and suprabasal keratinocytes. Phorbol myristate acetate (PMA), tumour necrosis factor alpha, and interferon gamma each activated NF-kappaB and inhibited keratinocyte proliferation. Lipopolysaccharide and dexamethasone did not activate NF-kappaB and had the least effect on proliferation. Finally, a high concentration of calcium (Ca2+) and retinoic acid each failed to activate NF-kappaB, but were potent inhibitors of keratinocyte proliferation, respectively. PMA-induced cell cycle arrest of keratinocytes was blocked by pretreatment with sodium salicylate. NF-kappaB is constitutively expressed in a resting state in both human cultured keratinocytes and the epidermis. Activation of NF-kappaB is required for PMA-induced keratinocyte growth arrest.


Identification and validation of a novel microRNA-like molecule derived from a cytoplasmic RNA virus antigenome by bioinformatics and experimental approaches.
It is generally believed that RNA virus replicating in the cell cytoplasm would not encode microRNAs (miRNAs) due to nucleus inaccessibility. Recent studies have described cytoplasmic RNA virus genome-derived miRNAs in West Nile virus (WNV) and Dengue virus (DENV). However, naturally occurring miRNAs derived from the antigenome of a cytoplasmic RNA virus have not been described. Hepatitis A virus (HAV) was served as a model virus to investigate whether the antigenome of a cytoplasmic RNA virus would be processed into miRNAs or miRNA-like small RNAs upon infection. HAV antigenome was queried for putative miRNA precursors (pre-miRNA) with the VMir analyzer program. Mature miRNA prediction was performed using MatureBayes and Bayes-SVM-MiRNA web server v1.0. Finally, multiple experimental approaches, including cloning and sequencing-, RNAi-, plasmid-based miRNA expression- and luciferase reporter assays, were performed to identify and validate naturally occurring viral antigenome-derived miRNAs. Using human HAV genotype IA (isolate H2) (HAVH2), a virally encoded miRNA-like small RNA was detected on the antigenome and named hav-miR-N1-3p. Transcription of viral pre-miRNA in KMB17 and HEK293T cells led to mature hav-miR-N1-3p production. In addition, silencing of the miRNA-processing enzyme Dicer or Drosha caused a dramatic reduction in miRNA levels. Furthermore, artificial target of hav-miR-N1-3p was silenced by synthesized viral miRNA mimics and the HAVH2 naturally-derived hav-miR-N1-3p. These results suggested that the antigenome of a cytoplasmic RNA virus could be processed into functional miRNAs. Our findings provide new evidence supporting the hypothesis that cytoplasmic RNA viruses naturally encode miRNAs through cellular miRNA processing machinery.


Amperometric determination of amino group on a solid support using glutaraldehyde.
A simple amperometric method has been developed for the determination of amino groups on a solid support. The method is based on oxygen consumption during the reaction between the bifunctional reagent gluaraldehyde and the amino groups on the solid support. Oxygen consumption was monitored with a Clark oxygen electrode. AHSepharose 4B, Affi-Gel 102, and Amino-Celluofine were used as amino-terminal solid supports. The concentration of amino groups on each support was calculated from a calibration curve of the standard compound. When hexamethylenediamine was used as the standard, the concentration of amino groups on AH-Sepharose 4B and Affi-Gel 102 by the present method showed good agreement with that by the conventional method. In the case of Amino-Cellulofine, ethanolamine had to be selected as the standard. The relative standard deviation for six successive determinations of AH-Sepharose 4B was 3.2%; the polymerization of glutaraldehyde showed no effect on the result. The spectral property of AH-Sepharose 4B treated with glutataraldehyde showed no effect on the result. The spectral property of AH-Sepharose 4B treated with lutaraldehyde suggested that the reaction mechanism of the present method was based on the oxygen consumption during the formation of the pyridinium salt on the support. A comparative study of the present method with a colorimetric method with Traut's regent is also included.


Clinical characteristics of imported malaria in Japan: analysis at a referral hospital.
Imported malaria remains an important problem in Japan. We have reviewed the medical records of 170 cases of malaria in our hospital, which corresponds to 14.9% of the total cases in Japan. The predominant malarial species was Plasmodium falciparum (52.3%), and the most frequent area of acquisition was Africa (54.2%), followed by Asia (20.9%) and Oceania (19.6%). The most common reason for travel among Japanese patients was business. A significant proportion (22.2%) of vivax malaria cases experienced relapse despite standard primaquine therapy. Most primaquine failures were from Oceania. We also found that a substantial number of Japanese patients contracted malaria without chemoprophylaxis and consulted medical facilities with an unfavorably long delay from initial symptoms (median: 3.0 days). Direct education of travelers and travel companies, in addition to health care providers, is likely necessary to improve outcomes of imported malaria.


In vitro susceptibilities of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum to sparfloxacin and PD 127391.
The in vitro activities of two investigational quinolones, sparfloxacin (previously designated AT 4140) and PD 127391, were determined for 30 strains each of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum and compared with those of ciprofloxacin, tetracycline, clindamycin, and erythromycin. Erythromycin was the most active compound against M. pneumoniae (maximum MIC, less than 0.008 microgram/ml). PD 127391 (MICs, less than 0.008 to 0.031 microgram/ml), sparfloxacin (MICs, less than 0.008 to 0.25 microgram/ml), clindamycin (MICs, less than 0.008 to 0.5 microgram/ml), and tetracycline (MICs, 0.063 to 0.25 microgram/ml) were superior to ciprofloxacin (MICs, 0.5 to 2 microgram/ml). Sparfloxacin and PD 127391 were active against M. hominis (MICs, less than 0.008 to 0.031 microgram/ml for each) at concentrations comparable to those of clindamycin (MICs, less than 0.008 to 0.063 microgram/ml) and at concentrations lower than those of ciprofloxacin (MICs, 0.125 to 0.5 microgram/ml). As expected, M. hominis was resistant to erythromycin (MICs, 32 to greater than or equal to 256 micrograms/ml). For U. urealyticum, PD 127391 (MICs, 0.031 to 0.5 microgram/ml) and sparfloxacin (MICs, 0.063 to 1 microgram/ml) were superior to erythromycin (MICs, 0.25 to 4 micrograms/ml), ciprofloxacin (MICs, 0.5 to 8 micrograms/ml), and clindamycin (MICs, 0.25 to 64 micrograms/ml. Both new quinolones were equally active against tetracycline-susceptible as well as resistant strains of M. hominis and U. urealyticum. The possible influence of medium components and/or pH on MICs was evaluated by testing a Staphylococcus aureus reference strain with each antibiotic in SP-4 broth and 10-B broth and comparing the results with published MICs for this strain. MICs determined in 10-B broth for erythromycin were affected most. This study shows that the activities of sparfloxacin and PD 127391 are similar to one another and comparable or superior to those of other drugs used to treat mycoplasmal infections. The MICs of both new quinolones were consistently 2 to several dilutions lower than those of ciprofloxacin for each species.


First in man study of [18F]fluoro-PEG-folate PET: a novel macrophage imaging technique to visualize rheumatoid arthritis.
Non-invasive imaging of arthritis activity in rheumatoid arthritis (RA) patients using macrophage PET holds promise for early diagnosis and therapeutic response monitoring. Previously obtained results with macrophage tracer (R)-[11C]PK11195 were encouraging, but the imaging signal could be further improved by reduction of background uptake. Recently, the novel macrophage tracer [18F]fluoro-PEG-folate was developed. This tracer showed excellent targeting of the folate receptor β on activated macrophages in synovial tissue in a preclinical arthritic rat model. We performed three substudies to investigate the biodistribution, potential for imaging arthritis and kinetic properties of [18F]fluoro-PEG-folate in RA patients. Firstly, biodistribution demonstrated fast clearance of [18F]fluoro-PEG-folate from heart and blood vessels and no dose limiting uptake in organs. Secondly, [18F]fluoro-PEG-folate showed uptake in arthritic joints with significantly lower background and hence significantly higher target-to-background ratios as compared to reference macrophage tracer (R)-[11C]PK11195. Lastly, dynamic scanning demonstrated fast tracer uptake in affected joints, reaching a plateau after 1 minute, co-existing with a rapid blood clearance. In conclusion, this first in man study demonstrates the potential of [18F]fluoro-PEG-folate to image arthritis activity in RA with favourable imaging characteristics of rapid clearance and low background uptake, that allow for detection of inflammatory activity in the whole body.


Loss of potentiating effect of hypoglycemia on the glucagon response to hyperaminoacidemia in IDDM.
IDDM subjects lose the ability to release glucagon during hypoglycemia. Because replacement of basal levels of amino acids enhances the glucagon response to hypoglycemia in healthy subjects, we tested whether raising amino acid levels during hypoglycemia could reverse the defective alpha-cell response in IDDM patients. For this purpose, 11 IDDM patients (HbA1 9.4 +/- 0.6%) and 8 healthy, nondiabetic subjects received two hypoglycemic insulin clamp studies (0.8 mU.kg-1 x min-1) in which plasma glucose was clamped at 55 mg/dl (3.08 mM) for 180 min. During one of the studies, an infusion of amino acids was superimposed between 120 and 180 min (0.3 g.kg-1 x h-1). This dose of amino acids had a small effect on plasma glucagon levels during euglycemic hyperinsulinemia that was comparable in normal and IDDM subjects. In healthy control subjects, plasma glucagon rose by 80% during the initial hypoglycemic phase of the study. The addition of amino acids produced a further sharp (200-250 ng/L, P < 0.02) rise in plasma glucagon, such a change did not occur in the absence of amino acids. In contrast, plasma glucagon in IDDM patients failed to increase during hypoglycemia alone and rose by only 40-50 ng/L (P < 0.05 vs. controls) when amino acid infusion was superimposed, even though plasma amino acid levels rose to the same extent in IDDM and control subjects. More importantly, the rise in glucagon produced by amino acids was comparable during hypoglycemic and euglycemic hyperinsulinemia in the IDDM patients, results strikingly different from those observed in nondiabetic control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)


Synthesis and biophysical properties of carbamate-locked nucleic acid (LNA) oligonucleotides with potential antisense applications.
Antisense oligonucleotides (ASOs) are becoming important drugs for hard to treat diseases. Modifications to their DNA backbones are essential to inhibit degradation in vivo, but they can reduce binding affinity to RNA targets. To address this problem we have combined the enzymatic resistance of carbamate (CBM) DNA backbone analogues with the thermodynamic stability conferred by locked nucleic acid sugars (LNA). Using a dinucleotide phosphoramidite strategy and automated solid phase synthesis, we have synthesised a set of oligonucleotides modified with multiple LNA-CBM units. The LNA sugars restore binding affinity to RNA targets, and in this respect LNA position with respect to the CBM linkage is important. Oligonucleotides containing carbamate flanked on its 5'and 3'-sides by LNA form stable duplexes with RNA and unstable duplexes with DNA, which is desirable for antisense applications. Carbamate-LNA modified oligonucleotides also show increased stability in the presence of snake venom and foetal bovine serum compared to LNA or CBM backbones alone.


Mechanism of the cell-penetrating peptide transportan 10 permeation of lipid bilayers.
The mechanism of the interaction between the cell-penetrating peptide transportan 10 (tp10) and phospholipid membranes was investigated. Tp10 induces graded release of the contents of phospholipid vesicles. The kinetics of peptide association with vesicles and peptide-induced dye efflux from the vesicle lumen were examined experimentally by stopped-flow fluorescence. The experimental kinetics were analyzed by directly fitting to the data the numerical solution of mathematical kinetic models. A very good global fit was obtained using a model in which tp10 binds to the membrane surface and perturbs it because of the mass imbalance thus created across the bilayer. The perturbed bilayer state allows peptide monomers to insert transiently into its hydrophobic core and cross the membrane, until the peptide mass imbalance is dissipated. In that transient state tp10 "catalyzes" dye efflux from the vesicle lumen. These conclusions are consistent with recent reports that used molecular dynamics simulations to study the interactions between peptide antimicrobials and phospholipid bilayers. A thermodynamic analysis of tp10 binding and insertion in the bilayer using water-membrane transfer hydrophobicity scales is entirely consistent with the model proposed. A small bilayer perturbation is both necessary and sufficient to achieve very good agreement with the model, indicating that the role of the lipids must be included to understand the mechanism of cell-penetrating and antimicrobial peptides.


Generalization of value in reinforcement learning by humans.
Research in decision-making has focused on the role of dopamine and its striatal targets in guiding choices via learned stimulus-reward or stimulus-response associations, behavior that is well described by reinforcement learning theories. However, basic reinforcement learning is relatively limited in scope and does not explain how learning about stimulus regularities or relations may guide decision-making. A candidate mechanism for this type of learning comes from the domain of memory, which has highlighted a role for the hippocampus in learning of stimulus-stimulus relations, typically dissociated from the role of the striatum in stimulus-response learning. Here, we used functional magnetic resonance imaging and computational model-based analyses to examine the joint contributions of these mechanisms to reinforcement learning. Humans performed a reinforcement learning task with added relational structure, modeled after tasks used to isolate hippocampal contributions to memory. On each trial participants chose one of four options, but the reward probabilities for pairs of options were correlated across trials. This (uninstructed) relationship between pairs of options potentially enabled an observer to learn about option values based on experience with the other options and to generalize across them. We observed blood oxygen level-dependent (BOLD) activity related to learning in the striatum and also in the hippocampus. By comparing a basic reinforcement learning model to one augmented to allow feedback to generalize between correlated options, we tested whether choice behavior and BOLD activity were influenced by the opportunity to generalize across correlated options. Although such generalization goes beyond standard computational accounts of reinforcement learning and striatal BOLD, both choices and striatal BOLD activity were better explained by the augmented model. Consistent with the hypothesized role for the hippocampus in this generalization, functional connectivity between the ventral striatum and hippocampus was modulated, across participants, by the ability of the augmented model to capture participants' choice. Our results thus point toward an interactive model in which striatal reinforcement learning systems may employ relational representations typically associated with the hippocampus.


End of life care for glioblastoma patients at a large academic cancer center.
Glioblastoma (GBM) is a universally fatal disease, complicated by significant cognitive and physical disabilities, inherent to the disease course. The purpose of this study was to retrospectively analyze end-of-life care for GBM patients at an academic center and compare utilization of these services to national quality of care guidelines, with the goal of identifying opportunities to improve end-of-life care. Single center retrospective cohort study of GBM patients at Johns Hopkins Hospital (JHH) between 2009 and 2014, using electronic medical records and hospice records. Comprehensive medical record review of 100 randomly selected patients with GBM, who were actively treated at JHH. Secondary analysis of all JHH GBM patients (n = 45) who received hospice care at Gilchrist Services, our largest provider, during this time period. Of 100 patients, 76 were referred to hospice. Despite the poor survival and changes in mental capacity associated with this disease, only 40% of individuals had documentation of code status and only 17% had any documentation of advance directives (ADs). None had documentation by a health care provider of a formal symptom, psychosocial, or spiritual assessment at greater than 50% of clinic visits. Only 17% used chemotherapy in their last month of life. 37% were hospitalized in the last month of life for an average of 9 days. Of the Gilchrist Services patients, the median length of stay in hospice was 21 days and 64% of these patients died in their residence with hospice services. Documentation of palliative care and end-of-life measures could improve quality of care for GBM patients, especially in the use of ADs, symptom, spiritual, and psychosocial assessments, with earlier use of hospice to prevent end-of-life hospitalizations.


Fas ligand (CD95L) and B7 expression on dendritic cells provide counter-regulatory signals for T cell survival and proliferation.
Activation of T cells is induced efficiently by dendritic cells (DC), but little is known about the role of DC in the regulation of T cell death. In this study, highly purified DC (DEC-205+, MHC class II(high), B7-1+ [CD80+], B7-2high [CD86high], CD40+, CD11c+) grown from normal mouse bone marrow in granulocyte-macrophage CSF + IL-4 were found to express FasL (CD95L) mRNA by reverse transcriptase PCR and to uniformly express FasL by both flow cytometric and immunocytochemical analyses. These cells, but not DC propagated from FasL-deficient (B6.gld) mice, induced dose-dependent increases in DNA fragmentation in Fas+ Jurkat T cells over 18 h coculture. Addition of mouse Fas-Fc fusion protein at the start of the cultures diminished this effect. Even at high relative concentrations, however, B7-2high DC induced only low levels of DNA fragmentation in Con A or alloactivated splenic T cells, as determined by radio- or spectrofluorometric assays and by in situ nick-end labeling. However, in the presence of CTLA4Ig, a molecule that blocks the B7-CD28 costimulatory pathway, DC that failed to stimulate in primary MLR induced markedly augmented levels of apoptosis in alloactivated T cells. CTLA4Ig treatment also increased the level of DNA fragmentation induced by FasL-deficient DC, indicating the existence of additional potential (Fas-independent) pathways of DC-induced T cell death. These findings suggest that the costimulatory (B7-CD28) and T cell death-inducing pathways may play important counter-regulatory roles in dictating the outcome of (allogeneic) DC-T cell interactions.


Affordable Care Act's Cadillac Tax Could Affect One-Fourth Of Workers With Employer Health Coverage By 2025.
The excise tax on high-cost health insurance plans (known as the Cadillac tax) under the Affordable Care Act (ACA) is an important part of the law's attempt to control rising health care costs. Analysts using different data sources have come to divergent estimates of how many people would be affected by this tax. We used the National Compensation Survey from the Bureau of Labor Statistics, which is better suited to this analysis because of its law-relevant details on employer-provided health benefits. Our research clarifies an important area of empirical uncertainty, thereby informing the debate about the ACA and its proposed replacements. Our base estimate of impact, 12 percent of workers participating in employer-provided health plans in 2020, lies in the middle of other estimates, but it is considerably more comprehensive, accurate, and delineated by worker characteristics (region, number of employees at the firm, industry, occupation, and so on) than others. Workers affected at the highest rate include those in education occupations and high-income workers, while those in industries involving manual labor and public safety are affected at some of the lowest rates.


Taxonomical over splitting in the Rhodnius prolixus (Insecta: Hemiptera: Reduviidae) clade: Are R. taquarussuensis (da Rosa et al., 2017) and R. neglectus (Lent, 1954) the same species?
The use of subtle features as species diagnostic traits in taxa with high morphological similarity sometimes fails in discriminating intraspecific variation from interspecific differences, leading to an incorrect species delimitation. A clear assessment of species boundaries is particularly relevant in disease vector organisms in order to understand epidemiological and evolutionary processes that affect transmission capacity. Here, we assess the validity of the recently described Rhodnius taquarussuensis (da Rosa et al., 2017) using interspecific crosses and molecular markers. We did not detect differences in hatching rates in interspecific crosses between R. taquarussuensis and R. neglectus (Lent, 1954). Furthermore, genetic divergence and species delimitation analyses show that R. taquarussuensis is not an independent lineage in the R. prolixus group. These results suggest that R. taquarussuensis is a phenotypic form of R. neglectus instead of a distinct species. We would like to stress that different sources of evidence are needed to correctly delimit species. We consider this is an important step in understanding vectorial Chagas disease spread and transmission.


Motility of cultured fish epidermal cells in the presence and absence of direct current electric fields.
The motile behavior and cytoskeletal structures of fish epidermal cells (keratocytes) in the presence and absence of direct current (DC) electric fields were examined. These cells spontaneously show highly directional locomotion in culture, migrating at rates of up to 1 micron/s. When DC electric fields between 0.5 and 15 V/cm are applied, single epidermal cells as well as cell clusters and cell sheets migrate towards the cathode. Cell clusters and sheets break apart into single migratory cells in the upper range of these field strengths. Cell shape and morphology are unaltered when the keratocytes are guided by an electric field. Neither the spontaneous locomotion nor the electrically guided motility were found to be microtubule dependent. 1 mM La3+, 10 mM Co2+, 50 microM verapamil, and 50 microM nitrendipine (calcium channel antagonists) reversibly inhibited lamellipod formation and cell locomotion in both spontaneously migrating and electrically guided cells. Ciba-Geigy Product 28392, which stimulates the opening of calcium channels, and is a competitive inhibitor of nitrendipine, has no effect on the locomotion of keratocytes. Cell motility was also unaffected by hyperpolarizing and depolarizing (low and high K+) media. It is argued that while a tissue cell may accommodate changes in resting membrane potential without becoming more or less motile, the cell may not be able to counterbalance the effects of depolarization and hyperpolarization simultaneously. In this context, a gradient of membrane potential, which is induced by an external DC electric field, will serve as a persistent stimulus for cell locomotion.


Incorporating skin care into a facial plastic surgery practice.
Starting a skin care practice takes patience and dedication, but it provides your patients with a necessary service for a comprehensive facial plastic and reconstructive surgery practice. This article discusses the differences between physician-directed skin care and spa-directed skin care and emphasizes procedures that may be performed by an aesthetician in a physician's office. Skin care practice can be classified into skin care regimens: chemical peels, microdermabrasion, and makeup. Optimal skin care regimens incorporate pharmaceutical-grade ingredients, including tretinoin, topical vitamin C, and hydroquinone. Microdermabrasion and superficial chemical peels, such as glycolic, salicylic, and trichloroacetic acid peels, are discussed. Noninvasive procedures by the physician, such as Botox and laser treatments, complement the procedures performed by the aesthetician. However, the physician is ultimately responsible for the philosophy of the skin care practice. Patient education, customer service, and skin health are key ingredients for success.


Integration and Task Allocation: Evidence from Patient Care.
Using the universe of patient transitions from inpatient hospital care to skilled nursing facilities and home health care in 2005, we show how integration eliminates task misallocation problems between organizations. We find that vertical integration allows hospitals to shift patient recovery tasks downstream to lower-cost organizations by discharging patients earlier (and in poorer health) and increasing post-hospitalization service intensity. While integration facilitates a shift in the allocation of tasks and resources, health outcomes either improved or were unaffected by integration on average. The evidence suggests that integration solves coordination problems that arise in market exchange through improvements in the allocation of tasks across care settings.


Adventitial red blood cells produce intimal platelet accumulation in cerebral arteries of cats following subarachnoid hemorrhage.
After dividing 21 cats into three equal groups, we exposed their right middle cerebral arteries transorbitally and adventitially irrigated them with 2 ml washed red blood cells, blood plasma, or saline. To determine arterial intimal platelet accumulation in each cat, we injected [111In]oxine-labeled platelets intravenously immediately before injecting the various experimental solutions. Animals were sacrificed 2 or 4 hours following the injection of labeled platelets. Irrigation with washed red blood cells produced a significantly greater intraluminal accumulation of platelets than irrigation with saline (p less than 0.05). Plasma tended to have less of an effect on platelet accumulation than washed red blood cells, but this difference was not significant. These data suggest that the adventitial blood fraction responsible for intimal platelet accumulation in cerebral arteries following subarachnoid hemorrhage may be derived mainly from the red blood cell fraction.


HIV protease inhibitors Nelfinavir and Lopinavir/Ritonavir markedly improve lung pathology in SARS-CoV-2-infected Syrian hamsters despite lack of an antiviral effect.
Nelfinavir is an HIV protease inhibitor that has been widely prescribed as a component of highly active antiretroviral therapy, and has been reported to exert in vitro antiviral activity against SARS-CoV-2. We here assessed the effect of Nelfinavir in a SARS-CoV-2 infection model in hamsters. Despite the fact that Nelfinavir, [50 mg/kg twice daily (BID) for four consecutive days], did not reduce viral RNA load and infectious virus titres in the lung of infected animals, treatment resulted in a substantial improvement of SARS-CoV-2-induced lung pathology. This was accompanied by a dense infiltration of neutrophils in the lung interstitium which was similarly observed in non-infected hamsters. Nelfinavir resulted also in a marked increase in activated neutrophils in the blood, as observed in non-infected animals. Although Nelfinavir treatment did not alter the expression of chemoattractant receptors or adhesion molecules on human neutrophils, in vitro migration of human neutrophils to the major human neutrophil attractant CXCL8 was augmented by this protease inhibitor. Nelfinavir appears to induce an immunomodulatory effect associated with increasing neutrophil number and functionality, which may be linked to the marked improvement in SARS-CoV-2 lung pathology independent of its lack of antiviral activity. Since Nelfinavir is no longer used for the treatment of HIV, we studied the effect of two other HIV protease inhibitors, namely the combination Lopinavir/Ritonavir (Kaletra™) in this model. This combination resulted in a similar protective effect as Nelfinavir against SARS-CoV2 induced lung pathology in hamsters.


Flexible polymer waveguide tunable lasers.
A flexible polymeric Bragg reflector is fabricated for the purpose of demonstrating widely tunable lasers with a compact simple structure. The external feedback of the Bragg reflected light into a superluminescent laser diode produces the lasing of a certain resonance wavelength. The highly elastic polymer device enables the direct tuning of the Bragg wavelength by controlling the imposed strain and provides a much wider tuning range than silica fiber Bragg gratings or thermo-optic tuned polymer devices. Both compressive and tensile strains are applied within the range from -36000 microepsilon to 35000 microepsilon, so as to accomplish the continuous tuning of the Bragg reflection wavelength over a range of up to 100 nm. The external feedback laser with the tunable Bragg reflector exhibits a repetitive wavelength tuning range of 80 nm with a side mode suppression ratio of 35 dB.


Early signal transduction in measles virus-infected lymphocytes is unaltered, but second messengers activate virus replication.
In order to understand measles virus-lymphocyte interactions, we have started to analyze factors and events which regulate measles virus infection in peripheral blood mononuclear cells (PBMC). We analyzed the initiation of cell proliferation, induced by phytohemagglutinin, in infected and control PBMC by measuring intracellular free Ca2+ by using fura-2. Measles virus-infected and control PBMC responded similarly with an increase in the amount of cytosolic free Ca2+, indicating that the early activation events are not affected and are not involved in immunosuppression. The activation signals, Ca2+ and protein kinase C, induced specifically and independently by Ca ionophore A23187 or 12-O-tetradecanoylphorbol-13-acetate (TPA), changed the restricted measles virus infection to a productive one. The combination of TPA and A23187 was the most potent activator of measles virus replication. TPA and A23187 operate through different activation mechanisms, and it is evident that measles virus replication depends on the activation of cellular signal pathways. Depletion of adherent cells enhanced virus replication, especially at the early stage of infection, indicating the inhibitory role of monocytes. Monocytes were strongly infected, but they supported complete measles virus replication only at a very low level, and virus replication could not be enhanced with TPA and/or A23187.


Immune Effector Functions of Human IgG2 Antibodies against EGFR.
Three FDA-approved epidermal growth factor receptor (EGFR) antibodies (cetuximab, panitumumab, necitumumab) are clinically available to treat patients with different types of cancers. Interestingly, panitumumab is of human IgG2 isotype, which is often considered to have limited immune effector functions. Unexpectedly, our studies unraveled that human IgG2 antibodies against EGFR mediated effective CDC when combined with another noncross-blocking EGFR antibody. This second antibody could be of human IgG1 or IgG2 isotype. Furthermore, EGFR antibodies of human IgG2 isotype were highly potent in recruiting myeloid effector cells such as M1 macrophages and PMN for tumor cell killing by ADCC. Tumor cell killing by PMN was more effective with IgG2 than with IgG1 antibodies if tumor cells expressed lower levels of EGFR. Additionally, lower expression levels of the "don't eat me" molecule CD47 on tumor cells enabled ADCC also by M2 macrophages, and improved PMN and macrophage-mediated ADCC. A TCGA enquiry revealed broadly varying CD47 expression levels across different solid tumor types. Together, these results demonstrate that human IgG2 antibodies against EGFR can promote significant Fc-mediated effector functions, which may contribute to their clinical efficacy. The future challenge will be to identify clinical situations in which myeloid effector cells can optimally contribute to antibody efficacy.


Radiation risk to children from computed tomography.
Imaging studies that use ionizing radiation are an essential tool for the evaluation of many disorders of childhood. Ionizing radiation is used in radiography, fluoroscopy, angiography, and computed tomography scanning. Computed tomography is of particular interest because of its relatively high radiation dose and wide use. Consensus statements on radiation risk suggest that it is reasonable to act on the assumption that low-level radiation may have a small risk of causing cancer. The medical community should seek ways to decrease radiation exposure by using radiation doses as low as reasonably achievable and by performing these studies only when necessary. There is wide agreement that the benefits of an indicated computed tomography scan far outweigh the risks. Pediatric health care professionals' roles in the use of computed tomography on children include deciding when a computed tomography scan is necessary and discussing the risk with patients and families. Radiologists should be a source of consultation when forming imaging strategies and should create specific protocols with scanning techniques optimized for pediatric patients. Families and patients should be encouraged to ask questions about the risks and benefits of computed tomography scanning. The information in this report is provided to aid in decision-making and discussions with the health care team, patients, and families.


Grey eosinophils in sighthounds: frequency in 3 breeds and comparison of eosinophil counts determined manually and with 2 hematology analyzers.
Grey eosinophils (GE) reported to occur in Greyhounds, and occasionally in other breeds, have clear granules, probably due to abnormal staining properties. The aims of this study were to investigate the frequency of GE in Greyhounds and 2 other sighthound breeds, and to assess the capacity of the ADVIA 120 and Sysmex XT-2000iV hematology analyzers to correctly identify GE. Blood samples from 20 Greyhounds, 29 Italian Greyhounds, and 24 Whippets were analyzed using the ADVIA and Sysmex hematology analyzers, and blood smears stained with May-Grünwald Giemsa were evaluated microscopically. The frequency of samples with GE detected on smears was recorded for each breed. Manual and automated eosinophil counts were compared using a Wilcoxon signed-rank test. Agreement between methods was assessed using Passing-Bablok and Bland-Altman plots. GE were detected in all 3 breeds: 9/20 Greyhounds (45.0%), 10/29 Italian Greyhounds (34.5%), and 5/24 Whippets (62.5%) with no significant differences in the frequency of GE among the breeds. In samples containing GE, both analyzers underestimated the percentage of eosinophils and occasionally eosinophils were not detected at all. When a novel "GE gate" was used, the percentage of eosinophils reported by the Sysmex was similar to that obtained by manual counting. GE are found in the blood of sighthounds other than Greyhounds. Hematology analyzers may underestimate the percentage of GE, probably due to their abnormal physical or chemical features. Underestimation is slight and usually clinically insignificant, but occasionally eosinophils are completely misclassified. Using the Sysmex analyzer, a GE gate can be designed to normalize the eosinophil count.


Polyphenol intake and metabolic syndrome risk in European adolescents: the HELENA study.
The role of polyphenol intake during adolescence to prevent metabolic syndrome (MetS) is little explored. This study aimed to evaluate the association between intake of total polyphenols, polyphenol classes and the 10 most consumed individual polyphenols with MetS risk in European adolescents. Of the cross-sectional HELENA study, 657 adolescents (54% girls; 14.8% overweight; 12.5-17.5 year) had a fasting blood sample and polyphenol intake data from two non-consecutive 24-h recalls matched with the Phenol-Explorer database. MetS was defined via the pediatric American Heart Association definition. Multilevel linear regressions examined the associations of polyphenol quartiles with MetS components, while logistic regression examined the associations with MetS risk. After adjusting for all potential confounders (socio-demographics and nine nutrients), total polyphenol intake, polyphenol classes and individual polyphenols were not associated with MetS risk. From all MetS components, only BMI z-score was modestly inversely associated with total polyphenol intake. Further sub analyses on polyphenol classes revealed that flavonoid intake was significantly associated with higher diastolic blood pressure and lower BMI, and phenolic acid intake was associated with higher low-density cholesterol. For individual polyphenols, the above BMI findings were often confirmed (not independent from dietary intake) and a few associations were found with insulin resistance. Higher intakes of total polyphenols and flavonoids were inversely associated with BMI. No consistent associations were found for other MetS components.


New isoform-specific monoclonal antibodies reveal different sub-cellular localisations for talin1 and talin2.
Talins are adaptor proteins that connect the integrin family of cell adhesion receptors to cytoskeletal actin. Vertebrates express two closely related talins encoded by separate genes, and while it is well established that talin1 plays a key role in cell adhesion and spreading, little is known about the role of talin2. To facilitate such studies, we report the characterisation of 4 new isoform-specific talin mouse monoclonal antibodies that work in Western blotting, immuno-precipitation, immuno-fluorescence and immuno-histochemistry. Using these antibodies, we show that talin1 and talin2 do not form heterodimers, and that they are differentially localised within the cell. Talin1 was concentrated in peripheral focal adhesions while talin2 was observed in both focal and fibrillar adhesions, and knock-down of talin2 compromised fibronectin fibrillogenesis. Although differentiated human macrophages express both isoforms, only talin1 showed discrete staining and was localised to the ring structure of podosomes. However, siRNA-mediated knock-down of macrophage talin2 led to a significant reduction in podosomal matrix degradation. We have also used the antibodies to localise each isoform in tissue sections using both cryostat and paraffin-embedded material. In skeletal muscle talin2 was localised to both myotendinous junctions and costameres while talin1 was restricted to the former structure. In contrast, both isoforms co-localised in kidney with staining of the glomerulus, and the tubular epithelial and interstitial cells of the cortex and medulla. We anticipate that these antibodies will form a valuable resource for future studies on the function of the two major talin isoforms.


Testing the habituation assumption underlying models of parasitoid foraging behavior.
Habituation, a form of non-associative learning, has several well-defined characteristics that apply to a wide range of physiological and behavioral responses in many organisms. In classic patch time allocation models, habituation is considered to be a major mechanistic component of parasitoid behavioral strategies. However, parasitoid behavioral responses to host cues have not previously been tested for the known, specific characteristics of habituation. In the laboratory, we tested whether the foraging behavior of the egg parasitoid Trissolcus basalis shows specific characteristics of habituation in response to consecutive encounters with patches of host (Nezara viridula) chemical contact cues (footprints), in particular: (i) a training interval-dependent decline in response intensity, and (ii) a training interval-dependent recovery of the response. As would be expected of a habituated response, wasps trained at higher frequencies decreased their behavioral response to host footprints more quickly and to a greater degree than those trained at low frequencies, and subsequently showed a more rapid, although partial, recovery of their behavioral response to host footprints. This putative habituation learning could not be blocked by cold anesthesia, ingestion of an ATPase inhibitor, or ingestion of a protein synthesis inhibitor. Our study provides support for the assumption that diminishing responses of parasitoids to chemical indicators of host presence constitutes habituation as opposed to sensory fatigue, and provides a preliminary basis for exploring the underlying mechanisms.


Trichain cationic lipids: the potential of their lipoplexes for gene delivery.
Lipoplexes (LDs) have been prepared from DNA and positively charged vesicles composed of the helper lipid, dioleoyl l-α-phosphatidylethanolamine (DOPE) and either a dichain (DC) oxyethylated cationic lipid or their corresponding novel trichain (TC) counterpart. This is the first study using the TC lipids for the preparation of LDs and their application. Here the results of biophysical experiments characterising the LDs have been correlated with the in vitro transfection activity of the complexes. Photon correlation spectroscopy, zeta potential measurements and transmission electron microscopy studies indicated that, regardless of the presence of a third chain, there were little differences between the size and charge of the TC and DC containing LDs. Small angle neutron scattering studies established however that there was a significant conformational re-arrangement of the lipid bilayer when in the form of a LD complex as opposed to the parent vesicles. This re-arrangement was particularly noticeable in LDs containing TC lipids possessing a third chain of C12 or a longer chain. These results suggested that the presence of a third hydrophobic chain had a significant effect on lipid packing in the presence of DNA. Picogreen fluorescence and gel electrophoresis studies showed that the TC lipids containing a third acyl chain of at least C12 were most effective at complexing DNA while the TC lipids containing an octanoyl chain and the DC lipids were least effective. The transfection efficacies of the TC lipids in the form of LDs were found to be higher than for the DC analogues, particularly when the third acyl chain was an octanoyl or oleoyl moeity. Little or no increase in transfection efficiency was observed when the third chain was a methyl, acetyl or dodecanoyl group. The large enhancement in transfection performance of the TC lipids can be attributed to their ability to complex their DNA payload. These studies indicate that presence of a medium or long third acyl chain was especially beneficial for transfection.


Antiviral activity of formyl peptide receptor 2 antagonists against influenza viruses.
Influenza viruses are one of the most important respiratory pathogens worldwide, causing both epidemic and pandemic infections. The aim of the study was to evaluate the effect of FPR2 antagonists PBP10 and BOC2 on influenza virus replication. We determined that these molecules exhibit antiviral effects against influenza A (H1N1, H3N2, H6N2) and B viruses. FPR2 antagonists used in combination with oseltamivir showed additive antiviral effects. Mechanistically, the antiviral effect of PBP10 and BOC2 is mediated through early inhibition of virus-induced ERK activation. Finally, our preclinical studies showed that FPR2 antagonists protected mice from lethal infections induced by influenza, both in a prophylactic and therapeutic manner. Thus, FPR2 antagonists might be explored for novel treatments against influenza.


An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart.
Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.


Mitochondrial haplogroups associated with elite Japanese athlete status.
It has been hypothesised that certain mitochondrial haplogroups, which are defined by the presence of a characteristic cluster of tightly linked mitochondrial DNA polymorphisms, would be associated with elite Japanese athlete status. To examine this hypothesis, the frequencies of mitochondrial haplogroups found in elite Japanese athletes were compared with those in the general Japanese population. Subjects comprised 139 Olympic athletes (79 endurance/middle-power athletes (EMA), 60 sprint/power athletes (SPA)) and 672 controls (CON). Two mitochondrial DNA fragments containing the hypervariable sequence I (m16024-m16383) of the major non-coding region and the polymorphic site at m.5178C>A within the NADH dehydrogenase subunit 2 gene were sequenced, and subjects were classified into 12 major mitochondrial haplogroups (ie, F, B, A, N9a, N9b, M7a, M7b, M*, G2, G1, D5 or D4). The mitochondrial haplogroup frequency differences among EMA, SPA and CON were then examined. EMA showed an excess of haplogroup G1 (OR 2.52, 95% CI 1.05 to 6.02, p=0.032), with 8.9% compared with 3.7% in CON, whereas SPA displayed a greater proportion of haplogroup F (OR 2.79, 95% CI 1.28 to 6.07, p=0.007), with 15.0% compared with 6.0% in CON. The results suggest that mitochondrial haplogroups G1 and F are associated with elite EMA and SPA status in Japanese athletes, respectively.


Species-Specific Differences in the in Vitro Metabolism of Lasiocarpine.
There are species-related differences in the toxicity of pyrrolizidine alkaloids (PAs) partly attributable to the hepatic metabolism of these alkaloids. In this study, the metabolism of lasiocarpine, a potent hepatotoxic and carcinogenic food contaminant, was examined in vitro with human, pig, rat, mouse, rabbit, and sheep liver microsomes. A total of 12 metabolites (M1-M12) were detected with the human liver microsomes, of which M1, M2, M4, and M6 were unstable in the presence of reduced glutathione (GSH). With the exception of M3 and M8, the formation of all metabolites of lasiocarpine was catalyzed by CYP3A4 in humans. Tandem mass spectra (MS/MS) detected several new metabolites, termed M4-M7; their toxicological significance is unknown. M9 (m/z 398), identified as a demethylation product, was the main metabolite in all species, although the relative dominance of this metabolite was lower in humans. The level of the reactive metabolites, as measured by M1 ((3H-pyrrolizin-7-yl)methanol) and the GSH conjugate, was higher with the liver microsomes of susceptible species (human, pig, rat, and mouse) than with the species (rabbit and sheep) resistant to PA intoxication. In general, in addition to the new metabolites (M4-M7) that could make humans more susceptible to lasiocarpine-induced toxicity, the overall metabolite fingerprint detected with the human liver microsomes differed from that of all other species, yielding high levels of GSH-reactive metabolites.


Pro-inflammatory cytokines predominate in the brains of dogs with visceral leishmaniasis: a natural model of neuroinflammation during systemic parasitic infection.
Visceral leishmaniasis is a multisystemic zoonotic disease that can manifest with several symptoms, including neurological disorders. To investigate the pathogenesis of brain alterations occurring during visceral leishmaniasis infection, the expression of the cytokines IL-1β, IL-6, IL-10, IL-12p40, IFN-γ, TGF-β and TNF-α and their correlations with peripheral parasite load were evaluated in the brains of dogs naturally infected with Leishmania infantum. IL-1β, IFN-γ and TNF-α were noticeably up-regulated, and IL-10, TGF-β and IL-12p40 were down-regulated in the brains of infected dogs. Expression levels did not correlate with parasite load suggestive that the brain alterations are due to the host's immune response regardless of the phase of the disease. These data indicate the presence of a pro-inflammatory status in the nervous milieu of dogs with visceral leishmaniasis especially because IL-1β and TNF-α are considered key factors for the initiation, maintenance and persistence of inflammation.


Measuring the focal length of optical systems by grating shearing interferometry.
Using grating shearing interferometry, a new and simple technique to measure the effective focal length of optical systems is described. The diffraction pattern of a phase grating positioned at the focal point of the lens under test is evaluated for this purpose. The relative lateral shift between the undiffracted zero order and the diffracted first orders caused by the grating is measured. By utilizing knowledge of the wavelength of light, the grating period, and the diameter of an aperture stop placed in front of the test lens, we can determine the effective focal length of the test lens. Results of measurements are presented and compared with calculated values. The dependence of the focal length on the wavelength of the light is shown by using two laser sources of different wavelengths. An analysis of the measurement accuracy is given.


Organizational climate, staffing, and safety equipment as predictors of needlestick injuries and near-misses in hospital nurses.
Recently passed federal legislation requires institutions to adopt safety equipment to prevent needlesticks, but there is little empirical evidence of the effectiveness of specific types of safety devices or the contribution of safety devices to reducing needlesticks relative to the contributions of staffing, organizational climate, and clinicians' experience. In 1998, 2287 medical-surgical unit nurses in 22 US hospitals were surveyed in regard to staffing and organizational climate in their hospitals and about patient and nurse outcomes, including needlestick injuries. Hospitals provided information about available protective devices at the time of the survey. Relationships between nurse and hospital characteristics and protective equipment and the likelihood of needlestick injuries and near-miss incidents were examined. Poor organizational climate and high workloads were associated with 50% to 2-fold increases in the likelihood of needlestick injuries and near-misses to hospital nurses. Capless-valve secondary intravenous set systems and use of any type of protective equipment for IV starts or blood draws were associated with 20% to 30% lowered risks of both event types. Nurse staffing and organizational climate are key determinants of needlestick risk and must be considered with the adoption of safety equipment to effectively reduce sharps injuries.


Detection of HSV-2 antigen in carcinoma cervix and premalignant conditions by immuno-cytochemistry.
One hundred and twelve cases of cervical pathology (58 paraffin sections and 54 cervical smears) were assessed by PAP technique and 30 cases by indirect immunofluorescent technique. Forty-two normal cervical smears from the age matched controls were stained by indirect immuno-fluorescent technique. HSV-2 antigen was detected by PAP method in 86 out of 112 cases (78.57%) i.e. 50/55 squamous cell carcinoma, 13/13 carcinoma in situ, 11/15 severe dysplasia, 3/4 moderate dysplasia and 9/16 mild dysplasia. The amount of antigen was maximum in squamous cell carcinoma and decreased in carcinoma in situ, severe, moderate and mild dysplasia in descending order. Three cases of adenocarcinoma cervix were negative. Only one case out of 42 controls was positive. HSV-2 antigen was detected by an indirect IF technique in 8/9 squamous cell carcinoma, 2/3 carcinoma in situ, 3/7 dysplasia, O/1 adenocarcinoma and 4/10 inflammatory cases. The above findings support the association between HSV-2 and squamous cell carcinoma cervix, as well as carcinoma in situ which is statistically significant.


Intersectin links WNK kinases to endocytosis of ROMK1.
With-no-lysine (WNK) kinases are a novel family of protein kinases characterized by an atypical placement of the catalytic lysine. Mutations of 2 family members, WNK1 and WNK4, cause pseudohypoaldosteronism type 2 (PHA2), an autosomal-dominant disease characterized by hypertension and hyperkalemia. WNK1 and WNK4 stimulate clathrin-dependent endocytosis of renal outer medullar potassium 1 (ROMK1), and PHA2-causing mutations of WNK4 increase the endocytosis. How WNKs stimulate endocytosis of ROMK1 and how mutations of WNK4 increase the endocytosis are unknown. Intersectin (ITSN) is a multimodular endocytic scaffold protein. Here we show that WNK1 and WNK4 interacted with ITSN and that the interactions were crucial for stimulation of endocytosis of ROMK1 by WNKs. The stimulation of endocytosis of ROMK1 by WNK1 and WNK4 required specific proline-rich motifs of WNKs, but did not require their kinase activity. WNK4 interacted with ROMK1 as well as with ITSN. Disease-causing WNK4 mutations enhanced interactions of WNK4 with ITSN and ROMK1, leading to increased endocytosis of ROMK1. These results provide a molecular mechanism for stimulation of endocytosis of ROMK1 by WNK kinases.