Proteomic comparison of human and great ape blood plasma reveals conserved glycosylation and differences in thyroid hormone metabolism.
Most blood plasma proteins are glycosylated. These glycoproteins typically carry sialic acid-bearing sugar chains, which can modify the observed molecular weights and isoelectric points of those proteins during electrophoretic analyses. To explore changes in protein expression and glycosylation that occurred during great ape and human evolution, we subjected multiple blood plasma samples from all these species to high-resolution proteomic analysis. We found very few species-specific differences, indicating a remarkable degree of conservation of plasma protein expression and glycosylation during approximately 12 million years of evolution. A few lineage-specific differences in protein migration were noted among the great apes. The only obvious differences between humans and all great apes were an apparent decrease in transthyretin (prealbumin) and a change in haptoglobin isoforms (the latter was predictable from prior genetic studies). Quantitative studies of transthyretin in samples of blood plasma (synthesized primarily by the liver) and of cerebrospinal fluid (synthesized locally by the choroid plexus of the brain) confirmed approximately 2-fold higher levels in chimpanzees compared to humans. Since transthyretin binds thyroid hormones, we next compared plasma thyroid hormone parameters between humans and chimpanzees. The results indicate significant differences in the status of thyroid hormone metabolism, which represent the first known endocrine difference between these species. Notably, thyroid hormones are known to play major roles in the development, differentiation, and metabolism of many organs and tissues, including the brain and the cranium. Also, transthyretin is known to be the major carrier of thyroid hormone in the cerebrospinal fluid, likely regulating delivery of this hormone to the brain. A potential secondary difference in retinoid (vitamin A) metabolism is also noted. The implications of these findings for explaining unique features of human evolution are discussed.


Results of a phase II upfront window of pharmacokinetically guided topotecan in high-risk medulloblastoma and supratentorial primitive neuroectodermal tumor.
To assess the antitumor efficacy of pharmacokinetically guided topotecan dosing in previously untreated patients with medulloblastoma and supratentorial primitive neuroectodermal tumors, and to evaluate plasma and CSF disposition of topotecan in these patients. After maximal surgical resection, 44 children with previously untreated high-risk medulloblastoma were enrolled, of which 36 were assessable for response. The topotecan window consisted of two cycles, administered initially as a 30-minute infusion daily for 5 days, lasting 6 weeks. Pharmacokinetic studies were conducted on day 1 to attain a topotecan lactone area under the plasma concentration-time curve (AUC) of 120 to 160 ng/mL.h. After 10 patients were enrolled, the infusion was modified to 4 hours, with dosage individualization. Of 36 assessable patients, four patients (11.1%) had a complete response and six (16.6%) showed a partial response, and disease was stable in 17 patients (47.2%). Toxicity was mostly hematologic, with only one patient experiencing treatment delay. The target plasma AUC was achieved in 24 of 32 studies (75%) in the 30-minute infusion group, and in 58 of 93 studies (62%) in the 4-hour infusion group. The desired CSF topotecan exposure was achieved in seven of eight pharmacokinetic studies when the topotecan plasma AUC was within target range. Topotecan is an effective agent against pediatric medulloblastoma in patients who have received no therapy other than surgery. Pharmacokinetically guided dosing achieved the target plasma AUC in the majority of patients. This drug warrants testing as part of standard postradiation chemotherapeutic regimens. Furthermore, these results emphasize the importance of translational research in drug development, which in this case identified an effective drug.


Noise performance of phase-insensitive frequency multicasting in parametric mixer with finite dispersion.
Noise performance of dual-pump, multi-sideband parametric mixer operated in phase-insensitive mode is investigated theoretically and experimentally. It is shown that, in case when a large number of multicasting idlers are generated, the noise performance is strictly dictated by the dispersion characteristics of the mixer. We find that the sideband noise performance is significantly degraded in anomalous dispersion region permitting nonlinear noise amplification. In contrast, in normal dispersion region, the noise performance converges to the level of four-sideband parametric process, rather than deteriorates with increased sideband creation. Low noise generation mandates precise dispersion-induced phase mismatch among pump and sideband waves in order to control the noise coupling. We measure the noise performance improvement for a many-sideband, multi-stage mixer by incorporating new design technique.


Reliability of ratings of perceived effort regulation of exercise intensity.
This study assessed the reliability of ratings of perceived exertion (RPE) for the prescription of exercise intensity during cycling. Ten healthy men (21 to 62 years) and six women (25 to 50 years) attended the laboratory four times, 5 to 7 days apart. On the first visit each subject's maximal oxygen uptake was measured and, subsequently, they cycled at constant work rates based on their perception of ratings 9, 13 and 17 of the Borg 6-20 scale by regulating the resistance of the ergometer without reference to the instrument display panel. Analysis of variance revealed no significant between-trial differences with regard to oxygen uptake (VO2) or heart rate for men or women. The relative exercise intensities corresponding to the 3 ratings of exertion did not differ between men and women. Between-trial correlations for VO2 were highest for visits two and three at RPE levels 9 and 13 (r = 0.83 and r = 0.94) and consistently high (r = 0.92 or greater) for the three trials at RPE17. These data suggest that RPE is a useful frame of reference for the regulation of high levels of exercise intensity in healthy men and women. Small amounts of practice with the scale improve its applicability at lower exercise levels.


Selective induction of interleukin-12 in reconstructed human epidermis by chemical allergens.
Keratinocytes play an important role in skin inflammatory and immunological reactions through the release of cytokines and response to them. These cells have been shown to direct T-cell priming by producing cytokines such as interleukin (IL)-10 and IL-12. The purpose of this work was to explore the potential use of IL-12 production to discriminate between skin irritants and contact allergens in vitro. Initially, a reconstituted human epidermis was treated with a known human skin irritant, sodium lauryl sulphate (SLS), and a known human contact allergen, 1-chloro-2,4-dinitrobenzene (DNCB). The expression of IL-12p40 was assessed at specific time intervals by the semi-quantitative reverse transcriptase-polymerase chain reaction (rt-PCR). The data obtained indicated that only DNCB induced an up-regulation of IL-12p40. This up-regulation occurred after exposure to DNCB for 3 hours. Importantly, the application of SLS or vehicles did not induce IL-12 mRNA up-regulation. An increase in total IL-12 protein content was detected in supernatants of allergen-stimulated, but not vehicle-stimulated, reconstituted epidermis. To confirm these results, the effects of benzalkonium chloride, oxazolone and eugenol were assessed. At concentrations that resulted in equivalent IL-1α release, only contact allergens increased IL-12 expression, which confirmed the previous results. These data suggest that IL-12, which is crucial for T-helper type 1 cell responses, could be a useful marker for discriminating between contact allergens and irritants.


Factors Influencing Non-sentinel Node Metastasis in Patients with Macrometastatic Sentinel Lymph Node Involvement and Validation of Three Commonly Used Nomograms.
Omitting axillary lymph node dissection (ALND) in a subgroup of patients with sentinel lymph node (SLN) metastasis is becoming a widely accepted practice. Avoiding the well-known complications of ALND is the sole aim without compromising the curative intention of surgery. The data were probed for breast cancer patients that were operated on between February 2014 and June 2016. SLN biopsies were performed in 507 patients and out of 157 patients who underwent ALND for a metastatic SLN, 151 were found eligible for the analyses as having macrometastatic (>2mm) SLN. MD Anderson, Memorial Sloan Kettering Cancer Center and Helsinki nomograms were also tested in our patient population. Pathologic tumor size greater than 2 cm, the ratio of metastatic SLN to dissected SLN, metastatic tumor greater than 1 cm and tumors that extended outside the SLN's capsule were found to be associated with non-sentinel node metastasis in both univariate and multivariate tests. MD Anderson nomogram performed well with an area under the curve (AUC) value of 0.72. Our results suggest that ALND should be considered in patients with macrometastatic SLN greater than 10 mm in size, have extracapsular extension, have metastatic SLNs at a rate of more than 50% and whose primary tumor is greater than 2 cm.


Gamma-aminobutyric acid and homovanillic acid concentration in the CSF of patients with senile dementia of Alzheimer's type.
The investigation of enzyme and neurotransmitter levels and/or their metabolites in the CSF of patients with senile dementia of Alzheimer's type (SDAT) could become a promising approach for a clinical research and diagnostic procedure. To learn more about the metabolic reflections of central metabolism in the CSF of patients with SDAT, we measured CSF levels of gamma-aminobutyric acid (GABA) and homovanillic acid (HVA), the dopamine metabolite. In 16 female patients with SDAT and in eight matched control patients, CSF GABA levels were measured by ion exchange with fluorimetric detection, and HVA levels were measured fluorimetrically. The GABA content of the CSF was significantly reduced in patients with SDAT, whereas the HVA level was unchanged.


Altered explorative strategies and reactive coping style in the FSL rat model of depression.
Modeling depression in animals is based on the observation of behaviors interpreted as analog to human symptoms. Typical tests used in experimental depression research are designed to evoke an either-or outcome. It is known that explorative and coping strategies are relevant for depression, however these aspects are generally not considered in animal behavioral testing. Here we investigate the Flinders Sensitive Line (FSL), a rat model of depression, compared to the Sprague-Dawley (SD) rat in three independent tests where the animals are allowed to express a more extensive behavioral repertoire. The multivariate concentric square field™ (MCSF) and the novel cage tests evoke exploratory behaviors in a novel environment and the home cage change test evokes social behaviors in the re-establishment of a social hierarchy. In the MCSF test, FSL rats exhibited less exploratory drive and more risk-assessment behavior compared to SD rats. When re-exposed to the arena, FSL, but not SD rats, increased their exploratory behavior compared to the first trial and displayed risk-assessment behavior to the same extent as SD rats. Thus, the behavior of FSL rats was more similar to that of SDs when the rats were familiar with the arena. In the novel cage test FSL rats exhibited a reactive coping style, consistent with the reduced exploration observed in the MCSF. Reactive coping is associated with less aggressive behavior. Accordingly, FSL rats displayed less aggressive behavior in the home cage change test. Taken together, our data show that FSL rats express altered exploratory behavior and reactive coping style. Reduced interest is a core symptom of depression, and individuals with a reactive coping style are more vulnerable to the disease. Our results support the use of FSL rats as an animal model of depression and increase our understanding of the FSL rat beyond the behavioral dimensions targeted by the traditional depression-related tests.


Deepening our understanding of immune sentinels in the skin.
Advances in our understanding of the skin immune system have a major impact on studies of skin autoimmunity, graft-versus-host disease, inflammation, and cancer as well as on the development of novel vaccines and immunotherapy approaches. In this issue of the JCI, Zaba et al. carefully dissected the complex network of DCs and macrophages residing in normal human skin and defined novel phenotypic markers for these immunocytes (see the related article beginning on page 2517). These studies provide the basis for better insight into the role of important immune sentinels contributing to the maintenance of skin tissue homeostasis and lay the foundation for future studies of the skin immune system.


Prognostic factors in metastatic neuroblastoma. A multivariate analysis of 182 cases.
The purpose of our study was to investigate the interrelationship of known and possible new risk factors in patients with metastatic neuroblastoma and to define groups at risk. The possible influence of 37 variables on event-free survival (EFS) was analyzed univariately in 308 consecutive patients using the Kaplan-Meier estimate. Fifteen factors were identified (p less than 0.05, logrank greater than 3.84) of whom eight showed a nonrandom correlation to several others (chi 2-test, p less than 0.05). Seven noncorrelated factors [lactate dehydrogenase (LDH) level, resectability of the primary tumor, histologic grade, leukopenia, presence of symptoms, general condition, and age at diagnosis] were included in the multivariate analysis of 182 patients according to the Cox model. The variables LDH (p = 0.0007), resectability (p = 0.0063), histologic grade (p = 0.0055), and leukopenia (p = 0.0470) were identified multivariately as prognostic factors for EFS. These results permitted the classification of patients into three prognostic groups. The 6 year event-free survival for group IV-A (LDH normal) was 0.37 +/- 0.12, for group IV-B (LDH abnormal, additional risk factors favorable) 0.18 +/- 0.10, and for group IV-C (LDH abnormal, 1-3 additional risk factors unfavorable) was 0.08 +/- 0.03. We conclude that the proposed clinicopathological classification may prove to be a reliable and easily applicable tool for estimating the outcome of metastatic neuroblastoma in children.


Diffractive optical elements designed for highly precise far-field generation in the presence of artifacts typical for pixelated spatial light modulators.
Diffractive optical elements (DOEs) realized by spatial light modulators (SLMs) often have features that distinguish them from most conventional, static DOEs: strong coupling between phase and amplitude modulation, a modulation versus steering parameter characteristic that may not be precisely known (and may vary with, e.g., temperature), and deadspace effects and interpixel cross talk. For an optimal function of the DOE, e.g. as a multiple-beam splitter, the DOE design must account for these artifacts. We present an iterative design method in which the optimal setting of each SLM pixel is carefully chosen by considering the SLM artifacts and the design targets. For instance, the deadspace-interpixel effects are modeled by dividing the pixel to be optimized, and its nearest neighbors, into a number of subareas, each with its unique response and far-field contribution. Besides the customary intensity control, the design targets can also include phase control of the optical field in one or more of the beams in the beam splitter. We show how this can be used to cancel a strong unwanted zeroth-order beam, which results from using a slightly incorrect modulation characteristic for the SLM, by purposely sending a beam in the same direction but with the opposite phase. All the designs have been implemented on the 256 x 256 central pixels of a reflective liquid crystal on silicon SLM with a selected input polarization state and a direction of transmission axis of the output polarizer such that for the available different pixel settings a phase modulation of ~2pi rad could be obtained, accompanied by an intensity modulation depth as high as >95%.


Cellular responses evoked by different surface characteristics of intraosseous titanium implants.
The properties of biomaterials, including their surface microstructural topography and their surface chemistry or surface energy/wettability, affect cellular responses such as cell adhesion, proliferation, and migration. The nanotopography of moderately rough implant surfaces enhances the production of biological mediators in the peri-implant microenvironment with consequent recruitment of differentiating osteogenic cells to the implant surface and stimulates osteogenic maturation. Implant surfaces with moderately rough topography and with high surface energy promote osteogenesis, increase the ratio of bone-to-implant contact, and increase the bonding strength of the bone to the implant at the interface. Certain features of implant surface chemistry are also important in enhancing peri-implant bone wound healing. It is the purpose of this paper to review some of the more important features of titanium implant surfaces which have an impact on osseointegration.


Engineered resistance in Aedes aegypti to a West African and a South American strain of yellow fever virus.
Double subgenomic Sindbis (dsSIN) viruses were engineered to transduce mosquito cells with antisense RNA derived either from the premembrane (prM) or polymerase (NS5) coding regions of the 17D vaccine strain of yellow fever virus (YFV). Aedes albopictus C6/36 cells were infected at high multiplicities of infection (MOI) with each dsSIN virus. Forty-eight hours later, the transduced cells were challenged with an MOI of 0.1 of the Asibi strain of YFV. At 72-hr postchallenge, the cells were assayed by immunofluorescence for the presence of YFV antigen. Cells transduced with prM or NS5 antisense RNAs derived from the YFV genome displayed no YFV-specific antigens. In contrast, cells infected with control dsSIN viruses that expressed no antisense RNA or dengue virus-derived antisense RNAs were permissive for the challenge virus. To analyze resistance in the mosquito, five log10 50% tissue culture infective doses (TCID50) of each dsSIN virus and three log10TCID50 of either a West African (BA-55) or South American (1899/81) strain of wild-type YFV were coinoculated into Ae. aegypti. Mosquitoes transduced with effector RNAs targeting the prM or NS5 gene regions did not transmit West African YFV and poorly transmitted the South American strain of YFV.


Similar efficacy of broad-range ITS PCR and conventional fungal culture for diagnosing fungal infections in non-immunocompromised patients.
Broad-range fungal inter spacer region (ITS) polymerase chain reaction (PCR) has been evaluated for the detection and identification of fungi in clinical specimens from severely immunocompromised patients, but not in non-selected patients. Thus, the aim of this study was to compare the diagnostic performance of ITS PCR with that of fungal culture and to investigate its clinical impact on the diagnosis of fungal infections in non-immunocompromised patients. The corresponding patients' data were retrieved by detailed medical chart reviews. Results from 251 specimens showed a high concordance of 89.6 % for ITS PCR and fungal culture. The analytical sensitivity and specificity of ITS PCR considering culture as gold standard were 87.7 and 90.3 %, respectively, the positive and negative predictive value (PPV and NPV) were 76 and 95.5 %, respectively. Assessing the clinical probability of a fungal infection based on detailed chart reviews, PCR had a clinical sensitivity of 88.9 %, a specificity of 86.3 %, a PPV of 64.0 % and a NPV of 96.6 %. The overall performance of fungal broad-range PCR was similar to that of culture. Our data show that, in non-selected and non-immunocompromised patients, the performance of ITS PCR is similar to that of culture for detecting fungal infections, not the least because sensitivity of culture in patients under antifungal treatment is surprisingly high. Compared to culture, PCR has the advantage of a rapid time-to-result (approximately two working days), proper identification of rare pathogens, prompt initiation of a species-targeted antifungal treatment, and prospects for automation.


Immunohistochemical demonstration of leucocyte elastase in human tissues.
A polyclonal antiserum, produced in sheep and reactive against purified human leucocyte elastase, has been applied to paraffin sections from a range of human tissues by means of the indirect immunoperoxidase method. Striking and consistent results have been obtained. Normal or inflammatory granulocytes were intensely positive, the reaction being "blocked" by pure elastase. Activity was not seen in other sites, including histiocytic reticulum and lymphoid cells, although some weaker reaction was present in gastric and ileal lining epithelium. Strong reactivity was also seen in the cells of acute and chronic myeloid leukaemia and in extramedullary haemopoiesis. The advantages of this technique are compared with those for muramidase (lysozyme), alpha 1-antitrypsin, and naphthol-AS-D-chloroacetate esterase.


Three mechanisms by which striatal denervation causes breakdown of dopamine signaling.
Progressive loss of nigrostriatal dopamine (DA) neurons is the neuropathological hallmark of Parkinson's disease (PD). Symptoms of the disease can often be treated by DA D2 agonists and thus seem related to disinhibition of the indirect striatal pathway. However, there is no evidence that symptoms arise by low extracellular DA concentration or are associated with reduced D2 receptor binding. Here I provide a theoretical analysis of the pathophysiology and postsynaptic adaptation resulting from striatal DA denervation. I found that progressive denervation may alter DA signaling by three independent mechanisms depending on degree of denervation and macroscopic morphology of the lesion. As long as the remaining innervation stays anatomically coherent, denervation reduces phasic variations in extracellular DA, but the DA tone is not changed. The reduction of phasic signaling can be partially compensated by upregulating postsynaptic signaling cascades. However, changes in DA dynamics evade compensation. With 80-99% denervation, a persistent aberrant signal develops in D2-regulated pathways caused by random fluctuations in tonic DA release. Permanent low DA levels occur in regions completely void of innervation. Simulation of l-dopa therapy reduced the aberrant D2 signal. With a high degree of denervation, l-dopa enhanced another aberrant signal, this time in the D1 pathway. This analysis provides a quantitative, physiologically consistent view of the early and late stages of PD, the effect of main therapeutic medications, and potential side effects. The mechanisms described here may also provide an explanation to currently inexplicable pathological phenomena such as psycho stimulant-induced contraversive rotations in animal models.


SU-E-J-17: Evaluation of Metal Artifact Reduction in MVCTs Using a Model Based Image Reconstruction Method.
To evaluate the performance of a model based image reconstruction in reducing metal artifacts in MVCT systems, and to compare with filtered-back projection (FBP) technique. Iterative maximum likelihood polychromatic algorithm for CT (IMPACT) is used with pair/triplet production process and the energy dependent response of detectors. The beam spectra for in-house bench-top and TomotherapyTM MVCT are modelled for use in IMPACT. The energy dependent gain of detectors is calculated using a constrained optimization technique and measured attenuation produced by 0 - 24 cm thick solid water slabs. A cylindrical (19 cm diameter) plexiglass phantom containing various central cylindrical inserts (relative electron density of 0.28-1.69) between two steel rods (2 cm diameter) is scanned in the bench-top [the bremsstrahlung radiation from 6 MeV electron beam passed through 4 cm solid water on the Varian Clinac 2300C] and TomotherapyTM MVCTs. The FBP reconstructs images from raw signal normalised to air scan and corrected for beam hardening using a uniform plexi-glass cylinder (20 cm diameter). IMPACT starts with FBP reconstructed seed image and reconstructs final image at 1.25 MeV in 150 iterations. FBP produces a visible dark shading in the image between two steel rods that becomes darker with higher density central insert causing 5-8 % underestimation of electron density compared to the case without the steel rods. In the IMPACT image the dark shading connecting the steel rods is nearly removed and the uniform background restored. The average attenuation coefficients of the inserts and the background are very close to the corresponding theoretical values at 1.25 MeV. The dark shading metal artifact due to beam hardening can be removed in MVCT using the iterative reconstruction algorithm such as IMPACT. However, the accurate modelling of detectors' energy dependent response and physical processes are crucial for successful implementation. Funding support for the research is obtained from "Vanier Canada Graduate Scholarship" and "Canadian Institute of Health Research".


Multi-infarct dementia and Alzheimer disease, contribution of cerebral circulation ultrasonography to pathogenesis and differential diagnosis. Value of microembolisation.
Dementias are one of the most serious health and socioeconomic issues. Multi-infarct dementia (MID) and Alzheimer´s type dementia (AD) exhibit differences in cerebrovascular blood flow velocity profiles and in presence of microemboli, detected by transcranial Doppler sonography. A group of 77 persons was divided into 4 subgroups: 1. subgroup of patients with MID (n=19; 10 male and 9 female, mean age was 74.32±8.30 years); 2. subgroup of patients with AD (n=19; 11 male and 8 female, mean age was 70.37±87.85 years); 3. subgroup of patients with hypertension (n=19; 11 male and 8 female, age adjusted) and 4. sex and age adjusted control group (CG) of 20 persons without hypertension or other serious risk factors. The duplex ultrasonographic examination of extracranial and intracranial circulation was preceded by neurologic, neuropsychological and psychiatric examination. The presence of microemboli was determined using Multi Dop X2 device (maker DWL), 60 minutes monitoring. All patients underwent brain computer tomography (CT) or magnetic resonance imaging (MRI). We found significantly higher incidence (68.4%, p=0.5267) of asymptomatic microemboli in ACM in the group of patients with MID compared to the AD group, the group of patients with hypertension and CG. The occurrence of "asymptomatic" emboli in the middle cerebral artery in patients with multi-infarct dementia is higher in the current study. Although these microemboli do not cause immediate symptoms, the evidence suggests, that they may be a risk factor for cognitive impairment, especially for multi-infarct dementia.


A cross-sectional analysis of physical activity and obesity indicators in European participants of the EPIC-PANACEA study.
Cross-sectional data suggest a strong association between low levels of physical activity and obesity. The EPIC-PANACEA (European Prospective Investigation into Cancer-Physical Activity, Nutrition, Alcohol, Cessation of Smoking, Eating out of home And obesity) project was designed to investigate the associations between physical activity and body mass index (BMI) and waist circumference based on individual data collected across nine European countries. In the European Prospective Investigation into Cancer and Nutrition (EPIC), 519 931 volunteers were recruited between 1992 and 2000, of whom 405 819 had data on main variables of interest. Height, body weight and waist circumference were measured using standardized procedures. Physical activity was assessed using a validated four-category index reflecting a self-reported usual activity during work and leisure time. The associations between physical activity and BMI and waist circumference were estimated using multilevel mixed effects linear regression models, adjusted for age, total energy intake, smoking status, alcohol consumption and educational level. A total of 125 629 men and 280 190 women with a mean age of 52.9 (s.d. 9.7) and 51.5 (s.d. 10.0) years, respectively were included. The mean BMI was 26.6 kg/m(2) (s.d. 3.6) in men and 25.0 kg/m(2) (s.d. 4.5) in women. Fifty percent of men and 30% of women were categorized as being active or moderately active. A one-category difference in the physical activity index was inversely associated with a difference of 0.18 kg/m(2) in the mean BMI (95% confidence interval, CI, 0.11, 0.24) and 1.04-cm (95% CI 0.82, 1.26) difference in waist circumference in men. The equivalent figures for women were 0.31 kg/m(2) (95% CI 0.23, 0.38) and 0.90 cm (95% CI 0.71, 1.08), respectively. Physical activity is inversely associated with both BMI and waist circumference across nine European countries. Although we cannot interpret the association causally, our results were observed in a large and diverse cohort independently from many potential confounders.


A conserved set of maternal genes? Insights from a molluscan transcriptome.
The early animal embryo is entirely reliant on maternal gene products for a 'jump-start' that transforms a transcriptionally inactive embryo into a fully functioning zygote. Despite extensive work on model species, it has not been possible to perform a comprehensive comparison of maternally-provisioned transcripts across the Bilateria because of the absence of a suitable dataset from the Lophotrochozoa. As part of an ongoing effort to identify the maternal gene that determines left-right asymmetry in snails, we have generated transcriptome data from 1 to 2-cell and ~32-cell pond snail (Lymnaea stagnalis) embryos. Here, we compare these data to maternal transcript datasets from other bilaterian metazoan groups, including representatives of the Ecydysozoa and Deuterostomia. We found that between 5 and 10% of all L. stagnalis maternal transcripts (~300-400 genes) are also present in the equivalent arthropod (Drosophila melanogaster), nematode (Caenorhabditis elegans), urochordate (Ciona intestinalis) and chordate (Homo sapiens, Mus musculus, Danio rerio) datasets. While the majority of these conserved maternal transcripts ("COMATs") have housekeeping gene functions, they are a non-random subset of all housekeeping genes, with an overrepresentation of functions associated with nucleotide binding, protein degradation and activities associated with the cell cycle. We conclude that a conserved set of maternal transcripts and their associated functions may be a necessary starting point of early development in the Bilateria. For the wider community interested in discovering conservation of gene expression in early bilaterian development, the list of putative COMATs may be useful resource.


BMP15 Mutations Associated With Primary Ovarian Insufficiency Reduce Expression, Activity, or Synergy With GDF9.
Bone morphogenetic protein (BMP)15 is an oocyte-specific growth factor, which, together with growth differentiation factor (GDF) 9, regulates folliculogenesis and ovulation rate. Multiple mutations in BMP15 have been identified in women with primary ovarian insufficiency (POI), supporting a pathogenic role; however, the underlying biological mechanism of many of these mutants remains unresolved. To determine how mutations associated with ovarian dysfunction alter the biological activity of human BMP15. The effects of 10 mutations in BMP15 on protein production, activation of granulosa cells, and synergy with GDF9 were assessed. Sequencing of 35 patients with POI identified both an unrecognized BMP15 variant (c.986G>A, R329H) and a variant (c.581T>C, F194S) previously associated with the condition. Assessing expression and activity of these and 8 other BMP15 mutants identified: (1) multiple variants, including L148P, F194S, and Y235C, with reduced mature protein production; (2) three variants (R138H, A180T, and R329H) with ∼fourfold lower activity than wild-type BMP15; and (3) 3 variants (R68W, F194S, and N196K) with a significantly reduced ability to synergize with GDF9. Mutations in BMP15 associated with POI reduce mature protein production, activity, or synergy with GDF9. The latter effect is perhaps most interesting given that interactions with GDF9 most likely underlie the physiology of BMP15 in the human ovary.


Nucleic acid, metabolic and histological changes in gilt mammary tissue during pregnancy and lactogenesis.
Changes in mammary gland histology, dry weights, nucleic acids and in vitro rates of substrate oxidation in incorporation into lipid were measured in mammary biopsies of three gilts each on d 30, 45, 60, 75, 90, 105 and 112 of pregnancy, and d 1 and 4 of lactation. Histological changes noted were progressive duct growth early in pregnancy followed by rapid lobulo-alveolar development between d 75 and 90 to complete mammogenesis. Colostrum and lipid were evident by d 105 with marked distension of alveolar lumina on d 112. Complete differentiation of the secretory process was apparent on the day of parturition. Concentrtion of dry, fat-free tissue (DFFT) and DNA changed little before d 60 but increased fourfold between d 75 and 90. No further increases in DFFT or DNA were noted. RNA concentrations paralleled DNA through d 90, after which they steadily increased. Rates of acetate and glucose oxidation increased transiently during midpregnancy then declined and remained low until initiation of lactogenesis. Substrate incorporation into lipid increased slightly at midpregnancy and again at d 105, after which it increased markedly. Collectively, results indicate that mammogenesis is complete by d 90, after which lactogenesis is initiated in a two-stage process. Metabolic rates expressed on a DNA basis indicated considerable rates of oxidation, but not of lipogenesis by proliferating mammary tissue. Preferential metabolism of acetate vs glucose near parturition suggests coordination of metabolism between the mammary gland and other maternal tissues.


From gene mutations to tumours--stem cells in gastrointestinal carcinogenesis.
Stem cells share many properties with malignant cells, such as the ability to self-renew and proliferate. Cancer is believed to be a disease of stem cells. The gastrointestinal tract has high cancer prevalence partly because of rapid epithelial cell turnover and exposure to dietary toxins. The molecular pathways of carcinogenesis differ according to the tissue. Work on hereditary cancer syndromes including familial adenomatous polyposis (FAP) has led to advances in our understanding of the events that occur in tumour development from a gastrointestinal stem cell. The initial mutation involved in the adenoma-carcinoma sequence is in the 'gatekeeper' tumour-suppressor gene adenomatous polyposis coli (APC). Somatic hits in this gene are non-random in FAP, with the type of mutation selected for by the position of the germline mutation. In the stomach, a metaplasia-dysplasia sequence occurs and is often related to Helicobacter pylori infection. Clonal expansion of mutated cells occurs by niche succession. Further expansion of the aberrant clone then occurs by the longitudinal division of crypts into two daughter units--crypt fission. Two theories seek to explain the early development of adenomas--the 'top down' and 'bottom up' hypotheses. Initial studies suggested that colorectal tumours were monoclonal; however, later work on chimeric mice and a sex chromosome mixoploid patient with FAP suggested that up to 76% of early adenomas were polyclonal. Introduction of a homozygous resistance allele has reduced tumour multiplicity in the mouse and has been used to rule out random collision of polyps as the cause of these observations. It is likely that short-range interaction between adjacent initiated crypts is responsible for polyclonality.


Pathways of Pathogenicity: Transcriptional Stages of Germination in the Fatal Fungal Pathogen Rhizopus delemar.
Rhizopus delemar is an invasive fungal pathogen responsible for the frequently fatal disease mucormycosis. Germination, a crucial mechanism by which infectious spores of Rhizopus delemar cause disease, is a key developmental process that transforms the dormant spore state into a vegetative one. The molecular mechanisms that underpin this transformation may be key to controlling mucormycosis; however, the regulation of germination remains poorly understood. This study describes the phenotypic and transcriptional changes that take place over the course of germination. This process is characterized by four distinct stages: dormancy, isotropic swelling, germ tube emergence, and hyphal growth. Dormant spores are shown to be transcriptionally unique, expressing a subset of transcripts absent in later developmental stages. A large shift in the expression profile is prompted by the initiation of germination, with genes involved in respiration, chitin, cytoskeleton, and actin regulation appearing to be important for this transition. A period of transcriptional consistency can be seen throughout isotropic swelling, before the transcriptional landscape shifts again at the onset of hyphal growth. This study provides a greater understanding of the regulation of germination and highlights processes involved in transforming Rhizopus delemar from a single-cellular to multicellular organism.IMPORTANCE Germination is key to the growth of many organisms, including fungal spores. Mucormycete spores exist abundantly within the environment and germinate to form hyphae. These spores are capable of infecting immunocompromised individuals, causing the disease mucormycosis. Germination from spore to hyphae within patients leads to angioinvasion, tissue necrosis, and often fatal infections. This study advances our understanding of how spore germination occurs in the mucormycetes, identifying processes we may be able to inhibit to help prevent or treat mucormycosis.


Reports of zoonotic disease outbreaks associated with animal exhibits and availability of recommendations for preventing zoonotic disease transmission from animals to people in such settings.
To assess the number of zoonotic disease outbreaks associated with animal exhibits and identify published recommendations for preventing zoonotic disease transmission from animals to people in exhibit settings. Literature review and survey of state public health veterinarians and state epidemiologists. MEDLINE and agriculture databases were searched from 1966 through 2000. Retrieved references and additional resources provided by the authors were reviewed. A survey was sent to state public health veterinarians and state epidemiologists to determine whether their states had written recommendations or guidelines for controlling zoonotic diseases in animal exhibition venues, whether their states maintained a listing of animal exhibitors in the state, and whether they had any information on recent outbreaks involving animals in exhibitions. 11 published outbreaks were identified. These outbreaks occurred in a variety of settings including petting zoos, farms, and a zoological park. An additional episode involving exposure to a potentially rabid bear required extensive public health resources. A survey of state public health veterinarians identified 16 additional unpublished outbreaks or incidents. Most states did not have written recommendations or guidelines for controlling zoonotic diseases or any means to disseminate educational materials to animal exhibitors. Recent outbreaks of zoonotic diseases associated with contact with animals in exhibition venues highlight concerns for disease transmission to public visitors. Only a handful of states have written guidelines for preventing zoonotic disease transmission in animal exhibition venues, and published recommendations currently available focus on preventing enteric diseases and largely do not address other zoonotic diseases or prevention of bite wounds.


Tolerance of low cerebral intracellular pH in rats during hyperbaric hypercapnia.
Brain acidosis from cerebral ischemia is characterized by average intracellular pH levels of approximately 5.8-6.2, which appear in turn to worsen cellular injury. We report that the brain is not injured when hypercapnia is used to reduce intracellular pH to about 6.2 during adequate oxygenation. A hyperbaric chamber is needed to achieve intracellular pH values so low because inspired CO2 tensions must be increased to approximately 1 atm. Using in vivo phosphorus-31 and proton nuclear magnetic resonance spectroscopy, we measured brain intracellular pH and lactate concentration of rats inside a nonmagnetic polycarbonate chamber at a barometric pressure of 1,500 mm Hg. Intubated rats were ventilated with a 50% O2/50% CO2 gas mixture for specific times. All six rats ventilated for 15 minutes with CO2 tensions of approximately 750 mm Hg woke up without neurological impairment, despite a decrease in intracellular pH to about 6.2. Higher CO2 tensions and longer exposures resulted in cardiovascular collapse and sudden death, followed by the postmortem appearance of brain lactate. Brain intracellular pH values near 6.2 can be induced briefly in vivo in ventilated rats without injury under hyperbaric hypercapnic conditions. If attempts are made to lower brain pH in vivo even further by increasing PCO2 beyond 750 mm Hg, mean arterial blood pressure and cerebral blood flow decrease to values incompatible with life.


Long-range energy transport in single supramolecular nanofibres at room temperature.
Efficient transport of excitation energy over long distances is a key process in light-harvesting systems, as well as in molecular electronics. However, in synthetic disordered organic materials, the exciton diffusion length is typically only around 10 nanometres (refs 4, 5), or about 50 nanometres in exceptional cases, a distance that is largely determined by the probability laws of incoherent exciton hopping. Only for highly ordered organic systems has the transport of excitation energy over macroscopic distances been reported--for example, for triplet excitons in anthracene single crystals at room temperature, as well as along single polydiacetylene chains embedded in their monomer crystalline matrix at cryogenic temperatures (at 10 kelvin, or -263 degrees Celsius). For supramolecular nanostructures, uniaxial long-range transport has not been demonstrated at room temperature. Here we show that individual self-assembled nanofibres with molecular-scale diameter efficiently transport singlet excitons at ambient conditions over more than four micrometres, a distance that is limited only by the fibre length. Our data suggest that this remarkable long-range transport is predominantly coherent. Such coherent long-range transport is achieved by one-dimensional self-assembly of supramolecular building blocks, based on carbonyl-bridged triarylamines, into well defined H-type aggregates (in which individual monomers are aligned cofacially) with substantial electronic interactions. These findings may facilitate the development of organic nanophotonic devices and quantum information technology.


Dual contributions of noradrenaline to behavioural flexibility and motivation.
While several theories have highlighted the importance of the noradrenergic system for behavioral flexibility, a number of recent studies have also shown a role for noradrenaline in motivation, particularly in effort processing. Here, we designed a novel sequential cost/benefit decision task to test the causal influence of noradrenaline on these two functions in rhesus monkeys. We manipulated noradrenaline using clonidine, an alpha-2 noradrenergic receptor agonist, which reduces central noradrenaline levels and examined how this manipulation influenced performance on the task. Clonidine had two specific and distinct effects: first, it decreased choice variability, without affecting the cost/benefit trade-off; and second, it reduced force production, without modulating the willingness to work. Together, these results support an overarching role for noradrenaline in facing challenging situations in two complementary ways: by modulating behavioral volatility, which would facilitate adaptation depending on the lability of the environment, and by modulating the mobilization of resources to face immediate challenges.


Muscle moment arms and sensitivity analysis of a mouse hindlimb musculoskeletal model.
Musculoskeletal modelling has become a valuable tool with which to understand how neural, muscular, skeletal and other tissues are integrated to produce movement. Most musculoskeletal modelling work has to date focused on humans or their close relatives, with few examples of quadrupedal animal limb models. A musculoskeletal model of the mouse hindlimb could have broad utility for questions in medicine, genetics, locomotion and neuroscience. This is due to this species' position as a premier model of human disease, having an array of genetic tools for manipulation of the animal in vivo, and being a small quadruped, a category for which few models exist. Here, the methods used to develop the first three-dimensional (3D) model of a mouse hindlimb and pelvis are described. The model, which represents bones, joints and 39 musculotendon units, was created through a combination of previously gathered muscle architecture data from microdissections, contrast-enhanced micro-computed tomography (CT) scanning and digital segmentation. The model allowed muscle moment arms as well as muscle forces to be estimated for each musculotendon unit throughout a range of joint rotations. Moment arm analysis supported the reliability of musculotendon unit placement within the model, and comparison to a previously published rat hindlimb model further supported the model's reliability. A sensitivity analysis performed on both the force-generating parameters and muscle's attachment points of the model indicated that the maximal isometric muscle moment is generally most sensitive to changes in either tendon slack length or the coordinates of insertion, although the degree to which the moment is affected depends on several factors. This model represents the first step in the creation of a fully dynamic 3D computer model of the mouse hindlimb and pelvis that has application to neuromuscular disease, comparative biomechanics and the neuromechanical basis of movement. Capturing the morphology and dynamics of the limb, it enables future dissection of the complex interactions between the nervous and musculoskeletal systems as well as the environment.


Exploring virtual worlds for scenario-based repeated team training of cardiopulmonary resuscitation in medical students.
Contemporary learning technologies, such as massively multiplayer virtual worlds (MMVW), create new means for teaching and training. However, knowledge about the effectiveness of such training is incomplete, and there are no data regarding how students experience it. Cardiopulmonary resuscitation (CPR) is a field within medicine in high demand for new and effective training modalities. In addition to finding a feasible way to implement CPR training, our aim was to investigate how a serious game setting in a virtual world using avatars would influence medical students' subjective experiences as well as their retention of knowledge. An MMVW was refined and used in a study to train 12 medical students in CPR in 3-person teams in a repeated fashion 6 months apart. An exit questionnaire solicited reflections over their experiences. As the subjects trained in 4 CPR scenarios, measurements of self-efficacy, concentration, and mental strain were made in addition to measuring knowledge. Engagement modes and coping strategies were also studied. Parametric and nonparametric statistical analyses were carried out according to distribution of the data. The majority of the subjects reported that they had enjoyed the training, had found it to be suitable, and had learned something new, although several asked for more difficult and complex scenarios as well as a richer virtual environment. The mean values for knowledge dropped during the 6 months from 8.0/10 to 6.25/10 (P = .002). Self-efficacy increased from before to after each of the two training sessions, from 5.9/7 to 6.5/7 (P = .01) after the first and from 6.0/7 to 6.7/7 (P = .03) after the second. The mean perceived concentration value increased from 54.2/100 to 66.6/100 (P = .006), and in general the mental strain was found to be low to moderate (mean = 2.6/10). Using scenario-based virtual world team training with avatars to train medical students in multi-person CPR was feasible and showed promising results. Although we found no evidence of stimulated recall of CPR procedures in our test-retest study, the subjects were enthusiastic and reported increased concentration during the training. We also found that subjects' self-efficacy had increased after the training. Despite the need for further studies, these findings imply several possible uses of MMVW technology for future emergency medical training.


Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs.
Canonical microRNAs (miRNAs) require two processing steps: the first by the Microprocessor, a complex of DGCR8 and Drosha, and the second by a complex of TRBP and Dicer. dgcr8Delta/Delta mouse embryonic stem cells (mESCs) have less severe phenotypes than dicer1Delta/Delta mESCs, suggesting a physiological role for Microprocessor-independent, Dicer-dependent small RNAs. To identify these small RNAs with unusual biogenesis, we performed high-throughput sequencing from wild-type, dgcr8Delta/Delta, and dicer1Delta/Delta mESCs. Several of the resulting DGCR8-independent, Dicer-dependent RNAs were noncanonical miRNAs. These derived from mirtrons and a newly identified subclass of miRNA precursors, which appears to be the endogenous counterpart of shRNAs. Our analyses also revealed endogenous siRNAs resulting from Dicer cleavage of long hairpins, the vast majority of which originated from one genomic locus with tandem, inverted short interspersed nuclear elements (SINEs). Our results extend the known diversity of mammalian small RNA-generating pathways and show that mammalian siRNAs exist in cell types other than oocytes.


High concentrations of hexavalent chromium in drinking water alter iron homeostasis in F344 rats and B6C3F1 mice.
Hexavalent chromium [Cr(VI)] induces hematological signs of microcytic anemia in rodents. Considering that Cr(VI) can oxidize ferrous (Fe(2+)) to ferric (Fe(3+)) iron, and that only the former is transported across the duodenum, we hypothesize that, at high concentrations, Cr(VI) oxidizes Fe(2+) in the lumen of the small intestine and perturbs iron absorption. Herein we report that 90-day exposure to Cr(VI) in drinking water resulted in dose-dependent decreases in Fe levels in the duodenum, liver, serum, and bone marrow. Toxicogenomic analyses from the duodenum indicate responses consistent with Fe deficiency, including significant induction of divalent metal transporter 1 (DMT1, Slc11a2) and transferrin receptor 1 (TFR1, Tfr1). In addition, at ⩾20mg Cr(VI)/L in drinking water, Cr RBC:plasma ratios in rats were increased and exceeded unity, indicating saturation of reductive capacity and intracellular absorption of Cr(VI) into red blood cells (RBCs). These effects occurred in both species but were generally more severe in rats. These data suggest that high concentrations of Cr(VI) in drinking limit Fe absorption and alter iron homeostasis. Furthermore, some effects observed at high doses in recent Cr(VI) chronic and subchronic bioassays may be explained, at least in part, by iron deficiency and disruption of homeostasis.


Using heart rate profiles during sleep as a biomarker of depression.
Abnormalities in heart rate during sleep linked to impaired neuro-cardiac modulation may provide new information about physiological sleep signatures of depression. This study assessed the validity of an algorithm using patterns of heart rate changes during sleep to discriminate between individuals with depression and healthy controls. A heart rate profiling algorithm was modeled using machine-learning based on 1203 polysomnograms from individuals with depression referred to a sleep clinic for the assessment of sleep abnormalities, including insomnia, excessive daytime fatigue, and sleep-related breathing disturbances (n = 664) and mentally healthy controls (n = 529). The final algorithm was tested on a distinct sample (n = 174) to categorize each individual as depressed or not depressed. The resulting categorizations were compared to medical record diagnoses. The algorithm had an overall classification accuracy of 79.9% [sensitivity: 82.8, 95% CI (0.73-0.89), specificity: 77.0, 95% CI (0.67-0.85)]. The algorithm remained highly sensitive across subgroups stratified by age, sex, depression severity, comorbid psychiatric illness, cardiovascular disease, and smoking status. Sleep-derived heart rate patterns could act as an objective biomarker of depression, at least when it co-occurs with sleep disturbances, and may serve as a complimentary objective diagnostic tool. These findings highlight the extent to which some autonomic functions are impaired in individuals with depression, which warrants further investigation about potential underlying mechanisms.


Characterization of the Council of Emergency Medicine Residency Directors' standardized letter of recommendation in 2011-2012.
The Council of Emergency Medicine Residency Directors (CORD) introduced the standardized letter of recommendation (SLOR) in 1997, and it has become a critical tool for assessing candidates for emergency medicine (EM) training. It has not itself been evaluated since the initial studies associated with its introduction. This study characterizes current SLOR use to evaluate whether it serves its intended purpose of being standardized, concise, and discriminating. This retrospective, multi-institutional study evaluated letters of recommendation from U.S. allopathic applicants to three EM training programs during the 2011-2012 Electronic Residency Application Service (ERAS) application cycle. Distributions of responses to each question on the SLOR were calculated, and the free-text responses were analyzed. Two pilots, performed on five applicants each, assisted in developing a strategy for limiting interrater reliability. Each of the three geographically diverse programs provided a complete list of U.S. allopathic applicants to their program. Upon randomization, each program received a list of coded applicants unique to their program randomly selected for data collection. The number of applicants was selected to reach a goal of approximately 200 SLORs per site (n = 602). Among this group, comprising 278 of 1,498 applicants (18.6%) from U.S. allopathic schools, a total of 1,037 letters of recommendation were written, with 724 (69.8%) written by emergency physicians. SLORs represented 57.9% (602/1037) of all LORs (by any kind of author) and 83.1% (602/724) of letters written by emergency physicians. Three hundred ninety-two of 602 SLORs had a single author (65.1%). For the question on "global assessment," students were scored in the top 10% in 234 of 583 of applications (40.1%; question not answered by some), and 485 of 583 (83.2%) of the applicants were ranked above the level of their peers. Similarly, >95% of all applicants were ranked in the top third compared to peers, for all but one section under "qualifications for emergency medicine." For 405 of 602 of all SLORs (67.2%), one or more questions were left unanswered, while 76 of all SLORs (12.6%) were "customized" or changed from the standard template. Finally, in 291 of 599 of SLORs (48.6%), the word count was greater than the recommended maximum of 200 words. Grade inflation is marked throughout the SLOR, limiting its ability to be discriminating. Furthermore, template customization and skipped questions work against the intention to standardize the SLOR. Finally, it is not uncommon for comments to be longer than guideline recommendations. As an assessment tool, the SLOR could be more discerning, concise, and standardized to serve its intended purpose.


Termination of persistent atrial fibrillation during pulmonary vein isolation: insight from the MAGIC-AF trial.
Controversy on the optimal ablation strategy for persistent atrial fibrillation (AF) exists with limited work evaluating a strategy of pulmonary vein isolation (PVI) alone when AF terminates during PVI. Thirty-five patients had AF termination during PVI in the Modified Ablation Guided by Ibutilide Use in Chronic Atrial Fibrillation (MAGIC-AF; ClinicalTrials.gov number: NCT01014741) study. The objective of the current study is to report the 1-year outcome after PVI alone in this unique patient group. The 1-year single procedure freedom from atrial arrhythmia off anti-arrhythmic drugs was reported for the 35 patients in the MAGIC-AF study with persistent AF termination during or upon completion of PVI. Freedom from recurrent atrial arrhythmia was achieved in 60% of patients where AF terminated during PVI. Cavotricuspid isthmus flutter was common when AF terminated to a macro re-entrant flutter during PVI, and responsible for 92% of all flutter circuits with AF termination. Persistent AF termination during PVI may identify a subgroup of patients who experience a similar long-term clinical outcome with PVI ablation alone when compared with other more extensive persistent AF ablation strategies. Pulmonary vein isolation alone may be an appropriate tactic in this subgroup of persistent AF patients.


3D Histopathology-a Lung Tissue Segmentation Workflow for Microfocus X-ray-Computed Tomography Scans.
Lung histopathology is currently based on the analysis of 2D sections of tissue samples. The use of microfocus X-ray-computed tomography imaging of unstained soft tissue can provide high-resolution 3D image datasets in the range of 2-10 μm without affecting the current diagnostic workflow. Important details of structural features such as the tubular networks of airways and blood vessels are contained in these datasets but are difficult and time-consuming to identify by manual image segmentation. Providing 3D structures permits a better understanding of tissue functions and structural interrelationships. It also provides a more complete picture of heterogeneous samples. In addition, 3D analysis of tissue structure provides the potential for an entirely new level of quantitative measurements of this structure that have previously been based only on extrapolation from 2D sections. In this paper, a workflow for segmenting such 3D images semi-automatically has been created using and extending the ImageJ open-source software and key steps of the workflow have been integrated into a new ImageJ plug-in called LungJ. Results indicate an improved workflow with a modular organization of steps facilitating the optimization for different sample and scan properties with expert input as required. This allows for incremental and independent optimization of algorithms leading to faster segmentation. Representation of the tubular networks in samples of human lung, building on those segmentations, has been demonstrated using this approach.


The M1 protein of Streptococcus pyogenes triggers an innate uptake mechanism into polarized human endothelial cells.
Serotype M1 Streptococcus pyogenes is a major human pathogen associated with severe invasive diseases causing high morbidity and mortality. In a substantial number of cases, invasive disease develops in previously healthy individuals with no obvious port of entry. This has led to the hypothesis that the source of streptococci in these cases is a transient bacteraemia. This study focuses on the analysis of interaction of tissue-invasive serotype M1 S. pyogenes with human endothelial cells (EC) of the vascular system. We identify the M1 surface protein of S. pyogenes as the EC invasin which is recognised by polarized human blood EC, thereby triggering rapid, phagocytosis-like uptake of streptococci into polarized EC layers. Upon internalization, the M1 S. pyogenes serotype is incorporated into phagosomes which traffic via the endosomal/lysosomal pathway. However, some of the streptococci successfully evade this innate killing process and hereby mediate their escape into the cytoplasm of the host cell. The results of this study demonstrate that blood EC possess an efficient uptake mechanism for serotype M1 S. pyogenes. Despite efficient phagocytosis, streptococcal survival within EC constitutes one potential mechanism which favours intracellular persistence and thus facilitates continuous infection and dissemination from the primary side of infection into deep tissue.


Estimating a state-space model from point process observations: a note on convergence.
Physiological signals such as neural spikes and heartbeats are discrete events in time, driven by continuous underlying systems. A recently introduced data-driven model to analyze such a system is a state-space model with point process observations, parameters of which and the underlying state sequence are simultaneously identified in a maximum likelihood setting using the expectation-maximization (EM) algorithm. In this note, we observe some simple convergence properties of such a setting, previously un-noticed. Simulations show that the likelihood is unimodal in the unknown parameters, and hence the EM iterations are always able to find the globally optimal solution.


Rational design of nanomaterials for water treatment.
The ever-increasing human demand for safe and clean water is gradually pushing conventional water treatment technologies to their limits. It is now a popular perception that the solutions to the existing and future water challenges will hinge upon further developments in nanomaterial sciences. The concept of rational design emphasizes on 'design-for-purpose' and it necessitates a scientifically clear problem definition to initiate the nanomaterial design. The field of rational design of nanomaterials for water treatment has experienced a significant growth in the past decade and is poised to make its contribution in creating advanced next-generation water treatment technologies in the years to come. Within the water treatment context, this review offers a comprehensive and in-depth overview of the latest progress in rational design, synthesis and applications of nanomaterials in adsorption, chemical oxidation and reduction reactions, membrane-based separation, oil-water separation, and synergistic multifunctional all-in-one nanomaterials/nanodevices. Special attention is paid to the chemical concepts related to nanomaterial design throughout the review.


The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization.
We sequenced and annotated the genome of the filamentous fungus Fusarium graminearum, a major pathogen of cultivated cereals. Very few repetitive sequences were detected, and the process of repeat-induced point mutation, in which duplicated sequences are subject to extensive mutation, may partially account for the reduced repeat content and apparent low number of paralogous (ancestrally duplicated) genes. A second strain of F. graminearum contained more than 10,000 single-nucleotide polymorphisms, which were frequently located near telomeres and within other discrete chromosomal segments. Many highly polymorphic regions contained sets of genes implicated in plant-fungus interactions and were unusually divergent, with higher rates of recombination. These regions of genome innovation may result from selection due to interactions of F. graminearum with its plant hosts.


Prediction of myocardial infarction by N-terminal-pro-B-type natriuretic peptide, C-reactive protein, and renin in subjects with cerebrovascular disease.
B-type natriuretic peptide (BNP), C-reactive protein (CRP), and renin are elevated in persons at risk for cardiovascular disease. However, data that directly compare these markers in the prediction of myocardial infarction (MI) are limited. N-terminal-proBNP (NT-proBNP), CRP, and renin were measured in baseline blood samples from a nested case-control study of the 6105 participants of the Perindopril Protection Against Recurrent Stroke Study (PROGRESS), a placebo-controlled study of a perindopril-based blood pressure-lowering regimen among individuals with previous stroke or transient ischemic attack. Each of 206 subjects who experienced MI, either fatal or nonfatal, during a mean follow-up of 3.9 years was matched to 1 to 3 control subjects. Most MI cases (67%) occurred in subjects without a history of coronary heart disease. NT-proBNP, CRP, and renin each predicted MI; the odds ratio for subjects in the highest compared with the lowest quarter was 2.2 (95% CI, 1.3 to 3.6) for NT-proBNP, 2.2 (95% CI, 1.3 to 3.6) for CRP, and 1.7 (95% CI, 1.1 to 2.8) for renin. NT-proBNP and renin, but not CRP, remained predictors of MI after adjustment for all other predictors, including LDL and HDL cholesterol levels. Individuals with both NT-proBNP and renin in their highest quarters had 4.5 times the risk of MI compared with subjects with both biological markers in their lowest quarters. NT-proBNP and renin, but not CRP, are independent predictors of MI risk after stroke or transient ischemic attack, providing information additional to that provided by classic risk factors, and may enable more effective targeting of MI prevention strategies.


Hyperexcitability in the olfactory bulb and impaired fine odor discrimination in the Fmr1 KO mouse model of fragile X syndrome.
Fragile X syndrome (FXS) is the single most common monogenetic cause of autism spectrum disorders in humans. FXS is caused by loss of expression of the Fragile X mental retardation protein (FMRP), an mRNA-binding protein encoded on the X chromosome involved in suppressing protein translation. Sensory processing deficits have been a major focus of studies of FXS in both humans and rodent models of FXS, but olfactory deficits remain poorly understood. Here we conducted experiments in wild-type and Fmr1 KO ( Fmr1 -/y ) mice (males) that lack expression of the gene encoding FMRP to assess olfactory circuit and behavioral abnormalities. In patch-clamp recordings conducted in slices of the olfactory bulb, output mitral cells (MCs) in Fmr1 KO mice displayed greatly enhanced excitation, as evidenced by a much higher rate of occurrence of spontaneous network-level events known as long-lasting depolarizations (LLDs). The higher probability of LLDs did not appear to reflect changes in inhibitory connections onto MCs but rather enhanced spontaneous excitation of external tufted cells (eTCs) that provide feedforward excitation onto MCs within glomeruli. In addition, in a go/no-go operant discrimination paradigm, we found that Fmr1 KO mice displayed impaired discrimination of odors in difficult tasks that involved odor mixtures but not altered discrimination of monomolecular odors. We suggest that the higher excitability of MCs in Fmr1 KO mice may impair fine odor discrimination by broadening odor tuning curves of MCs and/or altering synchronized oscillations through changes in transient inhibition. Fragile X syndrome (FXS) in humans is associated with a range of debilitating deficits including aberrant sensory processing. One sensory system that has received comparatively little attention in studies in animal models of FXS is olfaction. Here, we report the first comprehensive physiological analysis of circuit defects in the olfactory bulb in the commonly-used Fmr1 knockout (KO) mouse model of FXS. Our studies indicate that Fmr1 KO alters the local excitation/inhibition balance in the bulb - similar to what Fmr1 KO does in other brain circuits - but through a novel mechanism that involves enhanced feedforward excitatory drive. Furthermore, Fmr1 KO mice display behavioral impairments in fine odor discrimination, an effect that may be explained by enhanced neural excitability.


Fat Mass Is Associated with Foot Pain in Men: The Geelong Osteoporosis Study.
Foot pain is a common complaint in adults. Evidence suggests that body composition is involved in the development of foot pain. However, whether this is the case in men remains unclear because previous studies mainly examined women. The aim of this cross-sectional study was to determine the relationship between body composition and foot pain in men while accounting for important risk factors. Among 978 men (median age 60 yrs, range 24-98) from the Geelong Osteoporosis Study who participated in a followup study in 2006 to 2011, 796 provided responses to questions on health status and foot pain. Foot pain was determined using the Manchester Foot Pain and Disability Index, and body composition was measured using dual-energy x-ray absorptiometry. Of the 796 respondents, 177 (22%) had foot pain. Risk factors for foot pain were age (OR 1.03, 95% CI 1.02-1.04), self-reported depression (OR 2.05, 95% CI 1.30-3.20), decreased mobility (OR 1.54, 95% CI 1.05-2.24), and lower education (OR 1.47, 95% CI 1.03-2.09). Foot pain was associated with body mass index (OR 1.05, 95% CI 1.00-1.10), fat mass (OR 1.02, 95% CI 1.03-1.05), and fat mass index (OR 1.08, 95% CI 1.01-1.15), but not fat-free mass (OR 1.01, 95% CI 0.98-1.04) or fat-free mass index (OR 1.05, 95% CI 0.95-1.15) after appropriate adjustments were made. Fat mass is associated with foot pain in men. These findings complement those in studies that have mainly examined women, and provide further evidence for the relationship between obesity and foot pain.


[Complicated anesthetic management for esophageal atresia repair in a baby with total anomalous pulmonary venous connection diagnosed after esophageal surgery].
A male newborn infant born at 38 weeks of gestation and weighing 2,660 g, was diagnosed as esophageal atresia. Although there was mild cyanosis at birth, his initial ultrasonographic examination performed by neonatologist showed normal anatomy of the heart. He underwent a surgical repair of esophageal atresia under general anesthesia. Anesthesia was induced with fentanyl and pancuronium, and maintained with sevoflurane and 60% oxygen. Frequent desaturation occurred during the procedure, requiring manual hyperventilation with 100% oxygen. Since blood pressure during the operation was unstable, volume loading with albumin was attempted. Further echocardiography was performed by pediatric cardiologist after going back to NICU. This revealed total anomalous pulmonary venous connection (TAPVC). He underwent cardiac surgery for repair of TAPVC on the next day. Although TAPVC was not preoperatively diagnosed regardless of results of echocardiogram, cyanosis and unstable blood pressure should be considered as signs of potential cardiac disease. Fluid restriction, higher hemoglobin, lower inspired oxygen and slightly higher carbon dioxide rather than albumin administration and hyperventilation should have been attempted during the esophageal repair.


Happiness is related to cardiovascular risk: a cross-sectional study in Spain.
The study of cardiovascular risk factors has been deeply described in recent years, but the findings on the complex role of psychological indicators (i.e. happiness and depression) on cardiovascular health are mixed. The primary goal of our study was to examine the extent to which certain psychological aspects, namely happiness and depression, can predict cardiovascular risk. A sample of 173 (Mage = 44.9, SD = 14; 62% females) individuals from the general population who attended a public hospital of Lleida (Spain) participated voluntarily in the study. We measured happiness, depression and different clinical and sociodemographic variables. The sample reported low levels of depression and moderate levels of happiness, overweight levels of body mass index and mainly low levels of cardiovascular risk. Happiness was correlated positively to cardiovascular risk and negatively to depression. Increases in happiness, but not in depression, were associated with people being in a higher group of cardiovascular risk. Despite a body of literature indicates that subjective well-being has a protective role over cardiovascular health, the contradictory findings of our study might be explained by several factors. The present findings invite to consider the complex and indirect influence of happiness on physical health. Future research should investigate the potential biological and behavioral processes of happiness linked with increases in cardiovascular risk.


Laboratory-acquired parasitic infections from accidental exposures.
Parasitic diseases are receiving increasing attention in developed countries in part because of their importance in travelers, immigrants, and immunocompromised persons. The main purpose of this review is to educate laboratorians, the primary readership, and health care workers, the secondary readership, about the potential hazards of handling specimens that contain viable parasites and about the diseases that can result. This is accomplished partly through discussion of the occupationally acquired cases of parasitic infections that have been reported, focusing for each case on the type of accident that resulted in infection, the length of the incubation period, the clinical manifestations that developed, and the means by which infection was detected. The article focuses on the cases of infection with the protozoa that cause leishmaniasis, malaria, toxoplasmosis, Chagas' disease (American trypanosomiasis), and African trypanosomiasis. Data about 164 such cases are discussed, as are data about cases caused by intestinal protozoa and by helminths. Of the 105 case-patients infected with blood and tissue protozoa who either recalled an accident or for whom the likely route of transmission could be presumed, 47 (44.8%) had percutaneous exposure via a contaminated needle or other sharp object. Some accidents were directly linked to poor laboratory practices (e.g., recapping a needle or working barehanded). To decrease the likelihood of accidental exposures, persons who could be exposed to pathogenic parasites must be thoroughly instructed in safety precautions before they begin to work and through ongoing training programs. Protocols should be provided for handling specimens that could contain viable organisms, using protective clothing and equipment, dealing with spills of infectious organisms, and responding to accidents. Special care should be exercised when using needles and other sharp objects.


Molecularly imprinted polymers with synthetic dummy templates for the preparation of capsaicin and dihydrocapsaicin from chili peppers.
In this work, dummy molecularly imprinted polymers with high selectivity and affinity to capsaicin and dihydrocapsaicin are designed using N-vanillylnonanamide as a dummy template. The performance of dummy molecularly imprinted polymers and nonimprinted polymers was evaluated using adsorption isotherms, adsorption kinetics, and selective recognition capacity. Dummy molecularly imprinted polymers were found to exhibit good site accessibility, taking just 20 min to achieve adsorption equilibrium; they were also highly selective toward capsaicin and dihydrocapsaicin. We successfully used dummy molecularly imprinted polymers as a specific sorbent for selectively enriching capsaicin and dihydrocapsaicin from chili pepper samples. In a scaled-up experiment, the selective recovery of capsaicinoids was calculated to be 77.8% using solid-phase extraction. To the best of our knowledge, this is the first example of the use of N-vanillylnonanamide as a dummy template in molecularly imprinted polymers to simultaneously enrich capsaicin and dihydrocapsaicin.


Renovascular effects of neuropeptide-Y in the split hydronephrotic rat kidney: non-uniform pattern of vascular reactivity.
1. The renovascular effects of neuropeptide-Y (NPY) were examined in the split hydronephrotic rat kidney. 2. Systemic infusion of low non-pressor doses of NPY (0.2 micrograms kg-1 up to 5.0 micrograms kg-1) produced a non-uniform pattern of vascular reactivity. In general, a significant constriction of the proximal and distal arcuate artery was seen at all doses. No constriction was seen at the interlobular artery or the larger part of the afferent arteriole. These segments initially dilated during the lower dose infusions. The very distal part of the afferent arteriole adjacent to the glomerulus and the proximal efferent arteriole responded in a similar way to the arcuate arteries. 3. NPY, locally applied into the tissue bath at concentrations of 1 nmol l-1 up to 25 nmol l-1, produced non-uniform vascular reactions similar to those of intravenously infused NPY. At the considerably higher local dosage of 1.14 mumol l-1, all vascular segments revealed vasoconstriction. 4. NPY application did not attenuate effects of acetylcholine. This observation suggests that the mechanism of NPY-induced vasoconstriction does not rely upon antagonism of endothelium-derived vasodilatation. 5. The pattern of vascular reactivity to NPY was substantially different from that known for the vasoconstrictors noradrenaline and angiotensin II in our preparation.


Patients with postpartum hemorrhage admitted in intensive care unit: Patient condition, interventions, and outcome.
We conducted this study to analyze the data of patients admitted to intensive care unit (ICU) following postpartum hemorrhage (PPH) during one-year period, the interventions they received, and the outcome. Case records of patients admitted to ICU with PPH were analyzed. Data retrieved were as follows: Primary admission or referred case, duration between onset of PPH and arrival, condition at admission, resuscitative measures, procedures to manage PPH, presence of disseminated intravascular coagulation (DIC) and its management, duration of ICU stay, and the outcome. Of 21 patients with PPH, 15 were admitted in the ICU. All were referred from other hospitals. Duration between onset of PPH and arrival was 6 (15) hours (mean [standard deviation]). All were conscious on arrival. In 10 patients, blood pressure was not recordable. Tachycardia was a common feature. One patient had bradycardia (54 bpm/BP not recordable). Resuscitative measures included oxygen supplementation and fluid resuscitation. Eight patients underwent uterine artery embolization, 2 patients underwent embolization followed by surgery, and 11 patients underwent surgical intervention only. Twelve patients had DIC on admission which was managed with blood component therapy. Duration of stay in ICU was 12.6 (5.4) days (mean [standard deviation]). Two patients expired following intractable DIC and multiorgan dysfunction syndrome. Though these 2 patients had severe shock on presentation, they did not have DIC at the time of presentation. Despite early resuscitation and intensive care management, DIC is a major cause of mortality. Late onset DIC (onset after admission to ICU) was associated with poor outcome in this study.


Genome sequence of Desulfitobacterium hafniense DCB-2, a Gram-positive anaerobe capable of dehalogenation and metal reduction.
The genome of the Gram-positive, metal-reducing, dehalorespiring Desulfitobacterium hafniense DCB-2 was sequenced in order to gain insights into its metabolic capacities, adaptive physiology, and regulatory machineries, and to compare with that of Desulfitobacterium hafniense Y51, the phylogenetically closest strain among the species with a sequenced genome. The genome of Desulfitobacterium hafniense DCB-2 is composed of a 5,279,134-bp circular chromosome with 5,042 predicted genes. Genome content and parallel physiological studies support the cell's ability to fix N2 and CO2, form spores and biofilms, reduce metals, and use a variety of electron acceptors in respiration, including halogenated organic compounds. The genome contained seven reductive dehalogenase genes and four nitrogenase gene homologs but lacked the Nar respiratory nitrate reductase system. The D. hafniense DCB-2 genome contained genes for 43 RNA polymerase sigma factors including 27 sigma-24 subunits, 59 two-component signal transduction systems, and about 730 transporter proteins. In addition, it contained genes for 53 molybdopterin-binding oxidoreductases, 19 flavoprotein paralogs of the fumarate reductase, and many other FAD/FMN-binding oxidoreductases, proving the cell's versatility in both adaptive and reductive capacities. Together with the ability to form spores, the presence of the CO2-fixing Wood-Ljungdahl pathway and the genes associated with oxygen tolerance add flexibility to the cell's options for survival under stress. D. hafniense DCB-2's genome contains genes consistent with its abilities for dehalogenation, metal reduction, N2 and CO2 fixation, anaerobic respiration, oxygen tolerance, spore formation, and biofilm formation which make this organism a potential candidate for bioremediation at contaminated sites.


Fine needle aspiration cytology of papillary lesions of the breast: how accurate is the diagnosis?
Cytological diagnosis of mammary papillary lesions is difficult. To review the previous cytology diagnosis of 23 papillomas and 11 papillary carcinomas and specific cytological features that may assist in differentiating these entities. The cytology preparations were reviewed for: (i) overall cellularity; (ii) epithelial cell ball devoid of fibrovascular cores; (iii) background single cells; and (iv) papillary fragments and their morphology. The overall diagnostic accuracy was 59%, atypical rate was 24%, and the error (combined false positive and negative) rate was 17%. For overall cellularity, 6, 14 and 3 cases of papillomas, and 6, 3 and 2 cases of papillary carcinomas showed low, moderate and high cellularity, respectively. Cell balls were present in mild to moderate number in 20 papillomas and 10 papillary carcinomas. The background single cells were absent, or present in low or moderate to high numbers in 7, 10 and 6 papillomas, and 3, 3 and 5 papillary carcinomas, respectively. Papillary fragments were absent, or present in small, moderate or large quantities in 9, 4, 8 and 2 papillomas, and 6, 3, 1 and 1 papillary carcinomas, respectively. There was no demonstrable quantitative difference between papilloma and papillary carcinoma for all these parameters. Qualitatively, the cell balls and single cells showed a higher degree of atypia in papillary carcinoma, and the papillary fragments were more elaborate and slender. Cytological diagnosis of papillary lesions shows a significant error rate with overlapping features. Cellular atypia and fragments with long and slender papillae with ramifying edges favour papillary carcinoma.


Feasibility, Safety, and Efficacy of Posterior Wall Isolation During Atrial Fibrillation Ablation: A Systematic Review and Meta-Analysis.
The posterior left atrium is an arrhythmogenic substrate that contributes to the initiation and maintenance of atrial fibrillation (AF); however, the feasibility, safety, and efficacy of posterior wall isolation (PWI) as an AF ablation strategy has not been widely reported. We undertook a systematic review and meta-analysis of studies performing PWI to assess (1) acute procedural success including the ability to achieve PWI and the number of procedure-related complications, (2) Long-term, clinical success including rates of arrhythmia recurrence and posterior wall reconnection, and (3) The efficacy of PWI compared with pulmonary vein isolation on preventing arrhythmia recurrence. MEDLINE, EMBASE, and Web of Science databases were searched in May 2018 to retrieve relevant studies. Results were pooled using a random effects model. Seventeen studies (13 box isolation, 3 single ring isolation, and 1 debulking ablation) comprising 1643 patients (31.3% paroxysmal AF, left atrial diameter 41±3.1 mm) were included in the final analysis. In studies focusing specifically on PWI, the acute procedural success rate for achieving PWI was 94.1% (95% CI, 87.2%-99.3%). Single-procedure 12-month freedom from atrial arrhythmia was 65.3% (95% CI, 57.7%-73.9%) overall and 61.9% (54.2%-70.8%) for persistent AF. Randomized control trials comparing PWI to pulmonary vein isolation (3 studies, 444 patients) yielded conflicting results and could not confirm an incremental benefit to PWI. Fifteen major complications (0.1%), including 2 atrio-esophageal fistulas, were reported. PWI as an end point of AF ablation can be achieved in a large proportion of cases with good rates of 12-month freedom from atrial arrhythmia. Although the procedure-related complication rate is low, it did not eliminate the risk of atrio-esophageal fistula. Registration: URL: http://www.crd.york.ac.uk/prospero. PROSPERO registration number: CRD42018107212.


Social modulation of pain as evidence for empathy in mice.
Empathy is thought to be unique to higher primates, possibly to humans alone. We report the modulation of pain sensitivity in mice produced solely by exposure to their cagemates, but not to strangers, in pain. Mice tested in dyads and given an identical noxious stimulus displayed increased pain behaviors with statistically greater co-occurrence, effects dependent on visual observation. When familiar mice were given noxious stimuli of different intensities, their pain behavior was influenced by their neighbor's status bidirectionally. Finally, observation of a cagemate in pain altered pain sensitivity of an entirely different modality, suggesting that nociceptive mechanisms in general are sensitized.


Insomnia symptoms and risk of cardiovascular diseases among 0.5 million adults: A 10-year cohort.
To examine the associations of individual insomnia symptoms with risks of incident cardio-cerebral vascular diseases (CVD) and possible moderating factors among Chinese adults. The China Kadoorie Biobank is a prospective cohort study that recruited participants from 10 areas across China. Data from 487,200 adults 30 to 79 years of age who were free of stroke, coronary heart disease, and cancer at baseline were analyzed. Three insomnia symptoms were assessed with self-reported difficulties in initiating or maintaining sleep, early morning awakening, and daytime dysfunction for at least 3 d/wk at baseline. Incidences of CVD were followed up through disease registries and national health insurance databases until 2016. During a median of 9.6 years of follow-up, 130,032 cases of CVD were documented. Cox regressions showed that 3 insomnia symptoms were associated with increased risk of total CVD, with respective adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) of 1.09 (95% CI 1.07-1.11), 1.07 (95% CI 1.05-1.09), and 1.13 (95% CI 1.09-1.18). Participants with individual symptoms also had higher risks of ischemic heart disease (IHD; HR 1.13, 1.09, and 1.17) and ischemic stroke but not hemorrhagic stroke. Participants with all 3 symptoms were at an 18%, 22%, or 10% higher risk of CVD, IHD, or ischemic stroke compared to nonsymptomatic adults. Associations between 3 symptoms and CVD incidence were consistently stronger in younger adults or those without baseline hypertension (p for interaction <0.05). Individual and coexisting insomnia symptoms are independent risk factors for CVD incidence, especially among young adults or adults who have not developed hypertension.


A gating charge transfer center in voltage sensors.
Voltage sensors regulate the conformations of voltage-dependent ion channels and enzymes. Their nearly switchlike response as a function of membrane voltage comes from the movement of positively charged amino acids, arginine or lysine, across the membrane field. We used mutations with natural and unnatural amino acids, electrophysiological recordings, and x-ray crystallography to identify a charge transfer center in voltage sensors that facilitates this movement. This center consists of a rigid cyclic "cap" and two negatively charged amino acids to interact with a positive charge. Specific mutations induce a preference for lysine relative to arginine. By placing lysine at specific locations, the voltage sensor can be stabilized in different conformations, which enables a dissection of voltage sensor movements and their relation to ion channel opening.


An adapted anti-CTLA4 therapeutic aimed at mitigating the toxicities of checkpoint inhibition.
Antibodies that target immune checkpoint molecules, such as CTLA4, provide robust antitumor effects in a subset of patients. Unfortunately, not all patients respond to immune checkpoint inhibition, and some develop life-threatening immune-related adverse events (irAEs). The mechanisms that underlie irAEs from immune checkpoint inhibition are not fully understood, and treatment strategies are currently limited to targeting inflammatory mediators. In this issue of the JCI, Pai et al. report on their development of a modified CTLA4 antibody that shields the inner CTLA4-binding domain until the antibody is within the protease-rich tumor microenvironment. In a lymphopenic murine model reconstituted with naive CD4+ T cells, adapted anti-CTLA4 reduced the occurrence of irAEs and enhanced antitumor effects. This thought-provoking study lays the groundwork for further exploration of this adapted antibody in immunocompetent hosts and introduction of this adaptation to other immune checkpoint molecules. It also suggests that this approach may reduce the incidence of irAEs.


Primary hyperparathyroidism causing putty kidney with brown tumor located in the pubic ramus.
Primary hyperparathyroidism (pHPT) is a condition caused by excessive and uncontrolled secretion of parathyroid hormone. The classical presenting symptoms in primary hyperparathyroidism are renal stones, hypercalcemic crisis, soft tissue calcifications, and cystic bone disease. Although most cases of pHPT are detected early and before symptomatic lesions appear, some patients may present late in the course of their disease. In this report, we present a patient with extensive parenchymal calcifications forming a cast of the right kidney which is called as "putty kidney" with Brown tumor located in the pubic ramus.


A system for determination of 3D vessel tree centerlines from biplane images.
With the increasing number and complexity of therapeutic coronary interventions, there is an increasing need for accurate quantitative measurements. These interventions and measurements may be facilitated by accurate and reproducible magnifications and orientations of the vessel structures, specifically by accurate 3D vascular tree centerlines. A number of methods have been proposed to calculate 3D vascular tree centerlines from biplane images. In general, the calculated magnifications and orientations are accurate to within approximately 1-3% and 2-5 degrees, respectively. Here, we present a complete system for determination of the 3D vessel centerlines from biplane angiograms without the use of a calibration object. Subsequent to indication of the vessel centerlines, the imaging geometry and 3D centerlines are calculated automatically and within approximately 2 min. The system was evaluated in terms of the intra- and inter-user variations of the various calculated quantities. The reproducibilities obtained with this system are comparable to or better than the accuracies and reproducibilities quoted for other proposed methods. Based on these results and those reported in earlier studies, we believe that this system will provide accurate and reproducible vascular tree centerlines from biplane images while the patient is still on the table, and thereby will facilitate interventions and associated quantitative analyses of the vasculature.


A novel mechanism of inhibition by phenylthiourea on PvdP, a tyrosinase synthesizing pyoverdine of Pseudomonas aeruginosa.
The biosynthesis of pyoverdine, the major siderophore of Pseudomonas aeruginosa, is a well-organized process involving a discrete number of enzyme-catalyzed steps. The final step of this process involves the PvdP tyrosinase, which converts ferribactin into pyoverdine. Thus, inhibition of the PvdP tyrosinase activity provides an attractive strategy to interfere with siderophore synthesis to manage P. aeruginosa infections. Here, we report phenylthiourea as a non-competitive inhibitor of PvdP for which we solved a crystal structure in complex with PvdP. The crystal structure indicates that phenylthiourea binds to an allosteric binding site and thereby interferes with its tyrosinase activity. We further provide proofs that PvdP tyrosinase inhibition by phenylthiourea requires the C-terminal lid region. This provides opportunities to develop inhibitors that target the allosteric site, which seems to be confined to fluorescent pseudomonads, and not the tyrosinase active site. Furthermore, increases the chances to identify PvdP inhibitors that selectively interfere with siderophore synthesis.


Normalization of raised sodium absorption and raised calcium-mediated chloride secretion by adenovirus-mediated expression of cystic fibrosis transmembrane conductance regulator in primary human cystic fibrosis airway epithelial cells.
Cystic fibrosis airway epithelia exhibit a spectrum of ion transport properties that differ from normal, including not only defective cAMP-mediated Cl- secretion, but also increased Na+ absorption and increased Ca(2+)-mediated Cl- secretion. In the present study, we examined whether adenovirus-mediated (Ad5) transduction of CFTR can correct all of these CF ion transport abnormalities. Polarized primary cultures of human CF and normal nasal epithelial cells were infected with Ad5-CBCFTR at an moi (10(4)) which transduced virtually all cells or Ad5-CMV lacZ as a control. Consistent with previous reports, Ad5-CBCFTR, but not Ad5-CMV lacZ, corrected defective CF cAMP-mediated Cl- secretion. Basal Na+ transport rates (basal Ieq) in CF airway epithelial sheets (-78.5 +/- 9.8 microA/cm2) were reduced to levels measured in normal epithelial sheets (-30.0 +/- 2.0 microA/cm2) by Ad5-CBCFTR (-36.9 +/- 4.8 microA/cm2), but not Ad5-CMV lacZ (-65.8 +/- 6.1 microA/cm2). Surprisingly, a significant reduction in delta Ieq in response to ionomycin, a measure of Ca(2+)-mediated Cl- secretion, was observed in CFTR-expressing (corrected) CF epithelial sheets (-6.9 +/- 11.8 microA/cm2) when compared to uninfected CF epithelial sheets (-76.2 +/- 15.1 microA/cm2). Dose response effects of Ad5-CBCFTR on basal Na+ transport rates and Ca(2+)-mediated Cl- secretion suggest that the mechanism of regulation of these two ion transport functions by CFTR may be different. In conclusion, efficient transduction of CFTR corrects hyperabsorption of Na+ in primary CF airway epithelial cells and restores Ca(2+)-mediated Cl- secretion to levels observed in normal airway epithelial cells. Moreover, assessment of these ion transport abnormalities may represent important endpoints for testing the efficacy of gene therapy for cystic fibrosis.


Effect of Triflusal on Primary Vascular Dysregulation Compared with Aspirin: A Double-Blind, Randomized, Crossover Trial.
Primary vascular dysregulation (PVD) is a condition in which the response to cold temperature or external stimuli is abnormal. We investigated whether triflusal use results in amelioration of PVD symptoms and improvement of several related parameters compared with aspirin. Eighty-eight PVD patients (54% female, 56±8 years) were randomly selected to receive either triflusal (300 mg, b.i.d.) or aspirin (150 mg, b.i.d.) for a period of 6 weeks followed by crossover. PVD was defined as both red-blood-cell standstill in video-assisted microscopic capillaroscopy during cold stimulation using carbon dioxide gas and a score of more than 7 points in a validated questionnaire. Efficacy of treatment was assessed by 1) cold intolerance symptom severity (CISS) score, 2) finger Doppler indices, and 3) indocyanine green perfusion imaging. The use of triflusal resulted in a greater improvement in CISS score (44.5±18.4 vs. 51.9±16.2; p<0.001) and in mean radial peak systolic velocity (69.8±17.2 vs. 66.1±16.4; p=0.011) compared to aspirin. Furthermore, significant differences were also observed in perfusion rates on indocyanine green perfusion imaging between triflusal and aspirin (45.6±25.8 vs. 51.6±26.9; p=0.020). Triflusal was more effective and demonstrated a more consistent impact on the improvement of symptoms and blood flow in patients with PVD than aspirin.


The WNK1 and WNK4 protein kinases that are mutated in Gordon's hypertension syndrome phosphorylate and activate SPAK and OSR1 protein kinases.
Mutations in the human genes encoding WNK1 [with no K (lysine) protein kinase-1] and the related protein kinase WNK4 are the cause of Gordon's hypertension syndrome. Little is known about the molecular mechanism by which WNK isoforms regulate cellular processes. We immunoprecipitated WNK1 from extracts of rat testis and found that it was specifically associated with a protein kinase of the STE20 family termed 'STE20/SPS1-related proline/alanine-rich kinase' (SPAK). We demonstrated that WNK1 and WNK4 both interacted with SPAK as well as a closely related kinase, termed 'oxidative stress response kinase-1' (OSR1). Wildtype (wt) but not catalytically inactive WNK1 and WNK4 phosphorylated SPAK and OSR1 to a much greater extent than with other substrates utilized previously, such as myelin basic protein and claudin-4. Phosphorylation by WNK1 or WNK4 markedly increased SPAK and OSR1 activity. Phosphopeptide mapping studies demonstrated that WNK1 phosphorylated kinase-inactive SPAK and OSR1 at an equivalent residue located within the T-loop of the catalytic domain (Thr233 in SPAK, Thr185 in OSR1) and a serine residue located within a C-terminal non-catalytic region (Ser373 in SPAK, Ser325 in OSR1). Mutation of Thr185 to alanine prevented the activation of OSR1 by WNK1, whereas mutation of Thr185 to glutamic acid (to mimic phosphorylation) increased the basal activity of OSR1 over 20-fold and prevented further activation by WNK1. Mutation of Ser325 in OSR1 to alanine or glutamic acid did not affect the basal activity of OSR1 or its ability to be activated by WNK1. These findings suggest that WNK isoforms operate as protein kinases that activate SPAK and OSR1 by phosphorylating the T-loops of these enzymes, resulting in their activation. Our analysis also describes the first facile assay that can be employed to quantitatively assess WNK1 and WNK4 activity.