Mouse models of neointimal hyperplasia: techniques and applications.
Neointimal hyperplasia is a major cause of the failure in vascular reconstructive procedures such as angioplasty, vascular stenting, small caliber vascular graft, and vein graft. However, the underlying molecular mechanisms are not yet fully understood. The study of neointimal hyperplasia has relied heavily on the use of experimental animal models. Recent development in gene manipulation techniques in mice offers a unique opportunity to unravel the molecular basis of the neointimal response at the genetic level, which is critical to develop new strategies to prevent human neointimal hyperplasia. Several mouse models for studying neointimal hyperplasia have recently been established including blood-flow cessation, mechanical injury, and vein bypass graft. In an attempt to elaborate these models, this review highlights the characteristics, advantages, disadvantages, and applications of these mouse models in vascular disease. In addition, the difference between mouse models and human lesions is discussed. Thus, this review provides updated information and helps vascular surgeons and other vascular biologists in selecting appropriate mouse models for their research on neointimal hyperplasia.


Determination of critical nucleation number for a single nucleation amyloid-β aggregation model.
Aggregates of amyloid-β (Aβ) peptide are known to be the key pathological agents in Alzheimer disease (AD). Aβ aggregates to form large, insoluble fibrils that deposit as senile plaques in AD brains. The process of aggregation is nucleation-dependent in which the formation of a nucleus is the rate-limiting step, and controls the physiochemical fate of the aggregates formed. Therefore, understanding the properties of nucleus and pre-nucleation events will be significant in reducing the existing knowledge-gap in AD pathogenesis. In this report, we have determined the plausible range of critical nucleation number (n(*)), the number of monomers associated within the nucleus for a homogenous aggregation model with single unique nucleation event, by two independent methods: A reduced-order stability analysis and ordinary differential equation based numerical analysis, supported by experimental biophysics. The results establish that the most likely range of n(*) is between 7 and 14 and within, this range, n(*) = 12 closely supports the experimental data. These numbers are in agreement with those previously reported, and importantly, the report establishes a new modeling framework using two independent approaches towards a convergent solution in modeling complex aggregation reactions. Our model also suggests that the formation of large protofibrils is dependent on the nature of n(*), further supporting the idea that pre-nucleation events are significant in controlling the fate of larger aggregates formed. This report has re-opened an old problem with a new perspective and holds promise towards revealing the molecular events in amyloid pathologies in the future.


Repeated administration of MDMA causes transient down-regulation of serotonin 5-HT2 receptors.
The present study examined short- and long-term effects of MDMA (3,4-methylene-dioxymethamphetamine) on serotonin (5-HT2 and 5-HT1c) receptors in the brain of the rat. N1-Methyl-2-[125I]lysergic acid diethylamide ([125I]MIL) was used to label these receptors in vitro and in vivo. The usefulness of [125I]MIL for in vivo detection of changes in 5-HT2 receptors was confirmed in preliminary experiments in which rats were treated chronically with mianserin (5 mg/kg, once daily for 10 days). Decreases in specific in vivo binding of [125I]MIL, after treatment with mianserin were found to be of the same magnitude as those determined by others, using in vitro methods. The MDMA (8 doses; 5-20 mg/kg each) was administered to rats over a period of 4 days. At various times after administration of the last dose of MDMA, the binding of [125I]MIL was measured. Acutely, treatment with MDMA (20 mg/kg) reduced specific in vivo binding of [125I]MIL in all regions of brain studied. For example, in the frontal cortex, specific binding of [125I]MIL was decreased by 80% at 6 hr and by 62% at 24 hr, after cessation of treatment with MDMA. Twenty-one days after administration of MDMA however, the number of binding sites for [125I]MIL was back to control levels. Reductions in in vivo binding of [125I]MIL in frontal cortex were dependent on the dose of MDMA injected and were associated with decreases in the number of binding sites for [125I]MIL (Bmax values) in tissue homogenates of the same area. Autoradiographic studies of MDMA-treated rats confirmed the decreased density of 5-HT2 receptors and also suggested that the 5-HT1c receptor of the choroid plexus was not affected. These results indicate that repeated administration of MDMA caused transient down-regulation of 5-HT2 receptors in the brain of the rat. Further, they demonstrated that [125I]MIL is a suitable radioligand for labeling 5-HT2 receptors, both in vitro and in vivo. Once labeled with an appropriate radionuclide for SPECT (single photon emission computed tomography) or PET (positron emission tomography), MIL should prove useful for monitoring changes in the density of serotonin receptors in the living mammalian brain.


Synthesis of rigid p-terphenyl-linked carbohydrate mimetics.
An approach to β-D-2-aminotalose- and β-D-2-aminoidose-configured carbohydrate mimetics bearing a phenyl substituent is described. Unnatural divalent rigid p-terphenyl-linked C-aryl glycosides with 2.0 nm dimension are available using Suzuki cross-couplings. The key compound, a p-bromophenyl-substituted 1,2-oxazine, was prepared by a stereoselective [3 + 3]-cyclization of a D-isoascorbic acid-derived (Z)-nitrone and lithiated TMSE-allene. The Lewis acid-induced rearrangement of this heterocycle provided the corresponding bicyclic 1,2-oxazine derivative that may be regarded as internally protected amino sugar analogue. After subsequent reduction of the carbonyl group, the resulting bicyclic compound was used for Suzuki cross-couplings to form biphenyl aminopyran or p-terphenyl-linked dimers. Hydrogenolysis afforded new unnatural aminosugar mimetics. Zinc in the presence of acid or samarium diiodide were examined for the N-O bond cleavage in order to obtain the rigid p-terphenyl-linked C-glycosyl dimers.


Characterizing the Genetic Basis for Nicotine Induced Cancer Development: A Transcriptome Sequencing Study.
Nicotine is a known risk factor for cancer development and has been shown to alter gene expression in cells and tissue upon exposure. We used Illumina® Next Generation Sequencing (NGS) technology to gain unbiased biological insight into the transcriptome of normal epithelial cells (MCF-10A) to nicotine exposure. We generated expression data from 54,699 transcripts using triplicates of control and nicotine stressed cells. As a result, we identified 138 differentially expressed transcripts, including 39 uncharacterized genes. Additionally, 173 transcripts that are primarily associated with DNA replication, recombination, and repair showed evidence for alternative splicing. We discovered the greatest nicotine stress response by HPCAL4 (up-regulated by 4.71 fold) and NPAS3 (down-regulated by -2.73 fold); both are genes that have not been previously implicated in nicotine exposure but are linked to cancer. We also discovered significant down-regulation (-2.3 fold) and alternative splicing of NEAT1 (lncRNA) that may have an important, yet undiscovered regulatory role. Gene ontology analysis revealed nicotine exposure influenced genes involved in cellular and metabolic processes. This study reveals previously unknown consequences of nicotine stress on the transcriptome of normal breast epithelial cells and provides insight into the underlying biological influence of nicotine on normal cells, marking the foundation for future studies.


Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia.
Effective targeted cancer therapeutic development depends upon distinguishing disease-associated 'driver' mutations, which have causative roles in malignancy pathogenesis, from 'passenger' mutations, which are dispensable for cancer initiation and maintenance. Translational studies of clinically active targeted therapeutics can definitively discriminate driver from passenger lesions and provide valuable insights into human cancer biology. Activating internal tandem duplication (ITD) mutations in FLT3 (FLT3-ITD) are detected in approximately 20% of acute myeloid leukaemia (AML) patients and are associated with a poor prognosis. Abundant scientific and clinical evidence, including the lack of convincing clinical activity of early FLT3 inhibitors, suggests that FLT3-ITD probably represents a passenger lesion. Here we report point mutations at three residues within the kinase domain of FLT3-ITD that confer substantial in vitro resistance to AC220 (quizartinib), an active investigational inhibitor of FLT3, KIT, PDGFRA, PDGFRB and RET; evolution of AC220-resistant substitutions at two of these amino acid positions was observed in eight of eight FLT3-ITD-positive AML patients with acquired resistance to AC220. Our findings demonstrate that FLT3-ITD can represent a driver lesion and valid therapeutic target in human AML. AC220-resistant FLT3 kinase domain mutants represent high-value targets for future FLT3 inhibitor development efforts.


Estimation of Dynamic Sparse Connectivity Patterns From Resting State fMRI.
Functional connectivity (FC) estimated from functional magnetic resonance imaging (fMRI) time series, especially during resting state periods, provides a powerful tool to assess human brain functional architecture in health, disease, and developmental states. Recently, the focus of connectivity analysis has shifted toward the subnetworks of the brain, which reveals co-activating patterns over time. Most prior works produced a dense set of high-dimensional vectors, which are hard to interpret. In addition, their estimations to a large extent were based on an implicit assumption of spatial and temporal stationarity throughout the fMRI scanning session. In this paper, we propose an approach called dynamic sparse connectivity patterns (dSCPs), which takes advantage of both matrix factorization and time-varying fMRI time series to improve the estimation power of FC. The feasibility of analyzing dynamic FC with our model is first validated through simulated experiments. Then, we use our framework to measure the difference between young adults and children with real fMRI data set from the Philadelphia Neurodevelopmental Cohort (PNC). The results from the PNC data set showed significant FC differences between young adults and children in four different states. For instance, young adults had reduced connectivity between the default mode network and other subnetworks, as well as hyperconnectivity within the visual system in states 1 and 3, and hypoconnectivity in state 2. Meanwhile, they exhibited temporal correlation patterns that changed over time within functional subnetworks. In addition, the dSCPs model indicated that older people tend to spend more time within a relatively connected FC pattern. Overall, the proposed method provides a valid means to assess dynamic FC, which could facilitate the study of brain networks.


Uncertainty Class Activation Map (U-CAM) Using Gradient Certainty Method.
Understanding and explaining deep learning models is an imperative task. Towards this, we propose a method that obtains gradient-based certainty estimates that also provide visual attention maps. Particularly, we solve for visual question answering task. We incorporate modern probabilistic deep learning methods that we further improve by using the gradients for these estimates. These have two-fold benefits: a) improvement in obtaining the certainty estimates that correlate better with misclassified samples and b) improved attention maps that provide state-of-the-art results in terms of correlation with human attention regions. The improved attention maps result in consistent improvement for various methods for visual question answering. Therefore, the proposed technique can be thought of as a tool for obtaining improved certainty estimates and explanations for deep learning models. We provide detailed empirical analysis for the visual question answering task on all standard benchmarks and comparison with state of the art methods.


Absence of REM rebound after barbiturate withdrawal.
Administration of three different barbiturates reduced rapid eye movement (REM) sleep. Drug withdrawal led to a return to baseline REM] values without significant overshoot. Similar results are observed with administration of benzodiazepines in pharmacologically equivalent dosages; therefore, a distinction between these two drug classes on the basis of withdrawal effects on the sleep electroencephalogram appears unwarranted. Further investigation is required determine why high REM levels are sometimes associated with the withdrawal of sedative-hypnotic agents.


Prevalence of musculoskeletal injuries and a proposal for neuromuscular training to prevent lower limb injuries in Brazilian Army soldiers: an observational study.
The activities carried out by soldiers in the army involve great physical demands and require intense trainings to perform combat-specific tasks. Musculoskeletal injury is a potential threat to the health and physical integrity of the soldier. This study aimed to evaluate the prevalence of lower limb musculoskeletal injuries among soldiers and to propose a training protocol to prevent the most frequent injuries. This observational (cross-sectional) study recruited a sample of 103 soldiers who required medical attention, from a total 202 new battalion soldiers. The medical records (paper and online) had a form of running text. All data collected were recorded by the registered physicians of the battalion medical post. The records were analyzed by the following variables: medical diagnosis, injury site, mechanism, type of treatment, time loss, existence of previous injury, and recurring injury. A total of 112 musculoskeletal injuries were diagnosed in 71 soldiers, and other types of diseases/injuries were diagnosed in the other soldiers. Joint pain accounted for 55.4% of the diagnoses. The knee was the most affected site, while trauma and overload were the most common mechanisms of injury. Drug treatment was used most frequently, accounting for 58% of the cases. The majority of the sample obtained a temporary leave of absence for 1 to 6 days or not at all. Previous injuries and recurrence were not presented as risk factors for injury. With the data received, a protocol for the prevention of injuries to the lower limbs was proposed. This study indicated that the most frequent site of injury is the knee, and joint pain is the most common diagnosis. These results may support the necessity to develop a neuromuscular training protocol to prevent lower limb injuries, which we suggest to be applied in future studies.


Myxoma virus and the Leporipoxviruses: an evolutionary paradigm.
Myxoma virus (MYXV) is the type species of the Leporipoxviruses, a genus of Chordopoxvirinae, double stranded DNA viruses, whose members infect leporids and squirrels, inducing cutaneous fibromas from which virus is mechanically transmitted by biting arthropods. However, in the European rabbit (Oryctolagus cuniculus), MYXV causes the lethal disease myxomatosis. The release of MYXV as a biological control for the wild European rabbit population in Australia, initiated one of the great experiments in evolution. The subsequent coevolution of MYXV and rabbits is a classic example of natural selection acting on virulence as a pathogen adapts to a novel host species. Slightly attenuated mutants of the progenitor virus were more readily transmitted by the mosquito vector because the infected rabbit survived longer, while highly attenuated viruses could be controlled by the rabbit immune response. As a consequence, moderately attenuated viruses came to dominate. This evolution of the virus was accompanied by selection for genetic resistance in the wild rabbit population, which may have created an ongoing co-evolutionary dynamic between resistance and virulence for efficient transmission. This natural experiment was repeated on a continental scale with the release of a separate strain of MYXV in France and its subsequent spread throughout Europe. The selection of attenuated strains of virus and resistant rabbits mirrored the experience in Australia in a very different environment, albeit with somewhat different rates. Genome sequencing of the progenitor virus and the early radiation, as well as those from the 1990s in Australia and Europe, has shown that although MYXV evolved at high rates there was no conserved route to attenuation or back to virulence. In contrast, it seems that these relatively large viral genomes have the flexibility for multiple pathways that converge on a similar phenotype.


Hypertension in the military patient.
Hypertension and hypertension-related diseases are a leading cause of morbidity and mortality worldwide. A diagnosis of hypertension can have serious occupational implications for military personnel. This article examines the diagnosis and management of hypertension in military personnel, in the context of current international standards. We consider the consequences of hypertension in the military environment and potential military-specific issues relating to hypertension.


3D bioprinting of tissues and organs.
Additive manufacturing, otherwise known as three-dimensional (3D) printing, is driving major innovations in many areas, such as engineering, manufacturing, art, education and medicine. Recent advances have enabled 3D printing of biocompatible materials, cells and supporting components into complex 3D functional living tissues. 3D bioprinting is being applied to regenerative medicine to address the need for tissues and organs suitable for transplantation. Compared with non-biological printing, 3D bioprinting involves additional complexities, such as the choice of materials, cell types, growth and differentiation factors, and technical challenges related to the sensitivities of living cells and the construction of tissues. Addressing these complexities requires the integration of technologies from the fields of engineering, biomaterials science, cell biology, physics and medicine. 3D bioprinting has already been used for the generation and transplantation of several tissues, including multilayered skin, bone, vascular grafts, tracheal splints, heart tissue and cartilaginous structures. Other applications include developing high-throughput 3D-bioprinted tissue models for research, drug discovery and toxicology.


Superoxide dismutase isoenzymes in cerebrospinal fluid and plasma from patients with neuronal ceroid-lipofuscinoses.
The neuronal ceroid-lipofuscinoses is a group of diseases characterized by a widespread accumulation in the body of pigments believed to be end-products of lipid-peroxidation damaged organelles. It was recently shown that cerebrospinal fluid from patients with infantile and juvenile neuronal ceroid-lipofuscinosis were less protective against superoxide radical-induced hydroxyl radical formation compared with controls. The content of superoxide dismutase isoenzymes in cerebrospinal fluid and in plasma from patients with different forms of neuronal ceroid-lipofuscinosis was analysed. No significant difference from controls could be demonstrated in samples from patients with juvenile neuronal ceroid-lipofuscinosis. The few samples from patients with infantile and late infantile neuronal ceroid-lipofuscinosis analysed all fell within the range defined by the controls.


Digital training platform for interpreting radiographic images of the chest.
Time delays and errors exist which lead to delays in patient care and misdiagnosis. Reporting clinicians follow guidance to form their own search strategy. However, little research has tested these training guides. With the use of eye tracking technology and expert input we developed a digital training platform to be used in chest image interpretation learning. Two sections of a digital training platform were planned and developed; A) a search strategy training tool to assist reporters during their interpretation of images, and B) an educational tool to communicate the search strategies of expert viewers to trainees by using eye tracking technology. A digital training platform for use in chest image interpretation was created based on evidence within the literature, expert input and two search strategies previously used in clinical practice. Images and diagrams, aiding translation of the platform content, were incorporated where possible. The platform is structured to allow the chest image interpretation process to be clear, concise and methodical. A search strategy was incorporated within the tool to investigate its use, with the possibility that it could be recommended as an evidence based approach for use by reporting clinicians. Eye tracking, a checklist and voice recordings have been combined to form a multi-dimensional learning tool, which has never been used in chest image interpretation learning before. The training platform for use in chest image interpretation learning has been designed, created and digitised. Future work will establish the efficacy of the developed approaches.


p38 MAPK activation triggers pharmacologically-induced beta-adrenergic preconditioning, but not ischaemic preconditioning.
p38 Mitogen-activated protein kinase (p38 MAPK) is activated by short episodes of ischaemia-reperfusion as well as by sustained ischemia followed by reperfusion, Whether activation of this kinase is beneficial or deleterious to the ischaemic heart is still a subject of controversy. Since transient beta-adrenergic stimulation (5 min) stimulates p38 MAPK activation and mimics the cardioprotection of ischaemic preconditioning, it was used as a tool to further evaluate the role of this kinase in cardioprotection. The isolated perfused working rat heart, subjected to 25 min ischaemia and 30 min reperfusion was used as experimental model. p38 MAPK and ATF2 activation was determined using Western blots. The results showed that isoproterenol stimulated p38 MAPK in a dose- and time-dependent manner. Ischaemia-induced activation of p38 MAPK could be partially abolished by beta- and alpha1-adrenergic receptor blockade. Isoproterenol activation of the kinase could be abolished by alprenolol and verapamil, but not by 8-cyclopentyladenosine. p38 MAPK activation induced by either a multi-episode preconditioning protocol or isoproterenol (10(-7) M for 5 min) was associated with a significant reduction in p38 MAPK activation at all time intervals studied during 25 min global ischaemia and at 20 and 30 min of reperfusion, compared with the marked activation observed in untreated non-preconditioned hearts. In each case attenuation of p38 MAPK activation during ischaemia and during reperfusion was associated with improved functional recovery during reperfusion. Cyclic elevations in tissue cAMP during an ischaemic preconditioning protocol acted as trigger of cardioprotection, since pretreatment of such hearts with alprenolol abolished cardioprotection. Mechanical failure in such hearts was characterized by a significant stimulation of p38 MAPK activity during ischaemia and reperfusion. However, p38 MAPK activation during an ischaemic preconditioning protocol did not act as trigger: inhibition of p38 MAPK activation by SB 203580 during the preconditioning phase did not abolish cardioprotection. In fact, functional recovery was significantly better than that of untreated preconditioned hearts. On the other hand, SB 203580, when administered before and during the isoproterenol-preconditioning protocol abolished cardioprotection, suggesting that p38 MAPK activation by a beta -adrenergic-induced preconditioning protocol does act as trigger of cardioprotection. In addition, attenuation of p38 MAPK activity during sustained ischaemia and reperfusion as occurs in ischaemic- or isoproterenol-preconditioned hearts, is beneficial.


Investigation of LPG-SPR sensors using the finite element method and eigenmode expansion method.
As compared to the well-known traditional couple-mode theory, in this study, we proposed a visual, graphical, and simple numerical simulation method for long-period fiber-grating surface-plasmon-resonance (LPG-SPR) sensors. This method combines the finite element method and the eigenmode expansion method. The finite element method was used to solve for the guided modes in fiber structures, including the surface plasmon wave. The eigenmode expansion method was used to calculate the power transfer phenomenon of the guided modes in the fiber structure. This study provides a detailed explanation of the key reasons why the periodic structure of long-period fiber-grating (LPG) can achieve significantly superior results for our method compared to those obtained using other numerical methods, such as the finite-difference time-domain and beam propagation methods. All existing numerical simulation methods focus on large-sized periodic components; only the method established in this study has 3D design and analysis capabilities. In addition, unlike the offset phenomenon of the design wavelength λ(D) and the maximum transmission wavelength λ(max) of the traditional coupled-mode theory, the method established in this study has rapid scanning LPG period capabilities. Therefore, during the initial component design process, only the operating wavelength must be set to ensure that the maximum transmission wavelength of the final product is accurate to the original setup, for example, λ = 1550 nm. We verified that the LPG-SPR sensor designed in this study provides a resolution of ~-45 dB and a sensitivity of ~27000 nm/RIU (refractive index unit). The objective of this study was to use the combination of these two numerical simulation methods in conjunction with a rigorous, simple, and complete design process to provide a graphical and simplistic simulation technique that reduces the learning time and professional threshold required for research and applications of LPG-SPR sensors.


Ongoing burden of disease and mortality from HIV/CMV coinfection in Africa in the antiretroviral therapy era.
Human Cytomegalovirus (CMV) is a well-recognized pathogen in the context of HIV infection, but since the roll out of ART, clinical and scientific interest in the problem of HIV/CMV coinfection has diminished. However, CMV remains a significant cofactor in HIV disease, with an influence on HIV acquisition, disease progression, morbidity, and mortality. Disease manifestations may be a result of direct interplay between the two viruses, or may arise as a secondary consequence of immune dysregulation and systemic inflammation. The problem is most relevant when the rates of coinfection are high, most notably in sub-Saharan Africa, and in children at risk of acquiring both infections early in life. Understanding the interplay between these viruses and developing strategies to diagnose, treat and prevent CMV should be a priority.


Assessment of fine needle aspiration as a screening test for occult prostatic carcinoma.
As part of an ongoing study of objective parameters of prognostic value in prostatic carcinoma, a routine procedure was developed to aspirate all prostates prior to surgery. These targets were different from those of other workers in the field of prostatic fine needle aspiration (FNA), who generally advocate that FNA be confined to suspicious nodules. The aspirations were performed by a large group of practicing urologists who had no special training in prostatic FNA except for guidelines provided by their peers and information available in the literature. This approach permitted an assessment of the performance of FNA as a screening test rather than as a diagnostic procedure. During the period from January 1983 to February 1987, 1,683 patients had prostatic FNAs performed (plus subsequent histologic study). The following diagnoses were rendered: "inadequate/scanty specimen" in 625 cases (37%), "negative/atypical" in 844 cases (50%) and "suspicious/positive" in 214 cases (13%). Histologic examination showed stage A1 prostatic adenocarcinoma in 18 patients. The cytologic diagnoses on these 18 patients were inadequate/scanty in 3 (17%), negative/atypical in 13 (72%) and suspicious/positive in 2 (11%). Of the 214 patients with a positive/suspicious diagnosis by FNA, the diagnosis of prostatic carcinoma was confirmed by tissue evidence in 200; the other 14 patients had either no evidence of prostatic carcinoma on surgical biopsy (needle biopsy/transurethral resection/suprapubic prostatectomy) or had no surgical biopsy. Eight of the 14 patients developed clinical evidence of carcinoma, 1 died of urinary bladder carcinoma and 1 was lost to follow-up. In the remaining four patients, there is still no evidence of prostatic carcinoma after about one-and-one-half years of follow-up. These results indicate that (1) specialized training is required in order to obtain adequate smears by prostatic FNA; (2) prostatic FNA is not a good screening technique for detecting stage A1 prostatic carcinoma; and (3) a positive diagnosis by prostatic FNA, even when not confirmed by tissue biopsy, is still an indication of disease.


Mediterranean diet score is associated with greater allocentric processing in the EPAD LCS cohort: A comparative analysis by biogeographical region.
Background: Adherence to the Mediterranean diet (MedDiet), a primarily plant-based eating pattern, has been associated with lower dementia incidence. Much of the research has focused on Alzheimer's disease (AD) dementia and mild cognitive impairment (MCI), with less research looking at the preclinical symptomatically silent stages that pre-empt MCI and AD dementia. Although there is evidence from studies conducted globally, no studies have compared the effects of the MedDiet within and outside of the Mediterranean region in one cohort. Methods: Our study explored cross-sectional and longitudinal associations between MedDiet and cognition in the pan-European EPAD LCS, comparing those living within and outside of the Mediterranean region (as classified by European Union biogeographical definitions). After deriving MEDAS scores to quantify adherence to the MedDiet, we used linear regression and linear mixed effects models to test for associations between the MEDAS score and cognitive function measured by the Four Mountains Test (FMT) and the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). We additionally calculated MEDAS continuous and PYRAMID scores to provide alternative measures of MedDiet adherence. Results: We included 1826 participants, mean age 65.69 (±7.42) years, majority female (56.2%) with family history (65.8%) and minority APOEε4 carriers (38.9%). Higher MEDAS scores were associated with better performance on the FMT both cross-sectionally (n = 1,144, ß: -0.11, SE: 0.04, p = 0.007) and longitudinally (slope: 0.10, 95% CI: 0.04-0.17, p: 0.002). The effect was marginally greater in the Mediterranean region in the cross-sectional analysis, with a stronger effect emerging longitudinally. In exploratory analyses, the association between MEDAS and FMT scores was only seen in female participants. A sensitivity analysis excluding Toulouse and Perugia, as cities near, but not within, the biogeographical region, found significant associations between higher MEDAS and MEDAS continuous scores, and a number of RBANS total and index scores. Conclusion: MedDiet adherence is associated with better FMT scores, with effects seen most strongly in the Mediterranean region from longitudinal data. Our sensitivity analysis suggested a more global cognitive benefit of MedDiet adherence. This study highlights the need to further explore for whom and for what brain health outcomes the MedDiet confers benefit. This evidence would identify a window of opportunity in the life-course to maximise the benefit and better inform public health campaigns and patient-level interventions.


Protein-Templated Hit Identification through an Ugi Four-Component Reaction*.
Kinetic target-guided synthesis represents an efficient hit-identification strategy, in which the protein assembles its own inhibitors from a pool of complementary building blocks via an irreversible reaction. Herein, we pioneered an in situ Ugi reaction for the identification of novel inhibitors of a model enzyme and binders for an important drug target, namely, the aspartic protease endothiapepsin and the bacterial β-sliding clamp DnaN, respectively. Highly sensitive mass-spectrometry methods enabled monitoring of the protein-templated reaction of four complementary reaction partners, which occurred in a background-free manner for endothiapepsin or with a clear amplification of two binders in the presence of DnaN. The Ugi products we identified show low micromolar activity on endothiapepsin or moderate affinity for the β-sliding clamp. We succeeded in expanding the portfolio of chemical reactions and biological targets and demonstrated the efficiency and sensitivity of this approach, which can find application on any drug target.


alphaVbeta6 is a novel receptor for human fibrillin-1. Comparative studies of molecular determinants underlying integrin-rgd affinity and specificity.
Human fibrillin-1, the major structural protein of connective tissue 10-12 nm microfibrils, contains multiple calcium binding epidermal growth factor-like domains interspersed with transforming growth factor beta-binding protein-like (TB) domains. TB4 contains a flexible RGD loop that mediates cell adhesion via alphaVbeta3 and alpha5beta1 integrins. This study identifies integrin alphaVbeta6 as a novel cellular receptor for fibrillin-1 with a K(d) of approximately 0.45 mum. Analyses of this interaction by surface plasmon resonance and immunocytochemistry reveal different module requirements for alphaVbeta6 activation compared with those of alphaVbeta3, suggesting that a covalent linkage of an N-terminal calcium binding epidermal growth factor-like domain to TB4 can modulate alphaV integrin binding specificity. Furthermore, our data suggest alpha5beta1 is a low affinity fibrillin-1 receptor (K(d) > 1 mum), thus providing a molecular explanation for the different alpha5beta1 distribution patterns seen when human keratinocytes and fibroblasts are plated on recombinant fibrillin fragments versus those derived from the physiological ligand fibronectin. Non-focal contact distribution of alpha5beta1 suggests that its engagement by fibrillin-1 may elicit a lesser degree and/or different type of intracellular signaling compared with that seen with a high affinity ligand.


Risk of new-onset atrial fibrillation after drug-eluting stent implantation in patients with stable coronary artery disease.
New-onset atrial fibrillation (NOAF) is associated with adverse outcomes in patients with coronary artery disease (CAD). Although newer generation drug-eluting stents (NG-DESs) are more beneficial than bare-metal stents (BMSs) in reducing the risk of in-stent restenosis and revascularization, whether NG-DES implantation in patients with stable CAD reduces NOAF risk compared with BMS implantation remains unknown. This population-based cohort study was conducted using data from Taiwan's National Health Insurance Research Database. Propensity score matching was used to select 18,423 pairs of patients with stable CAD receiving NG-DES implantation and BMS implantation with similar baseline characteristics for evaluation. A competing risk model was used to evaluate the risk of NOAF between the NG-DES and BMS groups in which death was considered a competing risk. After adjustment for patients' clinical variables, the use of NG-DESs was associated with a decreased risk of NOAF at 1-year follow-up (adjusted subdistribution hazard ratio [SHR] = 0.79, 95% confidence interval [CI] = 0.68-0.93, P = 0.005) compared with the use of BMSs. Similar results indicated that NG-DESs were beneficial for reducing the risk of NOAF (adjusted SHR = 0.81, 95% CI = 0.67-0.97, P = 0.020) in patients with a CHA2DS2-VASc score of ≥2. These findings were also consistent with those for patients who received dual antiplatelet therapy for an undefined duration of >1 month following stent implantation. Our findings suggest that NG-DESs might reduce the risk of NOAF in patients with stable CAD.


Perlecan Diversely Regulates the Migration and Proliferation of Distinct Cell Types in vitro.
Perlecan is a multifunctional component of the extracellular matrix. It shows different effects on distinct cell types, and therefore it is thought to show potential for therapies targeting multiple cell types. However, the full range of multifunctionality of perlecan remains to be elucidated. We cultured various cell types, which were derived from epithelial/endothelial, connective and muscle tissues, in the presence of either antiserum against perlecan or exogenous perlecan, and examined the effects of perlecan on cell migration and proliferation. Cell migration was determined using a scratch assay. Blocking of perlecan by anti-perlecan antiserum inhibited the migration of vascular endothelial cells (VECs) and bone marrow-derived mesenchymal stem cells, and exogenous perlecan added to the culture medium promoted the migration of these cell types. The migration of other cell types was inhibited or was not promoted by exogenous perlecan. Cell proliferation was measured using a water-soluble tetrazolium dye. When cells were cultured at low densities, perlecan blocking inhibited the proliferation of VECs, and exogenous perlecan promoted the proliferation of keratinocytes. In contrast, the proliferation of fibroblasts, pre-adipocytes and vascular smooth muscle cells cultured at low densities was inhibited by exogenous perlecan. When cells were cultured at high densities, perlecan blocking promoted the proliferation of most cell types, with the exception of skeletal system-derived cells (chondrocytes and osteoblasts), which were inhibited by exogenous perlecan. Our results provide an overview of the multiple functions of perlecan in various cell types, and implicate a potential role of perlecan to inhibit undesirable activities, such as fibrosis, obesity and intimal hyperplasia.


Fresh Osteochondral Allograft Transplantation in the Knee: A Viability and Histologic Analysis for Optimizing Graft Viability and Expanding Existing Standard Processed Graft Resources Using a Living Donor Cartilage Program.
This study aims to (1) determine and validate living cartilage allograft transplantation as a novel source for viable osteochondral allograft (OCA) tissues and (2) perform histologic and viability comparisons of living donor cartilage tissues to currently available clinical-grade standard processed grafts. Using healthy cartilage from well-preserved contralateral compartments in 27 patients undergoing total knee arthroplasty (TKA) and 10 clinical-grade OCA specimens obtained immediately following operative implantation, standard and living donor OCA quality was evaluated at the time of harvest and following up to 3 weeks of storage on the basis of macroscopic International Cartilage Repair Society grade, histology, and viability. Osteochondral samples demonstrated a consistent decrease in viability and histologic quality over the first 3 weeks of storage at 37°C, supporting the utility of an OCA paradigm shift toward early implantation, as was the clinical standard up until recent adoption of transplantation at 14 to 35 days following donor procurement. Samples from the 10 clinical-grade OCAs, implanted at an average of 23 days following graft harvest demonstrated a mean viable cell density of 45.6% at implantation, significantly lower (P < 0.01) than the 93.6% viability observed in living donor allograft tissues. Osteochondral tissue viability and histologic quality progressively decreases with ex vivo storage, even when kept at physiologic temperatures. Currently available clinical OCAs are stored for 2 to 5 weeks prior to implantation and demonstrate inferior viability to that of fresh osteochondral tissues that can be made available through the use of a living donor cartilage program.


'Why must I wait?' The performance of legitimacy in a hospital emergency department.
This article examines the processes of negotiation that occur between patients and medical staff over accessing emergency medical resources. The field extracts are drawn from an ethnographic study of a UK emergency department (ED) in a large, inner city teaching hospital. The article focuses on the triage system for patient prioritisation as the first point of access to the ED. The processes of categorising patients for priority of treatment and care provide staff with the opportunities to maintain control over what defines the ED as a service, as types of work and as particular kinds of patients. Patients and relatives are implicated in this categorical work in the course of interactions with staff as they provide reasons and justifications for their attendance. Their success in legitimising their claim to treatment depends upon self-presentation and identity work that (re)produces individual responsibility as a dominant moral order. The extent to which people attending the ED can successfully perform as legitimate is shown to contribute to their placement into positive or negative staff-constituted patient categories, thus shaping their access to the resources of emergency medicine and their experience of care.


DNA polymerase beta can incorporate ribonucleotides during DNA synthesis of undamaged and CPD-damaged DNA.
Overexpression of the error-prone DNA polymerase beta (Pol beta) has been found to increase spontaneous mutagenesis by competing with the replicative polymerases during DNA replication. Here, we investigate an additional mechanism potentially used by Pol beta to enhance genetic instability via its ability to incorporate ribonucleotides into DNA. By using an in vitro primer extension assay, we show that purified human and calf thymus Pol beta can synthesize up to 8-mer long RNA. Moreover, Pol beta can efficiently incorporate rCTP opposite G in the absence of dCTP and, to a lesser extent, rATP opposite T in the absence of dATP and rGTP opposite C in the absence of dGTP. Recently, Pol beta was shown to catalyze in vitro translesion replication of a thymine cyclobutane pyrimidine dimer (CPD). Here, we investigate if ribonucleotides could be incorporated opposite the CPD damage and modulate the efficiency of the bypass process. We find that all four rNTPs can be incorporated opposite the CPD lesion, and that this process affects translesion synthesis. We discuss how incorporation of ribonucleotides into DNA may contribute to the high frequency of mutagenesis observed in Pol beta up-regulating cells.


Familial iris melanosis--a misnomer?
Iris melanosis is an unusual condition characterised by the presence of minute, discrete, pigmented elevations arising from the anterior surface of the iris. We encountered two unrelated Mexican families in which all children, but no parent, had varying degrees of the condition bilaterally. Some family members also gave findings suggestive of ocular hypertension. No family member had any other ocular or cutaneous pigmentary changes with the exception of a hairy naevus on the thigh of one member. To our knowledge these are the first families reported with more than one member having isolated iris melanosis. This is also the first report of a possible relationship with ocular hypertension. Finally we suggest that the term 'melanosis' may be a misnomer, since the condition is characterised not by abnormal iris hyperpigmentation but by discrete, round elevations on the anterior iris surface.


High- versus low-dose oxytocin for labor stimulation.
The number of cesarean births for dystocia has increased dramatically in the United States. Central to the management of dystocia is correction of ineffective labor by oxytocin administration, and contemporary obstetric practice is to stimulate labor with a low-dose oxytocin regimen. We prospectively compared a low-dose oxytocin regimen (1-mU/minute dosage increments) with a high-dose regimen (6-mU/minute dosage increments) in 2788 consecutive singleton cephalic pregnancies. The low-dose regimen was used first for 5 months in 1251 pregnancies, and the high-dose regimen in 1537 pregnancies during the subsequent 5 months. Indications for oxytocin stimulation were divided into augmentation (N = 1676) and induction (N = 1112). Labor stimulation was more than 3 hours shorter (P less than .0001) with the high-dose oxytocin regimen and associated with a reduction in neonatal sepsis (0.2 versus 1.3%; P less than .01). Uterine hyperstimulation was more common (55 versus 42%; P less than .0001) with the high-dose regimen, but no adverse fetal effects were observed. High-dose augmentation resulted in significantly fewer forceps deliveries (12 versus 16%; P = .03) and fewer cesareans for dystocia (9 versus 12%; P = .04). Similarly, failed induction was less frequent with high-dose compared with low-dose oxytocin (14 versus 19%; P = .05). Although the high-dose induction regimen was associated with a significantly increased cesarean incidence for fetal distress (6 versus 3%; P = .05), the incidence of umbilical artery cord blood acidemia was not increased in this subset. Induction of labor with high-dose oxytocin is problematic because of risk-benefit considerations. Although induction failed less frequently with the high-dose regimen, cesarean for fetal distress was performed more frequently. In contrast, high-dose oxytocin to augment ineffective spontaneous labor minimized the number of cesareans done for dystocia.


Factors contributing to late breast cancer presentation for health care amongst women in Kumasi, Ghana.
Delay in presenting breast cancer for health care is dangerous because it can increase the mortality rate amongst affected women. Delaying health care and treatment makes it difficult to manage advanced breast cancer successfully. Understanding the factors that contribute to delays in presentation for health care can save lives. The purpose of the study was to describe the factors which contribute to the late presentation of Ghanaian women with breast cancer for health care at a tertiary hospital in Kumasi, Ghana. A descriptive qualitative research design was utilised to answer the research question: 'What factors contribute to presenting with late breast cancer for health care amongst Ghanaian women who were treated for breast cancer at a tertiary hospital in Kumasi, Ghana?' A sample of 30 women diagnosed with breast cancer and presented with Stage II and Stage III participated in the study. Semi-structured interviews and field notes were conducted for data collection. Content data analysis was used in line with the research question. Five themes were discovered as findings. These were: lack of knowledge about breast cancer; fear of cancer treatment and its outcomes; poverty; traditional and spiritual beliefs and treatments and caring for others. We recommend the development of breast cancer awareness programmes and health education at primary health care level.


Prominent health problems, socioeconomic deprivation, and higher brain age in lonely and isolated individuals: A population-based study.
Loneliness is linked to increased risk for Alzheimer's disease, but little is known about factors potentially contributing to adverse brain health in lonely individuals. In this study, we used data from 24,867 UK Biobank participants to investigate risk factors related to loneliness and estimated brain age based on neuroimaging data. The results showed that on average, individuals who self-reported loneliness on a single yes/no item scored higher on neuroticism, depression, social isolation, and socioeconomic deprivation, performed less physical activity, and had higher BMI compared to individuals who did not report loneliness. In line with studies pointing to a genetic overlap of loneliness with neuroticism and depression, permutation feature importance ranked these factors as the most important for classifying lonely vs. not lonely individuals (ROC AUC = 0.83). While strongly linked to loneliness, neuroticism and depression were not associated with brain age estimates. Conversely, objective social isolation showed a main effect on brain age, and individuals reporting both loneliness and social isolation showed higher brain age relative to controls - as part of a prominent risk profile with elevated scores on socioeconomic deprivation and unhealthy lifestyle behaviours, in addition to neuroticism and depression. While longitudinal studies are required to determine causality, this finding may indicate that the combination of social isolation and a genetic predisposition for loneliness involves a risk for adverse brain health. Importantly, the results underline the complexity in associations between loneliness and adverse health outcomes, where observed risks likely depend on a combination of interlinked variables including genetic as well as social, behavioural, physical, and socioeconomic factors.


Safety and efficacy of continuous infusion of a combined factor VIII-von Willebrand factor (vWF) concentrate (Haemate-P) in patients with von Willebrand disease.
We studied the safety and efficacy of treatment with continuous infusion of a von Willebrand factor (vWF) concentrate Haemate-P in patients with von Willebrand disease (vWD). Three patients with mild and 5 patients with severe forms of vWD, were treated with continuous infusion of Haemate-P by minipump. The indications for treatment were: to prevent bleeding during 9 surgical procedures or 1 vaginal delivery in 6 patients and to treat 2 bleeding episodes in 2 patients. The patients were monitored daily for factor VIII (FVIII:C) and ristocetin cofactor (vWF: RCo) levels and the infusion rate was adjusted to maintain the desired therapeutic level of vWF:RCo. The treatment was effective in preventing surgical bleeding and controlling bleeding episodes. All factor VIII:C and most of the vWF:RCo levels measured during the study period were above the target therapeutic levels. A significant decrease in clearance of FVIII:C and vWF:RCo was observed over the treatment period. Haemate-P consumption averaged 24.3+/-7.9 vWF:RCo U/kg/day which is approximately half the expected dose had intermittent bolus injections been used. We suggest that continuous Haemate-P infusion is superior to intermittent bolus injections for the treatment of vWD patients by virtue of its efficiency, simplicity and considerable savings.


A Predictive-Reactive Approach with Genetic Programming and Cooperative Coevolution for the Uncertain Capacitated Arc Routing Problem.
The uncertain capacitated arc routing problem is of great significance for its wide applications in the real world. In the uncertain capacitated arc routing problem, variables such as task demands and travel costs are realised in real time. This may cause the predefined solution to become ineffective and/or infeasible. There are two main challenges in solving this problem. One is to obtain a high-quality and robust baseline task sequence, and the other is to design an effective recourse policy to adjust the baseline task sequence when it becomes infeasible and/or ineffective during the execution. Existing studies typically only tackle one challenge (the other being addressed using a naive strategy). No existing work optimises the baseline task sequence and recourse policy simultaneously. To fill this gap, we propose a novel proactive-reactive approach, which represents a solution as a baseline task sequence and a recourse policy. The two components are optimised under a cooperative coevolution framework, in which the baseline task sequence is evolved by an estimation of distribution algorithm, and the recourse policy is evolved by genetic programming. The experimental results show that the proposed algorithm, called Solution-Policy Coevolver, significantly outperforms the state-of-the-art algorithms to the uncertain capacitated arc routing problem for the ugdb and uval benchmark instances. Through further analysis, we discovered that route failure is not always detrimental. Instead, in certain cases (e.g., when the vehicle is on the way back to the depot) allowing route failure can lead to better solutions.


Afferent projections to the lateral and dorsomedial hypothalamus in a lizard, Gekko gecko.
Afferent projections to the lateral hypothalamic area and dorsomedial hypothalamic nucleus of the lizard Gekko gecko were studied after applications of wheat germ agglutinin conjugated to horseradish peroxidase. Large applications into the hypothalamus labeled several telencephalic populations not observed after smaller injections. These included the rostrolateral area of the dorsal cortex, a sheet of cells deep to the caudal pole of the lateral cortex, the external amygdala, and part of the dorsal ventricular ridge. Other populations were labeled in the diencephalon, including the supraoptic nucleus and nucleus ovalis; in the medulla the medial reticular area was labeled. Injections into the lateral hypothalamic area labeled neurons in the rostrolateral dorsal cortex, anterior, lateral, and dorsal septal nuclei, the striatoamygdalar area, nucleus accumbens, vertical limb of the diagonal band, nucleus of the accessory olfactory tract, the interstitial, ventral anterior, and ventral posterior amygdalar nuclei, several hypothalamic nuclei, and the posteroventral thalamic nucleus. Labeled brainstem populations included the ventral tegmental area, torus semicircularis, parvocellular and ventral isthmal nuclei, superior raphe, and the solitary nucleus. Injections in the dorsomedial hypothalamic nucleus labeled neurons in the rostral and caudolateral poles of the dorsal cortex, anterior septal nucleus, horizontal limb of the diagonal band, nucleus of the anterior commissure, several hypothalamic areas, the lateral habenula, the posteroventral thalamic nucleus, and cells scattered around the dorsolateral anterior thalamic nuclei. Labeled brainstem populations included the torus semicircularis, ventral tegmental area, superior raphe, parvocellular and ventral isthmal nuclei, and the lateral dorsal tegmental nucleus. The results of these studies are compared with findings in amphibians and mammals.


A Twitter campaign against pseudoscience: The sceptical discourse on complementary therapies in Spain.
The main objective of this article is to analyse the sceptical movement's discourse on complementary therapies in Spain, as well as comprehend its mobilisation against these therapies. Over the past 2 years, the Spanish sceptical movement, constituted by citizen's associations against unconventional therapies and in favour of evidence-based medicine, has increased its activism which, as a result, is now more familiar to the public. To perform this study, three sources of information were selected: (a) the #StopPseudociencias campaign, with a corpus of 6252 tweets; (b) 153 news articles published during the study timeline and (c) 7 interviews with members of the sceptical movement, journalists and politicians. The results of the content and discourse analyses have shown that the sceptical movement occupies a dominant discursive position on Twitter, while the perspective is more balanced in digital dailies.


Cellular automata-based modelling and simulation of biofilm structure on multi-core computers.
The article presents a mathematical model of biofilm growth for aerobic biodegradation of a toxic carbonaceous substrate. Modelling of biofilm growth has fundamental significance in numerous processes of biotechnology and mathematical modelling of bioreactors. The process following double-substrate kinetics with substrate inhibition proceeding in a biofilm has not been modelled so far by means of cellular automata. Each process in the model proposed, i.e. diffusion of substrates, uptake of substrates, growth and decay of microorganisms and biofilm detachment, is simulated in a discrete manner. It was shown that for flat biofilm of constant thickness, the results of the presented model agree with those of a continuous model. The primary outcome of the study was to propose a mathematical model of biofilm growth; however a considerable amount of focus was also placed on the development of efficient algorithms for its solution. Two parallel algorithms were created, differing in the way computations are distributed. Computer programs were created using OpenMP Application Programming Interface for C++ programming language. Simulations of biofilm growth were performed on three high-performance computers. Speed-up coefficients of computer programs were compared. Both algorithms enabled a significant reduction of computation time. It is important, inter alia, in modelling and simulation of bioreactor dynamics.


Operative technique for modified radical neck dissection in papillary thyroid carcinoma.
Papillary thyroid carcinoma is the most common endocrine malignancy. Recently, controversy has focused on the management of lymph node metastases, which represent approximately 90% of disease recurrences and may require considerable time, effort, and resources to diagnose and treat. Current intense postoperative surveillance by endocrinologists nationwide has the sensitivity to detect even minute lymph node metastases using ultrasonography, radioactive iodine scan, and thyroglobulin monitoring. To (1) present a succinct synopsis of the rationale and elements of our current surgical management strategy for papillary thyroid carcinoma and, within this context, (2) provide a detailed stepwise description of a compartment-oriented modified radical neck dissection. This description is combined with intraoperative photographs and a medical artist's illustrations to enhance and emphasize the most important points. With anatomically defined precise dissection, following the steps outlined and illustrated, a thorough lymphadenectomy can be accomplished safely, with reasonable cosmetic results, minimizing disease relapse.


How to read clinical journals: X. How to react when your colleagues haven't read a thing.
Physicians receive little instruction on how to interact with colleagues, and even less guidance on what to do when their colleagues are poorly informed. Eight techniques are presented here that may be of use in dealing with colleagues who have clearly not read the literature and are unable to maintain the facade that they have. If employed properly and used judiciously, these techniques may help avoid embarrassment for all and may also improve the exchange of valuable information between professionals.


Blimp1/Prdm1 governs terminal differentiation of endovascular trophoblast giant cells and defines multipotent progenitors in the developing placenta.
Developmental arrest of Blimp1/Prdm1 mutant embryos at around embryonic day 10.5 (E10.5) has been attributed to placental disturbances. Here we investigate Blimp1/Prdm1 requirements in the trophoblast cell lineage. Loss of function disrupts specification of the invasive spiral artery-associated trophoblast giant cells (SpA-TGCs) surrounding maternal blood vessels and severely compromises the ability of the spongiotrophoblast layer to expand appropriately, secondarily causing collapse of the underlying labyrinth layer. Additionally, we identify a population of proliferating Blimp1(+) diploid cells present within the spongiotrophoblast layer. Lineage tracing experiments exploiting a novel Prdm1.Cre-LacZ allele demonstrate that these Blimp1(+) cells give rise to the mature SpA-TGCs, canal TGCs, and glycogen trophoblasts. In sum, the transcriptional repressor Blimp1/Prdm1 is required for terminal differentiation of SpA-TGCs and defines a lineage-restricted progenitor cell population contributing to placental growth and morphogenesis.


Antibody Responses Against Plasmodium vivax TRAP Recombinant and Synthetic Antigens in Naturally Exposed Individuals From the Brazilian Amazon.
Thrombospondin-related adhesive protein (TRAP) is essential for sporozoite motility and the invasion of mosquitoes' salivary gland and vertebrate's hepatocyte and is, thus, considered a promising pre-erythrocytic vaccine candidate. Despite the existence of a few reports on naturally acquired immune response against Plasmodium vivax TRAP (PvTRAP), it has never been explored so far in the Amazon region, so results are conflicting. Here, we characterized the (IgG and IgG subclass) antibody reactivity against recombinant PvTRAP in a cross-sectional study of 299 individuals exposed to malaria infection in three municipalities (Cruzeiro do Sul, Mâncio Lima and Guajará) from the Acre state of the Brazilian Amazon. In addition, the full PvTRAP sequence was screened for B-cell epitopes using in silico and in vitro approaches. Firstly, we confirmed that PvTRAP is naturally immunogenic in the cohort population since 49% of the individuals were IgG-responders to it. The observed immune responses were mainly driven by cytophilic IgG1 over all other sublcasses and the IgG levels that was corelated with age and time of residence in the studied area (p < 0.05). Interestingly, only the levels of specific anti-TRAP IgG3 seemed to be associated with protection, as IgG3 responders presented a significantly higher time elapse since the last malaria episode than those recorded for IgG3 non-responders. Regarding the B-cell epitope mapping, among the 148 responders to PvTRAP, four predicted epitopes were confirmed by recognition of antibodies (PvTRAPR197-H227; PvTRAPE237-T258; PvTRAPP344-G374; and PvTRAPE439-K454). Nevertheless, the frequency of responders against these peptides were low and did not show a clear correlation with the antibody response against the corresponding antigen. Moreover, none of the linear confirmed epitopes were located in the binding regions of PvTRAP in respect to the host cell ligand. Collectively, our data confirm the PvTRAP immunogenicity among Amazon inhabitants, while suggesting that the main important B-cell epitopes are not linear.


Physical activity and risk of large bowel cancer in the Framingham Study.
We examined the relation between self-reported physical activity and large bowel cancer in a prospective cohort of men and women who participated in the Framingham Study. Self-assessments of physical activity were available from the fourth biennial examination on a total of 1906 men and 2308 women aged 30 to 62 yr in 1954. The cohort was followed for up to 28 yr and yielded 152 cases (73 men, 79 women) of large bowel cancer. Inactivity was associated with an increased risk of large bowel cancer among men but not among women. The relative risk estimates for large bowel cancer among men in the middle and lowest tertiles of a physical activity index (compared with the highest tertile) were 1.4 (95% confidence intervals, 0.8-2.6) and 1.8 (1.0-3.2), respectively. Among women the comparable estimates were 1.2 (0.7-2.1) and 1.1 (0.6-1.8), respectively. These findings were unchanged after adjustment for body mass index, serum cholesterol, alcohol, and other potentially confounding variables. The narrow range of physical activity and the minimal heavy activity reported by women in this cohort may have limited our ability to detect an association between physical activity and large bowel cancer among women.


Alcohol-related olfactory cues activate the nucleus accumbens and ventral tegmental area in high-risk drinkers: preliminary findings.
The mesocorticolimbic dopamine system is implicated in motivation and reward and may be involved in the development of alcoholism. We used functional magnetic resonance imaging to study the blood oxygen level-dependent (BOLD) response to alcohol-related olfactory stimuli (AROS; odors of beer and whiskey) and non-alcohol-related olfactory stimuli (NAROS; odors of grass and leather) in 10 high-risk (HR) drinkers (average drinks per week, 19.99; SD, 6.99; all with > or = 2 first- or second-degree alcoholic relatives) and 5 low-risk (LR) social drinking controls (drinks per week, 2.82; SD, 2.87; 1 subject had 1 second-degree alcoholic relative). Data were analyzed with SPM99 and random effects analysis by using regions of interest and corrected cluster statistics (p < 0.05) to focus on the nucleus accumbens (NAc) and ventral tegmental area (VTA). In HR subjects, there was a greater BOLD signal increase in the NAc during AROS than during clean air. BOLD signal increases during AROS were also greater in the NAc than the signal increases induced by NAROS. The AROS signal was significantly greater than the NAROS signal in a small number of voxels in the VTA. Finally, the AROS/NAROS difference signal was larger in HR drinkers in both the NAc and VTA. Alcoholic olfactory cues may invoke the dopaminergic mesocorticolimbic system to a greater degree than nonalcoholic odors and could be effective tools in exploring the role of the dopamine system in susceptibility to alcoholism.


Physical dependence and urinary catheters both strongly relate to physical inactivity in adults during hospital stay: a cross-sectional, observational study.
To determine which factors are associated with physical inactivity in hospitalized adults of all ages. A cross-sectional sample of 114 adults admitted to a gastrointestinal surgery, internal medicine or cardiology hospital ward (median age 60, length of stay 13 days) were observed during one random day from 8 am to 8 pm using wireless accelerometers and behavioral mapping protocols. Factors (e.g., comorbidities, self-efficacy, independence in mobility, functional restraints) were collected from medical records, surveys, and observations. Patients were physically active for median(IQR) 26 (13-52.3) min and were observed to lie in bed for 67.3%, sit for 25.2%, stand for 2.5%, and walk for 5.0% of the time. Multivariable regression analysis revealed that physical inactivity was 159.87% (CI = 89.84; 255.73) higher in patients dependent in basic mobility, and 58.88% (CI = 10.08; 129.33) higher in patients with a urinary catheter (adjusted R2 = 0.52). The fit of our multivariable regression analysis did not improve after adding hospital ward to the analysis (p > 0.05). Independence in mobility and urine catheter presence are two important factors associated with physical inactivity in hospitalized adults of all ages, and these associations do not differ between hospital wards. Routine assessments of both factors may therefore help to identify physically inactive patients throughout the hospital.IMPLICATIONS FOR REHABILITATIONHealthcare professionals should be aware that physical inactivity during hospital stay may result into functional decline.Regardless of which hospital ward patients are admitted to, once patients require assistance in basic mobility or have a urinary catheter they are at risk of physical inactivity during hospital stay.Implementing routine assessments on the independence of basic mobility and urine catheter presence may therefore assist healthcare professionals in identifying physically inactive patients before they experience functional decline.


Depsides: lichen metabolites active against hepatitis C virus.
A thorough phytochemical study of Stereocaulon evolutum was conducted, for the isolation of structurally related atranorin derivatives. Indeed, pilot experiments suggested that atranorin (1), the main metabolite of this lichen, would interfere with the lifecycle of hepatitis C virus (HCV). Eight compounds, including one reported for the first time (2), were isolated and characterized. Two analogs (5, 6) were also synthesized, to enlarge the panel of atranorin-related structures. Most of these compounds were active against HCV, with a half-maximal inhibitory concentration of about 10 to 70 µM, with depsides more potent than monoaromatic phenols. The most effective inhibitors (1, 5 and 6) were then added at different steps of the HCV lifecycle. Interestingly, atranorin (1), bearing an aldehyde function at C-3, inhibited only viral entry, whereas the synthetic compounds 5 and 6, bearing a hydroxymethyl and a methyl function, respectively, at C-3 interfered with viral replication.


X-ray microanalysis of HeLa S3 Cells. I. Instrumental calibration and analysis of randomly growing cultures.
Cryo-ultramicrotomy and X-ray microanalysis were used to study the elemental composition of HeLa S3 cells. Quantitation was achieved by reference to elemental standards of known concentration made up in 25% gelatin. Analysis of standards showed linear calibration for each of the elements studied: Na, P, S, Cl, K. Standardization was validated by comparing flame-photometric analysis of gelatin containing sodium potassium tartrate with that of X-ray microanalysis. Freeze-dried sections of cells showed good morphology and analysis of whole sections of the cells showed that K/Na varied in individual cells. Low K/Na could not be ascribed to cell damage or to the sequestering of Na in any particular subcompartment of the cells. Treatment with ouabain caused changes in levels of all the elements studied and resulted in a low K/Na ratio in all cells.


In vitro dentine permeability evaluation of HEMA-based (desensitizing) products using split-chamber model following in vivo application in the dog.
The aim of this study was to evaluate in vitro dentine permeability evaluation of 2-Hydroxyethyl methacrylate (HEMA)-based desensitizing products using split-chamber model following in vivo application in the dogs for three experimental time periods of 1 week, 1 month and 3 months. Buccal enamel of upper and lower canines of nine young dogs was removed and flat dentinal surface was obtained using a water-cooled diamond bur. The dentinal surface divided into four quarters, three experimental and a control. Health-Dent and Gluma Desensitizing agent and Single bond were applied to respective quarters of one tooth, according to manufacturers' instructions. The last quarters were left as control without any applications. For the each experimental period, 10 dentine samples from each group including control were used to measure the hydraulic conductance values. The dentine discs were placed pulp-side down in a split-chamber device in which the plastic spacers containing the rubber 0 rings have a surface area of 1 mm(2) and permeability was measured by fluid filtration. The data were expressed as hydraulic conductance (Lp). Differences in dentine permeability obtained for the desensitizing agents against the control were tested for statistical significance using Kruskal-Wallis one-way ANOVA and Bonferroni-adjusted Mann-Whitney U-tests. Differences in permeability in three time periods for each desensitizing agent were analysed using Friedman's anova and Wilcoxon signed rank tests. All desensitizing applications caused decreased Lp values compared with the control Lp value at the end of 1 week. In the 1-month samples, between the Lp values of Single bond and control, no statistical difference was found (P > 0.05). At the end of the 3-month period, the Gluma Desensitizing agent had the lowest the Lp value (P < 0.05). The result of this study is that the topical application of desensitizing agents led to decrease in dentine permeability in the dog model. The Gluma Desensitizing agent provide the more lasting tubule-occluding effect than the other material tested in this model.


[The measurement of maternal mind-mindedness: its relation to mother-infant interaction in natural settings].
The purpose of this study was to construct an original video-based measurement of maternal mind-mindedness (MM). MM is mother's proclivity to treat her infant as an individual with mind (Meins, 1997). Participants were 38 mothers and their six-month-old infants. The results of assessment suggested that there were large individual differences not only in frequency of maternal mental attributions, but also in the specific contents that mothers read from the filmed infants' behaviors. Moreover, it was revealed that these individual differences in MM were related to mother-infant interaction styles in natural settings, especially maternal comments on infant's internal state and the followings of infant's attention. Finally, the usefulness of this measurement in examining the effect of MM on the child's development of mind understanding was discussed.


Towards an understanding of women's brain aging: the immunology of pregnancy and menopause.
Women are at significantly greater risk of developing Alzheimer's disease and show higher prevalence of autoimmune conditions relative to men. Women's brain health is historically understudied, and little is therefore known about the mechanisms underlying epidemiological sex differences in neurodegenerative diseases, and how female-specific factors may influence women's brain health across the lifespan. In this review, we summarize recent studies on the immunology of pregnancy and menopause, emphasizing that these major immunoendocrine transition phases may play a critical part in women's brain aging trajectories.


Comparison of biocontaminant levels associated with hard vs. carpet floors in nonproblem schools: results of a year long study.
Choosing the appropriate floor surface for a school environment is a complex issue. To assist school personnel in determining which flooring is best for their school, we studied the biocontaminant levels associated with carpeted and hard surface flooring. Two schools were selected, one predominantly tiled and one predominantly carpeted, as similar as possible with the exception of their floor coverings. Neither school was a "problem" building. Multiple biocontaminants were measured. For flooring, there were statistically significant differences for all the tested biocontaminants except fungi. The carpeted surfaces, being strong sinks, generally had higher surface loadings of the biocontaminants, while the airborne levels were significantly higher over tiled floors. Significant differences in airborne levels were found for dust mass, spores, fungi, beta-1,3 glucans, and endotoxins. The results suggest that carpet flooring was not the major contributor to airborne levels of biocontaminants in these two nonproblem schools.


The effect of acetazolamide on regional cerebral blood flow in normal human subjects as measured by single-photon emission computed tomography.
Regional cerebral blood flow (rCBF) was evaluated in 15 normal, healthy volunteer control subjects before and after the administration of 1 g acetazolamide (ACZ) using a rotating four-detector single-photon emission computed tomograph (SPECT). ACZ, a carbonic anhydrase inhibitor, is a cerebral vasodilator. RCBF values in mL/minute/100 g were derived within eight cortical regions of interest (ROI), and from the whole slice as an expression of whole brain blood flow (WBF). ROI/WBF ratios were established for each ROI in each of the 15 subjects for both pre-ACZ and post-ACZ studies. ACZ produced a 30% +/- 17% increase in WBF. Studies were done in random order, with nine subjects undergoing the post-ACZ study first, and six the pre-ACZ, or baseline, study first. Statistical analysis showed no significant difference in any ROI that might be caused by sequence of test procedures. Ratios were then examined to determine whether rCBF elevation was proportionate in all ROI in all subjects. No significant difference was found in any ROI except for the left parietal, for which marginally significant change was identified. Subjects also were examined for possible age and sex differences in ACZ response, and no differences were found.


Anatomic distribution of deep vein thrombosis in pregnancy.
Prospective studies of nonpregnant patients have demonstrated that most deep vein thromboses of the lower extremity originate in the calf veins and progress proximally, but the anatomic distribution of thromboses in pregnant patients is unclear. An understanding of the anatomic distribution of deep vein thrombosis in pregnancy has important implications for optimizing diagnostic imaging protocols. We undertook this study to determine the anatomic distribution of deep vein thrombosis of the lower extremity in symptomatic pregnant patients. We systematically searched MEDLINE (1966 to January 2009), Embase (1980 to January 2009) and the Cochrane Library using prespecified criteria to identify articles providing objective diagnostic and anatomic information for unselected or consecutive symptomatic pregnant patients with deep vein thrombosis. Six articles from an initial list of 1098 titles met the inclusion criteria. These articles provided information for 124 pregnant women with a diagnosis of deep vein thrombosis. Overall, involvement of the left leg was reported in 84 (88%) of the 96 patients for which the side affected was known, and 87 (71%) of 122 thromboses were restricted to the proximal veins without involvement of the calf veins. Among these cases of proximal deep vein thrombosis, 64% (56/87) were restricted to the iliac and/or femoral vein. Despite a paucity of studies in this area, the results of our review suggest that the anatomic distribution of deep vein thrombosis in pregnant women differs from that for nonpregnant patients. In addition to what was previously known--that left-sided deep vein thrombosis is more common in pregnancy--we also found that proximal deep vein thrombosis restricted to the femoral or iliac veins is also more common (> 60% of cases). If confirmed by larger studies, these findings could affect our understanding of the pathophysiology and derivation of diagnostic algorithms for examination of pregnant women with suspected deep vein thrombosis.


Gene and protein nomenclature in public databases.
Frequently, several alternative names are in use for biological objects such as genes and proteins. Applications like manual literature search, automated text-mining, named entity identification, gene/protein annotation, and linking of knowledge from different information sources require the knowledge of all used names referring to a given gene or protein. Various organism-specific or general public databases aim at organizing knowledge about genes and proteins. These databases can be used for deriving gene and protein name dictionaries. So far, little is known about the differences between databases in terms of size, ambiguities and overlap. We compiled five gene and protein name dictionaries for each of the five model organisms (yeast, fly, mouse, rat, and human) from different organism-specific and general public databases. We analyzed the degree of ambiguity of gene and protein names within and between dictionaries, to a lexicon of common English words and domain-related non-gene terms, and we compared different data sources in terms of size of extracted dictionaries and overlap of synonyms between those. The study shows that the number of genes/proteins and synonyms covered in individual databases varies significantly for a given organism, and that the degree of ambiguity of synonyms varies significantly between different organisms. Furthermore, it shows that, despite considerable efforts of co-curation, the overlap of synonyms in different data sources is rather moderate and that the degree of ambiguity of gene names with common English words and domain-related non-gene terms varies depending on the considered organism. In conclusion, these results indicate that the combination of data contained in different databases allows the generation of gene and protein name dictionaries that contain significantly more used names than dictionaries obtained from individual data sources. Furthermore, curation of combined dictionaries considerably increases size and decreases ambiguity. The entries of the curated synonym dictionary are available for manual querying, editing, and PubMed- or Google-search via the ProThesaurus-wiki. For automated querying via custom software, we offer a web service and an exemplary client application.


Maximizing the success rate of minimal model insulin sensitivity measurement in humans: the importance of basal glucose levels.
Minimal model analysis of glucose and insulin concentrations in the intravenous glucose tolerance test (IVGTT) has been widely used to obtain a measure of insulin sensitivity in humans. Issues of model validity and IVGTT protocol have been explored extensively. Less attention has been paid, however, to the computer programming protocol for estimating the model parameters (programming implementation). Minimal model analysis of data from an IVGTT protocol involving a high glucose dose (0.5 g/kg) and a reduced sample schedule, employed in healthy pre- or post-menopausal women, healthy men or men with coronary heart disease or chronic heart failure (20 in each group), was undertaken according to 12 different programming implementations using a commercially available model-equation-solving program. The ability of the program to arrive at an acceptable solution to the model equations gave a success rate of between 39% and 96%, depending on the implementation. Variation in basal glucose assignment significantly affected the magnitude of estimates of insulin sensitivity. The maximum modelling success rate was achieved by introduction of an imputed glucose measurement at 360 min from the glucose injection, taking the basal glucose level as the fasting glucose concentration, and overweighting the initial glucose measurement after a delay for mixing. Use of this implementation to analyse data from a study comparing insulin sensitivities obtained using the minimal model and a euglycaemic clamp reference gave a correlation of 0.80 (P<0.001) between the two methods. Straightforward variations in programming implementation, involving appropriate assignment of the basal glucose concentration and use of an imputed glucose measurement signifying re-establishment of basal glucose levels following the IVGTT, can considerably improve modelling success rate.


The effect of d-cycloserine on brain processing of breathlessness over pulmonary rehabilitation: an experimental medicine study.
Pulmonary rehabilitation is the best treatment for chronic breathlessness in COPD but there remains an unmet need to improve efficacy. Pulmonary rehabilitation has strong parallels with exposure-based cognitive behavioural therapies (CBT), both clinically and in terms of brain activity patterns. The partial N-methyl-d-aspartate (NMDA)-receptor agonist d-cycloserine has shown promising results in enhancing efficacy of CBT, thus we hypothesised that it would similarly augment the effects of pulmonary rehabilitation in the brain. Positive findings would support further development in phase 3 clinical trials. 72 participants with mild-to-moderate COPD were recruited to a double-blind pre-registered (ClinicalTrials.gov identifier: NCT01985750) experimental medicine study running parallel to a pulmonary rehabilitation course. Participants were randomised to 250 mg d-cycloserine or placebo, administered immediately prior to the first four sessions of pulmonary rehabilitation. Primary outcome measures were differences between d-cycloserine and placebo in brain activity in the anterior insula, posterior insula, anterior cingulate cortices, amygdala and hippocampus following completion of pulmonary rehabilitation. Secondary outcomes included the same measures at an intermediate time point and voxel-wise difference across wider brain regions. An exploratory analysis determined the interaction with breathlessness anxiety. No difference between d-cycloserine and placebo groups was observed across the primary or secondary outcome measures. d-cycloserine was shown instead to interact with changes in breathlessness anxiety to dampen reactivity to breathlessness cues. Questionnaire and measures of respiratory function showed no group difference. This is the first study testing brain-active drugs in pulmonary rehabilitation. Rigorous trial methodology and validated surrogate end-points maximised statistical power. Although increasing evidence supports therapeutic modulation of NMDA pathways to treat symptoms, we conclude that a phase 3 clinical trial of d-cycloserine would not be worthwhile.


Radioiodinated Capsids Facilitate In Vivo Non-Invasive Tracking of Adeno-Associated Gene Transfer Vectors.
Viral vector mediated gene therapy has become commonplace in clinical trials for a wide range of inherited disorders. Successful gene transfer depends on a number of factors, of which tissue tropism is among the most important. To date, definitive mapping of the spatial and temporal distribution of viral vectors in vivo has generally required postmortem examination of tissue. Here we present two methods for radiolabeling adeno-associated virus (AAV), one of the most commonly used viral vectors for gene therapy trials, and demonstrate their potential usefulness in the development of surrogate markers for vector delivery during the first week after administration. Specifically, we labeled adeno-associated virus serotype 10 expressing the coding sequences for the CLN2 gene implicated in late infantile neuronal ceroid lipofuscinosis with iodine-124. Using direct (Iodogen) and indirect (modified Bolton-Hunter) methods, we observed the vector in the murine brain for up to one week using positron emission tomography. Capsid radioiodination of viral vectors enables non-invasive, whole body, in vivo evaluation of spatial and temporal vector distribution that should inform methods for efficacious gene therapy over a broad range of applications.


The GAA triplet-repeat is unstable in the context of the human FXN locus and displays age-dependent expansions in cerebellum and DRG in a transgenic mouse model.
Friedreich ataxia (FRDA) is caused by homozygosity for FXN alleles containing an expanded GAA triplet-repeat (GAA-TR) sequence. Patients have progressive neurodegeneration of the dorsal root ganglia (DRG) and in later stages the cerebellum may be involved. The expanded GAA-TR sequence is unstable in somatic cells in vivo, and although the mechanism of instability remains unknown, we hypothesized that age-dependent and tissue-specific somatic instability may be a determinant of the progressive pathology involving DRG and cerebellum. We show that transgenic mice containing the expanded GAA-TR sequence (190 or 82 triplets) in the context of the human FXN locus show tissue-specific and age-dependent somatic instability that is compatible with this hypothesis. Small pool PCR analysis, which allows quantitative analysis of repeat instability by assaying individual transgenes in vivo, showed age-dependent expansions specifically in the cerebellum and DRG. The (GAA)(190) allele showed some instability by 2 months, progressed at about 0.3-0.4 triplets per week, resulting in a significant number of expansions by 12 months. Repeat length was found to determine the age of onset of somatic instability, and the rate and magnitude of mutation. Given the low level of cerebellar instability seen by others in multiple transgenic mice with expanded CAG/CTG repeats, our data indicate that somatic instability of the GAA-TR sequence is likely mediated by unique tissue-specific factors. This mouse model will serve as a useful tool to delineate the mechanism(s) of disease-specific somatic instability in FRDA.


Eosinophilic Pneumonia Putatively Induced by Vancomycin: A Case Report.
BACKGROUND Herein, we describe a case of eosinophilic pneumonia that was likely to have been induced by vancomycin. CASE REPORT A 65-year-old man on maintenance hemodialysis presented with chest pain and dyspnea. He subsequently developed methicillin-resistant Staphylococcus aureus-positive acute pleural empyema in an evacuated right-sided pneumothorax. Surgical thoracoscopic curettage was ultimately performed, but dyspnea recurred postoperatively. Computed tomography depicted widespread reticular shadowing of the left lung, and peripheral eosinophilia was detected. The proportion of eosinophils found in bronchoalveolar lavage fluid was also remarkable (43%). All symptoms and the results of laboratory tests immediately improved after the discontinuation of vancomycin and initiation of prednisolone therapy. CONCLUSIONS We attribute this case of eosinophilic pneumonia to vancomycin, because all other candidate causes were ruled out, and only vancomycin fulfilled the criteria of both drug-induced eosinophilic pneumonia and drug-induced lung injury. If confirmed, this constitutes the first reported case of vancomycin-induced eosinophilic pneumonia.


A rhodium(III) complex for high-affinity DNA base-pair mismatch recognition.
A rhodium(III) complex, rac-[Rh(bpy)(2)phzi](3+) (bpy, 2,2'-bipyridine; phzi, benzo[a]phenazine-5,6-quinone diimine) has been designed as a sterically demanding intercalator targeted to destabilized mismatched sites in double-helical DNA. The complex is readily synthesized by condensation of the phenazine quinone with the corresponding diammine complex. Upon photoactivation, the complex promotes direct strand scission at single-base mismatch sites within the DNA duplex. As with the parent mismatch-specific reagent, [Rh(bpy)(2)(chrysi)](3+) [chrysene-5,6-quinone diimine (chrysi)], mismatch selectivity depends on the helix destabilization associated with mispairing. Unlike the parent chrysi complex, the phzi analogue binds and cleaves with high affinity and efficiency. The specific binding constants for CA, CC, and CT mismatches within a 31-mer oligonucleotide duplex are 0.3, 1, and 6 x 10(7) M(-1), respectively; site-specific photocleavage is evident at nanomolar concentrations. Moreover, the specificity, defined as the ratio in binding affinities for mispaired vs. well paired sites, is maintained. The increase in affinity is attributed to greater stability in the mismatched site associated with stacking by the heterocyclic aromatic ligand. The high-affinity complex is also applied in the differential cleavage of DNA obtained from cell lines deficient in mismatch repair vs. those proficient in mismatch repair. Agreement is found between photocleavage by the mismatch-specific probes and deficiency in mismatch repair. This mismatch-specific targeting, therefore, offers a potential strategy for new chemotherapeutic design.


Wnt signaling in planarians: new answers to old questions.
Wnts are secreted glycoproteins involved in a broad range of essential cell functions, including proliferation, migration and cell-fate determination. Recent years have seen substantial research effort invested in elucidating the role of the Wnt signaling pathway in planarians, flatworms with incredible regenerative capacities. In this review, we summarize current knowledge on the role of canonical (β-catenin-dependent) and non-canonical (β-catenin-independent) Wnt signaling in planarians, not only during regeneration, but also during normal homeostasis. We also describe some of the preliminary data that has been obtained regarding the role of these pathways during embryogenesis. Models are proposed to integrate the different results which have been obtained to date and highlight those questions that still remain to be answered.


Inflammatory mediators in breast cancer: coordinated expression of TNFα & IL-1β with CCL2 & CCL5 and effects on epithelial-to-mesenchymal transition.
The inflammatory chemokines CCL2 (MCP-1) & CCL5 (RANTES) and the inflammatory cytokines TNFα & IL-1β were shown to contribute to breast cancer development and metastasis. In this study, we wished to determine whether there are associations between these factors along stages of breast cancer progression, and to identify the possible implications of these factors to disease course. The expression of CCL2, CCL5, TNFα and IL-1β was determined by immunohistochemistry in patients diagnosed with: (1) Benign breast disorders (=healthy individuals); (2) Ductal Carcinoma In Situ (DCIS); (3) Invasive Ducal Carcinoma without relapse (IDC-no-relapse); (4) IDC-with-relapse. Based on the results obtained, breast tumor cells were stimulated by the inflammatory cytokines, and epithelial-to-mesenchymal transition (EMT) was determined by flow cytometry, confocal analyses and adhesion, migration and invasion experiments. CCL2, CCL5, TNFα and IL-1β were expressed at very low incidence in normal breast epithelial cells, but their incidence was significantly elevated in tumor cells of the three groups of cancer patients. Significant associations were found between CCL2 & CCL5 and TNFα & IL-1β in the tumor cells in DCIS and IDC-no-relapse patients. In the IDC-with-relapse group, the expression of CCL2 & CCL5 was accompanied by further elevated incidence of TNFα & IL-1β expression. These results suggest progression-related roles for TNFα and IL-1β in breast cancer, as indeed indicated by the following: (1) Tumors of the IDC-with-relapse group had significantly higher persistence of TNFα and IL-1β compared to tumors of DCIS or IDC-no-relapse; (2) Continuous stimulation of the tumor cells by TNFα (and to some extent IL-1β) has led to EMT in the tumor cells; (3) Combined analyses with relevant clinical parameters suggested that IL-1β acts jointly with other pro-malignancy factors to promote disease relapse. Our findings suggest that the coordinated expression of CCL2 & CCL5 and TNFα & IL-1β may be important for disease course, and that TNFα & IL-1β may promote disease relapse. Further in vitro and in vivo studies are needed for determination of the joint powers of the four factors in breast cancer, as well as analyses of their combined targeting in breast cancer.


Comprehensive analysis of DNA methylation and gene expression of rat liver in a 2-stage hepatocarcinogenesis model.
Recent studies have shown that epigenetic alterations correlate with carcinogenesis in various tissues. Identification of these alterations might help characterize the early stages of carcinogenesis. We comprehensively analyzed DNA methylation and gene expression in livers obtained from rats exposed to nitrosodiethylamine (DEN) followed by a promoter of hepatic carcinogenesis, phenobarbital (PB). The combination of DEN and PB induced marked increases in number and area of glutathione S-transferase-placental form (GST-P)-positive foci in the liver. In the liver of rats that received 30 mg/kg of DEN, pathway analysis revealed alterations of common genes in terms of gene expression and DNA methylation, and that these alterations were related to immune responses. Hierarchical clustering analysis of the expression of common genes from public data obtained through the Toxicogenomics Project-Genomics Assisted Toxicity Evaluation system (TG-GATEs) showed that carcinogenic compounds clustered together. MBD-seq and GeneChip analysis indicated that major histocompatibility complex class Ib gene RT1-CE5, which has an important role in antigen presentation, was hypomethylated around the promoter region and specifically induced in the livers of DEN-treated rats. Further, immunohistochemical analysis indicated that the co-localization of GST-P and protein homologous to RT1-CE5 was present at the foci of some regions. These results suggest that common genes were altered in terms of both DNA methylation and expression in livers, with preneoplastic foci indicating carcinogenic potential, and that immune responses are involved in early carcinogenesis. In conclusion, the present study identified a specific profile of DNA methylation and gene expression in livers with preneoplastic foci. Early epigenetic perturbations of immune responses might correlate with the early stages of hepatocarcinogenesis.


New domain motif: the structure of pectate lyase C, a secreted plant virulence factor.
Pectate lyases are secreted by pathogens and initiate soft-rot diseases in plants by cleaving polygalacturonate, a major component of the plant cell wall. The three-dimensional structure of pectate lyase C from Erwinia chrysanthemi has been solved and refined to a resolution of 2.2 angstroms. The enzyme folds into a unique motif of parallel beta strands coiled into a large helix. Within the core, the amino acids form linear stacks and include a novel asparagine ladder. The sequence similarities that pectate lyases share with pectin lyases, pollen and style proteins, and tubulins suggest that the parallel beta helix motif may occur in a broad spectrum of proteins.