Differential beta cell responses to hyperglycaemia and insulin resistance in two novel congenic strains of diabetes (FVB- Lepr (db)) and obese (DBA- Lep (ob)) mice.
Our goal was to identify genetic variants that determine the response to insulin resistance and hyperglycaemia. This report documents the diabetes syndrome of two new congenic strains of mice generated by the transfer of the Lepr(db) mutation to the FVB/NJ strain and the Lep(ob) mutation to the DBA/2J strain. Mice were characterised by measures of blood metabolites and hormones along with challenges with glucose and insulin injections. Histological examinations of the endocrine pancreas and the kidneys were also carried out. Obese mice of the FVB-db congenic strain show long-term hyperglycaemia that is primarily due to severe insulin resistance. The hyperglycaemia in the fed state persists despite escalating secretion of insulin and massive increase of pancreatic beta cells. Obese FVB-db mice show evidence of mesangial matrix expansion, a hallmark of diabetic nephropathy. Leptin-deficient mice of the DBA-ob strain have variable obesity-diabetes. In mice with high insulin (>10 ng/ml), DBA-ob/ob mice maintain their increased adiposity and have a large increase in beta-cell number. In mice with low insulin (<1 ng/ml) DBA-ob/ob mice have greatly diminished adiposity. These mice have atrophied islets with evidence of increased beta-cell neogenesis from the ductal epithelium. The strain-specific responses suggest the existence of genetic variants that control insulin sensitivity and beta-cell responses in the strains described in this report. These new models of obesity-diabetes should prove useful in dissecting the genetic control of beta-cell responses to hyperglycaemia and insulin resistance.


Earthworm effects without earthworms: inoculation of raw organic matter with worm-worked substrates alters microbial community functioning.
Earthworms are key organisms in organic matter decomposition because of the interactions they establish with soil microorganisms. They enhance decomposition rates through the joint action of direct effects (i.e. effects due to direct earthworm activity such as digestion, burrowing, etc) and indirect effects (i.e. effects derived from earthworm activities such as cast ageing). Here we test whether indirect earthworm effects affect microbial community functioning in the substrate, as when earthworms are present (i. e., direct effects). To address these questions we inoculated fresh organic matter (pig manure) with worm-worked substrates (vermicompost) produced by three different earthworm species. Two doses of each vermicompost were used (2.5 and 10%). We hypothesized that the presence of worm-worked material in the fresh organic matter will result in an inoculum of different microorganisms and nutrients. This inoculum should interact with microbial communities in fresh organic matter, thus promoting modifications similar to those found when earthworms are present. Inoculation of worm-worked substrates provoked significant increases in microbial biomass and enzyme activities (β-glucosidase, cellulase, phosphatase and protease). These indirect effects were similar to, although lower than, those obtained in pig manure with earthworms (direct and indirect earthworm effects). In general, the effects were not dose-dependent, suggesting the existence of a threshold at which they were triggered. Our data reveal that the relationships between earthworms and microorganisms are far from being understood, and suggest the existence of several positive feedbacks during earthworm activity as a result of the interactions between direct and indirect effects, since their combination produces stronger modifications to microbial biomass and enzyme activity.


A vision-based self-calibration method for robotic visual inspection systems.
A vision-based robot self-calibration method is proposed in this paper to evaluate the kinematic parameter errors of a robot using a visual sensor mounted on its end-effector. This approach could be performed in the industrial field without external, expensive apparatus or an elaborate setup. A robot Tool Center Point (TCP) is defined in the structural model of a line-structured laser sensor, and aligned to a reference point fixed in the robot workspace. A mathematical model is established to formulate the misalignment errors with kinematic parameter errors and TCP position errors. Based on the fixed point constraints, the kinematic parameter errors and TCP position errors are identified with an iterative algorithm. Compared to the conventional methods, this proposed method eliminates the need for a robot-based-frame and hand-to-eye calibrations, shortens the error propagation chain, and makes the calibration process more accurate and convenient. A validation experiment is performed on an ABB IRB2400 robot. An optimal configuration on the number and distribution of fixed points in the robot workspace is obtained based on the experimental results. Comparative experiments reveal that there is a significant improvement of the measuring accuracy of the robotic visual inspection system.


Do comprehensive deep learning algorithms suffer from hidden stratification? A retrospective study on pneumothorax detection in chest radiography.
To evaluate the ability of a commercially available comprehensive chest radiography deep convolutional neural network (DCNN) to detect simple and tension pneumothorax, as stratified by the following subgroups: the presence of an intercostal drain; rib, clavicular, scapular or humeral fractures or rib resections; subcutaneous emphysema and erect versus non-erect positioning. The hypothesis was that performance would not differ significantly in each of these subgroups when compared with the overall test dataset. A retrospective case-control study was undertaken. Community radiology clinics and hospitals in Australia and the USA. A test dataset of 2557 chest radiography studies was ground-truthed by three subspecialty thoracic radiologists for the presence of simple or tension pneumothorax as well as each subgroup other than positioning. Radiograph positioning was derived from radiographer annotations on the images. DCNN performance for detecting simple and tension pneumothorax was evaluated over the entire test set, as well as within each subgroup, using the area under the receiver operating characteristic curve (AUC). A difference in AUC of more than 0.05 was considered clinically significant. When compared with the overall test set, performance of the DCNN for detecting simple and tension pneumothorax was statistically non-inferior in all subgroups. The DCNN had an AUC of 0.981 (0.976-0.986) for detecting simple pneumothorax and 0.997 (0.995-0.999) for detecting tension pneumothorax. Hidden stratification has significant implications for potential failures of deep learning when applied in clinical practice. This study demonstrated that a comprehensively trained DCNN can be resilient to hidden stratification in several clinically meaningful subgroups in detecting pneumothorax.


Exploring the performance of nanostructured reagents with organic-group-defined morphology in cross-coupling reaction.
The great impact of the nanoscale organization of reactive species on their performance in chemical transformations creates the possibility of fine-tuning of reaction parameters by modulating the nano-level properties. This methodology is extensively applied for the catalysts development whereas nanostructured reactants represent the practically unexplored area. Here we report the palladium- and copper-catalyzed cross-coupling reaction involving nano-structured nickel thiolate particles as reagents. On the basis of experimental findings we propose the cooperative effect of nano-level and molecular-level properties on their reactivity. The high degree of ordering, small particles size, and electron donating properties of the substituents favor the product formation. Reactant particles evolution in the reaction is visualized directly by dynamic liquid-phase electron microscopy including recording of video movies. Mechanism of the reaction in liquid phase is established using on-line mass spectrometry measurements. Together the findings provide new opportunities for organic chemical transformations design and for mechanistic studies.


Defining 'low in fat' and 'high in fat' when applied to a food.
To describe four different methods of identifying indicator foods that are high, medium or low in fat with reference to dietary patterns and to use these indicator foods to test three sets of definitions of 'high', 'medium' and 'low' in fat from 'banding schemes' developed by the Coronary Prevention Group (CPG), the Food Standards Agency (FSA) and Sainsbury's. Indicator foods were developed using food intake data from the UK National Diet and Nutrition Survey and two parameters: (i) probability of the food being consumed by an individual with a high-fat diet (Method 1); and (ii) the contribution of the food to the fat intake of the average diet of consumers (Methods 3 and 4). Method 2 used both parameters. The three banding schemes were tested by assessing their levels of agreement with methods in categorising indicators. Sensitivity in identifying high, medium and low fat indicators was highest with the CPG banding scheme (high and medium fat indicators) and Sainsbury's scheme (low fat indicators) (Methods 2, 3 and 4). The levels of agreement (kappa coefficient) were 0.68 for the CPG scheme; 0.51 for the Sainsbury's scheme; and 0.41 for the FSA scheme (Method 3). It is possible to use indicator foods related to dietary patterns of a specific population to generate more rational definitions of 'high', 'medium' and 'low' in fat. This could be the starting point for the development of indicator foods for testing more complex nutrient profile models (i.e. those that consider more than one nutrient).


Aerosol deposition in the human lung: effect of high-frequency oscillation on the deposition characteristics of an inhaled nebulized aerosol.
1. Oral high-frequency oscillation (OHFO) may have important effects on aerosol deposition in the lungs. In order to investigate these, a technique was devised to measure regional deposition rates of a nebulized radiolabelled aerosol in the lungs during normal tidal breathing. 2. The effect of three frequencies of OHFO on pulmonary aerosol deposition rate (PADR) in four normal subjects and five patients with chronic airways obstruction (CAO) were assessed using the technique. 3. In separate experiments employing three normal subjects, the effect of OHFO was studied on the deposition rate of aerosol on the oropharynx and delivery apparatus, and on the amount and characteristics of aerosol inhaled by the subjects. 4. Total PADR was significantly reduced by OHFO at 8 Hz and 16 Hz in the normal subjects, and by all three frequencies of OHFO in the CAO patients. In the normal subjects, the regional distribution of aerosol deposition was unchanged, but in the CAO patients a larger proportion of total aerosol deposition occurred in peripheral lung. 5. OHFO reduced the oropharyngeal aerosol deposition rate, increased the loss of aerosol to the atmosphere before inhalation, and increased the deposition of aerosol on the delivery apparatus. The end result was a reduction in the amount of aerosol inhaled, and in the particle sizes measured at the mouthpiece. 6. We conclude that OHFO reduces the amount of aerosol inhaled, but may improve peripheral deposition of inhaled aerosol in patients with CAO. This effect may be of value in the clinical administration of nebulized drugs.


The success of surgery in the first 24 hours in patients with esophageal perforation.
Esophageal perforation (EP) is a critical and potentially life-threatening condition with considerable rates of morbidity and mortality. Despite many advances in thoracic surgery, the management of patients with EP is still controversial. We retrospectively reviewed 34 patients treated for EP, 62% male, mean age 53.9 years. Sixty-two percent of the EPs were iatrogenic. Spontaneous and traumatic EP rates were 26% and 6%, respectively. Three patients had EP in the cervical esophagus and 31 in the thoracic esophagus. Mean time to initial treatment was 34.2 hours. Twenty patients comprised the early group <24 h) and 14 patients the late group (>24 h). Management of the EP included primary closure in 30 patients, non-surgical treatment in two, stent in one and resection in one. Mortality occurred in nine of the 34 patients (26%). Mortality was EP-related in four patients. Three of the nine patients that died were in the early group (p<0.05). Mean hospital stay was 13.4 days. EP remains a potentially fatal condition and requires early diagnosis and accurate treatment to prevent the morbidity and mortality.


Synthesis and characterization of 4-methoxy-7-nitroindolinyl-D-aspartate, a caged compound for selective activation of glutamate transporters and N-methyl-D-aspartate receptors in brain tissue.
The D-isomer of aspartate is efficiently transported by high-affinity Na(+)/K(+)-dependent glutamate transporters and is an effective ligand of N-methyl-d-aspartate (NMDA) receptors. To facilitate analysis of the regulation of these proteins in their native membranes, we synthesized a photolabile analogue of D-aspartate, 4-methoxy-7-nitroindolinyl-D-aspartate (MNI-D-aspartate). This compound was photolyzed with a quantum efficiency of 0.09 at pH 7.4. Photorelease of d-aspartate in acute hippocampal slices through brief (1 ms) UV laser illumination of MNI-d-aspartate triggered rapidly activating currents in astrocytes that were inhibited by the glutamate transporter antagonist DL-threo-beta-benzyloxyaspartic acid (TBOA), indicating that they resulted from electrogenic uptake of D-aspartate. These transporter currents exhibited a distinct tail component that was approximately 2% of the peak current, which may result from the release of K(+) into the extracellular space during counter transport. MNI-D-aspartate was neither an agonist nor an antagonist of glutamate transporters at concentrations up to 500 muM and was stable in aqueous solution for several days. Glutamate transporter currents were also elicited in Bergmann glial cells and Purkinje neurons of the cerebellum in response to photolysis of MNI-D-aspartate, indicating that this compound can be used for monitoring the occupancy and regulation of glutamate transporters in different brain regions. Photorelease of D-aspartate did not activate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors or metabotropic glutamate receptors (mGluRs) in neurons, but resulted in the selective, but transient, activation of NMDA receptors in hippocampal pyramidal neurons; MNI-D-aspartate was not an antagonist of NMDA receptors. These results indicate that MNI-D-aspartate also may be useful for studying the regulation of NMDA receptors at excitatory synapses.


Ergonomics assessment of selected dust respirators: Their use in the tropics.
The suitability and effectiveness of four different types of British made respirators were studied with respect to comfort, convenience and fit on wearers in Sri Lanka (a developing country). Objective and subjective assessments were made to evaluate the degree of discomfort and interferences to the use of senses. The study revealed that factors such as breathing resistance, work-rate and activity period affected the physiological responses. The weight of the respirator and the skin temperature had no direct relationship with the cardiovascular stress. Positive-pressure respirators that gave lower face temperatures than negative-pressure masks gave this type of respirator an additional advantage in hot environments. Respirators that restricted jaw movement affected the speech intelligibility of the wearer. Orinasal masks restricted vision more than the other types. The problem of fit was found negligible though head and face dimensions significantly differed between the British and the Sri Lankans. Subjective assessment correlated well with objective tests.


Chemoenzymatic Synthesis of D-Glucitol-Based Non-Ionic Amphiphilic Architectures as Nanocarriers.
Newer non-ionic amphiphiles have been synthesized using biocompatible materials and by following a greener approach i.e., D-glucitol has been used as a template, and hydrophobic and hydrophilic segments were incorporated on it by using click chemistry. The hydrophilic segments in turn were prepared from glycerol using an immobilized Candida antarctica lipase (Novozym-435)-mediated chemoenzymatic approach. Surface tension measurements and dynamic light scattering studies reflect the self-assembling behavior of the synthesized amphiphilic architectures in the aqueous medium. The results from UV-Vis and fluorescence spectroscopy establish the encapsulation of guests in the hydrophobic core of self-assembled amphiphilic architectures. The results of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay indicate that the amphiphiles are well tolerated by the used A549 cell lines at all tested concentrations.


Characterization of a novel pterin intermediate formed in the catalytic cycle of tyrosine hydroxylase.
A novel pterin intermediate, in addition to the expected 4a-hydroxytetrahydrobiopterin (4a-OH-BH4) and quinonoid dihydrobiopterin, was generated during catalytic turnover of tyrosine hydroxylase (TH) with tetrahydrobiopterin as the cofactor. Based on chromatographic, spectroscopic and stability properties its structure is proposed to be similar to the product formed by the non-enzymic conversion of synthetic 4a-OH-BH4 [Bailey, Rebrin, Boerth and Ayling (1995) J. Am. Chem. Soc. 117, 10203-10211]. This compound was tentatively described as a 4a-adduct of a side-chain hydroxy group, i.e. the O2', 4a-cyclic-tetrahydrobiopterin (4a-Cyc-BH4). The intermediate generated in the TH reaction has a UV spectrum which is similar to that of 4a-OH-BH4, but elutes with a longer retention time (tR = 1.69 min compared with 1.06 min) on reversed-phase chromatography. Its conversion into quinonoid dihydrobiopterin is catalysed by pterin-4a-carbinolamine dehydratase (EC 188.8.131.52), although 4a-OH-BH4 is the preferred substrate for that enzyme. A precursor-product relationship was demonstrated between 4a-OH-BH4 and the putative 4a-Cyc-BH4 intermediate. The apparent stability of this compound is dependent on pH as well as on the nature of the buffer ions. At pH 8.0 a large amount was generated in Hepes and Tris, but little in phosphate buffer. At pH 7.0 in Hepes (standard assay conditions) and Tris buffer the putative 4a-Cyc-BH4, but no 4a-OH-BH4, was observed. None of the intermediates was observed at pH 6.0. The accumulation of these intermediates in the absence of dehydratase has important implications for the assay of TH and phenylalanine hydroxylase activities, and is also compatible with a possible physiological role of the dehydratase in the synthesis of catecholamines in vivo.


Cancer Exosomes as Mediators of Drug Resistance.
In the last decades, several studies demonstrated that the tumor microenvironment is a critical determinant not only of tumor progression and metastasis, but also of resistance to therapy. Exosomes are small membrane vesicles of endocytic origin, which contain mRNAs, DNA fragments, and proteins, and are released by many different cell types, including cancer cells. Mounting evidence has shown that cancer-derived exosomes contribute to the recruitment and reprogramming of constituents associated with the tumor microenvironment. Understanding how exosomes and the tumor microenvironment impact drug resistance will allow novel and better strategies to overcome drug resistance and treat cancer. Here, we describe a technique for exosome purification from cell culture, and fresh and frozen plasma, and further analysis by electron microscopy, NanoSight microscope, and Western blot.


Biodistribution of Agmatine to Brain and Spinal Cord after Systemic Delivery.
Agmatine, an endogenous polyamine, has been shown to reduce chronic pain behaviors in animal models and in patients. This reduction is due to inhibition of the GluN2B subunit of the N-methyl-D-aspartate receptor (NMDAR) in the central nervous system (CNS). The mechanism of action requires central activity, but the extent to which agmatine crosses biologic barriers such as the blood-brain barrier (BBB) and intestinal epithelium is incompletely understood. Determination of agmatine distribution is limited by analytical protocols with low sensitivity and/or inefficient preparation. This study validated a novel bioanalytical protocol using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) for quantification of agmatine in rat biologic matrices. These protocols were then used to determine the plasma pharmacokinetics of agmatine and the extent of distribution to the CNS. Precision and accuracy of the protocol met US Food and Drug Administration (FDA) standards in surrogate matrix as well as in corrected concentrations in appropriate matrices. The protocol also adequately withstood stability and dilution conditions. Upon application of this protocol to pharmacokinetic study, intravenous agmatine showed a half-life in plasma ranging between 18.9 and 14.9 minutes. Oral administration led to a prolonged plasma half-life (74.4-117 minutes), suggesting flip-flop kinetics, with bioavailability determined to be 29%-35%. Intravenous administration led to a rapid increase in agmatine concentration in brain but a delayed distribution and lower concentrations in spinal cord. However, half-life of agmatine in both tissues is substantially longer than in plasma. These data suggest that agmatine adequately crosses biologic barriers in rat and that brain and spinal cord pharmacokinetics can be functionally distinct. SIGNIFICANCE STATEMENT: Agmatine has been shown to be an effective nonopioid therapy for chronic pain, a significantly unmet medical necessity. Here, using a novel bioanalytical protocol for quantification of agmatine, we present the plasma pharmacokinetics and the first report of agmatine oral bioavailability as well as variable pharmacokinetics across different central nervous system tissues. These data provide a distributional rationale for the pharmacological effects of agmatine as well as new evidence for kinetic differences between brain and spinal cord.


Transcendental Meditation for the improvement of health and wellbeing in community-dwelling dementia caregivers [TRANSCENDENT]: a randomised wait-list controlled trial.
Dementia is a prevalent neurodegenerative disorder affecting an estimated 24.3 million people across the globe. The burden on those caring for people with dementia is substantial, with widespread implications for the caregiver, the care recipient and the community. Relaxation techniques, such as Transcendental Meditation® (TM), have been shown to reduce stress and anxiety in healthy workers; similar benefits are anticipated in dementia caregivers. The objective of this study was to ascertain whether TM can improve psychological stress, quality of life, affect and cognitive performance in dementia caregivers. The study was conducted as a pilot prospective, multi-centre, community-based, randomised wait-list controlled trial. Community-dwelling caregivers of persons with diagnosed dementia were randomly assigned to a 12-week (14-hour) TM training program or wait-list control. Participants were assessed for quality of life, stress, affect, cognitive performance and adverse effects. The feasibility of the study was also evaluated. Seventeen caregivers were recruited and randomised. Improvements in WebNeuro response speed scores over time were significantly (p = 0.03) greater in the TM group relative to control. Changes between groups over time in all other primary and secondary outcome measures did not reach statistical significance. However, there was a trend toward greater improvement in WebNeuro stress, depression and negativity bias scores in the TM group. Adverse events were reported amongst 63 % of TM-treated subjects; however, events were generally transient, of mild-moderate intensity and only 'possibly' related to TM. Dementia caregivers exposed to TM demonstrated varying degrees of improvement in several measures of cognitive function, mood, quality of life and stress following exposure to TM. However, as the pilot study was underpowered, no firm conclusions can be made about the effectiveness of TM in this caregiver population. Findings from full-scale trials are now warranted. Australian New Zealand Clinical Trial Registry ACTRN12613000184774 (Registered 15th February 2013).


Revealing the Virulence Potential of Clinical and Environmental Aspergillus fumigatus Isolates Using Whole-Genome Sequencing.
Aspergillus fumigatus is considered a common causative agent of human fungal infections. A restricted number of virulence factors have been described, and none of them lead to a differentiation in the virulence level among different strains. Variations in the virulence phenotype depending on the isolate origin, measured as survival percentage in animal infection models, have been previously reported. In this study, we analyzed the whole-genome sequence of A. fumigatus isolates from clinical and environmental origins to determine their virulence genetic content. The sample included four isolates sequenced at the University Medical Center Groningen (UMCG), three clinical (two of them isolated from the same patient) and the experimental strain B5233, and the draft genomes of one reference strain, two environmental and two clinical isolates obtained from a public database. The fungal genomes were screened for the presence of virulence-related genes (VRGs) using an in-house database of 244 genes related to thermotolerance, resistance to immune responses, cell wall formation, nutrient uptake, signaling and regulation, and production of toxins and secondary metabolites and allergens. In addition, we performed a variant calling analysis to compare the isolates sequenced at the UMCG and investigated their genetic relatedness using the TRESP (Tandem Repeats located within Exons of Surface Protein coding genes) genotyping method. We neither observed a difference in the virulence genetic content between the clinical isolates causing an invasive infection and a colonizing clinical isolate nor between isolates from the clinical and environmental origin. The four novel A. fumigatus sequences had a different TRESP genotype and a total number of genetic variants ranging from 48,590 to 68,352. In addition, a comparative genomics analysis showed the presence of single nucleotide polymorphisms in VRGs and repetitive genetic elements located next to VRG groups, which could influence the regulation of these genes. In conclusion, our genomic analysis revealed a high genetic diversity between environmental and clinical A. fumigatus isolates, as well as between clinical isolates from the same patient, indicating an infection with a mixed-population in the latter case. However, all isolates had a similar virulence genetic content, demonstrating their pathogenic potential at least at the genomic level.


Some opportunities for vibration analysis with time averaging in digital Fresnel holography.
Features offered by the combination of time averaging and digital Fresnel holography are investigated. In particular, we introduce the concept of the zero-crossing phase of Bessel fringes, which allows a highly contrasted determination of the dark fringes in the hologram. We discuss some particularities of the digital reconstruction and show how time-averaged digital holography can be used to study vibration drifts. Experiment results are presented in the case of a loudspeaker under a sinusoidal excitation; digital and analogical holography are also compared.


Characterization of monomeric L1 metallo-beta -lactamase and the role of the N-terminal extension in negative cooperativity and antibiotic hydrolysis.
The L1 metallo-beta-lactamase from Stenotrophomonas maltophilia is unique among this class of enzymes because it is tetrameric. Previous work predicted that the two regions of important intersubunit interaction were the residue Met-140 and the N-terminal extensions of each subunit. The N-terminal extension was also implicated in beta-lactam binding. Mutation of methionine 140 to aspartic acid results in a monomeric L1 beta-lactamase with a greatly altered substrate specificity profile. A 20-amino acid N-terminal deletion mutant enzyme (N-Del) could be isolated in a tetrameric form but demonstrated greatly reduced rates of beta-lactam hydrolysis and different substrate profiles compared with that of the parent enzyme. Specific site-directed mutations of individual N terminus residues were made (Y11S, W17S, and a double mutant L5A/L8A). All N-terminal mutant enzymes were tetramers and all showed higher K(m) values for ampicillin and nitrocefin, hydrolyzed ceftazidime poorly, and hydrolyzed imipenem more efficiently than ampicillin in contrast to wild-type L1. Nitrocefin turnover was significantly increased, probably because of an increased rate of breakdown of the intermediate species due to a lack of stabilizing forces. K(m) values for monomeric L1 were greatly increased for all antibiotics tested. A model of a highly mobile N-terminal extension in the monomeric enzyme is proposed to explain these findings. Tetrameric L1 shows negative cooperativity, which is not present in either the monomer or N-terminal deletion enzymes, suggesting that the cooperative effect is mediated via N-terminal intersubunit interactions. These data indicate that while the N terminus of L1 is not essential for beta-lactam hydrolysis, it is clearly important to its activity and substrate specificity.


ASPM promotes prostate cancer stemness and progression by augmenting Wnt-Dvl-3-β-catenin signaling.
Recurrent and hormone-refractory prostate cancer (PCA) exhibits aggressive behaviors while current therapeutic approaches show little effect of prolonging the survival of patients with PCA. Thus, a deeper understanding of the patho-molecular mechanisms underlying the disease progression in PCA is crucial to identify novel diagnostic and/or therapeutic targets to improve the outcome of patients. Recent evidence suggests that activation of Wnt signaling in cancer stem cells (CSCs) contributes to cancer progression in malignant tumors. Here, we report that a novel Wnt co-activator ASPM (abnormal spindle-like microcephaly associated) maintains the prostate CSC subpopulation by augmenting the Wnt-β-catenin signaling in PCA. ASPM expression is incrementally upregulated in primary and metastatic PCA, implicating its potential role in PCA progression. Consistently, downregulation of ASPM expression pronouncedly attenuated the proliferation, colony formation, and the invasive behavior of PCA cells, and dramatically reduced the number of ALDH+ CSCs and inhibited cancer stemness and tumorigenicity. Mechanistically, ASPM interacts with disheveled-3 (Dvl-3), a cardinal upstream regulator of canonical Wnt signaling, and inhibits its proteasome-dependent degradation, thereby increasing its protein stability and enabling the Wnt-induced β-catenin transcriptional activity in PCA cells. In keeping with the role of ASPM as a CSC-regulator, ASPM co-localizes with ALDH in PCA tissues and its expression exhibits high intra-tumoral heterogeneity. The proportion of high-ASPM-expressing cells in the tumor inversely correlates with the relapse-free survival of PCA patients. Collectively, our data points to ASPM as a novel oncoprotein and an essential regulator of Wnt signaling and cancer stemness in PCA, which has important clinical and therapeutic significance.


Towards the colloidal Laves phase from binary hard-sphere mixtures via sedimentation.
Colloidal photonic crystals, which show a complete band gap in the visible region, have numerous applications in fibre optics, energy storage and conversion, and optical wave guides. Intriguingly, two of the best examples of photonic crystals, the diamond and pyrochlore structure, can be self-assembled into the colloidal MgCu2 Laves phase crystal from a simple binary hard-sphere mixture. For these colloidal length scales thermal and gravitational energies are often comparable and therefore it is worthwhile to study the sedimentation phase behavior of these systems. For a multicomponent system this is possible through a theoretical construct known as a stacking diagram, which constitutes a set of all possible stacking sequences of phases in a sedimentation column, and uses as input the bulk phase diagram of the system in the chemical potential plane. We determine the stable phases for binary hard-sphere systems with varying diameter ratios using Monte Carlo simulations and analytical equations of state available in literature and calculate the corresponding stacking diagrams. We also discuss observations from event-driven Brownian dynamics simulations in relation to our theoretical stacking diagrams.


Impact of intestinal transplantation for intestinal failure in Japan.
The prognosis of intestinal failure has improved dramatically in the past few decades with the development of parenteral nutrition (PN). However, PN-dependent patients still have numerous complications. Intestinal transplantation can significantly improve their prognosis and quality of life. We report on the impact of intestinal transplantation for intestinal failure in Japan. Intestinal transplantations have been performed in Japan since 1996. Standardized forms were sent to all known intestinal transplantation programs, asking for information on intestinal transplantations performed between 1996 and June 31, 2012. All programs responded. Patient and graft survival estimates were obtained using the Kaplan-Meier method and analyzed with the Wilcoxon statistic. Five institutions provided data on 24 grafts in 21 patients. There were 12 cadaveric and 12 living related donor transplants. Causes of intestinal failure included short gut syndrome (n = 9), intestinal motility function disorders (n = 11), retransplantation (n = 3), and other (n = 1). The overall 1- and 5-year patient survival rates were 86% and 68%, respectively. In cases (n = 15) after 2006, the 1-year patient survival rate was 92%, and the 5-year survival rate was 83%. One- and five-year graft survival rates were 87% and 78%, respectively. More than 80% of all current survivors discontinued PN. Intestinal transplantation has become an effective therapy for patients with intestinal failure who cannot tolerate PN. After 2006, patient and graft survival rates approached rates associated with standard treatment for end-stage intestinal failure. Further improvements are expected with early referral due to suitable donor organ and pretransplant management.


Transmission of crayfish plague.
Two possible means of transmission of crayfish plague were investigated: via fish (as vectors), and via crayfish (as hosts or vectors when dead). The crayfish transmission experiments focussed on both the viability of the fungus in dead crayfish when kept in simulated field conditions, and on the treatments which kill viable forms of Aphanomyces astaci within the recently dead host (cadaver). It was found that A. astaci remains viable for 5 d, and possibly longer in crayfish kept in water at 21 degrees C after dying of crayfish plague. Heating (boiling for 1 min) was the quickest way of decontaminating crayfish cadavers. Freezing took considerably longer: after 48 h at -20 degrees C, viable stages were still present. It was also found that A. astaci is unlikely to survive passage of the gastrointestinal tract of either mammals or birds as no viable stages were found after 12 h at 37 degrees C. Two basic modes of transmission of crayfish plague via fish were investigated: (1) after passage of initially viable forms of A. astaci through the digestive tract of fish and (2) via fish skin. If A. astaci was fed to fish as infected abdominal cuticle, it was still viable after passage through the gastrointestinal tract. When pure mycelium or spores were fed to fish there was no indication of viable forms of A. astaci after passage through the gastrointestinal tract. Transmission via fish skin was not observed under the experimental conditions applied. The results on fish as vectors have practical importance for fish transport and stocking because the present study shows that there is a risk of transmission of crayfish plague via fish faeces. The investigation of crayfish as vectors delivers methods that could be used for treatment of crayfish imported for human consumption into crayfish-plague-free areas. This application is particularly important for the importation of American crayfish, which are in general suspected to carry A. astaci in their cuticle.


Corryocactus brevistylus (K. Schum. ex Vaupel) Britton & Rose (Cactaceae): Antioxidant, Gastroprotective Effects, and Metabolomic Profiling by Ultrahigh-Pressure Liquid Chromatography and Electrospray High Resolution Orbitrap Tandem Mass Spectrometry.
Corryocactus brevistylus (K. Schum. ex Vaupel) Britton & Rose (Cactaceae) is a shrubby or often arborescent cactus popularly known as "sancayo" and produce an edible fruit known as "Sanky" which is consumed in Arequipa-Perú. The purpose of this study was to report the gastroprotective activity and relate this activity to the antioxidant capacity and presence of phenolic compounds for the first time. A metabolomic profiling based on Ultrahigh-pressure liquid chromatography and electrospray high resolution mass spectrometry, and the antioxidant activities (DPPH, ABTS, and FRAP), ascorbic acid content, total phenolics and flavonoids contents, and the mode of gastroprotective action of the Sanky fruit including the involvement of prostaglandins, nitric oxide, and sulfhydryl compounds is reported. Thirty-eight compounds were detected in the ethanolic extract including 12 organic acids, nine hydroxycinnamic acids, three isoamericanol derivatives, six flavonoids, five fatty acids, and two sterols. The results of the biological tests showed that the ethanolic extract had antioxidant capacity and gastroprotective activity on the model of HCl/EtOH-induced gastric lesions in mice (at 10, 25, 50, and 100 mg/kg). The effect elicited by the extract at 50 mg/kg was reversed by indometacin and N-ethylmaleimide but not by NG-nitro-L-arginine methyl ester suggesting that sulfhydryl groups and prostaglandins are involved in the mode of gastroprotective action. In conclusion, our study proves that C. brevistylus pears have some gastroprotective and antioxidant capacities and consumption is recommended for the presence of several bioactive compounds.


Receptor palmitoylation and ubiquitination regulate anthrax toxin endocytosis.
The anthrax toxin is composed of three independent polypeptide chains. Successful intoxication only occurs when heptamerization of the receptor-binding polypeptide, the protective antigen (PA), allows binding of the two enzymatic subunits before endocytosis. We show that this tailored behavior is caused by two counteracting posttranslational modifications in the cytoplasmic tail of PA receptors. The receptor is palmitoylated, and this unexpectedly prevents its association with lipid rafts and, thus, its premature ubiquitination. This second modification, which is mediated by the E3 ubiquitin ligase Cbl, only occurs in rafts and is required for rapid endocytosis of the receptor. As a consequence, cells expressing palmitoylation-defective mutant receptors are less sensitive to anthrax toxin because of a lower number of surface receptors as well as premature internalization of PA without a requirement for heptamerization.


DDX5 and its associated lncRNA Rmrp modulate TH17 cell effector functions.
T helper 17 (TH17) lymphocytes protect mucosal barriers from infections, but also contribute to multiple chronic inflammatory diseases. Their differentiation is controlled by RORγt, a ligand-regulated nuclear receptor. Here we identify the RNA helicase DEAD-box protein 5 (DDX5) as a RORγt partner that coordinates transcription of selective TH17 genes, and is required for TH17-mediated inflammatory pathologies. Surprisingly, the ability of DDX5 to interact with RORγt and coactivate its targets depends on intrinsic RNA helicase activity and binding of a conserved nuclear long noncoding RNA (lncRNA), Rmrp, which is mutated in patients with cartilage-hair hypoplasia. A targeted Rmrp gene mutation in mice, corresponding to a gene mutation in cartilage-hair hypoplasia patients, altered lncRNA chromatin occupancy, and reduced the DDX5-RORγt interaction and RORγt target gene transcription. Elucidation of the link between Rmrp and the DDX5-RORγt complex reveals a role for RNA helicases and lncRNAs in tissue-specific transcriptional regulation, and provides new opportunities for therapeutic intervention in TH17-dependent diseases.


Tracking crop varieties using genotyping-by-sequencing markers: a case study using cassava (Manihot esculenta Crantz).
Accurate identification of crop cultivars is crucial in assessing the impact of crop improvement research outputs. Two commonly used identification approaches, elicitation of variety names from farmer interviews and morphological plant descriptors, have inherent uncertainty levels. Genotyping-by-sequencing (GBS) was used in a case study as an alternative method to track released varieties in farmers' fields, using cassava, a clonally propagated root crop widely grown in the tropics, and often disseminated through extension services and informal seed systems. A total of 917 accessions collected from 495 farming households across Ghana were genotyped at 56,489 SNP loci along with a "reference library" of 64 accessions of released varieties and popular landraces. Accurate cultivar identification and ancestry estimation was accomplished through two complementary clustering methods: (i) distance-based hierarchical clustering; and (ii) model-based maximum likelihood admixture analysis. Subsequently, 30% of the identified accessions from farmers' fields were matched to specific released varieties represented in the reference library. ADMIXTURE analysis revealed that the optimum number of major varieties was 11 and matched the hierarchical clustering results. The majority of the accessions (69%) belonged purely to one of the 11 groups, while the remaining accessions showed two or more ancestries. Further analysis using subsets of SNP markers reproduced results obtained from the full-set of markers, suggesting that GBS can be done at higher DNA multiplexing, thereby reducing the costs of variety fingerprinting. A large proportion of discrepancy between genetically unique cultivars as identified by markers and variety names as elicited from farmers were observed. Clustering results from ADMIXTURE analysis was validated using the assumption-free Discriminant Analysis of Principal Components (DAPC) method. We show that genome-wide SNP markers from increasingly affordable GBS methods coupled with complementary cluster analysis is a powerful tool for fine-scale population structure analysis and variety identification. Moreover, the ancestry estimation provides a framework for quantifying the contribution of exotic germplasm or older improved varieties to the genetic background of contemporary improved cultivars.


Associations between Salivary Testosterone Levels, Androgen-Related Genetic Polymorphisms, and Self-Estimated Ejaculation Latency Time.
Recently, testosterone (T) has been shown to be associated with premature ejaculation (PE) symptoms in the literature. Furthermore, studies suggest that the etiology of PE is partly under genetic control. The aim of this study was to reassess findings suggesting an association between testosterone (T) and a key symptom of PE, ejaculation latency time (ELT), as well as exploratively investigating associations between six androgen-related genetic polymorphisms and ELT. Statistical analyses were performed on a population-based sample of 1,429 Finnish men aged 18-45 years (M = 26.9, SD = 4.7). Genotype information was available for 1,345-1,429 of these (depending on the polymorphism), and salivary T samples were available from 384 men. Two androgen receptor gene-linked, two 5-alpha-reductase type 2-gene-linked, and two sex hormone-binding globuline gene-linked polymorphisms were genotyped. Ejaculatory function was assessed using self-reported ELT. We found no association between salivary T levels and ELT. We found a nominally significant association between a 5-alpha-reductase type 2-gene-linked polymorphism (rs2208532) and ELT, but this association did not remain significant after correction for multiple testing. One single nucleotide polymorphism in the sex hormone-binding globulin gene (rs1799941) moderated (significantly after correction for multiple testing) the association between salivary T and ELT, so that A:A genotype carriers had significantly lower salivary T levels as a function of increasing ELT compared with other genotype groups. We were unable to find support for the hypothesis suggesting an association between T levels and ELT, possibly because of the low number of phenotypically extreme cases (the sample used in the present study was population based). Our results concerning genetic associations should be interpreted with caution until replication studies have been conducted. Jern P, Westberg L, Ankarberg-Lindgren C, Johansson A, Gunst A, Sandnabba NK, and Santtila P. Associations between salivary testosterone levels, androgen-related genetic polymorphisms, and self-estimated ejaculation latency time. Sex Med 2014;2:107-114.


Genome-wide gene-environment analyses of major depressive disorder and reported lifetime traumatic experiences in UK Biobank.
Depression is more frequent among individuals exposed to traumatic events. Both trauma exposure and depression are heritable. However, the relationship between these traits, including the role of genetic risk factors, is complex and poorly understood. When modelling trauma exposure as an environmental influence on depression, both gene-environment correlations and gene-environment interactions have been observed. The UK Biobank concurrently assessed Major Depressive Disorder (MDD) and self-reported lifetime exposure to traumatic events in 126,522 genotyped individuals of European ancestry. We contrasted genetic influences on MDD stratified by reported trauma exposure (final sample size range: 24,094-92,957). The SNP-based heritability of MDD with reported trauma exposure (24%) was greater than MDD without reported trauma exposure (12%). Simulations showed that this is not confounded by the strong, positive genetic correlation observed between MDD and reported trauma exposure. We also observed that the genetic correlation between MDD and waist circumference was only significant in individuals reporting trauma exposure (rg = 0.24, p = 1.8 × 10-7 versus rg = -0.05, p = 0.39 in individuals not reporting trauma exposure, difference p = 2.3 × 10-4). Our results suggest that the genetic contribution to MDD is greater when reported trauma is present, and that a complex relationship exists between reported trauma exposure, body composition, and MDD.


Insulin-like growth factor-I induces renal cell hypertrophy via a calcineurin-dependent mechanism.
Insulin-like growth factor-I (IGF-I) may play an important role in the development of renal hypertrophy. In this study we determined the effect of IGF-I on cultured mesangial cells (MCs) and examined activation of key signaling pathways. IGF-I induced hypertrophy as determined by an increase in cell size and an increase in protein to DNA ratio and increased accumulation of extracellular matrix (ECM) proteins. IGF-I also activated both Erk1/Erk2 MAPK and phosphatidylinositol 3-kinase (PI3K) in MCs. Inhibition of either MAPK or PI3K, however, had no effect on IGF-I-induced hypertrophy or ECM production. Next, we examined the effect of IGF-I on activation of the calcium-dependent phosphatase calcineurin. IGF-I treatment stimulated calcineurin activity and increased the protein levels of calcineurin and the calcineurin binding protein, calmodulin. Cyclosporin A, an inhibitor of calcineurin, blocked both IGF-I-mediated hypertrophy and up-regulation of ECM. In addition, calcineurin resulted in sustained Akt activation, indicating possible cross-talk with other signaling pathways. Finally, IGF-I treatment resulted in the calcineurindependent nuclear localization of NFATc1. Therefore, IGF-I induces hypertrophy and increases ECM accumulation in MCs. IGF-I-mediated hypertrophy is associated with activation of Erk1/Erk2 MAPK and PI3K but does not require either of these pathways. Instead, IGF-I mediates hypertrophy via a calcineurin-dependent pathway.


A new surgical technique for scleral fixation using a "knot ball" for scleral-fixated intraocular lenses: impact on clinical outcomes.
Scleral fixation surgery is a pivotal procedure that depends on the availability of robust and innovative surgical fixation methods. There continues to be a need for innovation in suture fixation techniques, particularly for intraocular lens implantation. We conceived and designed a "knot ball" scleral fixation technique for suture burial in a retrospective sample of 108 patients with primary (n=40) or secondary (n=68) scleral-fixated intraocular lens. Importantly, our technique did not require additional scleral flap or tunnel procedures. We evaluated pre- and postoperative best-corrected visual aquity (BCVA) and postoperative complications. All data were analyzed and compared between groups. The preoperative mean BCVA improved significantly in both groups using the "knot ball" fixation technique (p<0.01). The extent of the improvement in the best-corrected visual acuity and late complications one month post-surgery were not significantly different between the groups (p>0.05). These clinical outcomes were consistent with those described in the ophthalmology literature. A "knot ball" scleral fixation technique is reported; to the best of our knowledge, this is the first report of such a technique, which offers a less invasive and simplified surgical procedure for transscleral fixation of scleral-fixated intraocular lenses. Moreover, the technique appears to display similar effectiveness and safety compared with existing scleral fixation techniques. We suggest that the "knot ball" technique warrants further research focus and clinical evaluation in future studies.


Interprotofilament interactions between Alzheimer's Abeta1-42 peptides in amyloid fibrils revealed by cryoEM.
Alzheimer's disease is a neurodegenerative disorder characterized by the accumulation of amyloid plaques in the brain. This amyloid primarily contains amyloid-beta (Abeta), a 39- to 43-aa peptide derived from the proteolytic cleavage of the endogenous amyloid precursor protein. The 42-residue-length Abeta peptide (Abeta(1-42)), the most abundant Abeta peptide found in plaques, has a much greater propensity to self-aggregate into fibrils than the other peptides and is believed to be more pathogenic. Synthetic human Abeta(1-42) peptides self-aggregate into stable but poorly-ordered helical filaments. We determined their structure to approximately 10-A resolution by using cryoEM and the iterative real-space reconstruction method. This structure reveals 2 protofilaments winding around a hollow core. Previous hairpin-like NMR models for Abeta(17-42) fit well in the cryoEM density map and reveal that the juxtaposed protofilaments are joined via the N terminus of the peptide from 1 protofilament connecting to the loop region of the peptide in the opposite protofilament. This model of mature Abeta(1-42) fibrils is markedly different from previous cryoEM models of Abeta(1-40) fibrils. In our model, the C terminus of Abeta forms the inside wall of the hollow core, which is supported by partial proteolysis analysis.


Missing potential opportunities to reduce repeat COPD exacerbations.
Long-acting beta-agonists (LABA) and/or inhaled corticosteroids (ICS) have been shown to reduce COPD exacerbation risk. Using data from a large integrated health-care system, we sought to examine whether these medication classes were initiated after an exacerbation of COPD. We identified patients who experienced an inpatient or outpatient COPD exacerbation within the Veterans Affairs Integrated Service Network (VISN)-20. We assessed the addition of a new inhaled therapy (an ICS, LABA or both) within 180 days after the exacerbation. We assessed independent predictors of adding treatment using logistic regression. We identified 45,780 patients with COPD, of whom 2,760 patients experienced an exacerbation of COPD. Of these individuals, 2,570 (93.1 %) were on either none or only one long-acting medication studied (LABA or ICS). In the subsequent 180-day period after their exacerbation, only 875 (34.1 %) patients had at least one of these additional therapies dispensed from a VA pharmacy. Among patients who were treated in the outpatient setting, older age [OR 0.98/year, 95 % CI (0.97-0.99)], current tobacco use [OR 0.74, 95 % CI (0.60-0.90)], greater use of ipratropium bromide [OR 0.97/canister, 95 % CI (0.96-0.98)], prior COPD exacerbation [OR 0.55, 95 % CI (0.46-0.67)], depression [OR 0.77, 95 % CI (0.61-0.98)], CHF [OR 0.74, 95 % CI (0.57-0.97)], and diabetes (OR 0.77 (0.60-0.99)] were associated with lower odds of additional therapy. Patients who were treated in the hospital had similar associated predictors. Among patients treated for an exacerbation of COPD, we found relatively few were subsequently prescribed inhaled therapies known to reduce exacerbations.


An electrophysiological study of task demands on concreteness effects: evidence for dual coding theory.
We examined ERP responses during the generation of word associates or mental images in response to concrete and abstract concepts. Of interest were the predictions of dual coding theory (DCT), which proposes that processing lexical concepts depends on functionally independent but interconnected verbal and nonverbal systems. ERP responses were time-locked to either stimulus onset or response to compensate for potential latency differences across conditions. During word associate generation, but not mental imagery, concrete items elicited a greater N400 than abstract items. A concreteness effect emerged at a later time point during the mental imagery task. Data were also analyzed using time-frequency analysis that investigated synchronization of neuronal populations over time during processing. Concrete words elicited an enhanced late going desynchronization of theta-band power (723-938 ms post stimulus onset) during associate generation. During mental imagery, abstract items elicited greater delta-band power from 800 to 1,000 ms following stimulus onset, theta-band power from 350 to 205 ms before response, and alpha-band power from 900 to 800 ms before response. Overall, the findings support DCT in suggesting that lexical concepts are not amodal and that concreteness effects are modulated by tasks that focus participants on verbal versus nonverbal, imagery-based knowledge.


Walking may be related to less vascular stiffness in the Activity Counseling Trial (ACT).
The ACT was a clinical trial of various patient education and counseling interventions to increase physical activity in sedentary primary care populations. It provided the opportunity to measure the effect of increasing physical activity on aortic pulse wave velocity (APWV), a measure of vascular stiffness, in a relatively healthy middle-aged population. The effects of the interventions, as well as the impact of walking and correlates such as older age and maximal oxygen uptake (VO2max), on APWV were assessed. The participants in this study were a subset of the 874 persons recruited for the ACT. Information about self-reported physical activity and disease status was collected at baseline (464 persons), 6-month (528 persons), and 24-month (555 persons) intervals. Physiological measures included APWV, systolic blood pressure, and other correlates. In multivariate analyses, the various treatment arms did not have a significant effect on APWV. However, walking in hours per day was associated with slower APWV times or less stiffness (P = .03). This was significant for women and consistent but not significant for men. In addition, age, clinic site, race, systolic blood pressure, and VO2max were independently associated with APWV. Increased walking frequency over a 24-month period was predictive of reduced vascular stiffness in ACT. The more significant result for walking frequency in women than in men might be caused by the presence of a low Vo2max or physical activity threshold for an effect of walking on APWV, which most women achieved but most men had surpassed at the start of the study. Although needing confirmation because this was a secondary analysis, modest physical activity may have a beneficial effect on large vessel structure.


Variable and changing trajectories in youth athlete development: further verification in advocating a long-term inclusive tracking approach.
Athlete development through adolescence can vary greatly because of maturational processes. For example, variation can be observed in anthropometric and fitness measures with later maturing individuals "catching up" their earlier maturing peers at later time points. This study examined a methodological issue concerning how best to assess anthropometric and fitness change (i.e., "across age categories" or "per year") relative to an age and skill-matched population (N = 1,172). Furthermore, it examined changes in anthropometric and fitness characteristics in 3 cases of youth rugby league players (aged 13-15) across a 2-year period. Findings identified the "per year" method as generating less deviated z-scores across anthropometric and fitness measures (e.g., mean change p < 0.001), suggesting less substantial change in case players relative to the population. When applied to additional players, z-score and radar graphs showed developmental variability and longitudinal change. The possibility of a "later maturing player" increasing anthropometric (e.g., height: player 4 = 3.3 cm; player 5 = 13.2 cm; and player 6 = 15.7 cm) and fitness (e.g., 30-m sprint: player 4 = -0.18 s, player 5 = -0.46 s, and player 6 = -0.59 s) characteristics compared with early maturing players was confirmed. Findings affirm the potential for variable and changing trajectories in adolescent athletes. Practical implications advocate a long-term inclusive tracking approach of athletes, the avoidance of (de)selection, and the reduction of a performance emphasis in adolescent stages of sport systems.


Polarized emission of quantum dots in microcavity and anisotropic Purcell factors.
We study the polarization properties of quantum dot (QD) emission coupled with the fundamental cavity modes. A rotation of polarization axis and a change of polarization degree are observed as the coupling is varied. To explain this observation, we derive an analytical model considering the polarization misalignment between QD dipole and cavity mode field. Our model also provides a new approach to extract the anisotropic Purcell factors by analyzing the polarization of detected quantum dot emission coupled to the cavity mode, which paves the way to develop high-efficiency polarized single photon sources.


Generating tissue topology through remodeling of cell-cell adhesions.
During tissue morphogenesis, cellular rearrangements give rise to a large variety of three-dimensional structures. Final tissue architecture varies greatly across organs, and many develop to include combinations of folds, tubes, and branched networks. To achieve these different tissue geometries, constituent cells must follow different programs that dictate changes in shape and/or migratory behavior. One essential component of these changes is the remodeling of cell-cell adhesions. Invasive migratory behavior and separation between tissues require localized breakdown of cadherin-mediated adhesions. Conversely, tissue folding and fusion require the formation and reinforcement of cell-cell adhesions. Cell-cell adhesion plays a critical role in tissue morphogenesis; its manipulation may therefore prove to be invaluable in generating complex topologies ex vivo. Recapitulating these shapes in engineered tissues would enable a better understanding of how these processes occur in vivo, and may lead to improved design of organs for clinical applications. In this review, we discuss work investigating the formation of folds, tubes, and branched networks with an emphasis on known or possible roles for cell-cell adhesion. We then examine recently developed tools that could be adapted to manipulate cell-cell adhesion in engineered tissues.


A randomized trial of mesenchymal stem cells in multiple system atrophy.
Neuroprotective or regenerative strategies are invaluable in multiple system atrophy (MSA) due to its rapid progression with fatal prognosis. We evaluated the efficacy of autologous mesenchymal stem cells (MSC) in patients with MSA-cerebellar type (MSA-C). Thirty-three patients with probable MSA-C and baseline unified MSA rating scale (UMSARS) scores ranging from 30 to 50 were randomly assigned to receive MSC (4 × 10(7) /injection) via intra-arterial and intravenous routes or placebo. The primary outcome was change in the total UMSARS scores from baseline throughout a 360-day follow-up period between groups. Secondary outcomes were changes in the UMSARS part II scores, cerebral glucose metabolism, gray matter density, and cognitive performance over a 360-day period. The mixed model analysis of neurological deficits revealed a significant interaction effect between treatment group and time, suggesting that the MSC group had a smaller increase in total and part II UMSARS scores compared with the placebo group (p = 0.047 and p = 0.008, respectively). Cerebral glucose metabolism and gray matter density at 360 days relative to the baseline were more extensively decreased in the cerebellum and the cerebral cortical areas, along with greater deterioration of frontal cognition in the placebo group compared with the MSC group. We found no serious adverse effects that were directly related to MSC treatment. However, intra-arterial infusion resulted in small ischemic lesions on magnetic resonance imaging. MSC therapy could delay the progression of neurological deficits in patients with MSA-C, suggesting the potential of MSC therapy as a treatment candidate of MSA.


Classification of polyhedral shapes from individual anisotropically resolved cryo-electron tomography reconstructions.
Cryo-electron tomography (cryo-ET) enables 3D imaging of macromolecular structures. Reconstructed cryo-ET images have a "missing wedge" of data loss due to limitations in rotation of the mounting stage. Most current approaches for structure determination improve cryo-ET resolution either by some form of sub-tomogram averaging or template matching, respectively precluding detection of shapes that vary across objects or are a priori unknown. Various macromolecular structures possess polyhedral structure. We propose a classification method for polyhedral shapes from incomplete individual cryo-ET reconstructions, based on topological features of an extracted polyhedral graph (PG). We outline a pipeline for extracting PG from 3-D cryo-ET reconstructions. For classification, we construct a reference library of regular polyhedra. Using geometric simulation, we construct a non-parametric estimate of the distribution of possible incomplete PGs. In studies with simulated data, a Bayes classifier constructed using these distributions has an average test set misclassification error of < 5 % with upto 30 % of the object missing, suggesting accurate polyhedral shape classification is possible from individual incomplete cryo-ET reconstructions. We also demonstrate how the method can be made robust to mis-specification of the PG using an SVM based classifier. The methodology is applied to cryo-ET reconstructions of 30 micro-compartments isolated from E. coli bacteria. The predicted shapes aren't unique, but all belong to the non-symmetric Johnson solid family, illustrating the potential of this approach to study variation in polyhedral macromolecular structures.


In vitro inhibition of adhesion of Candida albicans clinical isolates to human buccal epithelial cells by Fuc alpha 1----2Gal beta-bearing complex carbohydrates.
The role of cell surface glycoconjugates as possible adhesion receptors for Candida albicans yeasts on human buccal epithelial cells was investigated by using a quantitative radiometric assay involving 14C-metabolically labeled microorganisms. Various structurally defined soluble glycopeptides and oligosaccharides were tested at a low concentration (1 mg/ml) for their ability to competitively inhibit yeast adhesion to such exfoliated cells. Comparisons were also made with various molecular species previously proposed to act as adhesion molecules. A preparation of glycopeptides derived from pooled human newborn meconiums inhibited the attachment (up to 55%) of all three clinical isolates examined. The mild hydrolysis of fucosyl residues from the above mixture totally abolished its inhibitory potency. By using human milk oligosaccharide probes, the minimal structural requirement for activity was found to be the Fuc alpha 1----2Gal beta determinant (the H sugar sequence found on all blood group substances of the ABO [H] system). By contrast, the fucosylated determinants of the Lewis blood group system were found to be totally inactive. Total adhesion inhibitions were never obtained in the present experiments, suggesting that H disaccharide-bearing cell surface glycoconjugates could act as host receptors for C. albicans on human buccal epithelial cells as a part of a mechanism involving multireceptor specificities.


Mental Health Problems in Parents of Children with Congenital Heart Disease.
This review will provide a concise description of mental health problems in parents of children with a (non-syndromic) congenital heart disease (CHD) during different stressful periods. Predictors of these problems and also implications for clinical practice will be mentioned. Having a child with CHD can be very stressful for parents, who have to face overwhelming emotions and also extra physical, financial, and other practical challenges. Parental distress has been reported in 30-80% of parents and appears not to be related to severity of CHD. Parental mental health, parenting, the parent-child relationship, and parental quality of life can all be affected. Parents, and especially mothers, are at risk of psychological distress, anxiety, depression, somatization, hopelessness, and posttraumatic stress symptoms, which in turn may influence mother's responsiveness. In the long term, the majority of parents adapt successfully to living with a child with CHD, but approximately 40% report a need for psychosocial care. These families may be helped by early psychosocial interventions to alleviate stress and reduce children's emotional and behavioral problems. A holistic approach to early psychosocial interventions should aim at improving coping and enhance parenting. During routine medical checkups, medical professionals should ask about parental stress, family functioning, and psychosocial functioning of the child and, when needed, adequate psychosocial care should be provided.


Paracorporeal ventricular assist device as a bridge to transplant candidacy in the era of implantable continuous-flow ventricular assist device.
Ventricular assist devices (VADs) have long been used as bridge to transplant therapy (BTT). Nipro-Toyobo paracorporeal pulsatile-flow VAD (nt-VAD) was the only device available until April 2011, when implantable continuous-flow VADs (cf-VADs) became available. Although cf-VADs are central to BTT, nt-VAD remains a necessary option. We aimed to clarify the role of nt-VAD in an era of increasing cf-VAD use. We retrospectively reviewed patients who underwent VAD implantation at the National Cerebral and Cardiovascular Center from May 2011 to March 2013. Characteristics were compared between the nt-VAD and cf-VAD groups. Twenty-nine patients (mean age 37.7 ± 11.1 years, 23 males) underwent VAD implantation. Fifteen patients initially received nt-VADs, although 4 were converted to cf-VADs. Of these 15 patients, 3 were too small for cf-VADs and 2 needed bilateral ventricular support. The remaining 10 patients received nt-VADs (7 patients at INTERMACS level 1 and 3 at level 2). The nt-VAD group patients had significantly more preoperative mechanical circulatory support and were in a more critical condition before VAD implantation than the cf-VAD group. The 2-year survival rate was not significantly different. Despite the critical conditions of nt-VAD patients, their overall survival is not statistically inferior to that of cf-VAD patients. nt-VAD is a good option as a BTC for the patient with urgent and critical condition.


Antenna Deployment for the Localization of Partial Discharges in Open-Air Substations.
Partial discharges are ionization processes inside or on the surface of dielectrics that can unveil insulation problems in electrical equipment. The charge accumulated is released under certain environmental and voltage conditions attacking the insulation both physically and chemically. The final consequence of a continuous occurrence of these events is the breakdown of the dielectric. The electron avalanche provokes a derivative of the electric field with respect to time, creating an electromagnetic impulse that can be detected with antennas. The localization of the source helps in the identification of the piece of equipment that has to be decommissioned. This can be done by deploying antennas and calculating the time difference of arrival (TDOA) of the electromagnetic pulses. However, small errors in this parameter can lead to great displacements of the calculated position of the source. Usually, four antennas are used to find the source but the array geometry has to be correctly deployed to have minimal errors in the localization. This paper demonstrates, by an analysis based on simulation and also experimentally, that the most common layouts are not always the best options and proposes a simple antenna layout to reduce the systematic error in the TDOA calculation due to the positions of the antennas in the array.


Expansion of Circulating Tumor Cells from Patients with Locally Advanced Pancreatic Cancer Enable Patient Derived Xenografts and Functional Studies for Personalized Medicine.
Improvement in pancreatic cancer treatment represents an urgent medical goal that has been hampered by the lack of predictive biomarkers. Circulating Tumor Cells (CTCs) may be able to overcome this issue by allowing the monitoring of therapeutic response and tumor aggressiveness through ex vivo expansion. The successful expansion of CTCs is challenging, due to their low numbers in blood and the high abundance of blood cells. Here, we explored the utility of pancreatic CTC cultures as a preclinical model for treatment response. CTCs were isolated from ten patients with locally advanced pancreatic cancer using the Labyrinth, a biomarker independent, size based, inertial microfluidic separation device. Three patient-derived CTC samples were successfully expanded in adherent and spheroid cultures. Molecular and functional characterization was performed on the expanded CTC lines. CTC lines exhibited KRAS mutations, consistent with pancreatic cancers. Additionally, we evaluated take rate and metastatic potential in vivo and examined the utility of CTC lines for cytotoxicity assays. Patient derived expanded CTCs successfully generated patient derived xenograft (PDX) models with a 100% take rate. Our results demonstrate that CTC cultures are possible and provide a valuable resource for translational pancreatic cancer research, while also providing meaningful insight into the development of distant metastasis, as well as treatment resistance.


Disheveled hair and ear (Dhe), a spontaneous mouse Lmna mutation modeling human laminopathies.
Investigations of naturally-occurring mutations in animal models provide important insights and valuable disease models. Lamins A and C, along with lamin B, are type V intermediate filament proteins which constitute the proteinaceous boundary of the nucleus. LMNA mutations in humans cause a wide range of phenotypes, collectively termed laminopathies. To identify the mutation and investigate the phenotype of a spontaneous, semi-dominant mutation that we have named Disheveled hair and ear (Dhe), which causes a sparse coat and small external ears in heterozygotes and lethality in homozygotes by postnatal day 10. Genetic mapping identified a point mutation in the Lmna gene, causing a single amino acid change, L52R, in the coiled coil rod domain of lamin A and C proteins. Cranial sutures in Dhe/+ mice failed to close. Gene expression for collagen types I and III in sutures was deficient. Skulls were small and disproportionate. Skeletons of Dhe/+ mice were hypomineralized and total body fat was deficient in males. In homozygotes, skin and oral mucosae were dysplastic and ulcerated. Nuclear morphometry of cultured cells revealed gene dose-dependent blebbing and wrinkling. Dhe mice should provide a useful new model for investigations of the pathogenesis of laminopathies.


Three different regulatory mechanisms enable yeast hexose transporter (HXT) genes to be induced by different levels of glucose.
The HXT genes (HXT1 to HXT4) of the yeast Saccharomyces cerevisiae encode hexose transporters. We found that transcription of these genes is induced 10- to 300-fold by glucose. Analysis of glucose induction of HXT gene expression revealed three types of regulation: (i) induction by glucose independent of sugar concentration (HXT3); (ii) induction by low levels of glucose and repression at high glucose concentrations (HXT2 and HXT4); and (iii) induction only at high glucose concentrations (HXT1). The lack of expression of all four HXT genes in the absence of glucose is due to a repression mechanism that requires Rgt1p and Ssn6p. GRR1 seems to encode a positive regulator of HXT expression, since grr1 mutants are defective in glucose induction of all four HXT genes. Mutations in RGT1 suppress the defect in HXT expression caused by grr1 mutations, leading us to propose that glucose induces HXT expression by activating Grr1p, which inhibits the function of the Rgt1p repressor. HXT1 expression is also induced by high glucose levels through another regulatory mechanism: rgt1 mutants still require high levels of glucose for maximal induction of HXT1 expression. The lack of induction of HXT2 and HXT4 expression on high levels of glucose is due to glucose repression: these genes become induced at high glucose concentrations in glucose repression mutants (hxk2, reg1, ssn6, tup1, or mig1). Components of the glucose repression pathway (Hxk2p and Reg1p) are also required for generation of the high-level glucose induction signal for expression of the HXT1 gene. Thus, the glucose repression and glucose induction mechanisms share some of the same components and may share the same primary signal generated from glucose.


U.S. Medical Schools' Compliance With the Americans With Disabilities Act: Findings From a National Study.
Physician diversity improves care for underserved populations, yet there are few physicians with disabilities. The authors examined the availability of technical standards (TSs) from U.S. medical schools (MD- and DO-granting) and evaluated these relative to intent to comply with the Americans with Disabilities Act (ADA). Document analysis was conducted (2012-2014) on U.S. medical schools' TSs for hearing, visual, and mobility disabilities. Primary outcome measures were ease of obtaining TSs, willingness to provide reasonable accommodations, responsibility for accommodations, and acceptability of intermediaries or auxiliary aids. TSs were available for 161/173 (93%) schools. While 146 (84%) posted these on their Web sites, 100 (58%) were located easily. Few schools, 53 (33%), had TSs specifically supporting accommodating disabilities; 79 (49%) did not clearly state policies, 6 (4%) were unsupportive, and 23 (14%) provided no information. Most schools, 98 (61%), lacked information on responsibility for providing accommodations, 33 (27%) provided accommodations, and 10 (6%) had students assume some responsibility. Approximately 40% allowed auxiliary aids (e.g., motorized scooter), but < 10% allowed intermediaries (e.g., sign language interpreter). Supportive schools were more likely to allow accommodations (P < .001), assume responsibility for accommodations (P < .001), and accept intermediaries (P < .002). DO-granting schools were more supportive for students with mobility disabilities. Most medical school TSs do not support provision of reasonable accommodations for students with disabilities as intended by the ADA. Further study is needed to understand how schools operationalize TSs and barriers to achieving ADA standards.


The Clinical Relevance and Therapeutic Benefit of Established Active Ingredients Incorporated into Advanced Foam Vehicles: Vehicle Characteristics Can Influence and Improve Patient Outcomes
Topical delivery of therapeutic agents for skin diseases is a major advantage in dermatology. However, the efficacy and tolerability of topically applied therapies is dependent on several characteristics, including percutaneous penetration and permeation of active ingredient and lack of side effects, especially local tolerability reactions. Importantly, the ultimate performance of a topical product includes collectively the effects of the active ingredient and the impact that specific additives have on vehicle characteristics, such as penetration, permeation, epidermal barrier properties, relative irritancy, allergenicity potential, and patient acceptance/preference of the vehicle formulation used. Foam vehicles have evolved over time with the emergence of a menu of alcohol-based and aqueous-based variations that provide various advantages depending on clinical circumstances and the disease being treated. Aqueous-based foams have gained widespread acceptance and preference, especially due to favorable skin tolerability and the cosmetic elegance of the products. In this manuscript, data are presented supporting the efficacy, tolerability, and safety, of specific aqueous-based foam vehicles for calcipotriene used to treat plaque psoriasis, and for tazarotene used to treat acne vulgaris. Discussions include both vehicle-based properties that are relevant to clinical practice, and outcomes from the large-scale pivotal clinical trials that review efficacy and safety results and patient reported outcomes. The latter also discusses several practical subject assessments about use of the foam vehicle. J Drugs Dermatol. 2019;18(2 Suppl):s100-107.


Comparative anatomical distribution of neuronal calcium-binding protein (NECAB) 1 and -2 in rodent and human spinal cord.
Neuronal calcium-binding protein 1 and -2 (NECAB1/2) localize to multiple excitatory neuron populations in the mouse spinal cord. Here, we analyzed rat and human spinal cord, combining in situ hybridization and immunohistochemistry, complementing newly collated data on mouse spinal cord for direct comparisons. Necab1/2 mRNA transcripts showed complementary distribution in rodent's spinal cord. Multiple-labeling fluorescence histochemistry with neuronal phenotypic markers localized NECAB1 to a dense fiber plexus in the dorsal horn, to neurons mainly in superficial layers and to commissural interneurons in both rodent species. NECAB1-positive (+) motor neurons were only found in mice. NECAB1 distribution in the human spinal cord was similar with the addition of NECAB1-like immunoreactivity surrounding myelinated axons. NECAB2 was mainly present in excitatory synaptic boutons in the dorsal horn of all three species, and often in calbindin-D28k(+) neuronal somata. Rodent ependymal cells expressed calbindin-D28k. In humans, they instead were NECAB2(+) and/or calretinin(+). Our results reveal that the association of NECAB2 to excitatory neuronal circuits in the spinal cord is evolutionarily conserved across the mammalian species investigated so far. In contrast, NECAB1 expression is more heterogeneous. Thus, our study suggests that the phenotypic segregation of NECAB1 and -2 to respective excitatory and inhibitory spinal systems can underpin functional modalities in determining the fidelity of synaptic neurotransmission and neuronal responsiveness, and might bear translational relevance to humans.


Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus.
To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed. To evaluate the performance of multiplex ligaton-dependent probe amplification (MLPA) against Genotype® MTBDRplus to detect resistance to isoniazid (INHr) and rifampicin (RIFr). 96 culture isolates characterised for identification, drug susceptibility testing (DST) and sequencing of rpoB, katG, and inhA genes were evaluated by the MLPA and Genotype®MTBDRplus assays. With sequencing as a reference standard, sensitivity (SE) to detect INHr was 92.8% and 85.7%, and specificity (SP) was 100% and 97.5%, for MLPA and Genotype®MTBDRplus, respectively. In relation to RIFr, SE was 87.5% and 100%, and SP was 100% and 98.8%, respectively. Kappa value was identical between Genotype®MTBDRplus and MLPA compared with the standard DST and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)]. Compared to Genotype®MTBDRplus, MLPA showed similar sensitivity to detect INH and RIF resistance. The results obtained by the MLPA and Genotype®MTBDRplus assays indicate that both molecular tests can be used for the rapid detection of drug-resistant TB with high accuracy. MLPA has the added value of providing information on the circulating M. tuberculosis lineages.


Species decline under nitrogen fertilization increases community-level competence of fungal diseases.
The artificial fertilization of soils can alter the structure of natural plant communities and exacerbate pathogen emergence and transmission. Although the direct effects of fertilization on disease resistance in plants have received some research attention, its indirect effects of altered community structure on the severity of fungal disease infection remain largely uninvestigated. We designed manipulation experiments in natural assemblages of Tibetan alpine meadow vegetation along a nitrogen-fertilization gradient over 5 years to compare the relative importance of direct and indirect effects of fertilization on foliar fungal infections at the community level. We found that species with lower proneness to pathogens were more likely to be extirpated following fertilization, such that community-level competence of disease, and thus community pathogen load, increased with the intensity of fertilization. The amount of nitrogen added (direct effect) and community disease competence (indirect effect) provided the most parsimonious combination of parameters explaining the variation in disease severity. Our experiment provides a mechanistic explanation for the dilution effect in fertilized, natural assemblages in a highly specific pathogen-host system, and thus insights into the consequences of human ecosystem modifications on the dynamics of infectious diseases.


Mutations in the palmitoyl-protein thioesterase gene (PPT; CLN1) causing juvenile neuronal ceroid lipofuscinosis with granular osmiophilic deposits.
A subtype of neuronal ceroid lipofuscinosis (NCL) is well recognized which has a clinical course consistent with juvenile NCL (JNCL) but the ultrastructural characteristics of infantile NCL (INCL): granular osmiophilic deposits (GROD). Evidence supporting linkage of this phenotype, designated vJNCL/GROD, to the INCL region of chromosome 1p32 was demonstrated (pairwise lod score with D1S211 , Z max = 2.63, straight theta = 0.00). The INCL gene, palmitoyl-protein thioesterase (PPT ; CLN1), was therefore screened for mutations in 11 vJNCL/GROD families. Five mutations in the PPT gene were identified: three missense mutations, Thr75Pro, Asp79Gly, Leu219Gln, and two nonsense mutations, Leu10STOP and Arg151STOP. The missense mutation Thr75Pro accounted for nine of the 22 disease chromosomes analysed and the nonsense mutation Arg151STOP for seven. Nine out of 11 patients were shown to combine a missense mutation on one disease chromosome with a nonsense mutation on the other. Mutations previously identified in INCL were not observed in vJNCL/GROD families. Thioesterase activity in peripheral blood lymphoblast cells was found to be markedly reduced in vJNCL/GROD patients compared with controls. These results demonstrate that this subtype of JNCL is allelic to INCL and further emphasize the correlation which exists between genetic basis and ultrastructural changes in the NCLs.


Macrophage inflammatory markers are associated with subclinical carotid artery disease in women with human immunodeficiency virus or hepatitis C virus infection.
Infection with hepatitis C virus (HCV) or human immunodeficiency virus (HIV) may be associated with atherosclerosis and vascular disease. Macrophages are a major component of atherosclerotic plaque, and classically activated (M1) macrophages contribute to plaque instability. Our goal was to identify plasma biomarkers that reflect macrophage inflammation and are associated with subclinical atherosclerosis. We tested whether M1 macrophages produce galectin-3-binding protein in vitro. Then, we measured galectin-3-binding protein and the soluble macrophage biomarkers soluble cluster of differentiation (CD) 163 and soluble CD14 in 264 participants in the Women's Interagency HIV Study. Women were positive for HIV, HCV, both, or neither (66 in each group, matched for age, race/ethnicity, and smoking status). Carotid artery disease was assessed by ultrasound measurement of right distal common carotid artery intima-media thickness, distensibility, and presence of atherosclerotic lesions (intima-media thickness >1.5 mm). Plasma galectin-3-binding protein was higher in HCV+ than HCV- women (P<0.01) but did not differ by HIV status. The 3 inflammatory macrophage markers were significantly correlated with each other and negatively correlated with CD4+ counts in HIV-infected women. We defined a macrophage score as 1, 2, or 3 biomarkers elevated above the median. In models adjusted for traditional risk factors, higher macrophage scores were significantly associated with increased atherosclerotic lesions and lower carotid distensibility. Receiver-operator curve analysis of lesions revealed that the markers added predictive value beyond traditional risk factors and C-reactive protein. The macrophage inflammatory markers galectin-3-binding protein, soluble CD163, and soluble CD14 are significantly associated with carotid artery disease in the setting of HIV and HCV infection.


cnvCapSeq: detecting copy number variation in long-range targeted resequencing data.
Targeted resequencing technologies have allowed for efficient and cost-effective detection of genomic variants in specific regions of interest. Although capture sequencing has been primarily used for investigating single nucleotide variants and indels, it has the potential to elucidate a broader spectrum of genetic variation, including copy number variants (CNVs). Various methods exist for detecting CNV in whole-genome and exome sequencing datasets. However, no algorithms have been specifically designed for contiguous target sequencing, despite its increasing importance in clinical and research applications. We have developed cnvCapSeq, a novel method for accurate and sensitive CNV discovery and genotyping in long-range targeted resequencing. cnvCapSeq was benchmarked using a simulated contiguous capture sequencing dataset comprising 21 genomic loci of various lengths. cnvCapSeq was shown to outperform the best existing exome CNV method by a wide margin both in terms of sensitivity (92.0 versus 48.3%) and specificity (99.8 versus 70.5%). We also applied cnvCapSeq to a real capture sequencing cohort comprising a contiguous 358 kb region that contains the Complement Factor H gene cluster. In this dataset, cnvCapSeq identified 41 samples with CNV, including two with duplications, with a genotyping accuracy of 99%, as ascertained by quantitative real-time PCR.


Targeting the oxidative stress response system of fungi with redox-potent chemosensitizing agents.
The cellular antioxidant system is a target in the antifungal action of amphotericin B (AMB) and itraconazole (ITZ), in filamentous fungi. The sakAΔ mutant of Aspergillus fumigatus, a mitogen-activated protein kinase (MAPK) gene deletion mutant in the antioxidant system, was found to be more sensitive to AMB or ITZ than other A. fumigatus strains, a wild type and a mpkCΔ mutant (a MAPK gene deletion mutant in the polyalcohol sugar utilization system). Complete fungal kill (≥99.9%) by ITZ or AMB was also achieved by much lower dosages for the sakAΔ mutant than for the other strains. It appears msnA, an Aspergillus ortholog to Saccharomyces cerevisiaeMSN2 (encoding a stress-responsive C(2)H(2)-type zinc-finger regulator) and sakA and/or mpkC (upstream MAPKs) are in the same stress response network under tert-butyl hydroperoxide (t-BuOOH)-, hydrogen peroxide (H(2)O(2))- or AMB-triggered toxicity. Of note is that ITZ-sensitive yeast pathogens were also sensitive to t-BuOOH, showing a connection between ITZ sensitivity and antioxidant capacity of fungi. Enhanced antifungal activity of AMB or ITZ was achieved when these drugs were co-applied with redox-potent natural compounds, 2,3-dihydroxybenzaldehyde, thymol or salicylaldehyde, as chemosensitizing agents. We concluded that redox-potent compounds, which target the antioxidant system in fungi, possess a chemosensitizing capacity to enhance efficacy of conventional drugs.


Localization of Cacna1s to ON bipolar dendritic tips requires mGluR6-related cascade elements.
L-type voltage gated calcium channels in retina localize primarily at the presynaptic active zones of photoreceptors and bipolar cells where they modulate glutamate release. However, the pore forming subunit Cacna1s of certain L-type channels is also expressed postsynaptically at the tips of ON bipolar cell dendrites where it colocalizes with mGluR6, but has an unknown function. At these dendritic tips, the components of the mGluR6 signaling cascade cluster together in a macromolecular complex, and each one's localization often depends on that of the others. Thus, we explored if Cacna1s is part of the mGluR6 complex. We determined Cacna1s expression by PCR using an ON bipolar library, by Western blotting, and by standard immunohistochemistry. The PCR amplification confirmed expression of the transcript in ON bipolar cells, and Western blotting showed the expected bands. Immunostaining for Cacna1s was stronger in the dendritic tips of rod bipolar cells than in those of ON cone bipolar cells. This staining severely decreased in mice missing various mGluR6 cascade elements (Grm6(-/-), Gnao1(-/-), Gnb3(-/-), Gng13(-/-), and Trpm1(-/-)). During development, the ratio of the number of Cacna1s puncta to the number of presynaptic ribbons followed a sigmoidal pattern, rising rapidly from P13 to P17. The mGluR6 expression preceded that of Cacna1s and RGS11. Our results show that the localization and stability of Cacna1s depend on the expression of mGluR6 and its cascade components, and they suggest that Cacna1s is part of the mGluR6 complex. We hypothesize that Cacna1s contributes to light adaptation by permeating calcium.


Diversity of opines and opine-catabolizing bacteria isolated from naturally occurring crown gall tumors.
The diversity of opines from 43 naturally occurring crown gall tumors on several plant species was analyzed for the presence of agropine, chrysopine, iminodiacid, an unidentified leucinopine-like iminodiacid (IDA-B), mannopine, octopine, nopaline, DL- and LL-succinamopine, leucinopine and heliopine. Opine utilization patterns of agrobacteria and fluorescent pseudomonads resident in a tumor were then analyzed and compared for agreement with the opine isolated from that tumor. Nopaline was the most common opine found and was detected in tumors from cherry, blackberry, grape, and plum. Octopine was not found, although octopine-catabolizing bacteria were isolated from several tumors. A new, previously undescribed iminodiacid of the succinamopine-leucinopine type (provisionally designated IDA-B) was isolated from tumors of wild blackberry. Field tumors from apple, blueberry and grape yielded no detectable opines, even though opine-utilizing bacteria were present. Bacterial isolates from plum and cherry showed the best correspondence between the opine in tumors (nopaline) and the presence of bacteria that catabolized that opine. However, several unusual opine catabolic combinations were identified, including isolates that catabolized a variety of opines but were nonpathogenic. More variability was observed among isolates from field tumors on the remaining plant species. We isolated novel mannopine-nopaline type agrobacteria from field tumors of cherry, plum and blackberry that induced tumors containing either mannopine (plus agropine) or nopaline, but not both. Epidemiologically, the galled plants from an area were not of clonal origin (same Ti plasmid), indicating that the field tumors from a small area were incited by more than one type of Ti plasmid.


Crystal structure of the malaria vaccine candidate apical membrane antigen 1.
Apical membrane antigen 1 from Plasmodium is a leading malaria vaccine candidate. The protein is essential for host-cell invasion, but its molecular function is unknown. The crystal structure of the three domains comprising the ectoplasmic region of the antigen from P. vivax, solved at 1.8 angstrom resolution, shows that domains I and II belong to the PAN motif, which defines a superfamily of protein folds implicated in receptor binding. We also mapped the epitope of an invasion-inhibitory monoclonal antibody specific for the P. falciparum ortholog and modeled this to the structure. The location of the epitope and current knowledge on structure-function correlations for PAN domains together suggest a receptor-binding role during invasion in which domain II plays a critical part. These results are likely to aid vaccine and drug design.


Odd-even effect in two dimensions induced by the bicomponent blends of isobutenyl compounds.
Scanning tunneling microscopy (STM) investigation was performed for the blends of isobutenyl compounds, in which the long alkyl chains were connected with ester or carbamoyl linkages. Each component by itself did not show the odd-even effect of alkyl chain length, whereas after blending them, the 2D structures drastically changed and modulated to exhibit odd-even effect. Star, lozenge, twist-like, and linear structures were found, dependent on the blend ratio and alkyl chain length. The blend ratio dependence of 2D structures was explained in terms of homogeneous and heterogeneous dimerization due to the interdigitation of alkyl chains.


PD-1 Expression Status on CD8+ Tumour Infiltrating Lymphocytes Associates With Survival in Cervical Cancer.
Despite the expansion of PD-1 checkpoint blockade to multiple types of cancer, whether the programmed cell death 1 (PD-1) expression status on CD8+ tumour infiltrating lymphocytes (TILs) could be a prognostic factor in cervical cancer is still unclear. In this study, we performed ex vivo phenotypic analysis of PD-1 expression on CD8+ TILs by flow cytometry from 47 treatment-naïve cervical cancer patients. With a median follow-up of 26.1 months (95% confidence interval [CI], 24-28.2 months), we then linked the quantitative cellular expression results to progression-free survival and overall survival. Based on the intensity of PD-1 expression, we further categorised the cervical cancer patients into PD-1high expressers (29.8%, 14/47) and PD-1low expressers (70.2%, 33/47). Multivariate analysis revealed that PD-1high expressers are correlated with early recurrence (HR, 5.91; 95% CI, 1.03-33.82; P= 0.046). Univariate analysis also demonstrated that PD-1high expressers are associated with poor overall survival in cervical cancer (HR, 5.365; 95% CI, 1.55-18.6; P=0.008). Moreover, our study also demonstrated that CD8+/CD4+ TIL ratio and HPV infection status are risk factors for early relapse and mortality in cervical cancer patients. In conclusion, this study confirms that PD-1 expression status is an independent prognostic factor for progression free survival in cervical cancer. These findings could be important in predicting the relapse of cervical cancer as a cellular diagnosis method and could be important knowledge for the selection of prospective PD-1 blockade candidates.