Blocking the Nav1.5 channel using eicosapentaenoic acid reduces migration and proliferation of ovarian cancer cells.
Activity of the voltage-gated Nav1.5 sodium channel has been reported to be involved in cell proliferation, cancer invasion and gene expression. In addition, eicosapentaenoic acid (EPA) has recently been suggested to inhibit ovarian cancer cell growth and suppress tumor metastasis. The present study aimed to explore the association between EPA, the Nav1.5 sodium channel and ovarian cancer cells. Using patch-clamp technique and RNA interference approaches, sodium currents were recorded in epithelial ovarian cancer cells, and it was confirmed that the Nav1.5 channel carried the sodium currents. Furthermore, EPA effectively inhibited sodium currents in a dose-dependent manner, shifted the steady-state inactivation curve of sodium currents to the hyperpolarizing direction and reduced sodium window currents. In addition, EPA induced a shift in the inactivation curve in a dose-dependent manner. Inhibition of the sodium channel, either by EPA or by Nav1.5 knockdown, attenuated ovarian cancer cell migration and proliferation. To the best of our knowledge, the present study is the first to conduct sodium current recording in ovarian cancer cells, and revealed that EPA may inhibit Nav1.5-mediated ovarian cancer cell migration and growth. These findings not only present a potential prognostic biomarker for ovarian cancer, but also provide a strategy towards the development of novel pharmacological treatments for patients with ovarian cancer.


Tomato below ground-above ground interactions: Trichoderma longibrachiatum affects the performance of Macrosiphum euphorbiae and its natural antagonists.
Below ground and above ground plant-insect-microorganism interactions are complex and regulate most of the developmental responses of important crop plants such as tomato. We investigated the influence of root colonization by a nonmycorrhizal plant-growth-promoting fungus on direct and indirect defenses of tomato plant against aphids. The multitrophic system included the plant Solanum lycopersicum ('San Marzano nano'), the root-associated biocontrol fungus Trichoderma longibrachiatum strain MK1, the aphid Macrosiphum euphorbiae (a tomato pest), the aphid parasitoid Aphidius ervi, and the aphid predator Macrolophus pygmaeus. Laboratory bioassays were performed to assess the effect of T. longibrachiatum MK1, interacting with the tomato plant, on quantity and quality of volatile organic compounds (VOC) released by tomato plant, aphid development and reproduction, parasitoid behavior, and predator behavior and development. When compared with the uncolonized controls, plants whose roots were colonized by T. longibrachiatum MK1 showed quantitative differences in the release of specific VOC, better aphid population growth indices, a higher attractiveness toward the aphid parasitoid and the aphid predator, and a quicker development of aphid predator. These findings support the development of novel strategies of integrated control of aphid pests. The species-specific or strain-specific characteristics of these below ground-above ground interactions remain to be assessed.


Diagnosis and management of type I and type V hyperlipoproteinemia.
Both type I and type V hyperlipoproteinemia are characterized by severe hypertriglyceridemia due to an increase in chylomicrons. Type I hyperlipoproteinemia is caused by a decisive abnormality of the lipoprotein lipase (LPL)- apolipoprotein C-II system, whereas the cause of type V hyperlipoproteinemia is more complicated and more closely related to acquired environmental factors. Since the relationship of hypertriglyceridemia with atherosclerosis is not as clear as that of hypercholesterolemia, and since type I and V hyperlipoproteinemia are relatively rare, few guidelines for their diagnosis and treatment have been established; however, type I and V hyperlipoproteinemia are clinically important as underlying disorders of acute pancreatitis, and appropriate management is necessary to prevent or treat such complications. Against such a background, here we propose guidelines primarily concerning the diagnosis and management of type I and V hyperlipoproteinemia in Japanese.


Three Novel Spectrin Variants in Jaundiced Neonates.
Various mutations in the genes encoding alpha spectrin (SPTA1) or beta spectrin (SPTB) are known to cause erythrocyte membrane disorders, sometimes associated with severe neonatal jaundice and anemia. We used a next-generation sequencing panel to evaluate 3 unrelated neonates who had puzzling cases of nonimmune hemolytic jaundice. In each case, we identified novel mutations in either SPTA1 or SPTB. Correlating erythrocyte morphology, clinical course, and computational analysis, we submit that each of the 3 variants is a probable pathogenic cause of the hereditary hemolytic conditions in these patients. We hope other pediatric practitioners caring for neonates with what appears to be idiopathic severe neonatal hyperbilirubinemia will look for spectrin variants as a possible cause, because additional cases with these specific variants along with this clinical phenotype are needed to confirm our postulate that these 3 cases are indeed pathogenic mutations.


Association of the variant Cys139Arg at GRN gene to the clinical spectrum of frontotemporal lobar degeneration.
Progranulin protein (PGRN) is a cysteine-rich growth factor encoded by the progranulin gene (GRN). PGRN mutations were identified in patients with frontotemporal lobar degeneration (FTLD) and recently its role as risk factor has been described in patients with probable Alzheimer's disease (AD). To date, more than 100 genetic variants in GRN gene have been described and the pathogenic nature is still unclear for almost 36% of them. Here, we describe three clinical cases carrying the PGRN variation Cys139Arg in order to increase the knowledge on the association of this variant to the clinical spectrum of FTLD. The genetic analysis was performed using high resolution melting analysis. The Human Progranulin ELISA Kit was used in order to determine PGRN expression levels in the plasma samples. The three patients carrying the genetic variation showed three final different clinical diagnosis, respectively behavioral frontotemporal dementia, semantic dementia, and corticobasal syndrome, thus underlining the clinical heterogeneity typically associated with GRN mutations. All cases shared similar plasma PGRN levels that resulted intermediate between those measured in controls and in GRN null mutation carriers, showing a partial reduction of the protein in plasma. Moreover, according to the bioinformatics software, the Cys139Arg variation causes a decreased stability of the structure of the protein. We describe three new patients affected by neurological syndromes included in the clinical spectrum of FTLD carrying the Cys139Arg genetic variant, thus suggesting a possible implication in the pathogenesis of FTLD.


p53 mutations in colorectal cancer.
Immunohistological staining of primary colorectal carcinomas with antibodies specific to p53 demonstrated gross overexpression of the protein in approximately 50% of the malignant tumors examined. Benign adenomas were all negative for p53 overexpression. To determine the molecular basis for this overexpression we examined p53 protein expression in 10 colorectal cancer cell lines. Six of the cell lines expressed high levels of p53 in ELISA, cell-staining, and immunoprecipitation studies. Direct sequencing and chemical-mismatch-cleavage analysis of p53 cDNA by using the polymerase chain reaction in these cell lines showed that all cell lines that expressed high levels of p53 were synthesizing mRNAs that encoded mutant p53 proteins. In two of those four cell lines where p53 expression was lower, point mutations were still detected. Thus, we conclude that overexpression of p53 is synonymous with mutation, but some mutations would not be detected by a simple immunohistochemical analysis. Mutation of the p53 gene is one of the commonest genetic changes in the development of human colorectal cancer.


Preparation and in vivo evaluation of a gel-based nasal delivery system for risperidone.
The aim of this study was to prepare a nasal gel of risperidone and to investigate the pharmacokinetics and relative bioavailability of the drug in rats. Compared with oral dosing, the risperidone nasal gel exhibited very fast absorption and high bioavailability. Maximal plasma concentration (cmax) and the time to reach cmax (tmax) were 15.2 μg mL-1 and 5 min for the nasal gel, 3.6 μg mL-1 and 30 min for the oral drug suspension, respectively. Pharmacokinetic parameters such as tmax', cmax and AUC of oral and nasal routes were significantly different (p < 0.01). Relative bioavailability of the drug nasal preparation to the oral suspension was up to 1600.0 %. Further, the in vitro effect of the risperidone nasal gel on nasal mucociliary movement was also investigated using a toad palate model. The risperidone nasal formulation showed mild ciliotoxicity, but the adverse effect was temporary and reversible.


Human fecal water content of phenolics: the extent of colonic exposure to aromatic compounds.
Phenolic compounds are not completely absorbed in the small intestine and so enter the colon, where they might exert physiological effects. To identify phenolics that are present in normal human colon, fecal water was prepared from 5 free-living volunteers with no dietary restrictions and analyzed by gas chromatography-mass spectrometry. Daily measurements were also performed on a single individual to examine the variation more closely. Levels of polyphenols were variable between individuals. Naringenin and quercetin had mean concentrations of 1.20 and 0.63 microM. All other flavonoids examined were present < or =0.17 microM. Simple phenolic and other aromatic acids were present at much higher concentrations. The major components were phenylacetic acid, 479 microM; 3-phenylpropionic acid, 166 microM; 3-(4-hydroxy)-phenylpropionic acid, 68 microM; 3,4-dihydroxycinnamic acid, 52 microM; benzoic acid, 51 microM; 3-hydroxyphenylacetic acid, 46 microM; and 4-hydroxyphenylacetic acid, 19 microM. Other phenolic acids ranged from 0.04 to 7 microM. Decreased dietary phenolic intake caused a decrease in polyphenol and monophenolic acid concentration in fecal water 24 h later. This study is the first to measure the range of aromatic compounds in human fecal water and demonstrates that phenolic acid concentrations are high. The biological effects of phenolics may play an important role in colon function.


Stereoacuity in children with anisometropic amblyopia.
To determine factors associated with pretreatment and posttreatment stereoacuity in subjects with moderate anisometropic amblyopia. Data for subjects enrolled in seven studies conducted by the Pediatric Eye Disease Investigator Group were pooled. The sample included 633 subjects aged 3 to <18 years with anisometropic amblyopia, no heterotropia observed by cover test, and baseline amblyopic eye acuity of 20/100 or better. A subset included 248 subjects who were treated with patching or Bangerter filters and had stereoacuity testing at both the baseline and outcome examinations. Multivariate regression models identified factors associated with baseline stereoacuity and with outcome stereoacuity as measured by the Randot Preschool Stereoacuity test. Better baseline stereoacuity was associated with better baseline amblyopic eye acuity (P < 0.001), less anisometropia (P = 0.03), and anisometropia due to astigmatism alone (P < 0.001). Better outcome stereoacuity was associated with better baseline stereoacuity (P < 0.001) and better amblyopic eye acuity at outcome (P < 0.001). Among 48 subjects whose amblyopic eye visual acuity at outcome was 20/25 or better and within one line of the fellow eye, stereoacuity was worse than that of children with normal vision of the same age. In children with anisometropic amblyopia of 20/40 to 20/100 inclusive, better posttreatment stereoacuity is associated with better baseline stereoacuity and better posttreatment amblyopic eye acuity. Even if their visual acuity deficit resolves, many children with anisometropic amblyopia have stereoacuity worse than that of nonamblyopic children of the same age.


Skin Conductance Level Reactivity Moderates the Association Between Parental Psychological Control and Relational Aggression in Emerging Adulthood.
When studying factors that may heighten risk for relational aggression in youth, it is important to consider characteristics of both the individual and their environment. This research examined the associations between parental psychological control and reactive and proactive relational aggression in emerging adults in college. Given that sympathetic nervous system (SNS) activation may underlie differences between reactive and proactive aggression and has been shown to moderate the effects of parenting on youth development, the moderating role of SNS reactivity [indexed by skin conductance level reactivity (SCLR)] was also examined. Emerging adults (N = 180; 77.2 % female) self-reported on perceptions of parental psychological control and reactive and proactive relational aggression. SCLR was assessed in response to an interpersonal laboratory challenge task. Parental psychological control was positively associated with reactive relational aggression only for emerging adults who exhibited high SCLR. Parental psychological control was positively associated with proactive relational aggression only among emerging adults who showed low SCLR. This study extends previous research on parenting and aggression and suggests that parental psychological control is differentially associated with reactive versus proactive relational aggression, depending on emerging adults' SCLR to interpersonal stress.


The flipped classroom stimulates greater learning and is a modern 21st century approach to teaching today's undergraduates.
Many classrooms in higher education still rely on a transformative approach to teaching where students attend lectures and earn course grades through examination. In the modern age, traditional lectures are argued by some as obsolete and do not address the learning needs of today's students. An emerging pedagogical approach is the concept of the flipped classroom. The flipped classroom can simply be described as students viewing asynchronous video lectures on their own and then engaging in active learning during scheduled class times. In this study, we examined the flipped classroom teaching environment on student learning gains in an Introduction to Equine Science course. Students (n = 130) were asked to view 7.5 h of recorded lectures divided into 8 learning modules, take online quizzes to enforce lecture viewing, take 3 in-class exams, and prepare to participate in active learning during scheduled class times. Active learning approaches included individual activities, paired activities, informal small groups, and large group activities. When compared to students in the traditional lecture format in earlier years, students in the flipped format scored higher on all 3 exams (P < 0.05), with both formats taught by the same instructor. Analysis of ACT scores demonstrated no intellectual capacity differences between the student populations. To evaluate any gains in critical thinking, flipped format students were asked to take the Cornell Critical Thinking Exam (version X). Scores improved from the pretest (50.8 ± 0.57) to the posttest (54.4 ± 0.58; P < 0.01). In the flipped course, no correlations were found with student performance and interactions with online content. Students were asked in class to evaluate their experiences based on a 5-point Likert scale: 1 (strongly disagree) to 5 (strongly agree). The flipped classroom was ranked as an enjoyable learning experience with a mean of 4.4 ± 0.10, while students responded positively to other pointed questions. In formal course evaluations, flipped format students ranked the following higher (P < 0.05): instructor availability to assist students; encouragement of independent, creative, and critical thinking; and amount learned. Overall, the flipped classroom proved to be a positive learning experience for students. As the classroom continues to modernize, pedagogical approaches such as the flipped classroom should be considered for many lecture-style courses taught in the animal sciences.


Combination therapy using evening primrose oil and electrical stimulation to improve nerve function following a crush injury of sciatic nerve in male rats.
Peripheral nerve injuries with a poor prognosis are common. Evening primrose oil (EPO) has beneficial biological effects and immunomodulatory properties. Since electrical activity plays a major role in neural regeneration, the present study investigated the effects of electrical stimulation (ES), combined with evening primrose oil (EPO), on sciatic nerve function after a crush injury in rats. In anesthetized rats, the sciatic nerve was crushed using small haemostatic forceps followed by ES and/or EPO treatment for 4 weeks. Functional recovery of the sciatic nerve was assessed using the sciatic functional index. Histopathological changes of gastrocnemius muscle atrophy were investigated by light microscopy. Electrophysiological changes were assessed by the nerve conduction velocity of sciatic nerves. Immunohistochemistry was used to determine the remyelination of the sciatic nerve following the interventions. EPO + ES, EPO, and ES obviously improved sciatic nerve function assessed by the sciatic functional index and nerve conduction velocity of the sciatic nerve at 28 days after operation. Expression of the peripheral nerve remyelination marker, protein zero (P0), was increased in the treatment groups at 28 days after operation. Muscle atrophy severity was decreased significantly while the nerve conduction velocity was increased significantly in rats with sciatic nerve injury in the injury + EPO + ES group than in the EPO or ES group. Totally speaking, the combined use of EPO and ES may produce an improving effect on the function of sciatic nerves injured by a crush. The increased expression of P0 may have contributed to improving the functional effects of combination therapy with EPO and ES as well as the electrophysiological and histopathological features of the injured peripheral nerve.


Tris is a non-innocent buffer during intein-mediated protein cleavage.
Fusion protein purification systems based on self-cleavable protein splicing elements are well established nowadays and have the advantage of producing recombinant proteins with their native amino acid composition while abolishing the need of an additional proteolytic cleavage step for removal of a purification tag. However, a potential disadvantage is the concomitant generation of reactive thioester intermediates during the protein self-splicing process, which are prone to undergo side reactions yielding undesired adducts. We followed the formation of these adducts as well as ways to avoid them with electrospray ionization mass spectrometry using one of our target proteins, Triticum aestivum (wheat) E(c)-1, a plant metallothionein with the ability to bind a total of six zinc or cadmium ions in the form of metal-thiolate clusters. Our investigations show that one of the most commonly used buffer substances, tris(hydroxymethyl)aminomethane (Tris), has to be applied with caution in combination with the described purification system, as it can itself react with the thioester intermediate forming a yet unreported stable adduct. This makes Tris a so called non-innocent buffer during the protein isolation procedure. Additionally, the results presented open up an interesting possibility to directly couple the one-step purification strategy with selective carboxy-terminal protein or peptide modification, e.g. the addition of fluorophors or PEGylation of peptides. Unrelated to the purification system used, we further observed a high amount of N-formylmethionine in the mass spectra when the protein of interest was expressed in cadmium-supplemented growth media.


Is a single or double arm technique more advantageous in triple jumping?
Triple jumpers employ either an asymmetrical 'single-arm' action or symmetrical 'double-arm' action in the takeoff of each phase of the jump. This study investigated which technique is more beneficial in each phase using computer simulation. Kinematic data were obtained from an entire triple jump using a Vicon automatic motion capture system. A planar 13-segment torque-driven subject-specific computer simulation model was evaluated by varying torque generator activation timings using a genetic algorithm in order to match performance data. The matching produced a close agreement between simulation and performance, with differences of 3.8%, 2.7%, and 3.1% for the hop, step, and jump phases, respectively. Each phase was optimised for jump distance and an increase in jump distance beyond the matched simulations of 3.3%, 11.1%, and 8.2% was obtained for the hop, step, and jump, respectively. The optimised technique used symmetrical shoulder flexion whereas the triple jumper had used an asymmetrical arm technique. This arm action put the leg extensors into slower concentric conditions allowing greater extensor torques to be produced. The main increases in work came at the joints of the stance leg but the largest increases in angular impulse came at the shoulder joints, indicating the importance of both measures when assessing the impact of individual joint actions on changes in technique. Possible benefits of the double-arm technique include: cushioning the stance leg during impact; raising the centre of mass of the body at takeoff; facilitating an increase in kinetic energy at takeoff; allowing a re-orientation of the body during flight.


Pancreas adenocarcinoma: novel therapeutics.
Pancreatic ductal adenocarcinoma (PDAC) is the third highest cause of cancer-related deaths in the US, and is projected to be second only to non-small cell lung cancer (NSCLC) by the 2020s. Current therapies have a modest impact on survival and median overall survival (mOS) across all stages of disease remains under a year. Over the last decade, however, great strides have been made in the understanding of PDAC pathobiology including the role of the tumor microenvironment (TME), DNA damage repair and mechanism of immunosuppression. Exciting novel therapeutics are in clinical development targeting the TME to increase cytotoxic drug delivery, decrease immunosuppressive cell presence and attack cancer stem cells (CSCs). Immune checkpoint inhibitors, cancer vaccines and other immunotherapies are actively being studied and novel combinations of targeted agents are being pursued. There is a sense of optimism in the oncology community that these scientific advances will translate into improved outcomes for patients with PDAC in the proximate future. In this review, we examine various novel therapeutics under clinical development with a focus on stromal disrupting agents, immunotherapeutics and DNA damage repair strategies.


The effect of Nigella sativa oil against experimental allergic encephalomyelitis via nitric oxide and other oxidative stress parameters.
Reactive oxygen species (ROS) including nitric oxide (NO) are thought to be involved in inflammatory processes, exacerbating inflammation and tissue damage in multiple sclerosis (MS). The oil extracts of Nigella sativa (N. sativa) has been known as an antioxidant and antiinflammatory agent. The aim of the present study was to investigate the hypothesis that N. sativa components provide protection against oxidative stress induced by experimental autoimmune encephalomyelitis (EAE) in rats. For this purpose, EAE was induced in rats by using guinea pig myelin basic protein (MBP) in Freud's adjuvant with addition of heat-killed M. Tuberculosis H37Ra to test this hypothesis. In study groups, N. sativa was given by oral gavage to the rats. Treatment of the rats with N. sativa inhibited ROS production induced by EAE showing diminished levels of MDA of both brain and medulla spinalis tissues. Although there was a significant decrease in brain NO level, there was an increase in medulla spinalis NO level after EAE induction in rats. N. sativa regulated tissue NO levels in some extend when applied together with EAE. When N. sativa was given alone to the rats, no changes were shown in brain, medulla spinalis, and serum oxidant/antioxidant parameters. In conclusion, N sativa may protect brain and medulla spinalis tissues against oxidative stress induced by EAE. In addition, N. sativa display its antioxidant and regulatory effects via inflammatory cells rather than the host tissue (brain and medulla spinalis) for EAE in rats.


Characterization of the putative type III secretion ATPase CdsN (Cpn0707) of Chlamydophila pneumoniae.
Type III secretion (T3S) is utilized by a wide range of gram-negative bacterial pathogens to allow the efficient delivery of effector proteins into the host cell cytoplasm through the use of a syringe-like injectisome. Chlamydophila pneumoniae is a gram-negative, obligate intracellular pathogen that has the structural genes coding for a T3S system, but the functionality of the system has not yet been demonstrated. T3S is dependent on ATPase activity, which catalyzes the unfolding of proteins and the secretion of effector proteins through the injectisome. CdsN (Cpn0707) is predicted to be the T3S ATPase of C. pneumoniae based on sequence similarity to other T3S ATPases. Full-length CdsN and a C-terminal truncation of CdsN were cloned as glutathione S-transferase (GST)-tagged constructs and expressed in Escherichia coli. The GST-tagged C-terminal truncation of CdsN possessed ATPase activity, catalyzing the release of ADP and P(i) from ATP at a rate of 0.55 +/- 0.07 micromol min(-1) mg(-1) in a time- and dose-dependent manner. CdsN formed oligomers and high-molecular-weight multimers, as assessed by formaldehyde fixation and nondenaturing polyacrylamide gel electrophoresis. Using bacterial two-hybrid and GST pull-down assays, CdsN was shown to interact with CdsD, CdsL, CdsQ, and CopN, four putative structural components of the C. pneumoniae T3S system. CdsN also interacted with an unannotated protein, Cpn0706, a putative CdsN chaperone. Interactions between CdsN, CdsD, and CopN represent novel interactions not previously reported for other bacterial T3S systems and may be important in the localization and/or function of the ATPase at the inner membrane of C. pneumoniae.


International Federation of Clinical Chemistry (IFCC), Education Division. Guidelines (1988) for training in clinical laboratory management.
Trainees in laboratory medicine must develop skills in laboratory management. Guidelines are detailed for laboratory staff in training, directors responsible for staff development and professional bodies wishing to generate material appropriate to their needs. The syllabus delineates the knowledge base required and includes laboratory planning and organisation, control of operations, methodology and instrumentation, data management and statistics, financial management, clinical use of tests, communication, personnel management and training, and research and development. Methods for achievement of the skills required are suggested. A bibliography of IFCC publications and other material is provided to assist in training in laboratory management.


Alanine scan of the opioid peptide dynorphin B amide.
To date structure-activity relationship (SAR) studies of the dynorphins (Dyn), endogenous peptides for kappa opioid receptors (KOR), have focused almost exclusively on Dyn A with minimal studies on Dyn B. While both Dyn A and Dyn B have identical N-terminal sequences, their C-terminal sequences differ, which could result in differences in pharmacological activity. We performed an alanine scan of the non-glycine residues up through residue 11 of Dyn B amide to explore the roles of these side chains in the activity of Dyn B. The analogs were synthesized by fluorenylmethyloxycarbonyl (Fmoc)-based solid phase peptide synthesis and evaluated for their opioid receptor affinities and opioid potency and efficacy at KOR. Similar to Dyn A the N-terminal Tyr1 and Phe4 residues of Dyn B amide are critical for opioid receptor affinity and KOR agonist potency. The basic residues Arg6 and Arg7 contribute to the KOR affinity and agonist potency of Dyn B amide, while Lys10 contributes to the opioid receptor affinity, but not KOR agonist potency, of this peptide. Comparison to the Ala analogs of Dyn A (1-13) suggests that the basic residues in the C-terminus of both peptides contribute to KOR binding, but differences in their relative positions may contribute to the different pharmacological profiles of Dyn A and Dyn B. The other unique C-terminal residues in Dyn B amide also appear to influence the relative affinity of this peptide for KOR versus mu and delta opioid receptors. This SAR information may be applied in the design of new Dyn B analogs that could be useful pharmacological tools.


HMG-CoA reductase inhibitor improves endothelial dysfunction in spontaneous hypertensive rats via down-regulation of caveolin-1 and activation of endothelial nitric oxide synthase.
Hypertension is associated with endothelial dysfunction and increased cardiovascular risk. Caveolin-1 regulates nitric oxide (NO) signaling by modulating endothelial nitric oxide synthase (eNOS). The purpose of this study was to examine whether HMG-CoA reductase inhibitor improves impaired endothelial function of the aorta in spontaneous hypertensive rat (SHR) and to determine the underlying mechanisms involved. Eight-week-old male SHR were assigned to either a control group (CON, n=11) or a rosuvastatin group (ROS, n=12), rosuvastatin (10 mg/kg/day) administered for eight weeks. Abdominal aortic rings were prepared and responses to acetylcholine (10(-9)-10(-4) M) were determined in vitro. To evaluate the potential role of NO and caveolin-1, we examined the plasma activity of NOx, eNOS, phosphorylated-eNOS and expression of caveolin-1. The relaxation in response to acetylcholine was significantly enhanced in ROS compared to CON. Expression of eNOS RNA was unchanged, whereas NOx level and phosphorylated-eNOS at serine-1177 was increased accompanied with depressed level of caveolin-1 in ROS. We conclude that 3-Hydroxy-3-methylglutaryl Coenzyme-A (HMG-CoA) reductase inhibitor can improve impaired endothelial dysfunction in SHR, and its underlying mechanisms are associated with increased NO production. Furthermore, HMG-CoA reductase inhibitor can activate the eNOS by phosphorylation related to decreased caveolin-1 abundance. These results imply the therapeutic strategies for the high blood pressure-associated endothelial dysfunction through modifying caveolin status.


Clinical significance of CTNNB1 mutation and Wnt pathway activation in endometrioid endometrial carcinoma.
Endometrioid endometrial carcinoma (EEC) is the most common form of endometrial carcinoma. The heterogeneous clinical course of EEC is an obstacle to individualized patient care. We performed an integrated analysis on the multiple-dimensional data types including whole-exome and RNA sequencing, RPPA profiling, and clinical data from 271 EEC cases in The Cancer Genome Atlas (TCGA) to identify molecular fingerprints that may account for this clinical heterogeneity. Significance analysis of microarray was used to identify marker genes of each subtype that were subject to pathway analysis. Association of molecular subtypes with clinical features and mutation data was analyzed with the Mann Whitney, Chi-square, Fisher's exact, and Kruskal-Wallis tests. Survival analysis was evaluated with log-rank test. All statistical tests were two-sided. Four transcriptome subtypes with distinct clinicopathologic characteristics and mutation spectra were identified from the TCGA dataset and validated in an independent sample cohort of 184 EEC cases. Cluster II consisted of younger, obese patients with low-grade EEC but diminished survival. CTNNB1 exon 3 mutations were present in 87.0% (47/54) of Cluster II (P < .001) that exhibited a low overall mutation rate; this was statistically significantly associated with Wnt/β-catenin signaling activation (P < .001). High expression levels of CTNNB1 (P = .001), MYC (P = .01), and CCND1 (P = .01) were associated with poorer overall survival in low-grade EEC tumors. CTNNB1 exon 3 mutations are likely a driver that characterize an aggressive subset of low-grade and low-stage EEC occurring in younger women.


Thermochemical kinetics for multireference systems: addition reactions of ozone.
The 1,3-dipolar cycloadditions of ozone to ethyne and ethene provide extreme examples of multireference singlet-state chemistry, and they are examined here to test the applicability of several approaches to thermochemical kinetics of systems with large static correlation. Four different multireference diagnostics are applied to measure the multireference characters of the reactants, products, and transition states; all diagnostics indicate significant multireference character in the reactant portion of the potential energy surfaces. We make a more complete estimation of the effect of quadruple excitations than was previously available, and we use this with CCSDT/CBS estimation of Wheeler et al. (Wheeler, S. E.; Ess, D. H.; Houk, K. N. J. Phys. Chem. A 2008, 112, 1798.) to make new best estimates of the van der Waals association energy, the barrier height, and the reaction energy to form the cycloadduct for both reactions. Comparing with these best estimates, we present comprehensive mean unsigned errors for a variety of coupled cluster, multilevel, and density functional methods. Several computational aspects of multireference reactions are considered: (i) the applicability of multilevel theory, (ii) the convergence of coupled cluster theory for reaction barrier heights, (iii) the applicability of completely renormalized coupled cluster methods to multireference systems, (iv) the treatment by density functional theory, (v) the multireference perturbation theory for multireference reactions, and (vi) the relative accuracy of scaling-type multilevel methods as compared with additive ones. It is found that scaling-type multilevel methods do not perform better than the additive-type multilevel methods. Among the 48 tested density functionals, only M05 reproduces the best estimates within their uncertainty. Multireference perturbation theory based on the complete-active-space reference wave functions constructed using a small number of reaction-specific active orbitals gives accurate forward barrier heights; however, it significantly underestimates reaction energies.


Checkpoint kinase Chk2 controls renal Cyp27b1 expression, calcitriol formation, and calcium-phosphate metabolism.
Checkpoint kinase 2 (Chk2) is the main effector kinase of ataxia telangiectasia mutated (ATM) and responsible for cell cycle regulation. ATM signaling has been shown to upregulate interferon-regulating factor-1 (IRF-1), a transcription factor also expressed in the kidney. Calcitriol (1,25 (OH)2D3), a major regulator of mineral metabolism, is generated by 25-hydroxyvitamin D 1α-hydroxylase in the kidney. Since 25-hydroxyvitamin D 1α-hydroxylase expression is enhanced by IRF-1, the present study explored the role of Chk2 for calcitriol formation and mineral metabolism. Chk2-deficient mice (chk2 (-/-)) were compared to wild-type mice (chk2 (+/+)). Transcript levels of renal 25-hydroxyvitamin D 1α-hydroxylase, Chk2, and IRF-1 were determined by RT-PCR; Klotho expression by Western blotting; bone density by μCT analysis; serum or plasma 1,25 (OH)2D3, PTH, and C-terminal FGF23 concentrations by immunoassays; and serum, fecal, and urinary calcium and phosphate concentrations by photometry. The renal expression of IRF-1 and 25-hydroxyvitamin D 1α-hydroxylase as well as serum 1,25 (OH)2D3 and FGF23 levels were significantly lower in chk2 (-/-) mice compared to chk2 (+/+) mice. Plasma PTH was not different between the genotypes. Renal calcium and phosphate excretion were significantly higher in chk2 (-/-) mice than in chk2 (+/+) mice despite hypophosphatemia and normocalcemia. Bone density was not different between the genotypes. We conclude that Chk2 regulates renal 25-hydroxyvitamin D 1α-hydroxylase expression thereby impacting on calcium and phosphate metabolism.


Risk Management in Stalking Victims: A Multi-Agency Approach to Victim Advocacy.
A pilot Multi-Agency Stalking Intervention Programme (MASIP), introduced in three police forces in England, provided among a range of interventions, the delivery of safety planning advice, and needs-based support for stalking victims through a bespoke advocacy service. The ultimate aim of MASIP was to equip victims with tools to manage the variety of harms caused by stalking, as well as enable them to access the criminal justice system with adequate support. This study explores the personal needs of stalking victims from the perspectives of stalking victims, advocates and stakeholders involved in the intervention program, as part of a larger evaluation study conducted by the authors. Semi-structured interviews were conducted with a total of 10 stalking victims who participated in the MASIP, three advocates who directly interacted with the victims, and 19 MASIP stakeholders involved in the project. Findings revealed that overall, victims believed the advocacy service aided their ability to cope with the realities of stalking. Having a victim advocate as single point of contact made victims' journey through the justice system easier to navigate, provided them with the emotional support that they required to deal with the harms of stalking and the practical advice offered regarding their personal safety, and allowed them to feel in control of their own risk management. Advocates reported that the multi-agency context helped in risk assessment and ability to design and deliver bespoke support plans, which uniquely improved victims' engagement with the service. Due to the small size and possibly biased sample, our conclusions must be interpreted with caution.


A three-dimensional in vitro tumor platform for modeling therapeutic irreversible electroporation.
Irreversible electroporation (IRE) is emerging as a powerful tool for tumor ablation that utilizes pulsed electric fields to destabilize the plasma membrane of cancer cells past the point of recovery. The ablated region is dictated primarily by the electric field distribution in the tissue, which forms the basis of current treatment planning algorithms. To generate data for refinement of these algorithms, there is a need to develop a physiologically accurate and reproducible platform on which to study IRE in vitro. Here, IRE was performed on a 3D in vitro tumor model consisting of cancer cells cultured within dense collagen I hydrogels, which have been shown to acquire phenotypes and respond to therapeutic stimuli in a manner analogous to that observed in in vivo pathological systems. Electrical and thermal fluctuations were monitored during treatment, and this information was incorporated into a numerical model for predicting the electric field distribution in the tumors. When correlated with Live/Dead staining of the tumors, an electric field threshold for cell death (500 V/cm) comparable to values reported in vivo was generated. In addition, submillimeter resolution was observed at the boundary between the treated and untreated regions, which is characteristic of in vivo IRE. Overall, these results illustrate the advantages of using 3D cancer cell culture models to improve IRE-treatment planning and facilitate widespread clinical use of the technology.


Single-cell RNA sequencing reveals gene expression signatures of breast cancer-associated endothelial cells.
Tumor endothelial cells (TEC) play an indispensible role in tumor growth and metastasis although much of the detailed mechanism still remains elusive. In this study we characterized and compared the global gene expression profiles of TECs and control ECs isolated from human breast cancerous tissues and reduction mammoplasty tissues respectively by single cell RNA sequencing (scRNA-seq). Based on the qualified scRNA-seq libraries that we made, we found that 1302 genes were differentially expressed between these two EC phenotypes. Both principal component analysis (PCA) and heat map-based hierarchical clustering separated the cancerous versus control ECs as two distinctive clusters, and MetaCore disease biomarker analysis indicated that these differentially expressed genes are highly correlated with breast neoplasm diseases. Gene Set Enrichment Analysis software (GSEA) enriched these genes to extracellular matrix (ECM) signal pathways and highlighted 127 ECM-associated genes. External validation verified some of these ECM-associated genes are not only generally overexpressed in various cancer tissues but also specifically overexpressed in colorectal cancer ECs and lymphoma ECs. In conclusion, our data demonstrated that ECM-associated genes play pivotal roles in breast cancer EC biology and some of them could serve as potential TEC biomarkers for various cancers.


Intracellular Cl- fluxes play a novel role in Ca2+ handling in airway smooth muscle.
Intracellular Ca(2+) is actively sequestered into the sarcoplasmic reticulum (SR), whereas the release of Ca(2+) from the SR can be triggered by activation of the inositol 1,4,5-trisphosphate and ryanodine receptors. Uptake and release of Ca(2+) across the SR membrane are electrogenic processes; accumulation of positive or negative charge across the SR membrane could electrostatically hinder the movement of Ca(2+) into or out of the SR, respectively. We hypothesized that the movement of intracellular Cl(-) (Cl(i)(-)) across the SR membrane neutralizes the accumulation of charge that accompanies uptake and release of Ca(2+). Thus inhibition of SR Cl(-) fluxes will reduce Ca(2+) sequestration and agonist-induced release. The Cl(-) channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10(-4) M), previously shown to inhibit SR Cl(-) channels, significantly reduced the magnitude of successive acetylcholine-induced contractions of airway smooth muscle (ASM), suggesting a "run down" of sequestered Ca(2+) within the SR. Niflumic acid (10(-4) M), a structurally different Cl(-) channel blocker, had no such effect. Furthermore, NPPB significantly reduced caffeine-induced contraction and increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). Depletion of Cl(i)(-), accomplished by bathing ASM strips in Cl(-)-free buffer, significantly reduced the magnitude of successive acetylcholine-induced contractions. In addition, Cl(-) depletion significantly reduced caffeine-induced increases in [Ca(2+)](i). Together these data suggest a novel role for Cl(i)(-) fluxes in Ca(2+) handling in smooth muscle. Because the release of sequestered Ca(2+) is the predominate source of Ca(2+) for contraction of ASM, targeting Cl(i)(-) fluxes may prove useful in the control of ASM hyperresponsiveness associated with asthma.


Autoimmune haemolysis: an 18-year study of 865 cases referred to a regional transfusion centre.
Clinical and serological records of 865 patients with confirmed autoimmune haemolysis (AIH)--a much larger series than any previously reported--were critically reviewed nd analysed. A proposed new classification for AIH based on serological findings differs from traditional classifications in that a new category of "mixed" AIH has been defined in which both "warm" and "cold" autoantibodies are present, and both are capable of causing haemolysis. Patients in this mixed group tend to have severe disease that may run a chronic intermittent course. The presentation of cold agglutinin disease is much more variable than has been seen in previous studies, haemolysis due to low titre autoantibodies being common. The AIH associated with pregnancy, usually considered as being of bad prognosis, is often mild and self limiting in the absence of other associated disorders.


Alcohol-Paired Contextual Cues Produce an Immediate and Selective Loss of Goal-directed Action in Rats.
We assessed whether the presence of contextual cues paired with alcohol would disrupt rats' capacity to express appropriate goal-directed action control. Rats were first given differential context conditioning such that one set of contextual cues was paired with the injection of ethanol and a second, distinctive set of cues was paired with the injection of saline. All rats were then trained in a third, neutral context to press one lever for grain pellets and another lever for sucrose pellets. They were then given two extinction tests to evaluate their ability to choose between the two actions in response to the devaluation of one of the two food outcomes with one test conducted in the alcohol-paired context and the other conducted in the control (saline-paired) context. In the control context, rats exhibited goal-directed action control; i.e., they were able selectively to withhold the action that previously earned the now devalued outcome. However, these same rats were impaired when tested in the alcohol-paired context, performing both actions at the same rate regardless of the current value of their respective outcomes. Subsequent testing revealed that the rats were capable of overcoming this impairment if they were giving response-contingent feedback about the current value of the food outcomes. These results provide a clear demonstration of the disruptive influence that alcohol-paired cues can exert on decision-making in general and goal-directed action selection and choice in particular.


In utero meconium passage in fetuses and newborns with myelomeningocele.
The authors retrospectively investigated whether midgestational fetal myelomeningocele (fMMC) repair alters intrauterine meconium exposure. Prior to the National Institutes of Health Management of Myelomeningocele Study, 54 fetuses underwent fMMC repair at the authors' institution. Forty-six fMMC sacs were available for pathological examination and 53 MMC sacs from postnatally repaired MMCs (pMMCs) were available for comparison. The presence and distribution of meconium were blindly evaluated using a grading system defined as follows: absent (no meconium present), mild (<10 meconium-positive histiocytes [MPHs]/hpf), moderate (10-25 MPHs/hpf), and severe (>25 MPHs/hpf). Hall's bile stain was used to confirm meconium and Prussian blue and Fontana Masson stains to exclude hemosiderin and melanin, respectively. Compared to pMMCs (79%), meconium histiocytosis was less prevalent in fMMC sacs (57%; p=0.017). Meconium staining was completely absent in 43% of the fMMC sacs. Mild meconium histiocytosis was found in 35% fMMC and 61% pMMC sacs (p=0.035). There was no statistical difference between groups with moderate and severe meconium histiocytosis. Meconium passage in MMCs can occur early in fetal life. Fetal MMC repair may reduce the duration of meconium exposure, thereby potentially limiting the toxic injury to the vulnerable neural elements.


A novel missense variant in CUL3 shows altered binding ability to BTB-adaptor proteins leading to diverse phenotypes of CUL3-related disorders.
CUL3 forms Cullin-Ring ubiquitin ligases (CRL) with Ring-box protein and BTB-adaptor proteins. A variety of BTB-adaptor proteins have been reported to interact with the N-terminus of CUL3, which makes it possible to recognize various substrates for degradation. Regarding the association of CUL3 with neurodevelopmental disorders, a recent study reported three patients with global developmental delay, who carried de novo variants in CUL3. Here, we describe a novel de novo CUL3 variant (c.158G > A, p.Ser53Asn) identified in a patient with global developmental delay, who presented some novel dysmorphic features, including macrocephaly, characteristic facial features, and cutis marmorata. Immunoprecipitation and immunoblot analyses identified significantly weaker binding ability to some BTB proteins in CUL3-S53N compared to wild-type. Interestingly, label-free quantification proteomics analysis of samples immunoprecipitated by CUL3-S53N showed a significantly decreased interaction with some BTB proteins, while almost equal interaction or significantly increased interaction was observed with other BTB proteins. The binding between CUL3 and BTB proteins is essential for CRL substrate recognition, and alteration of their interaction is thought to result in the quantitative alteration in substrate proteins. It is possible that the difference of dysmorphic features between the present case and previously reported cases is caused by the distinctive effect of each CUL3 variant on substrate proteins. The clinical information of the present case will expand the picture of CUL3-related global developmental disorders, and subsequent cell biological analysis of the novel mutation will provide insight into the underlying molecular mechanism of how CUL3 pathogenic variants cause neurological disorders.


A high-throughput gateway-compatible yeast one-hybrid screen to detect protein-DNA interactions.
In recent years, new techniques have spurred the discovery of cis-regulatory DNA elements. These stretches of noncoding DNA contain combinations of recognition sites to which transcription factors (TFs) bind, and in doing so, these TFs can activate or repress transcription. These protein-DNA interactions form the core of gene regulatory networks (GRNs) that are responsible for the differential gene expression that allow diversification of cell types, developmental programs, and responses to the environment. The yeast one-hybrid system is a genetic assay to identify direct binding of proteins to DNA elements of interest and is, therefore, instrumental in uncovering these GRNs.


Revisiting comorbidities in gout: a cluster analysis.
The reciprocal links between comorbidities and gout are complex. We used cluster analysis to attempt to identify different phenotypes on the basis of comorbidities in a large cohort of patients with gout. This was a cross-sectional multicentre study of 2763 gout patients conducted from November 2010 to May 2011. Cluster analysis was conducted separately for variables and for observations in patients, measuring proximity between variables and identifying homogeneous subgroups of patients. Variables used in both analyses were hypertension, obesity, diabetes, dyslipidaemia, heart failure, coronary heart disease, renal failure, liver disorders and cancer. Comorbidities were common in this large cohort of patients with gout. Abdominal obesity, hypertension, metabolic syndrome and dyslipidaemia increased with gout duration, even after adjustment for age and sex. Five clusters (C1-C5) were found. Cluster C1 (n=332, 12%) consisted of patients with isolated gout and few comorbidities. In C2 (n=483, 17%), all patients were obese, with a high prevalence of hypertension. C3 (n=664, 24%) had the greatest proportion of patients with type 2 diabetes (75%). In C4 (n=782, 28%), almost all patients presented with dyslipidaemia (98%). Finally, C5 (n=502, 18%) consisted of almost all patients with a history of cardiovascular disease and renal failure, with a high rate of patients receiving diuretics. Cluster analysis of comorbidities in gout allowed us to identify five different clinical phenotypes, which may reflect different pathophysiological processes in gout.


Power quality control and design of power converter for variable-speed wind energy conversion system with permanent-magnet synchronous generator.
The control strategy and design of an AC/DC/AC IGBT-PMW power converter for PMSG-based variable-speed wind energy conversion systems (VSWECS) operation in grid/load-connected mode are presented. VSWECS consists of a PMSG connected to a AC-DC IGBT-based PWM rectifier and a DC/AC IGBT-based PWM inverter with LCL filter. In VSWECS, AC/DC/AC power converter is employed to convert the variable frequency variable speed generator output to the fixed frequency fixed voltage grid. The DC/AC power conversion has been managed out using adaptive neurofuzzy controlled inverter located at the output of controlled AC/DC IGBT-based PWM rectifier. In this study, the dynamic performance and power quality of the proposed power converter connected to the grid/load by output LCL filter is focused on. Dynamic modeling and control of the VSWECS with the proposed power converter is performed by using MATLAB/Simulink. Simulation results show that the output voltage, power, and frequency of VSWECS reach to desirable operation values in a very short time. In addition, when PMSG based VSWECS works continuously with the 4.5 kHz switching frequency, the THD rate of voltage in the load terminal is 0.00672%.


The interplay of past diversification and evolutionary isolation with present imperilment across the amphibian tree of life.
Human activities continue to erode the tree of life, requiring us to prioritize research and conservation. Amphibians represent key victims and bellwethers of global change, and the need for action to conserve them is drastically outpacing knowledge. We provide a phylogeny incorporating nearly all extant amphibians (7,238 species). Current amphibian diversity is composed of both older, depauperate lineages and extensive, more recent tropical radiations found in select clades. Frog and salamander diversification increased strongly after the Cretaceous-Palaeogene boundary, preceded by a potential mass-extinction event in salamanders. Diversification rates of subterranean caecilians varied little over time. Biogeographically, the Afro- and Neotropics harbour a particularly high proportion of Gondwanan relicts, comprising species with high evolutionary distinctiveness (ED). These high-ED species represent a large portion of the branches in the present tree: around 28% of all phylogenetic diversity comes from species in the top 10% of ED. The association between ED and imperilment is weak, but many species with high ED are now imperilled or lack formal threat status, suggesting opportunities for integrating evolutionary position and phylogenetic heritage in addressing the current extinction crisis. By providing a phylogenetic estimate for extant amphibians and identifying their threats and ED, we offer a preliminary basis for a quantitatively informed global approach to conserving the amphibian tree of life.


Selective cortical layering abnormalities and behavioral deficits in cortex-specific Pax6 knock-out mice.
The transcription factor Pax6 has been implicated in neocortical neurogenesis in vertebrates, including humans. Analyses of the role of Pax6 in layer formation and cognitive abilities have been hampered by perinatal lethality of Pax6 mutants. Here, we generated viable mutants exhibiting timed, restricted inactivation of Pax6 during early and late cortical neurogenesis using Emx1-Cre and hGFAP-Cre lines, respectively. The disruption of Pax6 at the onset of neurogenesis using Emx1-Cre line resulted in premature cell cycle exit of early progenitors, increase of early born neuronal subsets located in the marginal zone and lower layers, and a nearly complete absence of upper layer neurons, especially in the rostral cortex. Furthermore, progenitors, which accumulated in the enlarged germinal neuroepithelium at the pallial/subpallial border in the Pax6 mutants, produced an excess of oligodendrocytes. The inactivation of Pax6 after generation of the lower neuronal layers using hGFAP-Cre line did not affect specification or numbers of late-born neurons, indicating that the severe reduction of upper layer neurons in Pax6 deficiency is mostly attributable to a depletion of the progenitor pool, available for late neurogenesis. We further show that Pax6(fl/fl);Emx1-Cre mutants exhibited deficiencies in sensorimotor information integration, and both hippocampus-dependent short-term and neocortex-dependent long-term memory recall. Because a majority of the morphological and behavior disabilities of the Pax6 mutant mice parallel abnormalities reported for aniridia patients, a condition caused by PAX6 haploinsufficiency, the Pax6 conditional mutant mice generated here represent a valuable genetic tool to understand how the developmental cortical disruption can lead to a human behavior abnormality.


Partially supervised learning using an EM-boosting algorithm.
Training data in a supervised learning problem consist of the class label and its potential predictors for a set of observations. Constructing effective classifiers from training data is the goal of supervised learning. In biomedical sciences and other scientific applications, class labels may be subject to errors. We consider a setting where there are two classes but observations with labels corresponding to one of the classes may in fact be mislabeled. The application concerns the use of protein mass-spectrometry data to discriminate between serum samples from cancer and noncancer patients. The patients in the training set are classified on the basis of tissue biopsy. Although biopsy is 100% specific in the sense that a tissue that shows itself to have malignant cells is certainly cancer, it is less than 100% sensitive. Reference gold standards that are subject to this special type of misclassification due to imperfect diagnosis certainty arise in many fields. We consider the development of a supervised learning algorithm under these conditions and refer to it as partially supervised learning. Boosting is a supervised learning algorithm geared toward high-dimensional predictor data, such as those generated in protein mass-spectrometry. We propose a modification of the boosting algorithm for partially supervised learning. The proposal is to view the true class membership of the samples that are labeled with the error-prone class label as missing data, and apply an algorithm related to the EM algorithm for minimization of a loss function. To assess the usefulness of the proposed method, we artificially mislabeled a subset of samples and applied the original and EM-modified boosting (EM-Boost) algorithms for comparison. Notable improvements in misclassification rates are observed with EM-Boost.


Review of influential clinical factors in reducing the risk of unsuccessful MRI-guided HIFU treatment outcome of uterine fibroids.
Magnetic resonance imaging-guided high-intensity focused ultrasound (MRI-guided HIFU) is an effective noninvasive treatment option for symptomatic uterine fibroids. However, tissue characteristics of uterine fibroids and technical limitations can limit the patient population that can benefit from this therapy. In this article, we present our literature review focusing on the influential clinical factors that might reduce the risk of an unsuccessful MRI-guided HIFU treatment outcome of uterine fibroids.


Prenatal and postnatal exposure to persistent organic pollutants and attention-deficit and hyperactivity disorder: a pooled analysis of seven European birth cohort studies.
Attention-deficit/hyperactivity disorder (ADHD) is increasing worldwide for reasons largely unknown and environmental chemicals with neurotoxic properties, such as persistent organic pollutants (POPs), have been proposed to play a role. We investigated the association between prenatal and postnatal exposure to polychlorinated biphenyl-153 (PCB-153), p-p´-dichlorodiphenyldichloroethylene (p-p'-DDE) and hexachlorobenzene (HCB) and ADHD in childhood. We pooled seven European birth cohort studies encompassing 4437 mother-child pairs from the general population with concentrations of PCB-153, p-p´-DDE and HCB measured in cord blood, maternal blood or milk. We then calculated prenatal (birth) and postnatal (3, 6, 12 and 24 months) POP concentrations using a pharmacokinetic model. The operational definition of ADHD varied across cohorts and ranged from doctor diagnosis obtained from patient registries to maternal or teachers reports. We used multilevel (mixed) logistic regression models to estimate the associations between exposure to POPs at birth, 3, 6, 12 and 24 months and ADHD. The global prevalence of ADHD in our study was 6%. The mean age at assessment of ADHD was 5.8 years (range: 3.8-9.5 years). We found no association between exposure to PCB-153, p-p´-DDE and HCB at any age point between birth and 24 months and ADHD, in the pooled analyses (pooled odds ratios ranging from 1.00 to 1.01). A number of sensitivity analyses gave basically the same results. In the largest study to date of 4437 children in seven European birth cohorts, we did not observe any association between either pre- or postnatal exposure (up to 24 months) to PCB-153, p-p´-DDE and HCB and the risk of ADHD before the age of 10 years.


Optical Coherence Tomography Angiography Evaluation of Conjunctival Vessels During Filtering Surgery.
To evaluate the changes in conjunctival vascularization with optical coherence tomography angiography (OCT-A) before and after filtering surgery and to correlate these results with filtering surgery success. We evaluated 20 blebs of 20 patients after a first-time trabeculectomy. Conjunctival vascularization was quantified using ImageJ software. Eyes were classified into two groups according to the preoperative conjunctival vessel density: hypovascularized conjunctiva (HypoV; 10 eyes) and hypervascularized conjunctiva (HyperV; 10 eyes). The density of intraepithelial microcysts (0 to 3) was also analyzed. There were significantly more needling procedures in the HyperV group, with 70% of the eyes undergoing needling during follow-up compared to 20% in the HypoV group (P = 0.012). In the HyperV group, 50% of the eyes required IOP-lowering eyedrops after surgery, compared to 10% in the HypoV group (P = 0.029). HypoV showed significantly more intraepithelial microcysts than did HyperV at 1 week (1.1 vs. 0.4, P = 0.0215), 1 month (2.2 vs. 0.4, P = 0.0003), and 6 months postoperatively (2.0 vs. 0.7, P = 0.0068). A statistically significant correlation was found between preoperative conjunctival vascular density and mean IOP at 1 week (r = 0.483, P = 0.038), 1 month (r = 0.714, P = 0.001), and 6 months postoperatively (r = 0.471, P = 0.043). There was no statistically significant correlation between the preoperative conjunctival vascularization density and the eyedrop-year rate (r = 0.036, P = 0.8704) or the preservative-year rate (r = 0.1444, P = 0.5107). Poor conjunctival vascularization was associated with lower IOP and a higher number of intraepithelial microcysts evaluated with OCT-A. OCT-A provides a simple, noninvasive, and reproducible method to analyze and quantify bleb vessels before and after filtering surgery. Several studies have demonstrated that highly vascularized blebs might be associated with a higher risk of failure. OCT-A may provide a dye-free, noncontact method for monitoring conjunctival vascularization after filtering surgery.


Neutrophil:lymphocyte ratio is positively related to type 2 diabetes in a large-scale adult population: a Tianjin Chronic Low-Grade Systemic Inflammation and Health cohort study.
It is widely known that inflammation is related to type 2 diabetes (T2D), but few studies have shown a direct relationship between the immune system and T2D using a reliable biomarker. Neutrophil:lymphocyte ratio (NLR) is an easy-to-analyze inflammation biomarker, but few studies have assessed the relationship between NLR and T2D. In order to evaluate how NLR is related to T2D, we designed a large-scale cross-sectional and prospective cohort study in an adult population. Participants were recruited from the Tianjin Medical University General Hospital-Health Management Centre. Both a baseline cross-sectional (n=87,686) and a prospective (n=38,074) assessment were performed. Participants without a history of T2D were followed up for ∼ 6 years (with a median follow-up of 2.7 years). Adjusted logistic and Cox proportional hazards regression models were used to assess relationships between the quintiles of NLR and T2D (covariates: age, sex, BMI, smoking status, drinking status, hypertension, hyperlipidemia, and family history of cardiovascular disease, hypertension, hyperlipidemia, or diabetes). The prevalence and incidence of T2D were 4.9% and 6.8/1000 person-years respectively. The adjusted odds ratio and hazard ratio (95% CI) of the highest NLR quintile were 1.34 (1.21, 1.49) and 1.39 (1.09, 1.78) (both P for trend <0.01) respectively as compared to the lowest quintile of NLR. Leukocyte, neutrophil, and lymphocyte counts do not significantly predict the eventual development of T2D. The present study demonstrates that NLR is related to the prevalence and incidence of T2D, and it suggests that NLR may be an efficient and accurate prognostic biomarker for T2D.


Photoinduced single-crystal-to-single-crystal phase transition and photosalient effect of a gold(i) isocyanide complex with shortening of intermolecular aurophilic bonds.
We report the first photoinduced single-crystal-to-single-crystal (SCSC) phase transition of a gold complex that involves the shortening of intermolecular aurophilic bonds. This is also the first solid state photochromism of a gold complex. Upon UV irradiation, the blue-emitting crystals of the gold(i) isocyanide complex 1 (1B) transform into the weakly yellow-emitting polymorph 1Y. X-ray diffraction analyses reveal that this phase transition proceeds in an SCSC manner. After phase transition from 1B to 1Y, the intermolecular Au···Au separation decreases from 3.5041(14) to 3.2955(6) Å, resulting in a red-shifted emission. The photoinduced shortening of the aurophilic bond in the excited state initiates the change in the crystal structure from 1B to 1Y. Moreover, the crystal 1B showed a photosalient effect: the 1B crystals jump upon irradiation with strong UV light owing to the phase transition into 1Y. The aurophilic bond formation in the crystal generates the mechanical movement of the crystal.


Thiazide-sensitive Na+ -Cl- cotransporter (NCC) gene inactivation results in increased duodenal Ca2+ absorption, enhanced osteoblast differentiation and elevated bone mineral density.
Inactivation of the thiazide-sensitive sodium chloride cotransporter (NCC) due to genetic mutations in Gitelman's syndrome (GS) or pharmacological inhibition with thiazide diuretics causes hypocalciuria and increased bone mineral density (BMD) with unclear extrarenal calcium (Ca(2+) ) regulation. We investigated intestinal Ca(2+) absorption and bone Ca(2+) metabolism in nonsense Ncc Ser707X (S707X) homozygous knockin mice (Ncc(S707X/S707X) mice). Compared to wild-type and heterozygous knockin littermates, Ncc(S707X/S707X) mice had increased intestinal absorption of (45) Ca(2+) and expression of the active Ca(2+) transport machinery (transient receptor potential vanilloid 6, calbindin-D9K , and plasma membrane Ca(2+) ATPase isoform 1b). Ncc(S707X/S707X) mice had also significantly increased Ca(2+) content accompanied by greater mineral apposition rate (MAR) in their femurs and higher trabecular bone volume, cortical bone thickness, and BMD determined by μCT. Their osteoblast differentiation markers, such as bone alkaline phosphatase, procollagen I, osteocalcin, and osterix, were also significantly increased while osteoclast activity was unaffected. Analysis of marrow-derived bone cells, either treated with thiazide or directly cultured from Ncc S707X knockin mice, showed that the differentiation of osteoblasts was associated with increased phosphorylation of mechanical stress-induced focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). In conclusion, NCC inhibition stimulates duodenal Ca(2+) absorption as well as osteoblast differentiation and bone Ca(2+) storage, possibly through a FAK/ERK dependent mechanism.


Decoupled plant and insect diversity after the end-Cretaceous extinction.
Food web recovery from mass extinction is poorly understood. We analyzed insect-feeding damage on 14,999 angiosperm leaves from 14 latest Cretaceous, Paleocene, and early Eocene sites in the western interior United States. Most Paleocene floras have low richness of plants and of insect damage. However, a low-diversity 64.4-million-year-old flora from southeastern Montana shows extremely high insect damage richness, especially of leaf mining, whereas an anomalously diverse 63.8-million-year-old flora from the Denver Basin shows little damage and virtually no specialized feeding. These findings reveal severely unbalanced food webs 1 to 2 million years after the end-Cretaceous extinction 65.5 million years ago.


Cross-frequency transfer in a stochastically driven mesoscopic neuronal model.
The brain is known to operate in multiple coexisting frequency bands. Increasing experimental evidence suggests that interactions between those distinct bands play a crucial role in brain processes, but the dynamical mechanisms underlying this cross-frequency coupling are still under investigation. Two approaches have been proposed to address this issue. In the first one distinct nonlinear oscillators representing the brain rhythms involved are coupled actively (bidirectionally), whereas in the second one the oscillators are coupled unidirectionally and thus the driving between them is passive. Here we elaborate the latter approach by implementing a stochastically driven network of coupled neural mass models that operate in the alpha range. This model exhibits a broadband power spectrum with 1/f(b) form, similar to those observed experimentally. Our results show that such a model is able to reproduce recent experimental observations on the effect of slow rocking on the alpha activity associated with sleep. This suggests that passive driving can account for cross-frequency transfer in the brain, as a result of the complex nonlinear dynamics of its underlying oscillators.


Isolation by cell-column chromatography of immunoglobulins specific for cell surface carbohydrates.
A new method of affinity chromatography using glutaraldehyde-fixed cells immobilized on Sephadex beads has been used to isolate immunoglobulins (Ig's) specific for cell surface glycoproteins. Ig's that specifically bound and agglutinated the same cells as those originally fixed on the columns were isolated from nonimmune sera of various species. Periodate treatment of the cell-columns and the free cells destroyed their ability to bind the Ig's, and the binding of the Ig's to untreated cells was inhibited by monosaccharides such as D-galactose and sialic acid. The binding of antibodies directed against cell surfaces obtained by immunizing animals with the same mouse tumor cell lines used on the columns (P388 and EL4) was not inhibited by various saccharides. Surface glycoproteins obtained from the mouse tumor cells by immunoprecipitation with the column-isolated Ig's yielded specific electrophoretic patterns that differed from those obtained using Ig's from the sera of rabbits immunized with the tumor cells. The data suggest that the Ig's isolated by cell-column chromatography were directed against carbohydrates, probably those in terminal positions of the polysaccharide portions of the tumor cell surface glycoproteins. Column-isolated Ig's specific for carbohydrates were also useful in studies of cell interactions in nonmammalian systems including Dictyostelium discoideum and Saccharomyces cerevisiae. The cell-column method appears to be adaptable to the isolation of a variety of molecules in addition to antibodies.


Isoaspartate-dependent molecular switches for integrin-ligand recognition.
Integrins are cell-adhesion receptors that mediate cell-extracellular-matrix (ECM) and cell-cell interactions by recognizing specific ligands. Recent studies have shown that the formation of isoaspartyl residues (isoAsp) in integrin ligands by asparagine deamidation or aspartate isomerization could represent a mechanism for the regulation of integrin-ligand recognition. This spontaneous post-translational modification, which might occur in aged proteins of the ECM, changes the length of the peptide bond and, in the case of asparagine, also of the charge. Although these changes typically have negative effects on protein function, recent studies suggested that isoAsp formation at certain Asn-Gly-Arg (NGR) sites in ECM proteins have a gain-of-function effect, because the resulting isoAsp-Gly-Arg (isoDGR) sequence can mimic Arg-Gly-Asp (RGD), a well-known integrin-binding motif. Substantial experimental evidence suggests that the NGR-to-isoDGR transition can occur in vitro in natural proteins and in drugs containing this motif, thereby promoting integrin recognition and cell adhesion. In this Commentary, we review these studies and discuss the potential effects that isoAsp formation at NGR, DGR and RGD sites might have in the recognition of integrins by natural ligands and by drugs that contain these motifs, as well as their potential biological and pharmacological implications.


SWNT array resonant gate MOS transistor.
We show that thin horizontal arrays of single wall carbon nanotubes (SWNTs) suspended above the channel of silicon MOSFETs can be used as vibrating gate electrodes. This new class of nano-electromechanical system (NEMS) combines the unique mechanical and electronic properties of SWNTs with an integrated silicon-based motion detection. Its electrical response exhibits a clear signature of the mechanical resonance of SWNT arrays (120-150 MHz) showing that these thin horizontal arrays behave as a cohesive, rigid and elastic body membrane with a Young's modulus in the order of 1-10 GPa and ultra-low mass. The resonant frequency can be tuned by the gate voltage and its dependence is well understood within the continuum mechanics framework.


Elevation of glutathione in RAW 264.7 cells by low-dose gamma-ray irradiation and its responsibility for the appearance of radioresistance.
We examined the relationship between the induction of glutathione (GSH) level in macrophage-like RAW 264.7 cells by a low (adapting) dose gamma-rays and the cell damage caused by a lethal dose of gamma-rays at various intervals after the adapting dose. The reduced glutathione (GSH) level increased soon after exposure of the cells to 25 cGy of gamma-rays, peaked between 3 hr and 6 hr, and returned almost to the zero time (0 hr) level by 24 hr post-irradiation. Cell damage was assessed by measuring the 3H-thymidine (3H-TdR) incorporation into cellular DNA. gamma-Ray irradiation produced a dose-dependent cell damage in RAW cells, causing about 40% and 60% inhibition of 3H-TdR incorporation into DNA at 1.0 Gy and 2.0 Gy, respectively, as compared with non-irradiated cells. Treatment with the adapting dose of 25 cGy at 1 hr or 24 hr before the lethal irradiation was ineffective. However, pre-irradiation with 25 cGy at 3 hr or 6 hr prior to lethal irradiation inhibited the decrease of 3H-TdR incorporation into DNA, indicating a protective effect. GSH exogenously added to the medium also inhibited the cell damage induced by lethal doses of gamma-Rays in a dose-dependent manner. These results indicate that the induction of endogenous GSH in living cells immediately following low-dose gamma-Ray irradiation is at least partially responsible for the appearance of radioresistance to a subsequent lethal dose of radiation, and may make it possible to use higher doses of radiation in radiotherapy for tumor patients.


Extra virgin olive oil biophenols inhibit cell-mediated oxidation of LDL by increasing the mRNA transcription of glutathione-related enzymes.
It has been reported that oxidized LDL (oxLDL) are involved in the pathogenesis of atherosclerosis, and that macrophages as well as other cells of the arterial wall can oxidize LDL in vitro, depending on the balance between intracellular prooxidant generation and antioxidant defense efficiency. Because of their possible beneficial role in the prevention of atherosclerosis and other oxidative stress-associated diseases, phenolic compounds naturally occurring in vegetables, fruits, and beverages are receiving increased attention. In the present work, we investigated the mechanisms underlying the protective effect exerted by extra virgin olive oil biophenols, namely, protocatechuic acid and oleuropein, on LDL oxidation mediated by murine J774 A.1 macrophage-like cells. The biophenols were added to the cells with LDL and left in the medium during the entire experimental period, or for a period of 2 h and then removed from the medium before the addition of LDL. The effect of biophenols alone was also tested. In both experimental procedures, these antioxidants had the following effects: 1). completely prevented the J774 A.1-mediated oxidation of LDL; 2). counteracted the time-dependent variations in intracellular redox balance, inhibiting the production of O(2)(.-) and H(2)O(2) and the decrease in glutathione (GSH) content; 3). restored glutathione reductase (GR) and peroxidase (GPx) activities; and 4). restored the mRNA expression of gamma-glutamylcisteine synthetase (gammaGCS), GR, and GPx to control values. More importantly, we observed significant overtranscription and increased activities of two antioxidative enzymes, GPx and GR, compared with controls when the biophenols were present in the medium for 2 h and then removed before LDL exposure, or when the cells were exposed to the antioxidants alone for up to 24 h. Our findings suggest that the activation of mRNA transcription of GSH-related enzymes represents an important mechanism in phenolic antioxidative action.


Efficacy of melatonin with behavioural sleep-wake scheduling for delayed sleep-wake phase disorder: A double-blind, randomised clinical trial.
Delayed Sleep-Wake Phase Disorder (DSWPD) is characterised by sleep initiation insomnia when attempting sleep at conventional times and difficulty waking at the required time for daytime commitments. Although there are published therapeutic guidelines for the administration of melatonin for DSWPD, to our knowledge, randomised controlled trials are lacking. This trial tested the efficacy of 0.5 mg melatonin, combined with behavioural sleep-wake scheduling, for improving sleep initiation in clinically diagnosed DSWPD patients with a delayed endogenous melatonin rhythm relative to patient-desired (or -required) bedtime (DBT). This randomised, placebo-controlled, double-blind clinical trial was conducted in an Australian outpatient DSWPD population. Following 1-wk baseline, clinically diagnosed DSWPD patients with delayed melatonin rhythm relative to DBT (salivary dim light melatonin onset [DLMO] after or within 30 min before DBT) were randomised to 4-wk treatment with 0.5 mg fast-release melatonin or placebo 1 h before DBT for at least 5 consecutive nights per week. All patients received behavioural sleep-wake scheduling, consisting of bedtime scheduled at DBT. The primary outcome was actigraphic sleep onset time. Secondary outcomes were sleep efficiency in the first third of time in bed (SE T1) on treatment nights, subjective sleep-related daytime impairment (Patient Reported Outcomes Measurement Information System [PROMIS]), PROMIS sleep disturbance, measures of daytime sleepiness, clinician-rated change in illness severity, and DLMO time. Between September 13, 2012 and September 1, 2014, 307 participants were registered; 116 were randomised to treatment (intention-to-treat n = 116; n = 62 males; mean age, 29.0 y). Relative to baseline and compared to placebo, sleep onset occurred 34 min earlier (95% confidence interval [CI] -60 to -8) in the melatonin group. SE T1 increased; PROMIS sleep-related impairment, PROMIS sleep disturbance, insomnia severity, and functional disability decreased; and a greater proportion of patients showed more than minimal clinician-rated improvement following melatonin treatment (52.8%) compared to placebo (24.0%) (P < 0.05). The groups did not differ in the number of nights treatment was taken per protocol. Post-treatment DLMO assessed in a subset of patients (n = 43) was not significantly different between groups. Adverse events included light-headedness, daytime sleepiness, and decreased libido, although rates were similar between treatment groups. The clinical benefits or safety of melatonin with long-term treatment were not assessed, and it remains unknown whether the same treatment regime would benefit patients experiencing DSWPD sleep symptomology without a delay in the endogenous melatonin rhythm. In this study, melatonin treatment 1 h prior to DBT combined with behavioural sleep-wake scheduling was efficacious for improving objective and subjective measures of sleep disturbances and sleep-related impairments in DSWPD patients with delayed circadian phase relative to DBT. Improvements were achieved largely through the sleep-promoting effects of melatonin, combined with behavioural sleep-wake scheduling. This trial was registered with the Australian New Zealand Clinical Trials Registry, ACTRN12612000425897.


A microcalorimetric study of heat production in resting skeletal muscle from human subjects.
Different microcalorimetric techniques have been compared for the assessment in vitro of the total metabolic activity of resting skeletal muscle. Human fibre bundles were suspended in Krebs-Ringer-phosphate buffer containing glucose and insulin and the heat evolution was continuously monitored for 2-6 h. Palmitate as substrate was also tested. The power signals declined rapidly when a static calorimetric method (A) was used. Two different perfusion methods (B, C) gave higher power values. Long-lasting steady states were observed with method C, where the sample was contained in a cage acting as a stirrer. Significant differences were found for fibre bundles from different human muscles as well as age- and sex-related differences. The heat production in samples from the rectus abdominis muscle (method B), 0.73 mW/g muscle wet wt., was significantly higher than for the obliquus internus muscle, 0.44 mW/g, and the vastus lateralis muscle, 0.55 mW/g, but not different from the heat production value of vastus medialis, 0.66 mW/g. In method C particularly, the fibre bundles are believed to be in adequate contact with the surrounding medium. With the use of a multi-channel calorimeter it is possible to perform up to four experiments simultaneously, e.g. involving the calorigenic effects of pharmacological substances. The technique provides a new approach for detailed studies of muscle metabolism in physiological and pathological conditions.


Potential Markers of Dietary Glycemic Exposures for Sustained Dietary Interventions in Populations without Diabetes.
There is considerable interest in dietary and other approaches to maintaining blood glucose concentrations within the normal range and minimizing exposure to postprandial hyperglycemic excursions. The accepted marker to evaluate the sustained maintenance of normal blood glucose concentrations is glycated hemoglobin A1c (HbA1c). However, although this is used in clinical practice to monitor glycemic control in patients with diabetes, it has a number of drawbacks as a marker of efficacy of dietary interventions that might beneficially affect glycemic control in people without diabetes. Other markers that reflect shorter-term glycemic exposures have been studied and proposed, but consensus on the use and relevance of these markers is lacking. We have carried out a systematic search for studies that have tested the responsiveness of 6 possible alternatives to HbA1c as markers of sustained variation in glycemic exposures and thus their potential applicability for use in dietary intervention trials in subjects without diabetes: 1,5-anhydroglucitol (1,5-AG), dicarbonyl stress, fructosamine, glycated albumin (GA), advanced glycated end products (AGEs), and metabolomic profiles. The results suggest that GA may be the most promising for this purpose, but values may be confounded by effects of fat mass. 1,5-AG and fructosamine are probably not sensitive enough to the range of variation in glycemic exposures observed in healthy individuals. Use of measures based on dicarbonyls, AGEs, or metabolomic profiles would require further research into possible specific molecular species of interest. At present, none of the markers considered here is sufficiently validated and sensitive for routine use in substantiating the effects of sustained variation in dietary glycemic exposures in people without diabetes.


Activity-dependent interdomain dynamics of matrix metalloprotease-1 on fibrin.
The roles of protein conformational dynamics and allostery in function are well-known. However, the roles that interdomain dynamics have in function are not entirely understood. We used matrix metalloprotease-1 (MMP1) as a model system to study the relationship between interdomain dynamics and activity because MMP1 has diverse substrates. Here we focus on fibrin, the primary component of a blood clot. Water-soluble fibrinogen, following cleavage by thrombin, self-polymerize to form water-insoluble fibrin. We studied the interdomain dynamics of MMP1 on fibrin without crosslinks using single-molecule Forster Resonance Energy Transfer (smFRET). We observed that the distance between the catalytic and hemopexin domains of MMP1 increases or decreases as the MMP1 activity increases or decreases, respectively. We modulated the activity using (1) an active site mutant (E219Q) of MMP1, (2) MMP9, another member of the MMP family that increases the activity of MMP1, and (3) tetracycline, an inhibitor of MMP1. We fitted the histograms of smFRET values to a sum of two Gaussians and the autocorrelations to an exponential and power law. We modeled the dynamics as a two-state Poisson process and calculated the kinetic rates from the histograms and autocorrelations. Activity-dependent interdomain dynamics may enable allosteric control of the MMP1 function.


Disuse-related decline in trabecular bone structure.
Sedentary life style may degrade bone mass and microstructure resulting in osteoporosis. We characterized trabecular bone structural properties to determine if the LRP5 G171V mutation will protect against disuse-related bone loss. Forty-eight adult male mice representing three genotypes (WT = wild type, KO = LRP5-knockout +/-, HBM = High bone with the LRP5 G171V mutation) were each randomly divided between control and disuse (4 week hindlimb suspension) groups. Trabecular bone volume fraction (BV/TV) declined in all the three genotypes. Trabecular thickness was lower in the HBM and LRP5 (+/-) KO disuse groups when compared to their respective controls. While the remaining measures of bone structure (Trabecular number, connectivity density, apparent and tissue density) were lower, the trabecular separation increased in the LRP5 (+/-) with disuse. Although the absolute loss in BV/TV was similar, the relative loss due to disuse was far greater in the LRP5 (+/-) mice (67%) than in the HBM mice (14%). The disuse caused 20% decrease in trabecular number and thickness for LRP5 (+/-), while the decline was between 6 and 11% for the HBM and WT mice.


A decision-making tool to determine economic feasibility and break-even prices for artisan cheese operations.
Artisan cheese makers lack access to valid economic data to help them evaluate business opportunities and make important business decisions such as determining cheese pricing structure. The objective of this study was to utilize an economic model to evaluate the net present value (NPV), internal rate of return, and payback period for artisan cheese production at different annual production volumes. The model was also used to determine the minimum retail price necessary to ensure positive NPV for 5 different cheese types produced at 4 different production volumes. Milk type, cheese yield, and aging time all affected variable costs. However, aged cheeses required additional investment for aging space (which needs to be larger for longer aging times), as did lower yield cheeses (by requiring larger-volume equipment for pasteurization and milk handling). As the volume of milk required increased, switching from vat pasteurization to high-temperature, short-time pasteurization was necessary for low-yield cheeses before being required for high-yield cheeses, which causes an additional increase in investment costs. Because of these differences, high-moisture, fresh cow milk cheeses can be sold for about half the price of hard, aged goat milk cheeses at the largest production volume or for about two-thirds the price at the lowest production volume examined. For example, for the given model assumptions, at an annual production of 13,608kg of cheese (30,000 lb), a fresh cow milk mozzarella should be sold at a minimum retail price of $27.29/kg ($12.38/lb), whereas a goat milk Gouda needs a minimum retail price of $49.54/kg ($22.47/lb). Artisan cheese makers should carefully evaluate annual production volumes. Although larger production volumes decrease average fixed cost and improve production efficiency, production can reach volumes where it becomes necessary to sell through distributors. Because distributors might pay as little as 35% of retail price, the retail price needs to be higher to compensate. An artisan cheese company that has not achieved the recognition needed to achieve a premium price may not find distribution through distributors profitable.


Ivermectin sensitivity is an ancient trait affecting all ecdysozoa but shows phylogenetic clustering among sepsid flies.
Avermectins are potent and popular veterinary pharmaceuticals used globally to fight parasites of livestock and humans. By disturbing ion channel transport through the membrane, avermectins are effective against endo- and ectoparasitic round and horsehair worms (Nematoida), insects, or ticks (Arthropoda), but not against Plathelminthes, including flatworms (Trematoda) and tapeworms (Cestoda), or segmented worms (Annelida). Unfortunately, excreted avermectins have strong nontarget effects on beneficial arthropods such as the insect community decomposing livestock dung, ultimately impeding this important ecosystem function to the extent that regulators mandate standardized eco-toxicological tests of dung organisms worldwide. We show that the ancient phylogenetic pattern and qualitative mechanism of avermectin sensitivity is conserved and compatible with most recent phylogenomic hypotheses grouping the Nematoida with the Arthropoda as Ecdysozoa (molting animals). At the species level, we demonstrate phylogenetic clustering in ivermectin sensitivities of 23 species of sepsid dung flies (Diptera: Sepsidae). This clustered 500-fold quantitative variation in sensitivity may indicate recent lineage-specific responses to selection, but more likely reflects pre-existing genetic variation with pleiotropic effects on eco-toxicological responses to pollutants. Regardless, our results question the common practice in eco-toxicology of choosing single test species to infer detrimental effects on entire species communities, which should ideally assess a representative taxonomic sample.


Translational PK-PD modeling analysis of MCLA-128, a HER2/HER3 bispecific monoclonal antibody, to predict clinical efficacious exposure and dose.
Introduction MCLA-128 is a bispecific monoclonal antibody targeting the HER2 and HER3 receptors. Pharmacokinetics (PK) and pharmacodynamics (PD) of MCLA-128 have been evaluated in preclinical studies in cynomolgus monkeys and mice. The aim of this study was to characterize the PK and PD of MCLA-128 and to predict a safe starting dose and efficacious clinical dose for the First-In-Human study. Methods A PK-PD model was developed based on PK data from cynomolgus monkeys and tumor growth data from a mouse JIMT-1 xenograft model. Allometric scaling was used to scale PK parameters between species. Simulations were performed to predict the safe and efficacious clinical dose, based on AUCs, receptor occupancies and PK-PD model simulations. Results MCLA-128 PK in cynomolgus monkeys was described by a two-compartment model with parallel linear and nonlinear clearance. The xenograft tumor growth model consisted of a tumor compartment with a zero-order growth rate and a first-order dying rate, both affected by MCLA-128. Human doses of 10 to 480 mg q3wk were predicted to show a safety margin of >10-fold compared to the cynomolgus monkey AUC at the no-observed-adverse-effect-level (NOAEL). Doses of ≥360 mg resulted in predicted receptor occupancies above 99% (Cmax and Cave). These doses showed anti-tumor efficacy in the PK-PD model. Conclusions This analysis predicts that a flat dose of 10 to 480 mg q3wk is suitable as starting dose for a First-in-Human study with MCLA-128. Flat doses ≥360 mg q3wk are expected to be efficacious in human, based on receptor occupancies and PK-PD model simulations.


Role of cytochromes P450 and peroxidases in metabolism of the anticancer drug ellipticine: additional evidence of their contribution to ellipticine activation in rat liver, lung and kidney.
Ellipticine is a potent antineoplastic agent exhibiting multiple mechanisms of action. This anticancer agent should be considered a pro-drug, whose pharmacological efficiency and/or genotoxic side effects are dependent on its cytochrome P450 (CYP)- and/or peroxidase-mediated activation to species forming covalent DNA adducts. The target of this study was to investigate a role of CYP and peroxidase enzymes in ellipticine oxidative activation in rats, a suitable model mimicking the fate of ellipticine in humans, in details. The contribution of pulmonary and renal CYP- and peroxidase enzymes to ellipticine metabolic activation is investigated and compared with that found in the liver. Ellipticine oxidation and DNA adduct formation in vitro were investigated using microsomes isolated from liver, lung and kidney of rats, either control (untreated) or treated i.p. with a single dose of 40 mg of ellipticine per kg of body weight. HPLC with UV detection was employed for the separation and characterization of ellipticine metabolites. Inhibitors of CYPs and cyclooxygenase (prostaglandin H synthase, COX) were used to characterize the enzymes participating in ellipticine oxidative activation in rat liver, lung and kidney. Ellipticine-derived DNA adducts were detected by 32P-postlabeling. Using α-naphthoflavone, furafylline and ketoconazole, inhibitors of CYP1A, 1A2 and 3A, respectively, we found that the CYP1A and 3A enzymes play a major role in ellipticine activation to species forming DNA adducts in liver microsomes. Because of lower expression of these enzymes in lungs and kidneys, even after their induction by ellipticine, they play a minor role in ellipticine activation in these extrahepatic tissues. Arachidonic acid, a cofactor of COX, increased ellipticine activation in the microsomes of extrahepatic tissues. In addition, indomethacin, an inhibitor of COX, efficiently inhibited formation of ellipticine-derived DNA adduct in these microsomes. Based on these results, we attribute the higher activation of ellipticine in lung and kidney microsomes to COX than to CYP enzymes. The results demonstrate that whereas CYP enzymes of 1A and 3A subfamilies are the major enzymes activating ellipticine in rat livers, peroxidase COX plays a significant role in this process in lungs and kidneys.


Study of Medical Demand-Supply Balance for the Nankai Trough Earthquake.
The Nankai Trough, which marks the boundary between the Eurasian and Philippine Sea plates, is forecasted to create a catastrophic earthquake and tsunami within 30 years. The Japanese government believes that the number of casualties would be huge. However, the exact number of severely injured (SI) people who would need emergency and intensive care has not been identified. This study, therefore, aimed to clarify the gap between medical supplies and forecasted demand. The official data estimating the number of injured people were collected, together with the number of intensive care unit (ICU) and high care unit (HCU) beds from each prefecture throughout Japan. The number of SI cases was recalculated based on official data. The number of hospital beds was then compared with the number of SI people. The total number of hospitals in Japan is 8,493 with 893,970 beds, including 6,556 ICU and 5,248 HCU beds. When the Nankai Trough earthquake occurs, 187 of the 723 disaster base hospitals (DBHs) would be located in the areas with a seismic intensity of an upper six on the Japanese Seismic Intensity Scale (JSIS) of seven, and 79 DBHs would be located in the tsunami inundation area. The estimated total number of injured people would be 661,604, including 26,857 severe, 290,065 moderate, and 344,682 minor cases. Even if all ICU and HCU beds were available for severe patients, an additional 15,053 beds would be needed. If 80% of beds were used in non-disaster times, the available ICU and HCU beds would be only 2,361. The Cabinet Office of Japan (Chiyoda City, Tokyo, Japan) assumes that 60% of hospital beds would be unavailable in an area with an upper six on the JSIS. The number of ICU and HCU beds that would be usable during a disaster would thus further decrease. The beds needed for severe patients, therefore, would be significantly lacking when the Nankai Trough earthquake occurs. It would be necessary to start the treatment of those severe patients who are "more likely to be saved."