Transcriptional cross talk between orphan nuclear receptor ERRγ and transmembrane transcription factor ATF6α coordinates endoplasmic reticulum stress response.
Orphan nuclear receptor ERRγ is a member of nuclear receptor superfamily that regulates several important cellular processes including hepatic glucose and alcohol metabolism. However, mechanistic understanding of transcriptional regulation of the ERRγ gene remains to be elucidated. Here, we report that activating transcription factor 6α (ATF6α), an endoplasmic reticulum (ER)-membrane-bound basic leucine zipper (bZip) transcription factor, directly regulates ERRγ gene expression in response to ER stress. ATF6α binds to ATF6α responsive element in the ERRγ promoter. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) is required for this transactivation. Chromatin immunoprecipitation (ChIP) assay confirmed the binding of both ATF6α and PGC1α on the ERRγ promoter. ChIP assay demonstrated histone H3 and H4 acetylation occurs at the ATF6α and PGC1α binding site. Of interest, ERRγ along with PGC1α induce ATF6α gene transcription upon ER stress. ERRγ binds to an ERRγ responsive element in the ATF6α promoter. ChIP assay confirmed that both ERRγ and PGC1α bind to a site in the ATF6α promoter that exhibits histone H3 and H4 acetylation. Overall, for the first time our data show a novel pathway of cross talk between nuclear receptors and ER-membrane-bound transcription factors and suggest a positive feed-forward loop regulates ERRγ and ATF6α gene transcription.


Scientific evidence underlying the American College of Gastroenterology's clinical practice guidelines.
Clinical practice guidelines contain recommendations for physicians to determine the most appropriate care for patients. These guidelines systematically combine scientific evidence and clinical judgment, culminating in recommendations intended to optimize patient care. The recommendations in CPGs are supported by evidence which varies in quality. We aim to survey the clinical practice guidelines created by the American College of Gastroenterology, report the level of evidence supporting their recommendations, and identify areas where evidence can be improved with additional research. We extracted 1328 recommendations from 39 clinical practice guidelines published by the American College of Gastroenterology. Several of the clinical practice guidelines used the differing classifications of evidence for their recommendations. To standardize our results, we devised a uniform system for evidence. A total of 39 clinical practice guidelines were surveyed in our study. Together they account for 1328 recommendations. 693 (52.2%) of the recommendations were based on low evidence, indicating poor evidence or expert opinion. Among individual guidelines, 13/39 (33.3%) had no recommendations based on high evidence. Very few recommendations made by the American College of Gastroenterology are supported by high levels of evidence. More than half of all recommendations made by the American College of Gastroenterology are based on low-quality evidence or expert opinion.


Muscle organization in Caenorhabditis elegans: localization of proteins implicated in thin filament attachment and I-band organization.
The body wall muscle cells of Caenorhabditis elegans contain an obliquely striated myofibrillar lattice that is associated with the cell membrane through two structures: an M-line analogue in the A-band and a Z-disc analogue, or dense-body, in the I-band. By using a fraction enriched in these structures as an immunogen for hybridoma production, we prepared monoclonal antibodies that identify four components of the I-band as determined by immunofluorescence and Western transfer analysis. A major constituent of the dense-body is a 107,000-D polypeptide that shares determinants with vertebrate alpha-actinin. A second dense-body constituent is a more basic and antigenically distinct 107,000-D polypeptide that is localized to a narrow domain of the dense-body at or subjacent to the plasma membrane. This basic dense-body polypeptide is also found at certain cell boundaries where thin filaments in half-bands terminate at membrane-associated structures termed attachment plaques. A third, unidentified antigen is also found closely apposed to the cell membrane in regions of not only the dense-body and attachment plaque, but also the M-line analogue. Finally, a fourth high molecular weight antigen, composed of two polypeptides of approximately 400,000-D, is localized to the I-band regions surrounding the dense-body. The attachment of the dense-body to the cell surface and the differential localization of the dense-body-associated antigens suggest a model for their organization in which the unidentified antigen is a cell surface component, and the two 107,000-D polypeptides define different cytoplasmic domains of the dense-body.


The effect of a selective alpha 2-adrenergic receptor antagonist (midaglizole) on the cyclic 3',5'-adenosine monophosphate production in human mononuclear cells.
A selective alpha 2-adrenergic antagonist, midaglizole, has been recently reported to have efficacy in patients with asthma. To understand the mechanisms, we investigated the effect of midaglizole on the cyclic 3', 5'-adenosine monophosphate (cAMP) production in human mononuclear cells (MNCs). MNCs were separated by a histopaque gradient from 10 normal subjects and 10 subjects with asthma. cAMP was measured by RIA kits. Midaglizole (10 mumol/L) significantly enhanced isoproterenol-induced cAMP production in both groups, although midaglizole (from 1 to 100 mumol/L) did not increase the cAMP production by itself. The percent increase in cAMP was more in subjects with asthma (183.8%) than in normal subjects (140.4%); however, the absolute increase was not different. Platelet-activating factor has been demonstrated to inhibit beta-agonist-induced cAMP production in several mammalian tissues, including human MNCs. Midaglizole also prevented platelet-activating factor-induced desensitization of the cAMP response to isoproterenol in MNCs. These findings suggest that midaglizole may be a useful additional agent for the therapy of bronchial asthma through an enhancement of the cAMP production.


Copper-induced intra-specific oxidative damage and antioxidant responses in strains of the brown alga Ectocarpus siliculosus with different pollution histories.
Inter- and intra-specific variation in metal resistance has been observed in the ecologically and economically important marine brown macroalgae (Phaeophyceae), but the mechanisms of cellular tolerance are not well elucidated. To investigate inter-population responses of brown seaweeds to copper (Cu) pollution, the extent of oxidative damage and antioxidant responses were compared in three strains of the filamentous brown seaweed Ectocarpus siliculosus, the model organism for the algal class Phaeophyceae that diverged from other major eukaryotic groups over a billion year ago. Strains isolated from locations with different pollution histories (i.e. LIA, from a pristine site in Scotland; REP and Es524 from Cu-contaminated sites in England and Chile, respectively) were exposed to total dissolved Cu concentrations (CuT) of up to 2.4 μM (equivalent to 128 nM Cu(2+)) for 10 d. LIA exhibited oxidative stress, with increases in hydrogen peroxide (H2O2) and lipid peroxidation (measured as TBARS levels), and decreased concentrations of photosynthetic pigments. Es524 presented no apparent oxidative damage whereas in REP, TBARS increased, revealing some level of oxidative damage. Adjustments to activities of enzymes and antioxidant compounds concentrations in Es524 and REP were strain and treatment dependent. Mitigation of oxidative stress in Es524 was by increased activities of superoxide dismutases (SOD) at low CuT, and catalase (CAT) and ascorbate peroxidase (APX) at all CuT, accompanied by higher levels of antioxidants (ascorbate, glutathione, phenolics) at higher CuT. In REP, only APX activity increased, as did the antioxidants. For the first time evidence is presented for distinctive oxidative stress defences under excess Cu in two populations of a species of brown seaweed from environments contaminated by Cu.


Secondary autoimmune diseases occurring after HSCT for an autoimmune disease: a retrospective study of the EBMT Autoimmune Disease Working Party.
To specify the incidence and risk factors for secondary autoimmune diseases (ADs) after HSCT for a primary AD, we retrospectively analyzed AD patients treated by HSCT reported to EBMT from 1995 to 2009 with at least 1 secondary AD (cases) and those without (controls). After autologous HSCT, 29 of 347 patients developed at least 1 secondary AD within 21.9 (0.6-49) months and after allogeneic HSCT, 3 of 16 patients. The observed secondary ADs included: autoimmune hemolytic anemia (n = 3), acquired hemophilia (n = 3), autoimmune thrombocytopenia (n = 3), antiphospholipid syndrome (n = 2), thyroiditis (n = 12), blocking thyroid-stimulating hormone receptor antibody (n = 1), Graves disease (n = 2), myasthenia gravis (n = 1), rheumatoid arthritis (n = 2), sarcoidosis (n = 2), vasculitis (n = 1), psoriasis (n = 1), and psoriatic arthritis (n = 1). After autologous HSCT for primary AD, the cumulative incidence of secondary AD was 9.8% ± 2% at 5 years. Lupus erythematosus as primary AD, and antithymocyte globulin use plus CD34(+) graft selection were important risk factors for secondary AD by multivariate analysis. With a median follow-up of 6.2 (0.54-11) years after autologous HSCT, 26 of 29 patients with secondary AD were alive, 2 died during their secondary AD (antiphospholipid syndrome, hemophilia), and 1 death was HSCT-related. This European multicenter study underlines the need for careful management and follow-up for secondary AD after HSCT.


Hessian-based quantitative image analysis of host-pathogen confrontation assays.
Host-fungus interactions have gained a lot of interest in the past few decades, mainly due to an increasing number of fungal infections that are often associated with a high mortality rate in the absence of effective therapies. These interactions can be studied at the genetic level or at the functional level via imaging. Here, we introduce a new image processing method that quantifies the interaction between host cells and fungal invaders, for example, alveolar macrophages and the conidia of Aspergillus fumigatus. The new technique relies on the information content of transmitted light bright field microscopy images, utilizing the Hessian matrix eigenvalues to distinguish between unstained macrophages and the background, as well as between macrophages and fungal conidia. The performance of the new algorithm was measured by comparing the results of our method with that of an alternative approach that was based on fluorescence images from the same dataset. The comparison shows that the new algorithm performs very similarly to the fluorescence-based version. Consequently, the new algorithm is able to segment and characterize unlabeled cells, thus reducing the time and expense that would be spent on the fluorescent labeling in preparation for phagocytosis assays. By extending the proposed method to the label-free segmentation of fungal conidia, we will be able to reduce the need for fluorescence-based imaging even further. Our approach should thus help to minimize the possible side effects of fluorescence labeling on biological functions. © 2017 International Society for Advancement of Cytometry.


A topological mechanism for TRF2-enhanced strand invasion.
Telomeres can fold into t-loops that may result from the invasion of the 3' overhang into duplex DNA. Their formation is facilitated in vitro by the telomeric protein TRF2, but very little is known regarding the mechanisms involved. Here we reveal that TRF2 generates positive supercoiling and condenses DNA. Using a variety of TRF2 mutants, we demonstrate a strong correlation between this topological activity and the ability to stimulate strand invasion. We also report that these properties require the combination of the TRF-homology (TRFH) domain of TRF2 with either its N- or C-terminal DNA-binding domains. We propose that TRF2 complexes, by constraining DNA around themselves in a right-handed conformation, can induce untwisting of the neighboring DNA, thereby favoring strand invasion. Implications of this topological model in t-loop formation and telomere homeostasis are discussed.


Truncated FGFR2 is a clinically actionable oncogene in multiple cancers.
Somatic hotspot mutations and structural amplifications and fusions that affect fibroblast growth factor receptor 2 (encoded by FGFR2) occur in multiple types of cancer1. However, clinical responses to FGFR inhibitors have remained variable1-9, emphasizing the need to better understand which FGFR2 alterations are oncogenic and therapeutically targetable. Here we apply transposon-based screening10,11 and tumour modelling in mice12,13, and find that the truncation of exon 18 (E18) of Fgfr2 is a potent driver mutation. Human oncogenomic datasets revealed a diverse set of FGFR2 alterations, including rearrangements, E1-E17 partial amplifications, and E18 nonsense and frameshift mutations, each causing the transcription of E18-truncated FGFR2 (FGFR2ΔE18). Functional in vitro and in vivo examination of a compendium of FGFR2ΔE18 and full-length variants pinpointed FGFR2-E18 truncation as single-driver alteration in cancer. By contrast, the oncogenic competence of FGFR2 full-length amplifications depended on a distinct landscape of cooperating driver genes. This suggests that genomic alterations that generate stable FGFR2ΔE18 variants are actionable therapeutic targets, which we confirmed in preclinical mouse and human tumour models, and in a clinical trial. We propose that cancers containing any FGFR2 variant with a truncated E18 should be considered for FGFR-targeted therapies.


Implementing a mobile stroke unit program in the United States: why, how, and how much?
There are many ways a mobile stroke unit (MSU) might prove valuable for patients with ischemic and hemorrhagic stroke, such as earlier recognition, more accurate triage, improved management of blood pressure and other critical physiological variables, and eventually earlier implementation of effective therapies. The MSU may be particularly valuable for treatment of patients with acute ischemic stroke with tissue plasminogen activator (tPA) within 4.5 hours of symptom onset, the most evidence-based effective emergency treatment for the most prevalent stroke diagnosis. To review existing data on prehospital stroke treatment, especially relevant to MSU technology, to identify gaps in our understanding of MSU feasibility, especially relevant to applying the MSU strategy in the United States, and to describe the Houston MSU program and clinical trial. Published data from English-language journals in PubMed from 1995 to present reviewing early treatment with tPA and prehospital stroke evaluation and treatment. The MSU may result in an overall shift toward earlier evaluation and treatment with tPA, particularly into the first hour after symptom onset, leading to substantially better outcomes. As a result of improved clinical outcomes owing to earlier treatment, the costs of an MSU program may be offset by a reduction in the costs of long-term stroke care and an increase in quality-adjusted life-years, thereby supporting more widespread use of this technology. To make MSU deployment more practical, the vascular neurologist aboard the MSU must be replaced by a remote vascular neurologist connected to the MSU by telemedicine, reducing manpower requirements and costs. The MSU strategy could dramatically transform the way acute stroke is managed in the United States. A prospective study evaluating the logistics, outcomes, and cost-effectiveness of this approach is needed and under way.


First report of mites (Gamasida: Laelapidae) parasitic on wild rodents in Uruguay, with new host records.
Information is presented for the first time on laelapid mites associated with wild rodents in Uruguay. Specimens of the following species were identified: Laelapinae: Androlaelaps fahrenholzi (Berlese), Androlaelaps rotundus (Fonseca), Gigantolaelaps wolffsohni (Oudemans), Laelaps paulistanensis (Fonseca), Laelaps manguinhosi (Fonseca) and Mysolaelaps microspinosus Fonseca; Haemogamasinae: Eulaelaps stabularis (Koch). Most of the ectoparasite-host associations are reported for the first time. New host and locality records presented in this study are in accordance with previous findings on the same and/or related host species in nearby South American localities.


Youth Relationships in the Era of COVID-19: A Mixed-Methods Study Among Adolescent Girls and Young Women in Kenya.
Measures to mitigate COVID-19's impact may inhibit development of healthy youth relationships, affecting partnership quality and sexual and reproductive health (SRH) outcomes. We conducted a mixed-methods study to understand how COVID-19 affected girls' and young women's relationships in Kenya. Bivariate and multivariate logistic regression examined factors associated with relationship quality dynamics and SRH outcomes among 756 partnered adolescents aged 15-24 years. Qualitative data from in-depth interviews were analyzed using inductive thematic analysis to explore youth perceptions of how intimate relationships changed during COVID-19. Nearly three-quarters of youth described changes in relationship quality since COVID-19 began, with 24% reporting worsening. Reduced time with partners was the strongest predictor of changed relationship quality. Youth experiencing complete or partial COVID-19-related household income loss had heightened risk of deteriorating partnerships (relative risk ratio = 2.43 and 2.02; p < .05); those whose relationships worsened were more likely to experience recent intimate partner violence, relative to no relationship change (20.8% vs. 3.5%; p < .001). Qualitative analysis revealed how COVID-19 mitigation measures hindered intimate relationships, school closures accelerated marriage timelines, and economic hardships strained relationships, while increasing early pregnancy risk and girls' financial dependency on their partners. COVID-19 disrupted adolescent girls' and young women's romantic relationships, depriving some of partner emotional support and exposing others to sexual violence, early pregnancy, and economically motivated transactional relationships. Increased social support systems, including access to psychosocial services, are needed in low-income communities in Kilifi, Kisumu, and Nairobi, in particular the informal settlement areas, to mitigate COVID-19's consequences on girls' SRH.


Vaginoscopy Against Standard Treatment: a randomised controlled trial.
To evaluate whether vaginoscopy or standard hysteroscopy was more successful in the outpatient setting. Randomised controlled multicentre trial. Outpatient hysteroscopy clinics at two UK hospitals. 1597 women aged 16 or older undergoing an outpatient hysteroscopy. Women were allocated to vaginoscopy or standard hysteroscopy using third party randomisation stratified by menopausal status with no blinding of participants or clinicians. The primary outcome was 'success', a composite endpoint defined as: a complete procedure, no complications, a level of pain acceptable to the patient, and no sign of genitourinary tract infection 2 weeks after the procedure. Vaginoscopy was significantly more successful than standard hysteroscopy [647/726 (89%) versus 621/734 (85%), respectively; relative risk (RR) 1.05, 95% CI 1.01-1.10; P = 0.01]. The median time taken to complete vaginoscopy was 2 minutes compared with 3 minutes for standard hysteroscopy (P < 0.001). The mean pain score was 42.7 for vaginoscopy, which was significantly less than standard hysteroscopy 46.4 (P = 0.02). Operative complications occurred in five women receiving vaginoscopy and 19 women receiving standard hysteroscopy (RR 0.26, 95% CI 0.10-0.69). Vaginoscopy is quicker to perform, less painful, and more successful than standard hysteroscopy and therefore should be considered the technique of choice for outpatient hysteroscopy. Vaginoscopy is quicker to perform, less painful, and more successful than standard hysteroscopy.


Learning alters theta amplitude, theta-gamma coupling and neuronal synchronization in inferotemporal cortex.
How oscillatory brain rhythms alone, or in combination, influence cortical information processing to support learning has yet to be fully established. Local field potential and multi-unit neuronal activity recordings were made from 64-electrode arrays in the inferotemporal cortex of conscious sheep during and after visual discrimination learning of face or object pairs. A neural network model has been developed to simulate and aid functional interpretation of learning-evoked changes. Following learning the amplitude of theta (4-8 Hz), but not gamma (30-70 Hz) oscillations was increased, as was the ratio of theta to gamma. Over 75% of electrodes showed significant coupling between theta phase and gamma amplitude (theta-nested gamma). The strength of this coupling was also increased following learning and this was not simply a consequence of increased theta amplitude. Actual discrimination performance was significantly correlated with theta and theta-gamma coupling changes. Neuronal activity was phase-locked with theta but learning had no effect on firing rates or the magnitude or latencies of visual evoked potentials during stimuli. The neural network model developed showed that a combination of fast and slow inhibitory interneurons could generate theta-nested gamma. By increasing N-methyl-D-aspartate receptor sensitivity in the model similar changes were produced as in inferotemporal cortex after learning. The model showed that these changes could potentiate the firing of downstream neurons by a temporal desynchronization of excitatory neuron output without increasing the firing frequencies of the latter. This desynchronization effect was confirmed in IT neuronal activity following learning and its magnitude was correlated with discrimination performance. Face discrimination learning produces significant increases in both theta amplitude and the strength of theta-gamma coupling in the inferotemporal cortex which are correlated with behavioral performance. A network model which can reproduce these changes suggests that a key function of such learning-evoked alterations in theta and theta-nested gamma activity may be increased temporal desynchronization in neuronal firing leading to optimal timing of inputs to downstream neural networks potentiating their responses. In this way learning can produce potentiation in neural networks simply through altering the temporal pattern of their inputs.


Effectiveness of biphasic calcium phosphate block bone substitutes processed using a modified extrusion method in rabbit calvarial defects.
This study evaluated the mechanical and structural properties of biphasic calcium phosphate (BCP) blocks processed using a modified extrusion method, and assessed their in vivo effectiveness using a rabbit calvarial defect model. BCP blocks with three distinct ratios of hydroxyapatite (HA):tricalcium phosphate (TCP) were produced using a modified extrusion method:HA8 (8%:92%), HA48 (48%:52%), and HA80 (80%:20%). The blocks were examined using scanning electron microscopy, X-ray diffractometry, and a universal test machine. Four circular defects 8 mm in diameter were made in 12 rabbits. One defect in each animal served as a control, and the other three defects received the BCP blocks. The rabbits were sacrificed at either two weeks (n=6) or eight weeks (n=6) postoperatively. The pore size, porosity, and compressive strength of the three types of bone block were 140-170 µm, >70%, and 4-9 MPa, respectively. Histologic and histomorphometric observations revealed that the augmented space was well maintained, but limited bone formation was observed around the defect base and defect margins. No significant differences were found in the amount of new bone formation, graft material resorption, or bone infiltration among the three types of BCP block at either of the postoperative healing points. Block bone substitutes with three distinct compositions (i.e., HA:TCP ratios) processed by a modified extrusion method exhibited limited osteoconductive potency, but excellent space-maintaining capability. Further investigations are required to improve the processing method.


Development of simple sequence repeat (SSR) markers from a genome survey of Chinese bayberry (Myrica rubra).
Chinese bayberry (Myrica rubra Sieb. and Zucc.) is a subtropical evergreen tree originating in China. It has been cultivated in southern China for several thousand years, and annual production has reached 1.1 million tons. The taste and high level of health promoting characters identified in the fruit in recent years has stimulated its extension in China and introduction to Australia. A limited number of co-dominant markers have been developed and applied in genetic diversity and identity studies. Here we report, for the first time, a survey of whole genome shotgun data to develop a large number of simple sequence repeat (SSR) markers to analyse the genetic diversity of the common cultivated Chinese bayberry and the relationship with three other Myrica species. The whole genome shotgun survey of Chinese bayberry produced 9.01Gb of sequence data, about 26x coverage of the estimated genome size of 323 Mb. The genome sequences were highly heterozygous, but with little duplication. From the initial assembled scaffold covering 255 Mb sequence data, 28,602 SSRs (≥5 repeats) were identified. Dinucleotide was the most common repeat motif with a frequency of 84.73%, followed by 13.78% trinucleotide, 1.34% tetranucleotide, 0.12% pentanucleotide and 0.04% hexanucleotide. From 600 primer pairs, 186 polymorphic SSRs were developed. Of these, 158 were used to screen 29 Chinese bayberry accessions and three other Myrica species: 91.14%, 89.87% and 46.84% SSRs could be used in Myrica adenophora, Myrica nana and Myrica cerifera, respectively. The UPGMA dendrogram tree showed that cultivated Myrica rubra is closely related to Myrica adenophora and Myrica nana, originating in southwest China, and very distantly related to Myrica cerifera, originating in America. These markers can be used in the construction of a linkage map and for genetic diversity studies in Myrica species. Myrica rubra has a small genome of about 323 Mb with a high level of heterozygosity. A large number of SSRs were identified, and 158 polymorphic SSR markers developed, 91% of which can be transferred to other Myrica species.


Carbon monoxide releasing molecule-2 protects against particulate matter-induced lung inflammation by inhibiting TLR2 and 4/ROS/NLRP3 inflammasome activation.
Exposure to airborne particulate matter (PM) not only causes lung inflammation and chronic respiratory diseases, but also increases the incidence and mortality of cardiopulmonary diseases. The nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome activation has been shown to play a critical role in the formation of many chronic disorders. On the other hand, carbon monoxide (CO) has been shown to possess anti-inflammatory and antioxidant effects in many tissues and organs. Here, we investigated the effects and mechanisms of carbon monoxide releasing molecule-2 (CORM-2) on PM-induced inflammatory responses in human pulmonary alveolar epithelial cells (HPAEpiCs). We found that PM induced C-reactive protein (CRP) expression, NLRP3 inflammasome activation, IL-1β secretion, and caspase-1 activation, which were inhibited by pretreatment with CORM-2. In addition, transfection with siRNA of Toll-like receptor 2 (TLR2) or TLR4 and pretreatment with an antioxidant (N-acetyl-cysteine, NAC), the inhibitor of NADPH oxidase (diphenyleneiodonium, DPI), or a mitochondria-specific superoxide scavenger (MitoTEMPO) reduced PM-induced inflammatory responses. CORM-2 also inhibited PM-induced NADPH oxidase activity and NADPH oxidase- and mitochondria-derived ROS generation. However, pretreatment with inactivate CORM-2 (iCORM-2) had no effects on PM-induced inflammatory responses. Finally, we showed that CORM-2 inhibited PM-induced CRP, NLRP3 inflammasome, and ASC protein expression in the lung tissues of mice and IL-1β levels in the serum of mice. PM-enhanced leukocyte count in bronchoalveolar lavage fluid in mice was reduced by CORM-2. The results of this study suggested a protective role of CORM-2 in PM-induced lung inflammation by inhibiting the TLR2 and TLR4/ROS-NLRP3 inflammasome-CRP axial.


Neferine augments therapeutic efficacy of cisplatin through ROS- mediated non-canonical autophagy in human lung adenocarcinoma (A549 cells).
Combination of dietary components with chemotherapy drugs is an emerging new strategy for cancer therapy to increase antitumor responses. Neferine, major bisbenzylisoquinoline alkaloid isolated from the seed embryo of Nelumbo nucifera (Lotus). In the present study, we investigated the efficacy of the combinatorial regimen of neferine and cisplatin compared to cisplatin high dose in human lung adenocarcinoma (A549) cells. Co-treatment with neferine enhanced cisplatin-induced autophagy in A549 cells was accompanied by Acidic vesicular accumulation (AVO), enhanced generation of reactive oxygen species (ROS) and depletion of intracellular glutathione (GSH), down regulation of PI3K/AKT/mTOR pathway, conversion of LC3B-I to LC3B-II. This enhanced autophagy developed via a non-canonical mechanism that did not require Beclin-1, PI3KCIII. In conclusion, these results suggest that neferine enhances cisplatin -induced autophagic cancer cell death through downregulation of PI3K/Akt/mTOR signaling pro-survival pathway and ROS- mediated Beclin-1 and PI3K CIII independent autophagy in human lung adenocarcinoma (A549 cells).


Plasma membrane and internalized immunoglobulins of lymph node cells studied with conjugates of antibody or its Fab fragments with horseradish peroxidase.
Normal rat and mouse lymphoid cells were incubated at 0 degrees -4 degrees C for 1 h with purified rabbit or sheep antirat (mouse) immunoglobulin (Ig)-horseradish peroxidase (PO) conjugates or with Fab fragments of antibody coupled with peroxidase. Cells were subsequently washed and incubated in fresh medium, without labeled antibody or Fab fragments for 5-30 min at 20 degrees or 37 degrees C. With the use of the diaminobenzidine (DAB) method, distribution of peroxidase was studied in the light and electron microscopes. Fab fragments of antirat Ig antibody were iodinated with (125)I and subsequently coupled with horseradish PO. Plasma membrane and internalized immunoglobulins were detected by electron microscope autoradiography and peroxidase cytochemistry. Single- (Fab-PO), and double- ([(125)I]Fab-PO) labeled lymphoid cells showed identical patterns of surface or internal distribution of immunoglobulins. In the electron microscope, Fab-PO conjugates at 0 degrees -4 degrees C resulted in a diffuse specific staining of the plasmalemma of lymphocytes and plasma cells. Most of the small dark lymphocytes (T cells?) did not show plasma membrane Ig. Macrophages did not show plasmalemma staining, but displayed nonspecific cytoplasmic staining after incubation at 20 degrees or 37 degrees C with antibody or Fab-PO conjugates. Lymphocytes and plasma cells, after incubation with antibody-PO conjugates at 0 degrees -4 degrees C, had patchy deposits of oxidized DAB on their plasma membranes. Macrophages, similarly treated, had no plasmalemmal staining. Patch and cap formation on the plasma membrane of lymphocytes and plasma cells was seen regularly after antibody-PO incubation at 37 degrees C. Internalization patterns were different in lymphocytes and plasma cells. In lymphocytes, peroxidase staining was observed in small round or oval vesicles clustered at one pole of the cell (30 min at 37 degrees C). In plasma cells, peroxidase staining was seen in clusters of tubules resembling the Golgi apparatus. Internalization of plasma membrane IgG was less pronounced after antibody-PO labeling as compared to Fab-PO labeling.


ON AN ALLEGED TRUTH/FALSITY ASYMMETRY IN CONTEXT SHIFTING EXPERIMENTS.
Keith DeRose has argued that context shifting experiments should be designed in a specific way in order to accommodate what he calls a 'truth/falsity asymmetry'. I explain and critique DeRose's reasons for proposing this modification to contextualist methodology, drawing on recent experimental studies of DeRose's bank cases as well as experimental findings about the verification of affirmative and negative statements. While DeRose's arguments for his particular modification to contextualist methodology fail, the lesson of his proposal is that there is good reason to pay close attention to several subtle aspects of the design of context shifting experiments.


Revascularisation in older adult patients with non-ST-segment elevation acute coronary syndrome: effect and impact on 6-month mortality.
Although revascularisation in non-ST-segment elevation acute coronary syndrome (NSTEACS) is associated with better outcomes, its impact in older adult patients is unclear. This is a retrospective analyses of three national NSTEACS registries conducted during the past decade in Spain. Patients aged 75 years and older were included: DESCARTES (DES; year 2002; n=534), MASCARA (MAS; 2005; n=1736) and DIOCLES (DIO; 2012; n=593). The adjusted association between revascularisation and total (inhospital and 6-month) mortality was estimated by two-stage meta-analysis (pooled effect across the three registries with inverse-variability weights) and one-stage meta-analysis (multilevel model with random effects across studies). The impact of revascularisation was assessed comparing the observed and the expected mortality based on a logistic regression model in the pooled database. Although revascularisation was associated with a lower risk of mortality in meta-analyses (two-stage: odds ratio 0.44, 95% confidence interval 0.29-0.67; one-stage: odds ratio 0.54, 95% confidence interval 0.36-0.81) and the revascularisation rate increased steadily from 2002 (DES 14.2%) to 2012 (DIO 43.7%), its impact was not patent across registries, probably because this increase was concentrated in low and medium-risk GRACE strata (tertile 1, 2 and 3: MAS 59%, 20% and 6%; DIO 64%, 39% and 19%, respectively). In conclusion, a consistent increase of revascularisation in NSTEACS in older adults was not followed by a decrease in mortality at 6 months, probably because the impact of this strategy is limited to the higher risk population, the stratum with the lowest revascularisation rate in real life.


Real-Time Forecasting of Hand-Foot-and-Mouth Disease Outbreaks using the Integrating Compartment Model and Assimilation Filtering.
Hand-foot-and-mouth disease (HFMD) is a highly contagious viral infection, and real-time predicting of HFMD outbreaks will facilitate the timely implementation of appropriate control measures. By integrating a susceptible-exposed-infectious-recovered (SEIR) model and an ensemble Kalman filter (EnKF) assimilation method, we developed an integrated compartment model and assimilation filtering forecast model for real-time forecasting of HFMD. When applied to HFMD outbreak data collected for 2008-11 in Beijing, China, our model successfully predicted the peak week of an outbreak three weeks before the actual arrival of the peak, with a predicted maximum infection rate of 85% or greater than the observed rate. Moreover, dominant virus types enterovirus 71 (EV-71) and coxsackievirus A16 (CV-A16) may account for the different patterns of HFMD transmission and recovery observed. The results of this study can be used to inform agencies responsible for public health management of tailored strategies for disease control efforts during HFMD outbreak seasons.


Dynamic structure of unentangled polymer chains in the vicinity of non-attractive nanoparticles.
Using coarse-grained molecular dynamics simulation, we study the motion of unentangled polymer chains dynamically confined by non-attractive nanoparticles (NPs). Both normal mode and dynamic structure factor S(q, t) analysis are adopted to analyze chain's dynamics. Relaxation behaviors of chains are found to be significantly slowed down by NPs. The relaxation times of chain's normal modes are monotonically increasing with the NP volume fraction ϕ. At the same time, chains' dynamics are becoming non-Gaussian. Inspection of S(q, t) reveals that chain's dynamics can be attributed to two 'phases', a bulk polymer phase and a confined polymer phase between NPs. The dynamics of a confined polymer is slower than that of a bulk polymer, while still exhibiting high mobility. The amount of the bulk polymer phase is found to exponentially decay with increasing ϕ. With this figure at hand, we establish a simple relationship between NP and confined/interphase polymer volume fractions. This work seems to provide the first quantitative prediction on the relationship between NP and confined/interphase polymer volume fractions.


A method for flap monitoring based on bioelectrical-resistance analysis.
Axial pattern flaps are characterized by a single vascular pedicle that supplies the whole flap and carry the danger of flap loss. For this reason, monitoring and early identification of vascular perfusion is important for flap prognosis. The most commonly used method for flap monitoring is clinical assessment, but it has certain limitations. The bioelectrical properties of tissues can be affected by ischemic conditions, and a preliminary study showed that the bioelectrical resistance of blood was decreased under thrombotic conditions. In this controlled study, the resistance values of the axial flaps were calculated during thrombosis. A total of 28 rats were randomly divided into a control group (n = 14) and an experimental group (n = 14). Axial flaps were elevated based on the inferior epigastric pedicle. For the ischemic group, microvascular clamps were placed in the ischemic group to produce artificial thrombosis from day 1 of the experiment. Bioelectrical resistance was measured every 30 minutes for the first 6 hours and then daily after that. The resistance values from the ischemic group decreased progressively, and 150 minutes after the clamping, the difference became statistically significant (P < 0.05). The results showed that the assessment of flap resistance is a valuable tool and may allow early recognition of a compromise in the vascular system before clinical signs become obvious.


Radiocarbon Dating: Fictitious Results with Mollusk Shells.
Evidence is presented to show that modern mollusk shells from rivers can have anomalous radiocarbon ages, owing mainly to incorporation of inactive (carbon-14-deficient) carbon from humus, probably through the food web, as well as by the pathway of carbon dioxide from humus decay. The resultant effect, in addition to the variable contributions of atmospheric carbon dioxide, fermentative carbon dioxide from bottom muds, and, locally, of carbonate carbon from dissolving limestones, makes the initial carbon-14-activity of ancient fresh-water shell indeterminate, but within limits. Consequent errors of shell radiocarbon dates may be as large as several thousand years for river shells.


Is it possible to diagnose infectious oesophagitis without seeing the causative organism? A histopathological study.
We investigated the utility of using histological changes to diagnose infectious oesophagitis when causative organisms cannot be seen. Sixty-seven endoscopic biopsy specimens (51 Candida, 9 herpes simplex virus, 4 tuberculosis, and 3 cytomegalovirus oesophagitis) collected from 2000-2010 that matched the investigative criteria were included in the study. Cases were re-evaluated for histological changes observed in oesophagitis, and the findings were statistically compared using nonparametric tests. Thirty-nine cases occurred in male patients, and 28 occurred in female patients; the mean age of the patients was 51±20.1 years (range, 5-94 years). All cases showed lymphocytic and neutrophilic infiltration; while 27 (40.3%) showed eosinophilic infiltration. The density of lymphocytes and eosinophils were 8.43±6 and 1.07±1.62 per high power field, respectively, and these rates were higher in tuberculosis oesophagitis cases. Lamina propria infiltration was present in herpes simplex virus and Candida oesophagitis. Dense neutrophilic infiltration (>50/high power field) was noted in herpes simplex virus oesophagitis. Candida colonization was observed in 82% of cases with eosinophilic infiltration, and 80% of cases with erosion. Ulceration was present in all tuberculosis oesophagitis cases (p<0.001). Basal cell hyperplasia, papillary elongation, and dilated intercellular spaces were seen in all cases except for 2 Candida oesophagitis cases. Lamina propria fibrosis was especially noted in cytomegalovirus oesophagitis cases. It is not possible to distinguish infectious oesophagitis from other subtypes, especially reflux oesophagitis, if the causative organism is not detected. Clinicopathological correlation and control with repeat targeted biopsies are essential for diagnosis.


Use of ultrasounds in PD catheter-related infections: indications and clinical implications
Peritoneal dialysis (PD) related infections continue to be a major cause of morbidity and mortality in patients undertaking renal replacement therapy with PD. Nevertheless, despite the great effort invested in the prevention of PD infective episodes, almost one third of technical failures are still caused by peritonitis. Recent studies support the idea that there is a direct role of exit-site (ESIs) and tunnel infections (TIs) in causing peritonitis. Hence, both the prompt ESI/TI diagnosis and correct prognostic hypothesis would allow the timely start of an appropriate antibiotic therapy decreasing the associated complications and preserving the PD technique. The ultrasound exam (US) is a simple, rapid, non-invasive and widely available procedure for the tunnel evaluation in PD catheter-related infections. In case of ESI, the US possesses a greater sensibility in diagnosing a simultaneous TI compared to the clinical criterions. This peculiarity allows to distinguish the ESI episodes which will be healed with antibiotic therapy from those refractories to medical therapy. In case of TI, the US permits to localize the catheter portion involved in the infectious process obtaining significant prognostic information; while the US repetition after two weeks of antibiotic allows to monitor the patient responsiveness to the therapy. There is no evidence of the US usefulness as screening tool aimed to the precocious diagnosis of TI in asymptomatic PD patients.


Helping families raise children with special health care needs at home.
One goal of Healthy People 2010 is to reduce the number of people with disabilities in congregate care facilities, consistent with permanency-planning principles, to 0 by 2010 for persons aged 21 years and under (objective 6-7). Congregate care, in this regard, is defined as any setting in which 4 or more persons with disabilities reside, regardless of whether the residence is located in the community, such as a school, group home, nursing facility, or institution. Although this particular public health objective may reflect an unfamiliar concept for some pediatricians, the American Academy of Pediatrics supports the goals and objectives of Healthy People 2010 as well as the medical home and the provision of community-based, culturally effective, coordinated, and comprehensive care for children with special health care needs and their families. To advise families caring for children with special health care needs effectively, the pediatrician should be familiar with the principles of permanency planning and well informed of local family-support services. The pediatrician should also work with the family to identify the range of long-term supports and services available for their child. These supports may include respite for biological families as well as various additional parenting models such as shared parenting, foster care, alternate parents, and adoption. Although family-based supports are preferable, families may consider other out-of-home placements including group homes, placement in a nursing facility, or other forms of institutional care when sufficient family-based services are not available. Once all the options are understood, issues regarding quality of care can be individualized and judged by the parent or guardian, in close collaboration with the pediatrician and other professionals with expertise in permanency planning and long-term supports and services. The purpose of this clinical report is to educate physicians on the philosophy of providing a permanent family environment (permanency planning) for all children, including those with special health care needs, and the importance of adequate and accessible community services to support and maintain the well-being of all family members.


Generalized fuzzy C-means clustering algorithm with improved fuzzy partitions.
The fuzziness index m has important influence on the clustering result of fuzzy clustering algorithms, and it should not be forced to fix at the usual value m = 2. In view of its distinctive features in applications and its limitation in having m = 2 only, a recent advance of fuzzy clustering called fuzzy c-means clustering with improved fuzzy partitions (IFP-FCM) is extended in this paper, and a generalized algorithm called GIFP-FCM for more effective clustering is proposed. By introducing a novel membership constraint function, a new objective function is constructed, and furthermore, GIFP-FCM clustering is derived. Meanwhile, from the viewpoints of L(p) norm distance measure and competitive learning, the robustness and convergence of the proposed algorithm are analyzed. Furthermore, the classical fuzzy c-means algorithm (FCM) and IFP-FCM can be taken as two special cases of the proposed algorithm. Several experimental results including its application to noisy image texture segmentation are presented to demonstrate its average advantage over FCM and IFP-FCM in both clustering and robustness capabilities.


Weight fluctuations could increase blood pressure in android obese women.
Recent studies have documented a relationship between increased morbidity and mortality from cardiovascular diseases and a history of weight cycling (WC) in obese subjects. We performed a cross-sectional analysis in 96 weight-cycling android obese women, matched with 96 non-weight-cycling android obese women by age, body mass index (BMI) and waist-to-hip ratio (WHR), to evaluate any increase in blood pressure (BP) levels in association with WC. The patients were all between 20 and 45 years old, were non-menopausal, did not smoke, did not take any medication, had normal glucose tolerance and were otherwise healthy. A history of WC was established on the basis of at least five weight losses in the previous 5 years due to dieting, with a weight loss of at least 4.5 kg per cycle. We documented higher levels of casual BP in the weight-cycling obese compared with the non-weight-cycling subjects: 147+/-12/90+/-8 mmHg versus 125+/-14/79+/-8 mmHg (P<0.001). The women with WC showed a statistically significant positive correlation between BP and age, weight, BMI, waist circumference, WHR, total weight regained and weight cycling index (WCI). However, in a multiple regression model only the WHR and WCI contributed significantly to the BP variability. These findings could support the hypothesis that it is the combined exposure of central-type obesity and WC that strongly raises the prediction of hypertension.


The STAMPEDE trial: paradigm-changing data through innovative trial design.
Despite the numerous regulatory approvals for prostate cancer, metastatic prostate cancer remains a huge burden for men worldwide. In an exciting development, James et al. recently published data from the Systemic Therapy in Advanced or Metastatic Prostate Cancer: Evaluation of Drug Efficacy: a multi-stage multi-arm randomised control trial (STAMPEDE). This is an innovative multi-arm multi-stage (MAMS) trial that has utilized one control arm and several comparator arms in order to provide evidence for the inclusion of therapies beyond standard androgen deprivation alone. The patient population included: (I) men with high-risk, non-metastatic, node-negative disease; (II) men with distant-metastatic or node-positive disease; and (III) men with previously-treated prostate cancer by prostatectomy or definitive radiotherapy presenting with relapse. Men were to continue androgen deprivation for at least 2 years. The current data published by this group supports earlier results and provides additional evidence that docetaxel utilized in an up-front fashion provides a survival benefit in men with hormone-sensitive metastatic prostate cancer. Moreover, the initial results from STAMPEDE show how therapies without a demonstrated survival benefit can be efficiently excluded from further study once the likelihood of a benefit is ruled out by a predetermined analysis. In this piece, we will review the STAMPEDE data, contrast it with existing results, and provide our perspectives on how this will affect future trial conduct in the field of prostate cancer.


Resveratrol targeting of AKT and p53 in glioblastoma and glioblastoma stem-like cells to suppress growth and infiltration.
OBJECTIVE Glioblastoma multiforme (GBM) is an aggressive brain cancer with median survival of less than 2 years with current treatment. Glioblastomas exhibit extensive intratumoral and interpatient heterogeneity, suggesting that successful therapies should produce broad anticancer activities. Therefore, the natural nontoxic pleiotropic agent, resveratrol, was studied for antitumorigenic effects against GBM. METHODS Resveratrol's effects on cell proliferation, sphere-forming ability, and invasion were tested using multiple patient-derived GBM stem-like cell (GSC) lines and established U87 glioma cells, and changes in oncogenic AKT and tumor suppressive p53 were analyzed. Resveratrol was also tested in vivo against U87 glioma flank xenografts in mice by using multiple delivery methods, including direct tumor injection. Finally, resveratrol was delivered directly to brain tissue to determine toxicity and achievable drug concentrations in the brain parenchyma. RESULTS Resveratrol significantly inhibited proliferation in U87 glioma and multiple patient-derived GSC lines, demonstrating similar inhibitory concentrations across these phenotypically heterogeneous lines. Resveratrol also inhibited the sphere-forming ability suggesting anti-stem cell effects. Additionally, resveratrol blocked U87 glioma and GSC invasion in an in vitro Matrigel Transwell assay at doses similar to those mediating antiproliferative effects. In U87 glioma cells and GSCs, resveratrol reduced AKT phosphorylation and induced p53 expression and activation that led to transcription of downstream p53 target genes. Resveratrol administration via oral gavage or ad libitum in the water supply significantly suppressed GBM xenograft growth; intratumoral or peritumoral resveratrol injection further suppressed growth and approximated tumor regression. Intracranial resveratrol injection resulted in 100-fold higher local drug concentration compared with intravenous delivery, and with no apparent toxicity. CONCLUSIONS Resveratrol potently inhibited GBM and GSC growth and infiltration, acting partially via AKT deactivation and p53 induction, and suppressed glioblastoma growth in vivo. The ability of resveratrol to modulate AKT and p53, as well as reportedly many other antitumorigenic pathways, is attractive for therapy against a genetically heterogeneous tumor such as GBM. Although resveratrol exhibits low bioavailability when administered orally or intravenously, novel delivery methods such as direct injection (i.e., convection-enhanced delivery) could potentially be used to achieve and maintain therapeutic doses in the brain. Resveratrol's nontoxic nature and broad anti-GBM effects make it a compelling candidate to supplement current GBM therapies.


Investigating the functional anatomy of empathy and forgiveness.
Previous functional brain imaging studies suggest that the ability to infer the intentions and mental states of others (social cognition) is mediated by medial prefrontal cortex. Little is known about the anatomy of empathy and forgiveness. We used functional MRI to detect brain regions engaged by judging others' emotional states and the forgivability of their crimes. Ten volunteers read and made judgements based on social scenarios and a high level baseline task (social reasoning). Both empathic and forgivability judgements activated left superior frontal gyrus, orbitofrontal gyrus and precuneus. Empathic judgements also activated left anterior middle temporal and left inferior frontal gyri, while forgivability judgements activated posterior cingulate gyrus. Empathic and forgivability judgements activate specific regions of the human brain, which we propose contribute to social cohesion.


Classification of femoral periprosthetic fractures.
Classification systems for fractures of the femur with a prosthetic hip in situ are diverse and complex. Most of them are based on the site of the fracture, which is not the most important differentiating factor in treatment planning. The Coventry classification system groups the periprosthetic fractures into 'happy hips' and 'unhappy hips' based on whether or not the stem is loose. In 'happy' hips, treatment only needs to address the fracture itself, unless the fixation has been compromised by the fracture. In the 'unhappy hips', revision of the prosthesis is recommended.


Bile secretion and liver cell heterogeneity in the rat.
The contribution of hepatocytes of different acinar zones to bile salt transport and to the secretion of the BSNDF was studied in the rat. Changes in the removal of 14C-taurocholate from blood, in the biliary secretion of taurocholate, and in canalicular flow were determined after damage of the periportal (acinar zone 1) or centrilobular (acinar zone 3) areas by allyl alcohol or bromobenzene, respectively. The extent of cell necrosis was quantitated by light microscopy, and the quality of the intracellular damage was assessed by electron microscopy. After either periportal or centrilobular damage, surviving cells responded to an intravenous infusion of taurocholate by secreting bile salts into bile at a rate similar to controls. However, following the administration of 14C-taurocholate at high concentrations and as a single bolus, the rate of removal of this isotope from blood was slower than in controls. Both experiments suggested that periportal and centrilobular hepatocytes had the capability for bile salt transport. Consequently, since the concentration of bile salts in sinusoidal blood at each zone determines the relative contribution of hepatocytes to bile salt transport, periportal cells probably transport the largest amount of bile salts reaching the acinus. Canalicular bile flow, on the other hand, decreased following centrilobular cell damage, and this was associated with a high concentration of bile salts in bile. This suggested that at bile salt loads near physiological concentrations, the predominant contribution of centrilobular hepatocytes is to the secretion of the BSNDF.


The effect of transforming growth factor-beta on follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells.
Growth factors have been shown to modulate differentiation of cultured ovarian granulosa cells. Transforming growth factors (TGFs) constitute a family of polypeptide growth factors capable of reversibly inducing anchorage-independent growth in normal cells. Epidermal growth factor (EGF), which has significant structural homology with TGF alpha, has been shown to modulate differentiation of granulosa cells in vitro. Similarly, TGF beta (TGFB) has been found to have significant structural homology with ovarian follicular fluid inhibin. To examine whether TGFB might affect granulosa cell growth or differentiation, rat granulosa cells were cultured in serum-free medium containing insulin for up to 3 days with varying concentrations of TGFB in the presence or absence of FSH. TGFB caused a dose-dependent increase in FSH-stimulated LH/hCG receptor binding, but had no effect on binding in the absence of FSH; TGFB (10.0 ng/ml) further increased FSH-stimulated LH/hCG receptor binding by 48 +/- 8% (P less than 0.02). Similarly, FSH-stimulated progesterone production was increased by TGFB in a dose-dependent manner; TGFB (1.0-10.0 ng/ml) increased FSH-stimulated progesterone production 2- to 3-fold (P less than 0.02). In contrast, EGF (10.0 ng/ml) decreased FSH-stimulated LH/hCG receptor binding by 93 +/- 1% (P less than 0.02). Neither FSH-stimulated intracellular nor extracellular cAMP accumulations were affected by TGFB treatment. However, EGF (10.0 ng/ml) diminished extracellular and intracellular FSH-stimulated cAMP accumulation at 48 and 72 h of culture. Culture protein and DNA content were not significantly affected by TGFB. These results suggest that TGFB may enhance FSH-stimulated LH receptor induction and steroidogenesis by mechanisms that do not further increase net cellular cAMP accumulation; TGFB and EGF can have opposite effects on gonadotropin-dependent differentiation; and products of the TGFB/inhibin gene family may have a capacity for autocrine or paracrine modulation of granulosa cell differentiation.


"What" and "where" in auditory sensory processing: a high-density electrical mapping study of distinct neural processes underlying sound object recognition and sound localization.
Functionally distinct dorsal and ventral auditory pathways for sound localization (WHERE) and sound object recognition (WHAT) have been described in non-human primates. A handful of studies have explored differential processing within these streams in humans, with highly inconsistent findings. Stimuli employed have included simple tones, noise bursts, and speech sounds, with simulated left-right spatial manipulations, and in some cases participants were not required to actively discriminate the stimuli. Our contention is that these paradigms were not well suited to dissociating processing within the two streams. Our aim here was to determine how early in processing we could find evidence for dissociable pathways using better titrated WHAT and WHERE task conditions. The use of more compelling tasks should allow us to amplify differential processing within the dorsal and ventral pathways. We employed high-density electrical mapping using a relatively large and environmentally realistic stimulus set (seven animal calls) delivered from seven free-field spatial locations; with stimulus configuration identical across the "WHERE" and "WHAT" tasks. Topographic analysis revealed distinct dorsal and ventral auditory processing networks during the WHERE and WHAT tasks with the earliest point of divergence seen during the N1 component of the auditory evoked response, beginning at approximately 100 ms. While this difference occurred during the N1 timeframe, it was not a simple modulation of N1 amplitude as it displayed a wholly different topographic distribution to that of the N1. Global dissimilarity measures using topographic modulation analysis confirmed that this difference between tasks was driven by a shift in the underlying generator configuration. Minimum-norm source reconstruction revealed distinct activations that corresponded well with activity within putative dorsal and ventral auditory structures.


Nanomolar oxytocin synergizes with weak electrical afferent stimulation to activate the locomotor CpG of the rat spinal cord in vitro.
Synergizing the effect of afferent fibre stimulation with pharmacological interventions is a desirable goal to trigger spinal locomotor activity, especially after injury. Thus, to better understand the mechanisms to optimize this process, we studied the role of the neuropeptide oxytocin (previously shown to stimulate locomotor networks) on network and motoneuron properties using the isolated neonatal rat spinal cord. On motoneurons oxytocin (1 nM-1 μM) generated sporadic bursts with superimposed firing and dose-dependent depolarization. No desensitization was observed despite repeated applications. Tetrodotoxin completely blocked the effects of oxytocin, demonstrating the network origin of the responses. Recording motoneuron pool activity from lumbar ventral roots showed oxytocin mediated depolarization with synchronous bursts, and depression of reflex responses in a stimulus and peptide-concentration dependent fashion. Disinhibited bursting caused by strychnine and bicuculline was accelerated by oxytocin whose action was blocked by the oxytocin antagonist atosiban. Fictive locomotion appeared when subthreshold concentrations of NMDA plus 5HT were coapplied with oxytocin, an effect prevented after 24 h incubation with the inhibitor of 5HT synthesis, PCPA. When fictive locomotion was fully manifested, oxytocin did not change periodicity, although cycle amplitude became smaller. A novel protocol of electrical stimulation based on noisy waveforms and applied to one dorsal root evoked stereotypic fictive locomotion. Whenever the stimulus intensity was subthreshold, low doses of oxytocin triggered fictive locomotion although oxytocin per se did not affect primary afferent depolarization evoked by dorsal root pulses. Among the several functional targets for the action of oxytocin at lumbar spinal cord level, the present results highlight how small concentrations of this peptide could bring spinal networks to threshold for fictive locomotion in combination with other protocols, and delineate the use of oxytocin to strengthen the efficiency of electrical stimulation to activate locomotor circuits.


ATP production rate via creatine kinase or ATP synthase in vivo: a novel superfast magnetization saturation transfer method.
³¹P magnetization saturation transfer (MST) experiment is the most widely used method to study ATP metabolism kinetics. However, its lengthy data acquisition time greatly limits the wide biomedical applications in vivo, especially for studies requiring high spatial and temporal resolutions. We aimed to develop a novel superfast MST method that can accurately quantify ATP production rate constants (k(f)) through creatine kinase (CK) or ATP synthase (ATPase) with 2 spectra. The T₁(nom) (T₁ nominal) method uses a correction factor to compensate the partially relaxed MST experiments, thus allowing measurement of enzyme kinetics with an arbitrary repetition time and flip angle, which consequently reduces the data acquisition time of a transmurally differentiated CK k(f) measurement by 91% as compared with the conventional method with spatial localization. The novel T₁(nom) method is validated theoretically with numeric simulation, and further verified with in vivo swine hearts, as well as CK and ATPase activities in rat brain at 9.4 Tesla. Importantly, the in vivo data from swine hearts demonstrate, for the first time, that within an observation window of 30 minutes, the inhibition of CK activity by iodoacetamide does not limit left ventricular chamber contractile function. A novel MST method for superfast examination of enzyme kinetics in vivo has been developed and verified theoretically and experimentally. In the in vivo normal heart, redundant multiple supporting systems of myocardial ATP production, transportation, and utilization exist, such that inhibition of one mechanism does not impair the normal left ventricular contractile performance.


Four-membered heterocycles with a carbon-heteroatom exocyclic double bond at the 3-position: puckering potential and thermodynamic properties.
Despite the specific importance of four-membered heterocycles with a carbon-heteroatom double bond at the 3-position in organic and medicinal chemistry, little attention has been given up to now to theoretical computational studies on these molecules. However, the overall geometry, and degree of ring puckering especially, could significantly influence the reactivity and biological properties of these four-membered ring compounds. In this paper, focus is made on the influence of different substituents on the equilibrium geometry, ring puckering potential, and thermodynamic quantities. It was found that these properties are mainly affected by the heteroatom (oxygen, nitrogen, sulfur, phosphorus) contained in the ring skeleton. Moreover, the correct description of the puckering potential with the hindered rotor treatment leads to substantial corrections on the thermodynamic properties in the harmonic oscillator approximation.


Does a PEEK Femoral TKA Implant Preserve Intact Femoral Surface Strains Compared With CoCr? A Preliminary Laboratory Study.
Both the material and geometry of a total knee arthroplasty (TKA) component influence the induced periprosthetic bone strain field. Strain, a measure of the local relative deformation in a structure, corresponds to the mechanical stimulus that governs bone remodeling and is therefore a useful in vitro biomechanical measure for assessing the response of bone to new implant designs and materials. A polyetheretherketone (PEEK) femoral implant has the potential to promote bone strains closer to that of natural bone as a result of its low elastic modulus compared with cobalt-chromium (CoCr). In the present study, we used a Digital Image Correlation (DIC) technique to answer the following question: Does a PEEK TKA femoral component induce a more physiologically normal bone strain distribution than a CoCr component? To achieve this, a DIC test protocol was developed for periprosthetic bone strain assessment using an analog model; the protocol aimed to minimize errors in strain assessment through the selection of appropriate analysis parameters. Three synthetic bone femurs were used in this experiment. One was implanted with a CoCr femoral component and one with a PEEK femoral component. The third (unimplanted) femur was intact and used as the physiological reference (control) model. All models were subjected to standing loads on the corresponding polyethylene (ultrahigh-molecular-weight polyethylene) tibial component, and speckle image data were acquired for surface strain analysis using DIC in six repeat tests. The strain in 16 regions of interest on the lateral surface of each of the implanted bone models was plotted for comparison with the corresponding strains in the intact case. A Wilcoxon signed-rank test was used to test for difference at the 5% significance level. Surface analog bone strain after CoCr implantation indicated strain shielding (R2 = 0.6178 with slope, β = 0.4314) and was lower than the intact case (p = 0.014). The strain after implantation with the PEEK implant deviated less from the intact case (R2 = 0.7972 with slope β = 0.939) with no difference (p = 0.231). The strain shielding observed with the contemporary CoCr implant, consistent with clinical bone mineral density change data reported by others, may be reduced by using a PEEK implant. This bone analog in vitro study suggests that a PEEK femoral component could transfer more physiologically normal bone strains with a potentially reduced stress shielding effect, which may improve long-term bone preservation. Additional studies including paired cadaver tests are necessary to test the hypothesis further.


Developmental potential in the first 5 years for children in developing countries.
Many children younger than 5 years in developing countries are exposed to multiple risks, including poverty, malnutrition, poor health, and unstimulating home environments, which detrimentally affect their cognitive, motor, and social-emotional development. There are few national statistics on the development of young children in developing countries. We therefore identified two factors with available worldwide data--the prevalence of early childhood stunting and the number of people living in absolute poverty--to use as indicators of poor development. We show that both indicators are closely associated with poor cognitive and educational performance in children and use them to estimate that over 200 million children under 5 years are not fulfilling their developmental potential. Most of these children live in south Asia and sub-Saharan Africa. These disadvantaged children are likely to do poorly in school and subsequently have low incomes, high fertility, and provide poor care for their children, thus contributing to the intergenerational transmission of poverty.


Extensive Distribution of the Lyme Disease Bacterium, Borrelia burgdorferi Sensu Lato, in Multiple Tick Species Parasitizing Avian and Mammalian Hosts across Canada.
Lyme disease, caused by the spirochetal bacterium, Borrelia burgdorferi sensu lato (Bbsl), is typically transmitted by hard-bodied ticks (Acari: Ixodidae). Whenever this tick-borne zoonosis is mentioned in medical clinics and emergency rooms, it sparks a firestorm of controversy. Denial often sets in, and healthcare practitioners dismiss the fact that this pathogenic spirochetosis is present in their area. For distribution of Bbsl across Canada, we conducted a 4-year, tick⁻host study (2013⁻2016), and collected ticks from avian and mammalian hosts from Atlantic Canada to the West Coast. Overall, 1265 ticks representing 27 tick species belonging to four genera were collected. Of the 18 tick species tested, 15 species (83%) were positive for Bbsl and, of these infected ticks, 6 species bite humans. Overall, 13 of 18 tick species tested are human-biting ticks. Our data suggest that a 6-tick, enzootic maintenance cycle of Bbsl is present in southwestern B.C., and five of these tick species bite humans. Biogeographically, the groundhog tick, Ixodes cookei, has extended its home range from central and eastern Canada to southwestern British Columbia (B.C.). We posit that the Fox Sparrow, Passerella iliaca, is a reservoir-competent host for Bbsl. The Bay-breasted Warbler, Setophaga castanea, and the Tennessee Warbler, Vermivora peregrina, are new host records for the blacklegged tick, Ixodes scapularis. We provide the first report of a Bbsl-positive Amblyomma longirostre larva parasitizing a bird; this bird parasitism suggests that a Willow Flycatcher is a competent reservoir of Bbsl. Our findings show that Bbsl is present in all provinces, and that multiple tick species are implicated in the enzootic maintenance cycle of this pathogen. Ultimately, Bbsl poses a serious public health contagion Canada-wide.


No association between habitat, autogeny and genetics in Moroccan Culex pipiens populations.
Mosquitoes of the Culex pipiens complex are found across the globe and are the focus of many research studies. Among the temperate species C. pipiens sensu stricto (s.s.), two forms are usually described: molestus and pipiens. These two forms are indistinguishable in terms of morphology but show behavioral and physiological differences that may have consequences for their associated epidemiology. The two forms are well defined in the northern part of the species distribution, where autogeny is strictly associated with the molestus form. However, whether the two remain distinct and show the characteristic differences in behavior is less clear in North Africa, at the southern edge of their range. The association between autogeny, as determined by ovarian dissection, and molecular forms, based on the CQ11 microsatellite marker, was studied in six Moroccan populations of C. pipiens. An overall low prevalence of autogeny was found at three of the Moroccan regions studied, although it reached 17.5% in the Agadir population. The prevalence of form-specific CQ11 alleles was quite similar across all populations, with the molestus allele being rarer (approx. 15%), except in the Agadir population where it reached 43.3%. We found significant deficits in heterozygotes at the diagnostic CQ11 locus in three populations, but the three other populations showed no significant departure from panmixia, which is in line with the results of a retrospective analysis of the published data. More importantly, we found no association between the autogeny status and CQ11 genotypes, despite the many females analyzed. There was limited evidence for two discrete forms in Morocco, where individuals carrying pipiens and molestus alleles breed and mate in the same sites and are equally likely to be capable of autogeny. These observations are discussed in the epidemiological context of Morocco, where C. pipiens is the main vector of several arboviruses.


Effect of Elliptical High Intensity Interval Training on Metabolic Risk Factor in Pre- and Type 2 Diabetes Patients: A Pilot Study.
The purpose of the current study was to examine the impact of 12 weeks of elliptical high intensity interval training (HIIT) on metabolic risk factors and body composition in pre- and type 2 diabetes patients. Sixteen pre- (n = 8) and type 2 diabetes (n = 8) participants completed this study. Fasting blood glucose, HbA1c, anthropometric measurements, body composition (DXA), blood pressure, resting heart rate, VO2max, and dietary factors, as well as total and physical activity energy expenditure, were measured. The HIIT program on the elliptical was performed 3 times a week for 12 weeks. After the intervention, we observed a significant improvement for fasting blood glucose, waist and hip circumference, appendicular fat mass, leg lean body mass and appendicular lean body mass, systolic blood pressure, resting heart rate, and VO2max (P < .05). In addition, we noted a lower tendency for leg fat mass (P = .06) and diastolic blood pressure (P = .05) as well as a higher tendency for total energy expenditure (P = .06) after the intervention. The current study indicates that elliptical HIIT seems to improve metabolic risk factors and body composition in pre- and type 2 diabetes patients.


Onset of anthrax toxin pore formation.
Protective antigen (PA) is the anthrax toxin protein recognized by capillary morphogenesis gene 2 (CMG2), a transmembrane cellular receptor. Upon activation, seven ligand-receptor units self-assemble into a heptameric ring-like complex that becomes endocytozed by the host cell. A critical step in the subsequent intoxication process is the formation and insertion of a pore into the endosome membrane by PA. The pore conversion requires a change in binding between PA and its receptor in the acidified endosome environment. Molecular dynamics simulations totaling approximately 136 ns on systems of over 92,000 atoms were performed. The simulations revealed how the PA-CMG2 complex, stable at neutral conditions, becomes transformed at low pH upon protonation of His-121 and Glu-122, two conserved amino acids of the receptor. The protonation disrupts a salt bridge important for the binding stability and leads to the detachment of PA domain II, which weakens the stability of the PA-CMG2 complex significantly, and subsequently releases a PA segment needed for pore formation. The simulations also explain the great strength of the PA-CMG2 complex achieves through extraordinary coordination of a divalent cation.


UroVysion testing can lead to early identification of intravesical therapy failure in patients with high risk non-muscle invasive bladder cancer.
In this study, we investigated the ability of UroVysion to assess response to intravesical therapy in patients with high risk superficial bladder tumors. We performed a retrospective review of patients undergoing intravesical therapy for high risk superficial bladder tumors. Urine specimens were collected for UroVysion analysis before and immediately after a course of intravesical therapy. Cytology and cystoscopy were performed six weeks after treatment, using either a positive cytology or visible abnormality on cystoscopy as a prompt for biopsy. The operating characteristics of the UroVysion test were then determined. 41 patients were identified in whom 47 cycles of induction and 41 cycles of maintenance intravesical therapy were given during the study period. This yielded a total of 88 treatment and evaluation cycles. Median follow-up was 9 months per induction (range 1-21 months) and 13 months per patient (range 1-25 months). A total of 133 urine samples were collected for UroVysionTM of which 40 were positive. Based upon standard clinical evaluation, 41 biopsies were performed which detected 20 recurrences. UroVysionTM testing performed immediately upon completion of therapy for the 41 patients undergoing biopsy yielded a sensitivity, specificity, and accuracy of 85%, 61%, and 71%. The use of UroVysionTM following intravesical therapy for high-risk superficial bladder tumors helps to identify patients at high risk of refractory or recurrent disease who should undergo immediate biopsy under anesthesia.


Human Puumala hantavirus infection in northern Sweden; increased seroprevalence and association to risk and health factors.
The rodent borne Puumala hantavirus (PUUV) causes haemorrhagic fever with renal syndrome in central and northern Europe. The number of cases has increased and northern Sweden has experienced large outbreaks in 1998 and 2006-2007 which raised questions regarding the level of immunity in the human population. A randomly selected population aged between 25 and 74 years from northern Sweden were invited during 2009 to participate in a WHO project for monitoring of trends and determinants in cardiovascular disease. Health and risk factors were evaluated and sera from 1,600 participants were available for analysis for specific PUUV IgG antibodies using a recombinant PUUV nucleocapsid protein ELISA. The overall seroprevalence in the investigated population was 13.4 %, which is a 50 % increase compared to a similar study only two decades previously. The prevalence of PUUV IgG increased with age, and among 65-75 years it was 22 %. More men (15.3 %) than women (11.4 %) were seropositive (p < 0.05). The identified risk factors were smoking (OR = 1.67), living in rural areas (OR = 1.92), and owning farmland or forest (OR = 2.44). No associations were found between previous PUUV exposure and chronic lung disease, diabetes, hypertension, renal dysfunction, stroke or myocardial infarction. PUUV is a common infection in northern Sweden and there is a high life time risk to acquire PUUV infection in endemic areas. Certain risk factors as living in rural areas and smoking were identified. Groups with increased risk should be targeted for future vaccination when available, and should also be informed about appropriate protection from rodent secreta.


The global consequence of disruption of the AcrAB-TolC efflux pump in Salmonella enterica includes reduced expression of SPI-1 and other attributes required to infect the host.
The mechanisms by which RND pumps contribute to pathogenicity are currently not understood. Using the AcrAB-TolC system as a paradigm multidrug-resistant efflux pump and Salmonella enterica serovar Typhimurium as a model pathogen, we have demonstrated that AcrA, AcrB, and TolC are each required for efficient adhesion to and invasion of epithelial cells and macrophages by Salmonella in vitro. In addition, AcrB and TolC are necessary for Salmonella to colonize poultry. Mutants lacking acrA, acrB, or tolC showed differential expression of major operons and proteins involved in pathogenesis. These included chemotaxis and motility genes, including cheWY and flgLMK and 14 Salmonella pathogenicity island (SPI)-1-encoded type III secretion system genes, including sopE, and associated effector proteins. Reverse transcription-PCR confirmed these data for identical mutants in two other S. Typhimurium backgrounds. Western blotting showed reduced production of SipA, SipB, and SipC. The absence of AcrB or TolC also caused widespread repression of chemotaxis and motility genes in these mutants, and for acrB::aph, this was associated with decreased motility. For mutants lacking a functional acrA or acrB gene, the nap and nir operons were repressed, and both mutants grew poorly in anaerobic conditions. All phenotypes were restored to that of the wild type by trans-complementation with the wild-type allele of the respective inactivated gene. These data explain how mutants lacking a component of AcrAB-TolC are attenuated and that this phenotype is a result of decreased expression of numerous genes encoding proteins involved in pathogenicity. The link between antibiotic resistance and pathogenicity establishes the AcrAB-TolC system as fundamental to the biology of Salmonella.


The Arabidopsis leaf transcriptome reveals distinct but also overlapping responses to colonization by phyllosphere commensals and pathogen infection with impact on plant health.
Plants are colonized by a variety of bacteria, most of which are not pathogenic. Currently, the plant responses to phyllosphere commensals or to pathogen infection in the presence of commensals are not well understood. Here, we examined the transcriptional response of Arabidopsis thaliana leaves to colonization by common commensal bacteria in a gnotobiotic system using RNA sequencing and conducted plant mutant assays. Arabidopsis responded differently to the model bacteria Sphingomonas melonis Fr1 (S.Fr1) and Methylobacterium extorquens PA1 (M.PA1). Whereas M.PA1 only marginally affected the expression of plant genes (< 10), S.Fr1 colonization changed the expression of almost 400 genes. For the latter, genes related to defense responses were activated and partly overlapped with those elicited by the pathogen Pseudomonas syringae DC3000 (Pst). As S.Fr1 is able to mediate plant protective activity against Pst, we tested plant immunity mutants and found that the pattern-recognition co-receptor mutant bak1/bkk1 showed attenuated S.Fr1-dependent plant protection. The experiments demonstrate that the plant responds differently to members of its natural phyllosphere microbiota. A subset of commensals trigger expression of defense-related genes and thereby may contribute to plant health upon pathogen encounter.


Analysis of the immunomodulatory properties of two heat-killed mycobacterial preparations in a human whole blood model.
The significant role played by mycobacteria in modulating immune responses through enhancing the crosstalk between innate and adaptive immunity has been highlighted in several studies. Owing to their unique antigenic profile, heat killed (HK) preparations of rapid-growing mycobacteria, currently undergoing clinical development, have been assessed as adjuvant therapy in various diseases. The purpose of this study is to investigate the regulation of leukocyte surface receptors, in whole blood from healthy donors, following in vitro stimulation with HK Mycobacterium vaccae (M. vaccae) or M. obuense. We have demonstrated the ability of both mycobacterial preparations to target monocytes and neutrophils and to regulate the surface expression of selected adhesion receptors, antigen-presenting and costimulatory receptors, pattern recognition receptors, complement and Fc receptors, as well as cytokine/chemokine receptors. Toll-like receptors (TLRs) 1 and 2 were also shown to be involved in mediating the M. obuense-induced upregulation of selected surface receptors on monocytes. Whole blood stimulation with M. vaccae or M. obuense resulted in a significant increase in the secretion of a specific set of cytokines and chemokines. Both mycobacterial preparations induced strong antigen-specific proliferative responses in peripheral blood mononuclear cells. Collectively, our data shows that M. vaccae and M. obuense have the potential to act as potent immunomodulators. Future research based on these findings may reveal novel immune pathways induced by these preparations with potential implication for their use in diverse immunotherapeutic approaches.


Heparin-induced thrombocytopenia: analysis of risk factors in medical inpatients.
Heparin-induced thrombocytopenia (HIT) is an unpredictable reaction to heparin characterized by thrombocytopenia and increased risk of life-threatening venous and/or arterial thrombosis. Data are lacking regarding additional risk factors that may be associated with the development of HIT. This study aimed to identify the risk factors that may be associated with HIT in medical inpatients receiving heparin. Twenty five thousand six hundred and fifty-three patients admitted to the medicine service who received heparin product were reviewed retrospectively. The diagnosis of HIT was confirmed if the platelet count dropped >50% from baseline and there was a positive laboratory HIT assay. Fifty-five cases of in-hospital HIT were observed. Multivariate analysis identified the administration of full anticoagulation dose with unfractionated heparin or exposure to heparin products for more than 5 d with an increased risk of HIT. Moreover, patients who were on haemodialysis, carried a diagnosis of autoimmune disease, gout or heart failure were also at increased risk. The results suggest that when using heparin products in these patient cohorts, increased surveillance for HIT is necessary.


Altered avr-14B gene transcription patterns in ivermectin-resistant isolates of the cattle parasites, Cooperia oncophora and Ostertagia ostertagi.
Ivermectin (IVM) resistance is an emerging problem for the control of gastrointestinal nematodes of cattle such as Cooperia oncophora and Ostertagia ostertagi. Although there is still a poor understanding of the molecular basis of macrocyclic lactone (ML)-resistance, it is clear that IVM exerts its activity by binding to glutamate-gated chloride (GluCl) channels within the parasite's neuromuscular system. One of the GluCl genes (avr-14) encodes, via alternative splicing, two subunits, AVR-14A and AVR-14B; the latter is suggested to be the main target for IVM. The genomic DNA (gDNA) sequence of avr-14 in C. oncophora contains 21 exons separated by 20 introns and spans approximately 10 kb of gDNA. Intron 13 contains a sequence with high homology to a mammalian mariner transposase. The L256F polymorphism in the avr-14 gene, which was shown to be associated with IVM resistance in a UK isolate of C. oncophora, was not found in the IVM-resistant C. oncophora and O. ostertagi isolates investigated in this study. However, genetic analyses on C. oncophora indicated a loss in allelic diversity of the avr-14 gene in the resistant isolates compared with the susceptible isolate. This suggests that the avr-14 gene, or another genetically linked locus, is under selection in these Belgian C. oncophora isolates. Comparison of the full-length avr-14B coding sequence in the susceptible and resistant C. oncophora isolates did not show any polymorphisms specifically linked to IVM resistance, although a decrease in the number of avr-14B isoforms was observed in the resistant isolates compared with the susceptible one. Measuring the transcription levels of avr-14B in adult male and female C. oncophora and O. ostertagi worms showed significantly lower levels in resistant worms compared with susceptible ones. Whether the down-regulation of this IVM target actually contributes to the resistance mechanism in these worms remains unclear.


Genome-wide analysis identifies MYND-domain protein Mub1 as an essential factor for Rpn4 ubiquitylation.
The proteasome homeostasis in Saccharomyces cerevisiae is regulated by a negative feedback circuit in which the Rpn4 transcription factor upregulates the proteasome genes and is rapidly degraded by the proteasome. Previous work has identified Ubr2 and Rad6 as the cognate E3 and E2 enzymes for Rpn4 ubiquitylation. However, our recent attempts to ubiquitylate Rpn4 using purified Ubr2 and Rad6 proteins in a reconstitution system have been unsuccessful, suggesting that an additional factor is required for Rpn4 ubiquitylation. Here, we screened the entire collection of the single-gene-deletion yeast mutants generated by the Saccharomyces Genome Deletion Project and identified the mub1Delta mutant defective in ubiquitin-dependent degradation of Rpn4. An in vitro reconstitution ubiquitylation assay confirms that Mub1 is the missing factor for Rpn4 ubiquitylation. We further show that Mub1 directly interacts with Ubr2 and Rpn4. The MYND domain of Mub1 may play an important role in Rpn4 ubiquitylation. Interestingly, Mub1 itself is a short-lived protein and its degradation is dependent on the Ubr2/Rad6 ubiquitin ligase. Together, these data suggest that Mub1 and Ubr2 cooperate to transfer ubiquitin to Rpn4 from Rad6 and that Mub1 may switch from a partner to a substrate of the Ubr2/Rad6 ubiquitin ligase.


Energy cost of wheel running in house mice: implications for coadaptation of locomotion and energy budgets.
Laboratory house mice (Mus domesticus) that had experienced 10 generations of artificial selection for high levels of voluntary wheel running ran about 70% more total revolutions per day than did mice from random-bred control lines. The difference resulted primarily from increased average velocities rather than from increased time spent running. Within all eight lines (four selected, four control), females ran more than males. Average daily running distances ranged from 4.4 km in control males to 11.6 km in selected females. Whole-animal food consumption was statistically indistinguishable in the selected and control lines. However, mice from selected lines averaged approximately 10% smaller in body mass, and mass-adjusted food consumption was 4% higher in selected lines than in controls. The incremental cost of locomotion (grams food/revolution), computed as the partial regression slope of food consumption on revolutions run per day, did not differ between selected and control mice. On a 24-h basis, the total incremental cost of running (covering a distance) amounted to only 4.4% of food consumption in the control lines and 7.5% in the selected ones. However, the daily incremental cost of time active is higher (15.4% and 13.1% of total food consumption in selected and control lines, respectively). If wheel running in the selected lines continues to increase mainly by increases in velocity, then constraints related to energy acquisition are unlikely to be an important factor limiting further selective gain. More generally, our results suggest that, in small mammals, a substantial evolutionary increase in daily movement distances can be achieved by increasing running speed, without remarkable increases in total energy expenditure.


Prevalence of respiratory pathogens in diseased, non-vaccinated, routinely medicated veal calves.
The prevalence of respiratory pathogens in diseased veal calves was determined in 24 respiratory disease outbreaks in 15 herds in Belgium. Bacteria were cultured from nasopharyngeal swabs and seroconversion against viruses and Mycoplasma bovis was determined on paired sera. At the individual calf level, Mycoplasma species, Mannheimia haemolytica and Pasteurella multocida, were isolated from 70.5 per cent, 21.5 per cent and 26.0 per cent of swabs, respectively. At the herd level, the presence of M bovis could be confirmed in 84.6 per cent of the herds examined. Seroconversion against bovine viral diarrhoea virus (BVDV) was present in 71.4 per cent of herds, parainfluenzavirus type 3 in 53.3 per cent, bovine respiratory syncytial virus in 40.0 per cent, bovine adenovirus type 3 in 46.7 per cent, bovine coronavirus in 30.0 per cent, and bovine herpesvirus type 1 in 26.7 per cent. At postmortem examination, Mycoplasma species could be cultured from 61.9 per cent of pneumonic lungs (n=21). Sixty per cent of calves tested were positive for BVDV (n=20), and 20.0 per cent were positive for bovine respiratory syncytial virus (n=16).


Effect of breast cancer surgery on chest tightness and upper limb dysfunction.
This study aimed to investigate the possible changes in anterior chest tightness after breast cancer surgery. We also try to investigate whether anterior chest tightness is associated with upper-limb dysfunction after breast cancer surgery. Eighty-three women who underwent breast cancer surgery were evaluated before and 2 weeks, 3 months, and 9 months after surgery. Anterior chest tightness was measured using the length of the pectoralis minor muscle through 2 methods (length from the coracoid process to the fourth rib and linear distance from the table to the posterior acromion with supine position). Shoulder range of motion and the K-DASH (Korean version of Disability Arm and Shoulder Questionnaire) score were measured to quantify functional performance of upper limb. Anterior chest tightness of patients with breast cancer significantly increased after surgery. Upper limb dysfunction was observed such as reduced shoulder range-of-motion and increased K-DASH score over time. Increase in chest tightness was correlated with shoulder range-of-motion reduction. Chest tightness was not correlated with K-DASH score directly. However, shoulder range-of-motion reduction was significantly correlated with K-DASH score. Chest tightness and upper limb dysfunction increased in breast cancer survivor. Increase in chest tightness after surgery is associated with upper limb dysfunction and careful attention is required.


Surveillance of physical activity in the UK is flawed: validation of the Health Survey for England Physical Activity Questionnaire.
Public health surveillance of physical activity in children in the UK depends on a parent-reported physical activity questionnaire which has not been validated. We aimed to validate this questionnaire against measurement of physical activity using accelerometry in 6-7-year-old children. In 130 children aged 6-7 years (64 boys, 66 girls) we estimated habitual moderate-vigorous intensity physical activity (MVPA) using the Health Survey for England parent-report questionnaire for physical activity. For the same time period and the same children, we measured MVPA objectively using 7-day accelerometry with the Actigraph accelerometer. The questionnaire over-estimated MVPA significantly (paired t test, p<0.01). Mean error (bias) when using the questionnaire was 122 min/day (95% CI 124 to 169). Mean time spent in MVPA was 146 min/day (95% CI 124 to 169) using the questionnaire and 24 min/day (95% CI 22 to 26) using the accelerometer. Rank order correlations between MVPA measured by accelerometer and estimated by the questionnaire were not statistically significant. Public health surveillance of physical activity should not rely on this questionnaire. Levels of habitual physical activity in children are likely to be substantially lower than those reported in UK health surveys.


A homeopathic remedy from arnica, marigold, St. John's wort and comfrey accelerates in vitro wound scratch closure of NIH 3T3 fibroblasts.
Drugs of plant origin such as Arnica montana, Calendula officinalis or Hypericum perforatum have been frequently used to promote wound healing. While their effect on wound healing using preparations at pharmacological concentrations was supported by several in vitro and clinical studies, investigations of herbal homeopathic remedies on wound healing process are rare. The objective of this study was to investigate the effect of a commercial low potency homeopathic remedy Similasan® Arnica plus Spray on wound closure in a controlled, blind trial in vitro. We investigated the effect of an ethanolic preparation composed of equal parts of Arnica montana 4x, Calendula officinalis 4x, Hypericum perforatum 4x and Symphytum officinale 6x (0712-2), its succussed hydroalcoholic solvent (0712-1) and unsuccussed solvent (0712-3) on NIH 3T3 fibroblasts. Cell viability was determined by WST-1 assay, cell growth using BrdU uptake, cell migration by chemotaxis assay and wound closure by CytoSelect ™Wound Healing Assay Kit which generated a defined "wound field". All assays were performed in three independent controlled experiments. None of the three substances affected cell viability and none showed a stimulating effect on cell proliferation. Preparation (0712-2) exerted a stimulating effect on fibroblast migration (31.9%) vs 14.7% with succussed solvent (0712-1) at 1:100 dilutions (p < 0.001). Unsuccussed solvent (0712-3) had no influence on cell migration (6.3%; p > 0.05). Preparation (0712-2) at a dilution of 1:100 promoted in vitro wound closure by 59.5% and differed significantly (p < 0.001) from succussed solvent (0712-1), which caused 22.1% wound closure. Results of this study showed that the low potency homeopathic remedy (0712-2) exerted in vitro wound closure potential in NIH 3T3 fibroblasts. This effect resulted from stimulation of fibroblasts motility rather than of their mitosis.


Quantitative analysis of multiple urinary biomarkers of carcinoid tumors through gold-nanoparticle-assisted laser desorption/ionization time-of-flight mass spectrometry.
A simple technique for quantitative analysis of four urinary biomarkers, tryptophan (TRP), 5-hydroxytryptophan (5-HTP), 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) of carcinoid tumors is developed using gold nanoparticles as the assisted matrix in surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF MS). The optimal SALDI conditions for the efficient ionization of those biomarkers are systematically explored by the adjustments of the concentrations of gold nanoparticles and internal standards. The mass spectra with strong signals and minimal background noise are obtained using 1-naphthaleneacetamide (NAD) as the internal standard. The calibration curves of the biomarker concentrations are determined using SALDI-TOF MS and the high linearity is obtained in all samples. For future clinical testing, multiplexed detection of those biomarkers in the urine samples of healthy males is performed. The successful quantitative detections of TRP, 5-HTP, 5-HT and 5-HIAA indicate that our technique provided great potentials to be developed a simple and rapid platform for the tumor biomarker detections.


Future savings from measles eradication in industrialized countries.
Estimates are made of monetary savings associated with measles eradication in seven industrialized countries. Three scenarios were studied: First, changing from the present two-dose measles-mumps-rubella (MMR) immunization schedule to one-dose of MMR; second, the use of an MMR and mumps-rubella schedule; or third, continuing the present schedule. Results show that the largest savings (US $623 million) would be achieved by changing to a one-dose MMR schedule with an assumption of a 3% discount rate and measles eradication in 2010. The smallest overall savings would result from option 3, by use of a 5% discount rate and the assumption that measles eradication occurs in 2020 ($10 million). These savings are less than previously estimated for the United States, partly because of the assumption that measles vaccines will continue to be delivered in response to possible bioterrorism threats.