Deficiency of either cyclooxygenase (COX)-1 or COX-2 alters epidermal differentiation and reduces mouse skin tumorigenesis.
Nonsteroidal anti-inflammatory drugs are widely reported to inhibit carcinogenesis in humans and in rodents. These drugs are believed to act by inhibiting one or both of the known isoforms of cyclooxygenase (COX). However, COX-2, and not COX-1, is the isoform most frequently reported to have a key role in tumor development. Here we report that homozygous deficiency of either COX-1 or COX-2 reduces skin tumorigenesis by 75% in a multistage mouse skin model. Reduced tumorigenesis was observed even though the levels of stable 7,12-dimethylbenz(a)anthracene-DNA adducts were increased about 2-fold in the COX-deficient mice compared with wild-type mice. The premature onset of keratinocyte terminal differentiation appeared to be the cellular event leading to the reduced tumorigenesis because keratin 1 and keratin 10, two keratins that indicate the commitment of keratinocytes to differentiate, were expressed 8-13-fold and 10-20-fold more frequently in epidermal basal cells of the COX-1-deficient and COX-2-deficient mice, respectively, than in wild-type mice. Papillomas on the COX-deficient mice also displayed the premature onset of keratinocyte terminal differentiation. However, loricrin, a late marker of epidermal differentiation, was not significantly altered, suggesting that it was the early stages of keratinocyte differentiation that were primarily affected by COX deficiency. Because keratin 5, a keratin associated with basal cells, was detected differently in papillomas of COX-1-deficient as compared with COX-2-deficient mice, it appears that the isoforms do not have identical roles in papilloma development. Interestingly, apoptosis, a cellular process associated with nonsteroidal anti-inflammatory drug-induced inhibition of tumorigenesis, was not significantly altered in the epidermis or in papillomas of the COX-deficient mice. Thus, both COX-1 and COX-2 have roles in keratinocyte differentiation, and we propose that the absence of either isoform causes premature terminal differentiation of initiated keratinocytes and reduced tumor formation.


Boni mores and consent for child research in South Africa.
Consent is required for almost all health research. In order for consent to be valid a number of requirements must be met including that the consent cannot be contra bonos mores or contrary to public policy. This principle has its roots in the common law and it is used to ensure that the consent to harm, or the risk of harm, is permitted or ought to be permitted by the legal order. Recently, it has also become a statutory requirement embedded in the consent obligations relating to non-therapeutic health research with minors. Section 71 of the National Health Act provides that the Minister of Health (or potentially his or her delegated authority) must provide consent to non-therapeutic research with minors. However, such consent may not be granted if 'the reasons for the consent to the research or experimentation are contrary to public policy'. Limited work has been done on how to determine when consent to health research with children would be contrary to public policy. This article attempts to begin the debate by describing the boni mores principle, setting out some of the general factors that could be used to assess whether consent is consistent with it and suggesting how they could be applied to health research. The article concludes by stating that simply requiring proxy consent for non-therapeutic health research with children is insufficient as it cannot always be assumed that proxy consenters will act in the best interests of the child. Thus the boni mores principle acts as a limit on autonomy in order to protect the child participant. It is further submitted that establishing when consent to health research is consistent with public policy requires an assessment of whether the research is consistent with constitutional values, prevailing legal norms regarding children, and an assessment of the legal convictions of the community.


Cytoprotective function of heme oxygenase 1 induced by a nitrated cyclic nucleotide formed during murine salmonellosis.
Signaling mechanisms of NO-mediated host defense are yet to be elucidated. In this study, we report a unique signal pathway for cytoprotection during Salmonella infection that involves heme oxygenase 1 (HO-1) induced by a nitrated cyclic nucleotide, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP). Wild-type C57BL/6 mice and C57BL/6 mice lacking inducible NO synthase (iNOS) were infected with Salmonella enterica serovar Typhimurium LT2. HO-1 was markedly up-regulated during the infection, the level being significantly higher in wild-type mice than in iNOS-deficient mice. HO-1 up-regulation was associated with 8-nitro-cGMP formation detected immunohistochemically in Salmonella-infected mouse liver and peritoneal macrophages. 8-Nitro-cGMP either exogenously added or formed endogenously induced HO-1 in cultured macrophages infected with Salmonella. HO-1 inhibition by polyethylene glycol-conjugated zinc-protoporphyrin IX impaired intracellular killing of bacteria in mouse liver and in both RAW 264 cells and peritoneal macrophages. Infection-associated apoptosis was also markedly increased in polyethylene glycol-conjugated zinc-protoporphyrin IX-treated mouse liver cells and cultured macrophages. This effect of HO-1 inhibition was further confirmed by using HO-1 short interfering RNA in peritoneal macrophages. Our results suggest that HO-1 induced by NO-mediated 8-nitro-cGMP formation contributes, via its potent cytoprotective function, to host defense during murine salmonellosis.


High incidence of in-hospital pulmonary embolism following joint arthroplasty with dalteparin prophylaxis.
The in-hospital incidence of pulmonary embolism (PE) in patients undergoing elective joint arthroplasty who receive a minimum of 10 days of dalteparin prophylaxis is reported to be less than 1%. Recent clinical experience raised suspicion that the incidence of PE was significantly higher at our tertiary care institution. It was the objective of this study to determine the incidence of in-hospital PE and symptomatic deep-vein thrombosis following elective joint arthroplasty in patients who received a minimum of 10 days of dalteparin prophylaxis. Consecutive charts of patients who underwent elective joint arthroplasty at our institution between January 2008 and June 2008 were reviewed. Data on risk factors for venous thromboembolism, objectively documented venous thromboembolic events, and signs and symptoms of PE were abstracted. Patients who received concomitant warfarin in the postoperative period were excluded. The study population consisted of 437 knee arthroplasty and 246 hip arthroplasty patients. The incidence of in-hospital PE following knee arthroplasty and hip arthroplasty was 4.6% and 0.4%, respectively. One out of every 10 knee patients, and one out of every 20 hip patients underwent testing for pulmonary embolism. Pulmonary embolism was diagnosed a median of 3.5 days after knee arthroplasty. The incidence of in-hospital PE in knee arthroplasty patients who received dalteparin prophylaxis was significantly higher than expected. Potential explanations for this finding include poor efficacy of dalteparin started 12-24 hours postoperatively and/or a low threshold for ordering diagnostic imaging for PE. Studies to clarify these issues are needed.


Quantity and Diversity: Simulating Early Word Learning Environments.
The words in children's language learning environments are strongly predictive of cognitive development and school achievement. But how do we measure language environments and do so at the scale of the many words that children hear day in, day out? The quantity and quality of words in a child's input are typically measured in terms of total amount of talk and the lexical diversity in that talk. There are disagreements in the literature whether amount or diversity is the more critical measure of the input. Here we analyze the properties of a large corpus (6.5 million words) of speech to children and simulate learning environments that differ in amount of talk per unit time, lexical diversity, and the contexts of talk. The central conclusion is that what researchers need to theoretically understand, measure, and change is not the total amount of words, or the diversity of words, but the function that relates total words to the diversity of words, and how that function changes across different contexts of talk.


Population genetic variation in gene expression is associated with phenotypic variation in Saccharomyces cerevisiae.
The relationship between genetic variation in gene expression and phenotypic variation observable in nature is not well understood. Identifying how many phenotypes are associated with differences in gene expression and how many gene-expression differences are associated with a phenotype is important to understanding the molecular basis and evolution of complex traits. We compared levels of gene expression among nine natural isolates of Saccharomyces cerevisiae grown either in the presence or absence of copper sulfate. Of the nine strains, two show a reduced growth rate and two others are rust colored in the presence of copper sulfate. We identified 633 genes that show significant differences in expression among strains. Of these genes, 20 were correlated with resistance to copper sulfate and 24 were correlated with rust coloration. The function of these genes in combination with their expression pattern suggests the presence of both correlative and causative expression differences. But the majority of differentially expressed genes were not correlated with either phenotype and showed the same expression pattern both in the presence and absence of copper sulfate. To determine whether these expression differences may contribute to phenotypic variation under other environmental conditions, we examined one phenotype, freeze tolerance, predicted by the differential expression of the aquaporin gene AQY2. We found freeze tolerance is associated with the expression of AQY2. Gene expression differences provide substantial insight into the molecular basis of naturally occurring traits and can be used to predict environment dependent phenotypic variation.


Post-stroke cognitive impairment: epidemiology, mechanisms and management.
Post-stroke cognitive impairment occurs frequently in the patients with stroke. The prevalence of post-stroke cognitive impairment ranges from 20% to 80%, which varies for the difference between the countries, the races, and the diagnostic criteria. The risk of post-stroke cognitive impairment is related to both the demographic factors like age, education and occupation and vascular factors. The underlying mechanisms of post-stroke cognitive impairment are not known in detail. However, the neuroanatomical lesions caused by the stroke on strategic areas such as the hippocampus and the white matter lesions (WMLs), the cerebral microbleeds (CMBs) due to the small cerebrovascular diseases and the mixed AD with stroke, alone or in combination, contribute to the pathogenesis of post-stroke cognitive impairment. The treatment of post-stroke cognitive impairment may benefit not only from the anti-dementia drugs, but also the manage measures on cerebrovascular diseases. In this review, we will describe the epidemiological features and the mechanisms of post-stroke cognitive impairment, and discuss the promising management strategies for these patients.


Individual differences in reproductive costs examined using multi-state methods.
1. Trade-offs among life-history traits are common because individuals have to partition limited resources between multiple traits. Reproductive costs are generally assumed to be high, resulting in reduced survival and fecundity in the following year. However, it is common to find positive rather than negative correlations between life-history traits. 2. Here, we use a data set from the individual-based study of red deer on the Isle of Rum to examine how these costs vary between individuals and at different ages, using multi-state mark-recapture methodology. 3. Females that had reproduced frequently in the past incurred lower costs of reproduction in terms of survival in the following year and were more likely to reproduce in two consecutive years. Older individuals and those that had not reproduced frequently exhibited higher costs. 4. These results highlight the importance of considering heterogeneity and individual quality when examining trade-offs and demonstrate the effectiveness of using detailed long-term data sets to explore life-history strategies using multi-state mark-recapture models.


Enhanced Bioavailability and Dissolution of Atorvastatin Calcium from Floating Microcapsules using Minimum Additives.
Atorvastatin calcium, a lipid-lowering drug, is much less bioavailable because of reduced solubility in acidic media. Multiple-unit floating microcapsules of Atorvastatin calcium (ATC) were developed to expand the gastric residence time of the drug, as ATC has maximum rate of absorption in the upper GI tract. Floating microcapsules were prepared by Emulsion-solvent evaporation technique through incorporation of dioctyl sodium sulphosuccinate (DSS) as a dissolution enhancer. The microcapsules were assessed for shape, size, drug entrapment efficiency, stability and in-vitro drug dissolution rate and were subjected to SEM, DSC and PXRD studies. The ATC-loaded floating microcapsules were spherical in shape and had the particle size of about 28.10 μm and drug-loading efficiency of about 96.55 %. The floating microspheres containing DSS had significantly higher drug dissolution rates than those without DSS. The best formulation, AT4, consisting of Ethyl cellulose, DSS and Poly Ox®, had a maximum drug dissolution rate of 97.86 %, as compared to Storvas 80 mg (Ranbaxy Ltd, as a reference) which had a rate of only 54% during a period of 12 h in acidic media. A pharmacokinetic study performed on albino rabbits illustrates that the bioavailability of AT4 floating microcapsules significantly increased to nearly 1.7 times that of Storvas 80 mg. The present study indicates that the use of multi-unit floating microcapsules for delivery of ATC can improve its bioavailability.


The clinical and molecular spectrum of QRICH1 associated neurodevelopmental disorder.
De novo variants in QRICH1 (Glutamine-rich protein 1) has recently been reported in 11 individuals with intellectual disability (ID). The function of QRICH1 is largely unknown but it is likely to play a key role in the unfolded response of endoplasmic reticulum stress through transcriptional control of proteostasis. In this study, we present 27 additional individuals and delineate the clinical and molecular spectrum of the individuals (n = 38) with QRICH1 variants. The main clinical features were mild to moderate developmental delay/ID (71%), nonspecific facial dysmorphism (92%) and hypotonia (39%). Additional findings included poor weight gain (29%), short stature (29%), autism spectrum disorder (29%), seizures (24%) and scoliosis (18%). Minor structural brain abnormalities were reported in 52% of the individuals with brain imaging. Truncating or splice variants were found in 28 individuals and 10 had missense variants. Four variants were inherited from mildly affected parents. This study confirms that heterozygous QRICH1 variants cause a neurodevelopmental disorder including short stature and expands the phenotypic spectrum to include poor weight gain, scoliosis, hypotonia, minor structural brain anomalies, and seizures. Inherited variants from mildly affected parents are reported for the first time, suggesting variable expressivity.


Objective hot flashes are negatively related to verbal memory performance in midlife women.
To test the hypothesis that hot flashes specifically relate to verbal memory performance by examining the relationship between objective hot flashes and cognitive test performance in women with moderate to severe vasomotor symptoms. In an observational study, 29 midlife women (mean age, 53 y) with moderate to severe hot flashes provided measures of objective hot flashes with an ambulatory hot flash monitor, subjective hot flashes with a diary and questionnaire, and objective measures of verbal memory and other cognitive functions with standardized neuropsychological tests. The mean number of objective hot flashes was 19.5 per day (range, 6 to 35), including 15.3 (range, 6 to 35) during waking hours and 4.2 (range, 0 to 9) during sleep. The mean sensitivity (ie, subjective detection of objectively measured hot flashes) was 60%. Regression analyses revealed that total number of objective hot flashes, sleep duration, and verbal knowledge were significant predictors of delayed verbal memory. Verbal fluency correlated positively with objective daytime hot flashes. Hot flashes did not predict performance on any of the other secondary cognitive measures (ie, attention, working memory, visual memory), although poor sleep predicted worse performance on several outcome measures. Highly symptomatic women underreport the number of objective hot flashes that they experience by 43%. Verbal memory performance relates significantly to the objective number of hot flashes women experience but not to the number of hot flashes that they report. These findings suggest that physiological factors related to hot flashes, rather than psychological factors, predict poorer verbal memory function.


Comparison of coronary artery calcium presence, carotid plaque presence, and carotid intima-media thickness for cardiovascular disease prediction in the Multi-Ethnic Study of Atherosclerosis.
Presence of coronary artery calcium (CAC), carotid plaque, and increased carotid intima-media thickness (IMT) may indicate elevated cardiovascular disease (CVD) risk; however, no large studies have compared them directly. This study compares predictive uses of CAC presence, carotid artery plaque presence, and high IMT for incident CVD events. Participants were from the Multi-Ethnic Study of Atherosclerosis (MESA). Predictive values of carotid plaque, IMT, and CAC presence were compared using Cox proportional hazards models, c-statistics, and net reclassification indices. The 6779 participants were mean (SD) 62.2 (10.2) years old; 49.9% had CAC, and 46.7% had carotid plaque. The mean left and right IMT were 0.754 (0.210) mm and 0.751 (0.187) mm, respectively. After 9.5 years (mean), 538 CVD events, 388 coronary heart disease (CHD) events, and 196 stroke/transient ischemic attacks were observed. CAC presence was a stronger predictor of incident CVD and CHD than carotid ultrasound measures. Mean IMT≥75th percentile (for age, sex, and race) alone did not predict events. Compared with traditional risk factors, c-statistics for CVD (c=0.756) and CHD (c=0.752) increased the most by the addition of CAC presence (CVD, 0.776; CHD, 0.784; P<0.001) followed by carotid plaque presence (CVD, c=0.760; CHD, c=0.757; P<0.05). Compared with risk factors (c=0.782), carotid plaque presence (c=0.787; P=0.045) but not CAC (c=0.785; P=0.438) improved prediction of stroke/transient ischemic attacks. In adults without CVD, CAC presence improves prediction of CVD and CHD more than carotid plaque presence or high IMT. CAC and carotid ultrasound parameters performed similarly for stroke/transient ischemic attack event prediction.


Cytotoxicity of ZnO Nanoparticles Can Be Tailored by Modifying Their Surface Structure: A Green Chemistry Approach for Safer Nanomaterials.
ZnO nanoparticles (NP) are extensively used in numerous nanotechnology applications; however, they also happen to be one of the most toxic nanomaterials. This raises significant environmental and health concerns and calls for the need to develop new synthetic approaches to produce safer ZnO NP, while preserving their attractive optical, electronic, and structural properties. In this work, we demonstrate that the cytotoxicity of ZnO NP can be tailored by modifying their surface-bound chemical groups, while maintaining the core ZnO structure and related properties. Two equally sized (9.26 ± 0.11 nm) ZnO NP samples were synthesized from the same zinc acetate precursor using a forced hydrolysis process, and their surface chemical structures were modified by using different reaction solvents. X-ray diffraction and optical studies showed that the lattice parameters, optical properties, and band gap (3.44 eV) of the two ZnO NP samples were similar. However, FTIR spectroscopy showed significant differences in the surface structures and surface-bound chemical groups. This led to major differences in the zeta potential, hydrodynamic size, photocatalytic rate constant, and more importantly, their cytotoxic effects on Hut-78 cancer cells. The ZnO NP sample with the higher zeta potential and catalytic activity displayed a 1.5-fold stronger cytotoxic effect on cancer cells. These results suggest that by modifying the synthesis parameters/conditions and the surface chemical structures of the nanocrystals, their surface charge density, catalytic activity, and cytotoxicity can be tailored. This provides a green chemistry approach to produce safer ZnO NP.


Quantifying the sensitivity of the network structure and properties from simultaneous measurements during photopolymerization.
We present a method that combines experimental and computational approaches to assess a comprehensive set of structural and functional evolution during a network formation process via photopolymerization. Our work uses the simultaneous measurement of the degree of conversion, polymerization stress, the change in reaction temperature, and shrinkage strain in situ. These measurements are combined with the theory of viscoelastic materials to deduce the relaxation time and frequency-dependent modulus of the polymerizing network. The relaxation time and degree of conversion are used to demonstrate the effect of processing parameters (e.g. curing protocol adjusted by the light intensity) in creating different network structures for the same initial resin. We describe experimental trends using effective medium calculations on a cross-linked polymer network model. In particular, we show that the effect of curing conditions on the spatial heterogeneity in crosslink density can be quantified using multiparametric measurements and modeling. Collectively, the present method is a way to examine holistically the complex structural and functional evolution of the network formation process.


In situ infrared emission spectroscopy for quantitative gas-phase measurement under high temperature reaction conditions: an analytical method for methane by means of an innovative small-volume flowing cell.
We have used infrared emission spectroscopy (IRES) in order to perform in situ studies under flowing gas-phase conditions. When the small-volume cell developed herein is used, we can (1) observe emission spectra from a hot gas-phase sample having an effective volume much less than one milliliter, (2) observe spectra of typical molecular species present, and (3) observe spectra of the more important molecular species down to below 10% and in some cases even as low as 1%. In addition, an analytical method has been derived in order to conduct quantitative studies under typical reaction conditions. We show that simplifications can be made in the data acquisition and handling for a direct linear correlation between band intensity and concentration with only simple background correction. The practical lower limit for methane in the present setup is approximately 0.5-1% v/v depending on the selected temperature. Our data were collected at 500, 600, and 700 degrees C, respectively. The major features of the present cell design are fairly simple and basically formed by a quartz tube (outer diameter=6 mm, inner diameter=4 mm) inside a metal pipe and two tubular ceramic heaters. This simple setup has advantages and attractive features that have extended the application of IRES to new fields and, in particular, for in situ studies of hydrocarbon reactions at different residence times at high temperature.


Membrane type 1 matrix metalloproteinase is a crucial promoter of synovial invasion in human rheumatoid arthritis.
A hallmark of rheumatoid arthritis (RA) is invasion of the synovial pannus into cartilage, and this process requires degradation of the collagen matrix. The aim of this study was to explore the role of one of the collagen-degrading matrix metalloproteinases (MMPs), membrane type 1 MMP (MT1-MMP), in synovial pannus invasiveness. The expression and localization of MT1-MMP in human RA pannus were investigated by Western blot analysis of primary synovial cells and immunohistochemical analysis of RA joint specimens. The functional role of MT1-MMP was analyzed by 3-dimensional (3-D) collagen invasion assays and a cartilage invasion assay in the presence or absence of tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, or GM6001. The effect of adenoviral expression of a dominant-negative MT1-MMP construct lacking a catalytic domain was also examined. MT1-MMP was highly expressed at the pannus-cartilage junction in RA joints. Freshly isolated rheumatoid synovial tissue and isolated RA synovial fibroblasts invaded into a 3-D collagen matrix in an MT1-MMP-dependent manner. Invasion was blocked by TIMP-2 and GM6001 but not by TIMP-1. Invasion was also inhibited by the overexpression of a dominant-negative MT1-MMP, which inhibits collagenolytic activity and proMMP-2 activation by MT1-MMP on the cell surface. Synovial fibroblasts also invaded into cartilage in an MT1-MMP-dependent manner. This process was further enhanced by removing aggrecan from the cartilage matrix. MT1-MMP serves as an essential collagen-degrading proteinase during pannus invasion in human RA. Specific inhibition of MT1-MMP-dependent invasion may represent a novel therapeutic strategy for RA.


Intercellular redistribution of cAMP underlies selective suppression of cancer cell growth by connexin26.
Connexins (Cx), which constitute gap junction intercellular channels in vertebrates, have been shown to suppress transformed cell growth and tumorigenesis, but the mechanism(s) still remain largely speculative. Here, we define the molecular basis by which Cx26, but less frequently Cx43 or Cx32, selectively confer growth suppression on cancer cells. Functional intercellular coupling is shown to be required, producing partial blocks of the cell cycle due to prolonged activation of several mitogenic kinases. PKA is both necessary and sufficient for the Cx26 induced growth inhibition in low serum and the absence of anchorage. Activation of PKA was not associated with elevated cAMP levels, but appeared to result from a redistribution of cAMP throughout the cell population, eliminating the cell cycle oscillations in cAMP required for efficient cell cycle progression. Cx43 and Cx32 fail to mediate this redistribution as, unlike Cx26, these channels are closed during the G2/M phase of the cell cycle when cAMP levels peak. Comparisons of tumor cell lines indicate that this is a general pattern, with growth suppression by connexins occurring whenever cAMP oscillates with the cell cycle, and the gap junction remain open throughout the cell cycle. Thus, gap junctional coupling, in the absence of any external signals, provides a general means to limit the mitotic rate of cell populations.


Longitudinal study on oral shedding of herpes simplex virus 1 and varicella-zoster virus in individuals infected with HIV.
Primary herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) infection leads to a life-long latent infection of ganglia innervating the oral mucosa. HSV-1 and VZV reactivation is more common in immunocompromised individuals and may result in viral shedding in saliva. We determined the kinetics and quantity of oral HSV-1 and VZV shedding in HSV-1 and VZV seropositive individuals infected with HIV and to assess whether HSV-1 shedding involves reactivation of the same strain intra-individually. HSV-1 and VZV shedding was determined by real-time PCR of sequential daily oral swabs (n = 715) collected for a median period of 31 days from 22 individuals infected with HIV. HSV-1 was genotyped by sequencing the viral thymidine kinase gene. Herpesvirus shedding was detected in 18 of 22 participants. Shedding of HSV-1 occurred frequently, on 14.3% of days, whereas solely VZV shedding was very rare. Two participants shed VZV. The median HSV-1 load was higher compared to VZV. HSV-1 DNA positive swabs clustered into 34 shedding episodes with a median duration of 2 days. The prevalence, duration and viral load of herpesvirus shedding did not correlate with CD4 counts and HIV load. The genotypes of the HSV-1 viruses shed were identical between and within shedding episodes of the same person, but were different between individuals. One-third of the individuals shed an HSV-1 strain potentially refractory to acyclovir therapy. Compared to HSV-1, oral VZV shedding is rare in individuals infected with HIV. Recurrent oral HSV-1 shedding is likely due to reactivation of the same latent HSV-1 strain.


Acquired activated protein C resistance is associated with lupus anticoagulants and thrombotic events in pediatric patients with systemic lupus erythematosus.
Acquired activated protein C resistance (APCR) has been hypothesized as a possible mechanism by which antiphospholipid antibodies (APLAs) cause thrombotic events (TEs). However, available evidence for an association of acquired APCR with APLAs is limited. More importantly, an association of acquired APCR with TEs has not been demonstrated. The objective of the study was to determine, in pediatric patients with systemic lupus erythematosus (SLE), whether (1) acquired APCR is associated with the presence of APLAs, (2) APCR is associated with TEs, and (3) there is an interaction between APCR and APLAs in association with TEs. A cross-sectional cohort study of 59 consecutive, nonselected children with SLE was conducted. Primary clinical outcomes were symptomatic TEs, confirmed by objective radiographic tests. Laboratory testing included lupus anticoagulants (LAs), anticardiolipin antibodies (ACLAs), APC ratio, protein S, protein C, and factor V Leiden. The results revealed that TEs occurred in 10 (17%) of 59 patients. Acquired APCR was present in 18 (31%) of 58 patients. Acquired APCR was significantly associated with the presence of LAs but not ACLAs. Acquired APCR was also significantly associated with TEs. There was significant interaction between APCR and LAs in the association with TEs. Presence of both APCR and LAs was associated with the highest risk of a TE. Protein S and protein C concentrations were not associated with the presence of APLAs, APCR, or TEs. Presence of acquired APCR is a marker identifying LA-positive patients at high risk of TEs. Acquired APCR may reflect interference of LAs with the protein C pathway that may represent a mechanism of LA-associated TEs. (Blood. 2001;97:844-849)


Oxidative stability of dispersions prepared from purified marine phospholipid and the role of α-tocopherol.
The objective of this study was to investigate the oxidative stability of dispersions prepared from different levels of purified marine phospholipid (PL) obtained by acetone precipitation, with particular focus on the interaction between α-tocopherol and PL in dispersions. This also included the investigation of nonenzymatic browning in purified marine PL dispersions. Dispersions were prepared by high-pressure homogenizer. The oxidative and hydrolytic stabilities of dispersions were investigated by determination of hydroperoxides, secondary volatile oxidation products, and free fatty acids, respectively, during 32 days of storage at 2 °C. Nonenzymatic browning was investigated through measurement of Strecker aldehydes, color changes, and pyrrole content. Dispersions containing α-tocopherol or higher levels of purified marine PL showed a lower increment of volatiles after 32 days storage. The results suggested that tocopherol is an efficient antioxidant in PL dispersions or that the presence of α-tocopherol and pyrroles may be the main reason for the high oxidative stability of purified marine PL dispersions.


The expression of memorization in early childhood.
This experiment examined the expression and effectiveness of memorization in young children. Sixty children at each of the ages 4, 5, and 6 were randomly assigned to a memory group or to one of two control groups. All of the children were told that they could play with a group of toys during a brief activity period; the children in the memory condition were also instructed to memorize a specified subset of the toys. Mnemonic mediators were identified on the basis of differences in the activity period behaviors of children given memory and play instructions. Relative to the children in the play groups, the children in the memory conditions played with the toys less; further, their use of naming and visual examination as mnemonic mediators differentiated the groups at all ages and increased with age. Only the oldest subjects given memory instructions, however, demonstrated superior recall. The relationships between activity period behavior and recall among the different conditions were explored with regression techniques. although previous research has focused on identifying the earliest use of memory strategies, the present findings underscore the importance of examining the development of these skills.


Clinical survey of ischemic cerebrovascular disease in children in a district of Japan.
Ischemic cerebrovascular disease in children is relatively rare. To clarify the clinical features of ischemic stroke occurring in infants and children, we evaluated 54 cases of cerebral infarction, excluding cases of moyamoya disease, in patients less than 16 years old at 24 clinics in the Tohoku (northeast) district of Japan. We observed two incidence peaks, one in little children and the other in junior high school students. Infection and minor head trauma were more frequently seen prior to ischemic strokes than was heart disease. The middle cerebral artery region, including the basal ganglia, was most commonly affected (49 patients, 91%) on computed tomograms. Angiography was performed in 48 patients (89%) and showed various types of occlusive lesions, mostly affecting the middle cerebral artery. Hemiparesis was the most common form of disability following ischemic strokes (48 patients, 89%). Surgical treatment was carried out in seven patients (13%). The clinical course of these cases showed that the recovery of children after a stroke tends to be better than that of adults, but that permanent disabilities, such as hemiparesis or mental retardation, occur commonly. Further investigation of juvenile cerebrovascular disease is important to prevent ischemic strokes in children.


The Global Phosphorylation Landscape of SARS-CoV-2 Infection.
The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.


A lateral ridge augmentation study to evaluate a synthetic membrane for guided bone regeneration: an experiment in the canine mandible.
To evaluate guided bone regeneration outcomes in defects protected with an in situ formed polyethylene glycol (PEG) hydrogel membrane as compared to a non-cross-linked collagen membrane (CM). Four mandibular alveolar ridge defects were created in eight hound dogs. Regenerative procedures were randomly allocated to one of four groups consisting of freeze-dried bone allograft, which is referred to in this study as freeze-dried bone xenograft (FDBX) + PEG, autogenous bone (AB) + PEG, AB + CM, and AB alone. After 8 weeks, titanium dental implants were placed into augmented sites. After 8 weeks of allowed time for osseointegration, the animals were sacrificed to harvest block specimens for bone-to-implant contact (BIC) and ridge width histomorphometric analysis. Polyethylene glycol membranes had an exposure rate of 50% as compared to 12.5% for sites grafted with CM. Regenerative outcomes with respect to implant placement were least favorable for FDBX + PEG which had implants placed in 37.5% of augmented sites compared to 100% implant placement for all other groups. No statistically significant differences were noted between groups for ridge width measurements in implant and non-implant histologic sections (P > 0.05). Buccal BIC (%) values between treatment groups also failed to reach statistical significant difference (FDBX + PEG [60.2 ± 9.4]; AB + PEG [58.8 ± 8.5]; AB + CM [57.9 ± 12.8]; AB [61.0 ± 10.2]). When used in conjunction with FDBX, PEG had unpredictable bone formation and in most cases negatively impacted future implant placement.


Increased delivery of tumor-specific monoclonal antibodies to brain after osmotic blood-brain barrier modification in patients with melanoma metastatic to the central nervous system.
We evaluated the delivery of melanoma-specific radiolabeled monoclonal antibody (MAb) Fab fragments in a pilot study of three patients with melanoma metastatic to the central nervous system. Tumor samples demonstrated excellent immunohistochemical reactivity with Fab 96.5, specific for a 97,000-molecular-weight melanoma antigen (p97), or Fab 48.7, specific for a melanoma-associated proteoglycan antigen. All three patients received 131I-labeled tumor-specific Fab (5 to 7 mg, 1 mCi/mg) intravenously. On a separate occasion, two patients received 131I-labeled nonspecific Fab (5 to 7 mg, 1 mCi/mg). There was no uptake of either antibody into the region of the tumor (as documented by gamma camera brain images). However, there was increased uptake in the blood-brain barrier (BBB)-modified areas in all three patients when radiolabeled tumor-specific MAb was administered intravenously in conjunction with osmotic BBB opening. In one patient, the estimated cerebrovascular permeability X capillary surface area (PA) for the tumor-bearing hemisphere 3 hours after disruption was 1.16 X 10(-6) sec-1 compared to the PA of 0.395 X 10(-6) sec-1 in the nondisrupted hemisphere. Serial brain scans showed that greater than 90% of the radiolabeled antibody cleared from the brain by 72 hours. The highest radiation doses (rads) calculated per 7 mCi injection were: left brain (barrier-modified hemisphere), 5.46; right brain (non-barrier modified hemisphere), 1.68; thyroid, 98; stomach, 9.1; kidney, 39.9; and total body, 1.33. There seemed to be increased uptake of antibody in the tumor region after barrier modification in one patient, but antibody clearance from that region occurred at the same rate as from surrounding and apparently tumor-free brain. In one patient who had carcinomatous meningitis, we demonstrated antibody bound to only a fraction of the antigen binding sites on tumor cells in the cerebrospinal fluid after BBB modification. We have not shown distinct, persistent localization of antibody in brain tumor; studies investigating MAb dose and other parameters as the basis for this problem are under way.


GRM1 is upregulated through gene fusion and promoter swapping in chondromyxoid fibroma.
Glutamate receptors are well-known actors in the central and peripheral nervous systems, and altered glutamate signaling is implicated in several neurological and psychiatric disorders. It is increasingly recognized that such receptors may also have a role in tumor growth. Here we provide direct evidence of aberrant glutamate signaling in the development of a locally aggressive bone tumor, chondromyxoid fibroma (CMF). We subjected a series of CMFs to whole-genome mate-pair sequencing and RNA sequencing and found that the glutamate receptor gene GRM1 recombines with several partner genes through promoter swapping and gene fusion events. The GRM1 coding region remains intact, and 18 of 20 CMFs (90%) showed a more than 100-fold and up to 1,400-fold increase in GRM1 expression levels compared to control tissues. Our findings unequivocally demonstrate that direct targeting of GRM1 is a necessary and highly specific driver event for CMF development.


Recovery of Drug Delivery Nanoparticles from Human Plasma Using an Electrokinetic Platform Technology.
The effect of complex biological fluids on the surface and structure of nanoparticles is a rapidly expanding field of study. One of the challenges holding back this research is the difficulty of recovering therapeutic nanoparticles from biological samples due to their small size, low density, and stealth surface coatings. Here, the first demonstration of the recovery and analysis of drug delivery nanoparticles from undiluted human plasma samples through the use of a new electrokinetic platform technology is presented. The particles are recovered from plasma through a dielectrophoresis separation force that is created by innate differences in the dielectric properties between the unaltered nanoparticles and the surrounding plasma. It is shown that this can be applied to a wide range of drug delivery nanoparticles of different morphologies and materials, including low-density nanoliposomes. These recovered particles can then be analyzed using different methods including scanning electron microscopy to monitor surface and structural changes that result from plasma exposure. This new recovery technique can be broadly applied to the recovery of nanoparticles from high conductance fluids in a wide range of applications.


Primary care providers' experiences with and perceptions of personalized genomic medicine.
To assess primary care providers' (PCPs') experiences with, perceptions of, and desired role in personalized medicine, with a focus on cancer. Qualitative study involving focus groups. Urban and rural interprofessional primary care team practices in Alberta and Ontario. Fifty-one PCPs. Semistructured focus groups were conducted and audiorecorded. Recordings were transcribed and analyzed using techniques informed by grounded theory including coding, interpretations of patterns in the data, and constant comparison. Five focus groups with the 51 participants were conducted; 2 took place in Alberta and 3 in Ontario. Primary care providers described limited experience with personalized medicine, citing breast cancer and prenatal care as main areas of involvement. They expressed concern over their lack of knowledge, in some circumstances relying on personal experiences to inform their attitudes and practice. Participants anticipated an inevitable role in personalized medicine primarily because patients seek and trust their advice; however, there was underlying concern about the magnitude of information and pace of discovery in this area, particularly in direct-to-consumer personal genomic testing. Increased knowledge, closer ties to genetics specialists, and relevant, reliable personalized medicine resources accessible at the point of care were reported as important for successful implementation of personalized medicine. Primary care providers are prepared to discuss personalized medicine, but they require better resources. Models of care that support a more meaningful relationship between PCPs and genetics specialists should be pursued. Continuing education strategies need to address knowledge gaps including direct-to-consumer genetic testing, a relatively new area provoking PCP concern. Primary care providers should be mindful of using personal experiences to guide care.


Immunization delivery in British Columbia: perspectives of primary care physicians.
To explore the experiences of family physicians and pediatricians delivering immunizations, including perceived barriers and supports. Qualitative study using focus groups. Ten cities throughout British Columbia. A total of 46 family physicians or general practitioners, 10 pediatricians, and 2 residents. A semistructured dialogue guide was used by a trained facilitator to explore participants' experiences and views related to immunization delivery in British Columbia. Verbatim transcriptions were independently coded by 2 researchers. Key themes were analyzed and identified in an iterative manner using interpretive description. Physicians highly valued vaccine delivery. Factors facilitating physician-delivered immunizations included strong beliefs in the value of vaccines and having adequate information. Identified barriers included the large time commitment and insufficient communication about program changes, new vaccines, and the adult immunization program in general. Some physicians reported good relationships with local public health, while others reported the opposite experience, and this varied by geographic location. These findings suggest that physicians are supportive of delivering vaccines. However, there are opportunities to improve the sustainability of physician-delivered immunizations. While compensation schemes remain under the purview of the provincial governments, local public health authorities can address the information needs of physicians.


Global Foot-and-Mouth Disease Research Update and Gap Analysis: 3 - Vaccines.
This study assessed research knowledge gaps in the field of FMDV (foot-and-mouth disease virus) vaccines. The study took the form of a literature review (2011-15) combined with research updates collected in 2014 from 33 institutes from across the world. Findings were used to identify priority areas for future FMD vaccine research. Vaccines play a vital role in FMD control, used both to limit the spread of the virus during epidemics in FMD-free countries and as the mainstay of disease management in endemic regions, particularly where sanitary controls are difficult to apply. Improvements in the performance or cost-effectiveness of FMD vaccines will allow more widespread and efficient disease control. FMD vaccines have changed little in recent decades, typically produced by inactivation of whole virus, the quantity and stability of the intact viral capsids in the final preparation being key for immunogenicity. However, these are exciting times and several promising novel FMD vaccine candidates have recently been developed. This includes the first FMD vaccine licensed for manufacture and use in the USA; this adenovirus-vectored FMD vaccine causes in vivo expression of viral capsids in vaccinated animals. Another promising vaccine candidate comprises stabilized empty FMDV capsids produced in vitro in a baculovirus expression system. Recombinant technologies are also being developed to improve otherwise conventionally produced inactivated vaccines, for example, by creating a chimeric vaccine virus to increase capsid stability and by inserting sequences into the vaccine virus for desired antigen expression. Other important areas of ongoing research include enhanced adjuvants, vaccine quality control procedures and predicting vaccine protection from immune correlates, thus reducing dependency on animal challenge studies. Globally, the degree of independent vaccine evaluation is highly variable, and this is essential for vaccine quality. Previously neglected, the importance of evaluating vaccination programme effectiveness and impact is increasingly being recognized.


Lack of association between intact/deletion polymorphisms of the APOBEC3B gene and HIV-1 risk.
The human APOBEC3 family of proteins potently restricts HIV-1 replication APOBEC3B, one of the family genes, is frequently deleted in human populations. Two previous studies reached inconsistent conclusions regarding the effects of APOBEC3B loss on HIV-1 acquisition and pathogenesis. Therefore, it was necessary to verify the effects of APOBEC3B on HIV-1 infection in vivo. Intact (I) and deletion (D) polymorphisms of APOBEC3B were analyzed using PCR. The syphilis, HBV and HCV infection rates, as well as CD4(+) T cell counts and viral loads were compared among three APOBEC3B genotype groups (I/I, D/I, and D/D). HIV-1 replication kinetics was assayed in vitro using primary cells derived from PBMCs. A total of 248 HIV-1-infected Japanese men who have sex with men (MSM) patients and 207 uninfected Japanese MSM were enrolled in this study. The genotype analysis revealed no significant differences between the APOBEC3B genotype ratios of the infected and the uninfected cohorts (p = 0.66). In addition, HIV-1 disease progression parameters were not associated with the APOBEC3B genotype. Furthermore, the PBMCs from D/D and I/I subjects exhibited comparable HIV-1 susceptibility. Our analysis of a population-based matched cohort suggests that the antiviral mechanism of APOBEC3B plays only a negligible role in eliminating HIV-1 in vivo.


A scar-like lesion is apparent in basement membrane after wound repair in vivo.
Basement membrane is a highly conserved sheet-like extracellular matrix in animals, underlying simple and complex epithelia, and wrapping around tissues like muscles and nerves. Like the tissues they support, basement membranes become damaged by environmental insults. Although it is clear that basement membranes are repaired after damage, virtually nothing is known about this process. For example, it is not known how repaired basement membranes compare to undamaged ones, whether basement membrane components are necessary for epithelial wound closure, or whether there is a hierarchy of assembly that repairing basement membranes follow, similar to the hierarchy of assembly of embryonic basement membranes. In this report, we address these questions using the basement membrane of the Drosophila larval epidermis as a model system. By analyzing the four main basement membrane proteins - laminin, collagen IV, perlecan, and nidogen - we find that although basement membranes are repaired within a day after mechanical damage in vivo, thickened and disorganized matrix scars are evident with all four protein components. The new matrix proteins that repair damaged basement membranes are provided by distant adipose and muscle tissues rather than by the local epithelium, the same distant tissues that provide matrix proteins for growth of unwounded epithelial basement membranes. To identify a hierarchy of repair, we tested the dependency of each of the basement membrane proteins on the others for incorporation after damage. For proper incorporation after damage, nidogen requires laminin, and perlecan requires collagen IV, but surprisingly collagen IV does not to depend on laminin. Thus, the rules of basement membrane repair are subtly different than those of de novo assembly.


Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay.
Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world.


Feature Selection for Automatic Tuberculosis Screening in Frontal Chest Radiographs.
To detect pulmonary abnormalities such as Tuberculosis (TB), an automatic analysis and classification of chest radiographs can be used as a reliable alternative to more sophisticated and technologically demanding methods (e.g. culture or sputum smear analysis). In target areas like Kenya TB is highly prevalent and often co-occurring with HIV combined with low resources and limited medical assistance. In these regions an automatic screening system can provide a cost-effective solution for a large rural population. Our completely automatic TB screening system is processing the incoming CXRs (chest X-ray) by applying image preprocessing techniques to enhance the image quality followed by an adaptive segmentation based on model selection. The delineated lung regions are described by a multitude of image features. These characteristics are than optimized by a feature selection strategy to provide the best description for the classifier, which will later decide if the analyzed image is normal or abnormal. Our goal is to find the optimal feature set from a larger pool of generic image features, -used originally for problems such as object detection, image retrieval, etc. For performance evaluation measures such as under the curve (AUC) and accuracy (ACC) were considered. Using a neural network classifier on two publicly available data collections, -namely the Montgomery and the Shenzhen dataset, we achieved the maximum area under the curve and accuracy of 0.99 and 97.03%, respectively. Further, we compared our results with existing state-of-the-art systems and to radiologists' decision.


Identification of novel quinazoline derivatives as potent antiplasmodial agents.
Despite the recent reductions in the global burden of malaria, this disease remains a devastating cause of death in tropical and subtropical regions. As there is no broadly effective vaccine for malaria, prevention and treatment still rely on chemotherapy. Unfortunately, emerging resistance to the gold standard artemisinin combination therapies means that new drugs with novel modes of action are urgently needed. In this context, Plasmodium histone modifying enzymes have emerged as potential drug targets, prompting us to develop and optimize compounds directed against such epigenetic targets. A panel of 51 compounds designed to target different epigenetic enzymes were screened for activity against Plasmodium falciparum parasites. Based on in vitro activity against drug susceptible and drug-resistant P. falciparum lines, selectivity index criterion and favorable pharmacokinetic properties, four compounds, one HDAC inhibitor (1) and three DNMT inhibitors (37, 43 and 45), were selected for preclinical studies in a mouse model of malaria. In vivo data showed that 37, 43 and 45 exhibited oral efficacy in the mouse model of Plasmodium berghei infection. These compounds represent promising starting points for the development of novel antimalarial drugs.


Gas exchange rates and chlorophyll content of epi- and endolithic lichens from the Trieste Karst (NE Italy).
The photosynthetic activity of calcicolous endo- and epilithic lichens from the Trieste Karst area (NE Italy) was investigated. The data consist of: (a) gas exchange rates of Acrocordia conoidea, Petractis clausa, Rinodina immersa (endolithic), and Aspicilia calcarea (epilithic), recorded at different combinations of temperature, water content and radiation flux values, (b) chlorophyll content values of nine species, and (c)δ13 C values measured in 24 lichen species with different growth-forms. The results show that: (1) the maximum photosynthetic rates of endolithic lichens are rather small, ranging between 0·2 and 1·5μmol CO2 m-2 h-1 at optimal conditions; (2) the relation between thallus water content and photosynthesis differs from that of foliose and fruticose lichens; the optimum water content of endolithic lichens is particularly small, when expressed in g H2 O cm-2 ; (3) the algae of endolithic lichens, which belong to different systematic groups, are light-saturated at a small radiation flux; (4) chlorophyll contents of endolithic lichens are rather large, being similar to those of some parmelioid lichens. The ecological implications of the endolithic growth-form are briefly discussed. Endolithic lichens should be regarded as slow-growing, stress-tolerant organisms, which are rather similar in their physiology to epilithic crustose lichens; they have a high resistance to CO2 diffusion, saturation being reached only at a very large CO2 concentration.


Gene expression during the development of experimentally induced cerebral aneurysms.
Cerebral aneurysms are a major cause of subarachnoid hemorrhage, but the mechanism of their development remains unclear. In this study, we investigated candidate genes whose expression is significantly changed during the development of experimentally induced cerebral aneurysms using adaptor-tagged competitive polymerase chain reaction (ATAC-PCR). Twenty-four rats received sham operation (control) or the operation for the induction of experimental cerebral aneurysms. Rats were sacrificed at time 0, 2 weeks, 1 month and 3 months after the operation (n = 6 for each group). RNAs from right anterior cerebral artery/olfactory artery (ACA/OA) bifurcations were assessed via a 191-gene data matrix expression profile by ATAC-PCR. We identified 15 genes whose expression is significantly altered during cerebral aneurysm formation, including major heparan sulfate proteoglycan, cathepsin B, hevin and beta(4)-integrin. We also confirmed protein expression of beta(4)-integrin in rat cerebral aneurysms by quantitative real-time PCR and immunohistochemistry. The ATAC-PCR revealed temporal changes in gene expression during the development of experimental cerebral aneurysms. The genes that were significantly changed in this study would be the candidates for future studies concerning the development of cerebral aneurysms.


Glottic-modulated lung ventilation during continuous transtracheal gas insufflation: an experimental study.
We investigated a new method of pulmonary ventilation that included a minitracheostomy, a reverse thrust catheter to deliver continuous flow of gas to the carina, and a threshold valve to avoid lung overinflation. In six lightly sedated healthy sheep, at a continuous flow of 5, 10, or 15 L/min and a threshold valve of 5, 10, 15, or 20 cm H(2)O, we observed a novel respiratory pattern that was characterized either by active lung inflation followed by passive and prolonged inspiratory hold (mixed pattern) or by an absence of all active inspiratory effort and only passive inflation of the lungs (passive pattern). We correlated airway pressure changes with direct visualization of the glottic opening through a fiberoptic bronchoscope. We measured airway pressures at the level of the carina, the subglottic level, and in the pleural space, and respiratory events were monitored through inductive plethysmography. An increase in continuous flow, threshold valve, or both resulted in 1) an increase in glottic breathing; 2) a decrease in respiratory rate, with a decrease in inspiratory pleural pressure excursion; or 3) an increased inspiratory/expiratory ratio and mean airway pressure. During transtracheal gas insufflation, as in this study, a novel respiratory pattern evolved that was modulated by the glottis, accompanied by a decreased effort of breathing; coughing and swallowing remained, and vocalization remained unimpaired.


Opposing effects of Tcf3 and Tcf1 control Wnt stimulation of embryonic stem cell self-renewal.
The co-occupancy of Tcf3 with Oct4, Sox2 and Nanog on embryonic stem cell (ESC) chromatin indicated that Tcf3 has been suggested to play an integral role in a poorly understood mechanism underlying Wnt-dependent stimulation of mouse ESC self-renewal of mouse ESCs. Although the conventional view of Tcf proteins as the β-catenin-binding effectors of Wnt signalling suggested Tcf3-β-catenin activation of target genes would stimulate self-renewal, here we show that an antagonistic relationship between Wnt3a and Tcf3 on gene expression regulates ESC self-renewal. Genetic ablation of Tcf3 replaced the requirement for exogenous Wnt3a or GSK3 inhibition for ESC self-renewal, demonstrating that inhibition of Tcf3 repressor is the necessary downstream effect of Wnt signalling. Interestingly, both Tcf3-β-catenin and Tcf1-β-catenin interactions contributed to Wnt stimulation of self-renewal and gene expression, and the combination of Tcf3 and Tcf1 recruited Wnt-stabilized β-catenin to Oct4 binding sites on ESC chromatin. This work elucidates the molecular link between the effects of Wnt and the regulation of the Oct4/Sox2/Nanog network.


Serum Creatinine and Chronic Kidney Disease-Epidemiology Estimated Glomerular Filtration Rate: Independent Predictors of Renal Replacement Therapy following Cardiac Surgery.
Most studies evaluating predictors of renal replacement therapy (RRT) following cardiac surgery use arbitrary defined limits of preoperative serum creatinine. The aim of this study was to evaluate the effect of preoperative renal function using either estimated-glomerular filtration rate (eGFR) derived using Chronic Kidney Disease-Epidemiology (CKD-EPI) or serum creatinine alone as a predictor for RRT after cardiac surgery. In this prospective cohort study, baseline, intraoperative, and postoperative data of all patients who underwent an elective, urgent, or emergency cardiac surgery between 2012 and 2016 in a single center were analyzed in order to identify multivariate parameters determining the need for RRT after surgery. For preoperative renal function, we used serum creatinine levels and eGFR-derived CKD-EPI equation. We also divided our cohort into eGFR groups following the thresholds of the currently proposed CKD classification. From the 1,614 patients (mean age: 65.4 ± 10.6 years; male: 77.6%) that constituted the study population, 42 (2.6%) underwent RRT postoperatively. EUROSCORE II, cardiopulmonary bypass time, cross clamp time, red blood cell (RBC) units transfused, type and urgency of surgery, combined/non combined operation, peripheral vascular disease, heart failure, chronic obstructive pulmonary disease, dyslipidemia, and preoperative renal function were all univariately associated with RRT use. Multivariate regression with bootstrap utilization indicated that CKD-EPI eGFR (OR 0.979; 95% CI 0.956-0.998), heart failure with the New York Heart Association class ≥2 (OR 4.695; 95% CI 1.756-14.061) and RBC units transfused (OR 1.287; 95% CI 1.081-1.850) were independently associated with RRT need. When serum creatinine (OR 2.920, 95% CI1.056-8.074) was used in the model, the associations with RRT were also significant. Preoperative renal function, defined by serum creatinine or eGFR by CKD-EPI, NYHA class II-IV, and the number of blood units transfused were all independent predictors of RRT postoperatively.


Distinctive aspects of consent in pilot and feasibility studies.
Prior to a main randomized clinical trial, investigators often carry out a pilot or feasibility study in order to test certain trial processes or estimate key statistical parameters, so as to optimize the design of the main trial and/or determine whether it can feasibly be run. Pilot studies reflect the design of the intended main trial, whereas feasibility studies may not do so, and may not involve allocation to different treatments. Testing relative clinical effectiveness is not considered an appropriate aim of pilot or feasibility studies. However, consent is no less important than in a main trial as a means of morally legitimizing the investigator's actions. Two misperceptions are central to consent in clinical studies-therapeutic misconception (a tendency to conflate research and therapy) and therapeutic misestimation (a tendency to overestimate possible benefits and/or underestimate possible harms associated with participation). These phenomena may take a distinctive form in pilot and feasibility studies, owing to potential participants' likely prior unfamiliarity with the nature and purposes of such studies. Thus, participants may confuse the aims of a pilot or feasibility study (developing or optimizing trial design and processes) with those of a main trial (testing treatment effectiveness) and base consent on this misconstrual. Similarly, a misunderstanding of the ability of pilot and feasibility studies to provide information that will inform clinical care, or the underdeveloped nature of interventions included in such studies, may lead to inaccurate assessments of the objective possibility of benefit, and weaken the epistemic basis of consent accordingly. Equipoise may also be particularly challenging to grasp in the context of a pilot study. The consent process in pilot and feasibility studies requires a particular focus, and careful communication, if it is to carry the appropriate moral weight. There are corresponding implications for the process of ethical approval.


Synergistic induction of apoptosis in human myeloid leukemia cells by phorbol 12-myristate 13-acetate and flavopiridol proceeds via activation of both the intrinsic and tumor necrosis factor-mediated extrinsic cell death pathways.
Previous studies have shown that coexposure to marginally toxic concentrations of phorbol 12-myristate 13-acetate (PMA; 10 nM) and the cyclin-dependent kinase inhibitor flavopiridol (FP; 100-200 nM) synergistically induces apoptosis in human myeloid leukemia cells U937 and HL-60 (i.e., >50% apoptotic at 24 h). Attempts have now been made to characterize the cell death pathway(s) involved in this phenomenon. In contrast to cytochrome c release and caspase-3 activation, which occur within 2.5 h of PMA/FP coexposure, caspase-8 activation and Bid cleavage appeared as later events. Such findings implicate the mitochondria-dependent pathway in the initial induction of apoptosis by PMA/FP. However, U937 cells ectopically expressing CrmA, dominant-negative caspase-8, or dominant-negative Fas-associated death domain that were highly resistant to tumor necrosis factor (TNF)/cycloheximide-induced lethality displayed significant, albeit incomplete, resistance to PMA/FP-induced apoptosis after 24 h. Furthermore, coadministration of TNF soluble receptor significantly attenuated PMA/FP-induced apoptosis in U937 (p < 0.02) and HL-60 (p < 0.03) cells at 24 h. PMA/FP coadministration also triggered substantial increases in TNFalpha mRNA and protein secretion compared with the effects of PMA administered alone. The protein kinase C (PKC) inhibitor bisindolylmaleimide (1 microM) completely blocked PMA/FP-induced TNFalpha secretion in U937 cells and attenuated apoptosis. Taken together, these results suggest that coadministration of PMA with FP in myeloid leukemia cells initially triggers mitochondrial damage, an event followed by the PKC-dependent induction and release of TNFalpha, supporting a model in which the synergistic induction of leukemic cell apoptosis by this drug combination proceeds via both mitochondrial- and TNF receptor-related apoptotic pathways.


The encounter with the unknown: Nurses lived experiences of their responsibility for the care of the patient in the Swedish ambulance service.
Registered nurses (RNs) have, according to the Swedish National Board of Health and Welfare, the overall responsibility for the medical care in the ambulance care setting. Bringing RNs into the ambulance service are judged, according to earlier studies, to lead to a degree of professionalism with a higher quality of medical care. Implicitly in earlier studies, the work in the ambulance service involves interpersonal skills. The aim of this study was to describe RNs' experiences of being responsible for the care of the patient in the Swedish ambulance service. A reflective lifeworld approach within the perspective of caring science was used. Five RNs with at least five years experience from care in the ambulance care setting were interviewed. The findings show that the essence of the phenomenon is to prepare and create conditions for care and to accomplish care close to the patient. Three meaning constituents emerged in the descriptions: prepare and create conditions for the nursing care, to be there for the patient and significant others and create comfort for the patient and significant others. The responsibility is a complex phenomenon, with a caring perspective, emerging from the encounter with the unique human being.


Physical activity is associated with reduced risk of gastric cancer: a systematic review and meta-analysis.
Physical activity may be associated with reduced risk of gastric cancer. We performed a systematic review and meta-analysis to evaluate the magnitude of the association and the quality of supporting evidence. After a comprehensive search of bibliographic databases and conference proceedings through February 2013 for observational studies that examined associations between recreational and/or occupational physical activity and gastric cancer risk, we identified 16 studies (seven cohort, nine case control) reporting 11,111 cases of gastric cancer among 1,606,760 patients. Summary adjusted-OR estimates with 95% confidence intervals (CI) were estimated using the random-effects model. Meta-analysis demonstrated that the risk of gastric cancer was 21% lower among the most physically active people as compared with the least physically active people (OR = 0.79; 95% CI, 0.71-0.87) with moderate heterogeneity among studies (I(2) = 55%). This protective effect was seen for gastric cancers in the cardia (four studies; OR = 0.80; 95% CI, 0.63-1.00) and distal stomach (five studies; OR = 0.63; 95% CI, 0.52-0.76). The effect size was significantly smaller in high-quality studies (six studies; OR = 0.86; 95% CI, 0.75-0.99), as compared with low-quality studies (10 studies; OR = 0.74; 95% CI, 0.69-0.81). The results were consistent across sex, study quality, study design, and geographic location. In conclusion, meta-analysis of published observational studies indicates that physical activity is associated with reduced risk of gastric cancer. Lifestyle interventions focusing on increasing physical activity may decrease the global burden of gastric cancer, in addition to a myriad of other health benefits.


Fatty acid-induced mitochondrial uncoupling in adipocytes is not a promising target for treatment of insulin resistance unless adipocyte oxidative capacity is increased.
The release of fatty acids from white adipose tissue is regulated at several levels. We have examined the suggestion that fatty acid release might be diminished by upregulation of mitochondrial fatty acid oxidation in the adipocyte, through increasing mitochondrial uncoupling. The intrinsic oxidative capacity of white adipose tissue is low, and older studies suggest that there is little fatty acid oxidation in white adipocytes, human or rodent. We have examined data on fatty acid metabolism and O(2) consumption in human white adipose tissue in vivo, and conclude that increasing fatty acid oxidation within the oxidative capacity of the tissue would produce only small changes (a few percent) in fatty acid release. The major locus of control of fatty acid release beyond the stimulation of lipolysis is the pathway of fatty acid esterification, already probably targeted by the thiazolidinedione insulin-sensitising agents. An alternative approach would be to upregulate the mitochondrial capacity of the adipocyte. We review proof-of-concept studies in which the phenotype of the white adipocyte has been changed to resemble that of the brown adipocyte by expression of peroxisome proliferator-activated receptor coactivator-1alpha. This increases oxidative capacity and also leads to fatty acid retention through upregulation of glycerol-3-phosphate production, and hence increased fatty acid re-esterification. We conclude that prevention or treatment of insulin resistance through alteration of adipocyte fatty acid handling will require more than a simple alteration of the activity of mitochondrial beta-oxidation within normal limits.


Multiomics Analyses Reveal Microbiome-Gut-Brain Crosstalk Centered on Aberrant Gamma-Aminobutyric Acid and Tryptophan Metabolism in Drug-Naïve Patients with First-Episode Schizophrenia.
Schizophrenia (SCZ) is associated with complex crosstalk between the gut microbiota and host metabolism, but the underlying mechanism remains elusive. Investigating the aberrant neurotransmitter processes reflected by alterations identified using multiomics analysis is valuable to fully explain the pathogenesis of SCZ. We conducted an integrative analysis of multiomics data, including the serum metabolome, fecal metagenome, single nucleotide polymorphism data, and neuroimaging data obtained from a cohort of 127 drug-naïve, first-episode SCZ patients and 92 healthy controls to characterize the microbiome-gut-brain axis in SCZ patients. We used pathway-based polygenic risk score (PRS) analyses to determine the biological pathways contributing to genetic risk and mediation effect analyses to determine the important neuroimaging features. Additionally, a random forest model was generated for effective SCZ diagnosis. We found that the altered metabolome and dysregulated microbiome were associated with neuroactive metabolites, including gamma-aminobutyric acid (GABA), tryptophan, and short-chain fatty acids. Further structural and functional magnetic resonance imaging analyses highlighted that gray matter volume and functional connectivity disturbances mediate the relationships between Ruminococcus_torgues and Collinsella_aerofaciens and symptom severity and the relationships between species Lactobacillus_ruminis and differential metabolites l-2,4-diaminobutyric acid and N-acetylserotonin and cognitive function. Moreover, analyses of the Polygenic Risk Score (PRS) support that alterations in GABA and tryptophan neurotransmitter pathways are associated with SCZ risk, and GABA might be a more dominant contributor. This study provides new insights into systematic relationships among genes, metabolism, and the gut microbiota that affect brain functional connectivity, thereby affecting SCZ pathogenesis.


Synthesis of antimicrobial peptides using the SPOT technique.
Developing new lead structures for drugs against multiresistant bacteria is an urgent need for modern medicine. Antimicrobial peptides are a class of drugs that can be used to discover such structures. In order to support development of this research, a fast, easy, and inexpensive method to synthesize peptides is necessary. The SPOT synthesis has the potential to produce the required peptide arrays, synthesizing up to 8,000 peptides, peptide mixtures, or other organic compounds on cellulose or other planar surfaces in a positionally addressable and multiple manner. Protocols for the preparation of cellulose membranes and the SPOT synthesis as well as cleavage of peptides from the support are described.


Screen time and young children: Promoting health and development in a digital world.
The digital landscape is evolving more quickly than research on the effects of screen media on the development, learning and family life of young children. This statement examines the potential benefits and risks of screen media in children younger than 5 years, focusing on developmental, psychosocial and physical health. Evidence-based guidance to optimize and support children's early media experiences involves four principles: minimizing, mitigating, mindfully using and modelling healthy use of screens. Knowing how young children learn and develop informs best practice strategies for health care providers.


Effectiveness of the Chinese herbal formula TongXieYaoFang for irritable bowel syndrome: a systematic review.
Irritable bowel syndrome (IBS) is a common problem, but treatment is unsatisfactory. Although Chinese herbal medicines have been tried, there are limited data to support their usage. The authors set out to systematically review the effectiveness of the Chinese herbal medicine TongXieYaoFang (TXYF) and TXYF with different Chinese herbal additions (TXYF-A) in the management of IBS in order to make evidence-based recommendations. The authors searched the literature to identify randomized trials of TXYF or TXYF-A for patients with IBS by using MEDLINE, EMBASE, the Cochrane Controlled Trials Register (CCTR), the Cochrane Complementary Medicine Fields Specialized Register from 1966 to 2004, the CMB Disc database from January 1978 to December 1988, and the Full Text Chinese Journal database from January 1994 to December 2004. Standard forms regarding study design, treatment course, outcome measures, and adverse events were used to abstract data. Studies were assigned a quality score based on published methodology. Relative risk (RR) and 95% confidence interval (CI) were analyzed. Twelve studies (n = 1125) that met the criteria for review were identified. There was heterogeneity in the studies with regard to herbal formulas, control medicine, treatment course, and outcome measurement. The quality of all studies was low. The pooled analyses of effectiveness for the near-term (immediately after finishing the treatment course), short-term (3 months after finishing the treatment course), and long-term (6 months or more after finishing the treatment course) effectiveness showed that the effect of TXYF-A was better than that of conventional medicine in four, two, and six studies, respectively. The RRs were 1.34 (95% CI 1.16-1.54, p < 0.05), 1.39 (95% CI 1.17-1.64, p < 0.05), and 1.34 (95% CI 1.12-1.61, p < 0.05), respectively. Overall, combining all data from the 12 studies, the effectiveness of TXYF-As was higher than that in control groups, with statistical significance and RR of 1.35 (95% CI 1.21-1.50, p < 0.05). There is evidence to indicate the potential usefulness of TXYF-A for IBS patients. The results were limited by the poor quality and heterogeneity of these studies. Further studies with carefully designed, randomized double-blinded placebo-controlled trials will be needed to confirm the effectiveness of TXYF or TXYF-A for IBS.


EST sequencing and gene expression profiling of defence-related genes from Persea americana infected with Phytophthora cinnamomi.
Avocado (Persea americana) belongs to the Lauraceae family and is an important commercial fruit crop in over 50 countries. The most serious pathogen affecting avocado production is Phytophthora cinnamomi which causes Phytophthora root rot (PRR). Root pathogens such as P. cinnamomi and their interactions with hosts are poorly understood and despite the importance of both the avocado crop and the effect Phytophthora has on its cultivation, there is a lack of molecular knowledge underpinning our understanding of defence strategies against the pathogen. In order to initiate a better understanding of host-specific defence we have generated EST data using 454 pyrosequencing and profiled nine defence-related genes from Pc-infected avocado roots. 2.0 Mb of data was generated consisting of ~10,000 reads on a single lane of the GS FLX platform. Using the Newbler assembler 371 contigs were assembled, of which 367 are novel for Persea americana. Genes were classified according to Gene Ontology terms. In addition to identifying root-specific ESTs we were also able to identify and quantify the expression of nine defence-related genes that were differentially regulated in response to P. cinnamomi. Genes such as metallothionein, thaumatin and the pathogenesis related PsemI, mlo and profilin were found to be differentially regulated. This is the first study in elucidating the avocado root transcriptome as well as identifying defence responses of avocado roots to the root pathogen P. cinnamomi. Our data is currently the only EST data that has been generated for avocado rootstocks, and the ESTs identified in this study have already been useful in identifying defence-related genes as well as providing gene information for other studies looking at processes such as ROS regulation as well as hypoxia in avocado roots. Our EST data will aid in the elucidation of the avocado transcriptome and identification of markers for improved rootstock breeding and screening. The characterization of the avocado transcriptome will furthermore form a basis for functional genomics of basal angiosperms.


Running Economy: Neuromuscular and Joint-Stiffness Contributions in Trained Runners.
It is debated whether running biomechanics make good predictors of running economy, with little known about the neuromuscular and joint-stiffness contributions to economical running gait. To understand the relationship between certain neuromuscular and spatiotemporal biomechanical factors associated with running economy. Thirty trained runners performed a 6-min constant-speed running set at 3.3 m·s-1, where oxygen consumption was assessed. Overground running trials were also performed at 3.3 m·s-1 to assess kinematics, kinetics, and muscle activity. Spatiotemporal gait variables, joint stiffness, preactivation, and stance-phase muscle activity (gluteus medius, rectus femoris, biceps femoris, peroneus longus, tibialis anterior, and gastrocnemius lateralis and medius) were variables of specific interest and thus determined. In addition, preactivation and ground contact of agonist-antagonist coactivation were calculated. More economical runners presented with short ground-contact times (r = .639, P < .001) and greater stride frequencies (r = -.630, P < .001). Lower ankle and greater knee stiffness were associated with lower oxygen consumption (r = .527, P = .007 and r = .384, P = .043, respectively). Only lateral gastrocnemius-tibialis anterior coactivation during stance was associated with lower oxygen cost of transport (r = .672, P < .0001). Greater muscle preactivation and biarticular muscle activity during stance were associated with more economical runners. Consequently, trained runners who exhibit greater neuromuscular activation prior to and during ground contact, in turn optimizing spatiotemporal variables and joint stiffness, will be the most economical runners.


One-stage vs. two-stage brachio-basilic arteriovenous fistula for dialysis access: a systematic review and a meta-analysis.
A brachiobasilic arteriovenous fistula (BB-AVF) can provide access for haemodialysis in patients who are not eligible for a more superficial fistula. However, it is unclear whether one- or two-stage BB-AVF is the best option for patients. To systematically assess the difference between both procedures in terms of access maturation, patency and postoperative complications. Online search for randomised controlled trials (RCTs) and observational studies that compared the one-stage versus the two-stage technique for creating a BB-AVF. Eight studies were included (849 patients with 859 fistulas), 366 created using a one-stage technique, while 493 in a two-stage approach. There was no statistically significant difference between the two groups in the rate of successful maturation (Pooled risk ratio = 0.95 [0.82, 1.11], P = 0.53). Similarly, the incidence of postoperative haematoma (Pooled risk ratio = 0.73 [0.34, 1.58], P = 0.43), wound infection (Pooled risk ratio = 0.77 [0.35, 1.68], P = 0.51) and steal syndrome (Pooled risk ratio = 0.65 [0.27, 1.53], P = 0.32) were statistically comparable. Although more studies seem to favour the two-stage BVT approach, evidence in the literature is not sufficient to draw a final conclusion as the difference between the one-stage and the two-stage approaches for creation of a BB-AVF is not statistically significant in terms of the overall maturation rate and postoperative complications. Patency rates (primary, assisted primary and secondary) were comparable in the majority of studies. Large randomised properly conducted trials with superior methodology and adequate sub-group analysis are needed before making a final recommendation.


CD44 expression in plexiform lesions of idiopathic pulmonary arterial hypertension.
Plexiform lesions in pulmonary arteries are a characteristic histological feature for idiopathic pulmonary arterial hypertension (IPAH). The pathogenesis of the plexiform lesion is not fully understood, although it may be related to endothelial cell dysfunction and local inflammation. CD44 is a cell adhesion molecule and it is also involved in angiogenesis, endothelial cell proliferation and migration. The expression of CD44 was examined in lung plexiform lesions obtained from patients with IPAH (IPAH group, n= 7) and pulmonary arterial hypertension associated with atrial septal defect (ASD-PAH group, n= 4). Expression of CD44 was detected in 49 out of 52 plexiform lesions (93%) from all patients in the IPAH group, whereas 31 plexiform lesions obtained from the ASD-PAH group lacked CD44 positivity by immunohistochemistry. In the IPAH group, CD44 was localized in the endothelial cells of microvessels within plexiform lesions and activated T cells in and around the lesions. Furthermore, T cell infiltration and endothelial cell proliferation activity were prominent in the plexiform lesions of the IPAH group, compared to those of the ASD-PAH group. These findings suggest that CD44 and activated T cell infiltration play an important role in the development of plexiform lesions particularly in IPAH.


The Protective Role of Tanshinone IIA in Silicosis Rat Model via TGF-β1/Smad Signaling Suppression, NOX4 Inhibition and Nrf2/ARE Signaling Activation.
Silicosis is an occupational disease caused by inhalation of silica and there are no effective drugs to treat this disease. Tanshinone IIA (Tan IIA), a traditional natural component, has been reported to possess anti-inflammatory, antioxidant, and anti-fibrotic properties. The current study's purpose was to examine Tan IIA's protective effects against silica-induced pulmonary fibrosis and to explore the underlying mechanisms. 48 male SD rats were randomly divided into four groups (n=12): i) Control group; ii) Silicosis group; iii) Tan IIA group; iv) Silicosis +Tan IIA group. Two days after modeling, the rats of Tan IIA group and Silicosis +Tan IIA group were given intraperitoneal administration 25 mg/kg/d Tan IIA for 40 days. Then, the four groups of rats were sacrificed and the lung inflammatory responses were measured by ELISA, lung damage and fibrosis were analyzed by hematoxylin and eosin (H&E) staining and Masson staining, the expression levels of collagen I, fibronectin and α-smooth muscle actin (α-SMA) were measured by immunohistochemistry. The markers of oxidative stress were measured by commercial kits, and the activity of the TGF-β1/Smad and NOX4, Nrf2/ARE signaling pathways were measured by RT-PCR and Western blotting. The silica-induced pulmonary inflammtory responses, structural damage and fibrosis were significantly attenuated by Tan IIA treatment. In addition, treatment with Tan IIA decreased collagen I, fibronectin and α-SMA expression, and inhibited TGF-β1/Smad signaling in the lung tissue. The upregulated levels of oxidative stress markers in silicosis rats were also markedly restored following Tan IIA treatment. Furthermore, treatment with Tan IIA reduced NOX4 expression and enhanced activation of the Nrf2/ARE pathway in the lung tissue of silicosis rats. These findings suggest that Tan IIA may protect lung from silica damage via the suppression of TGF-β1/Smad signaling, inhibition of NOX4 expression and activation of the Nrf2/ARE pathway.


Peroral transgastric endoscopic primary repair of a ventral hernia in a porcine model.
Multiple studies have demonstrated the feasibility of peroral transgastric endoscopic procedures in animal models. The aim of the study was to evaluate the feasibility of a peroral transgastric endoscopic approach to repair abdominal wall hernias. We performed acute experiments under general anesthesia with endotracheal intubation using 50-kg pigs. Following peroral intubation an incision of the gastric wall was made and the endoscope was advanced into the peritoneal cavity. An internal anterior abdominal wall incision was performed with a needle knife to create an animal model of a ventral hernia. After hernia creation an endoscopic suturing device was used for primary repair of the hernia. After completion of the hernia repair the endoscope was withdrawn into the stomach and the gastric wall incision was closed with endoscopic clips. Then the animals were killed for necropsy. Two acute experiments were performed. Incision of the gastric wall was easily achieved with a needle knife and a pull-type sphincterotome. A large (3 x 2 cm) defect of the abdominal wall (ventral hernia model) was closed with five or six sutures using the endoscopic suturing device. Postmortem examination revealed complete closure of the hernia without any complications. Transgastric endoscopic primary repair of ventral hernias in a porcine model is feasible and may be technically simpler than laparoscopic surgery.


Remotely controlled chemomagnetic modulation of targeted neural circuits.
Connecting neural circuit output to behaviour can be facilitated by the precise chemical manipulation of specific cell populations1,2. Engineered receptors exclusively activated by designer small molecules enable manipulation of specific neural pathways3,4. However, their application to studies of behaviour has thus far been hampered by a trade-off between the low temporal resolution of systemic injection versus the invasiveness of implanted cannulae or infusion pumps2. Here, we developed a remotely controlled chemomagnetic modulation-a nanomaterials-based technique that permits the pharmacological interrogation of targeted neural populations in freely moving subjects. The heat dissipated by magnetic nanoparticles (MNPs) in the presence of alternating magnetic fields (AMFs) triggers small-molecule release from thermally sensitive lipid vesicles with a 20 s latency. Coupled with the chemogenetic activation of engineered receptors, this technique permits the control of specific neurons with temporal and spatial precision. The delivery of chemomagnetic particles to the ventral tegmental area (VTA) allows the remote modulation of motivated behaviour in mice. Furthermore, this chemomagnetic approach activates endogenous circuits by enabling the regulated release of receptor ligands. Applied to an endogenous dopamine receptor D1 (DRD1) agonist in the nucleus accumbens (NAc), a brain area involved in mediating social interactions, chemomagnetic modulation increases sociability in mice. By offering a temporally precise control of specified ligand-receptor interactions in neurons, this approach may facilitate molecular neuroscience studies in behaving organisms.


Adverse Events in the Efficacy of Crotalidae Polyvalent Immune Fab Antivenom vs Placebo in Recovery from Copperhead Snakebite Trial.
To compare the incidence of hypersensitivity reactions following copperhead envenomation treated with Fab antivenom (FabAV) or placebo. Patients with copperhead snakebites received treatment and follow-up in a prospective, randomized, double-blind, placebo-controlled trial of FabAV or placebo. The treatment allocation ratio was 2:1 (FabAV:placebo). All of the included patients received at least one dose of study treatment. We reviewed all treatment-emergent adverse events (AEs) using a previously published scale to classify likely hypersensitivity reactions as mild, moderate, or severe. We enrolled 74 patients at 13 sites. Forty-five patients received FabAV, and 29 patients received placebo. Five FabAV patients and 4 placebo patients had moderate envenomations; the rest were mild. Twenty-five FabAV patients and 8 placebo patients had at least 1 AE. Mild skin reactions occurred in 11 (24%) FabAV patients (pruritis, urticaria, rash, ecchymosis, erythema) and 1 (3%) placebo patient (pruritis). Moderate gastrointestinal AEs occurred in 7 (16%) FabAV patients (nausea, vomiting, constipation, diarrhea, oral paresthesia) and in 2 (7%) placebo patients (nausea). Respiratory AEs occurred in 3 (7%) FabAV patients (dyspnea, pulmonary embolism, nasal congestion, sneezing) and no placebo patients. Hypotension occurred in 1 patient in each group. In a randomized controlled trial of FabAV for copperhead bites, the incidence of hypersensitivity reactions was low. Most reactions were mild skin reactions.


Distinct prognostic value of circulating anti-telomerase CD4+ Th1 immunity and exhausted PD-1+/TIM-3+ T cells in lung cancer.
Despite the critical roles of Th1-polarised CD4+ T cells in cancer immunosurveillance, the translation of their potential to clinical use remains challenging. Here, we investigate the clinical relevance of circulating antitumor Th1 immunity in non-small cell lung cancer (NSCLC). The circulating antitumor Th1 response was assessed by the ELISpot assay in 170 NSCLC patients using a mixture of HLA class II-restricted peptides from telomerase (TERT). Phenotyping of blood immune cells was performed by flow cytometry. TERT-reactive CD4 T-cell response was detected in 35% of NSCLC patients before any treatment. Functional analysis showed that these cells were effector memory and Th1 polarised capable to produce effector cytokines, such as IFN-γ, TNF-α and IL-2. The presence of anti-TERT Th1 response was inversely correlated with the level of exhausted PD-1+/TIM-3+CD4 T cells. The level of these two immune parameters differentially affected the survival, so that increased level of anti-TERT Th1 response and low rate of exhausted PD-1+TIM-3+CD4+ T cells were associated with a better prognosis. Systemic anti-TERT Th1 response plays a strong antitumor protective role in NSCLC. This study underlines the potential interest of monitoring circulating antitumor Th1 response for patients' stratification and therapy decision.


Free energy contributions to direct readout of a DNA sequence.
The energetic contributions of individual DNA-contacting side chains to specific DNA recognition in the human papillomavirus 16 E2C-DNA complex is small (less than 1.0 kcal mol(-1)), independent of the physical and chemical nature of the interaction, and is strictly additive. The sum of the individual contributions differs 1.0 kcal mol(-1) from the binding energy of the wild-type protein. This difference corresponds to the contribution from the deformability of the DNA, known as "indirect readout." Thus, we can dissect the energetic contribution to DNA binding into 90% direct and 10% indirect readout components. The lack of high energy interactions indicates the absence of "hot spots," such as those found in protein-protein interfaces. These results are compatible with a highly dynamic and "wet" protein-DNA interface, yet highly specific and tight, where individual interactions are constantly being formed and broken.


Inhibition of mTOR signaling reduces PELP1-mediated tumor growth and therapy resistance.
Proline, Glutamic acid-, and Leucine-rich Protein 1 (PELP1) is a proto-oncogene that modulates estrogen receptor (ER) signaling. PELP1 expression is upregulated in breast cancer, contributes to therapy resistance, and is a prognostic marker of poor survival. In a subset of breast tumors, PELP1 is predominantly localized in the cytoplasm and PELP1 participates in extranuclear signaling by facilitating ER interactions with Src and phosphoinositide 3-kinase (PI3K). However, the mechanism by which PELP1 extranuclear actions contributes to cancer progression and therapy resistance remains unclear. In this study, we discovered that PELP1 cross-talked with the serine/threonine protein kinase mTOR and modulated mTOR signaling. PELP1 knockdown significantly reduced the activation of mTOR downstream signaling components. Conversely, PELP1 overexpression excessively activated mTOR signaling components. We detected the presence of the mTOR signaling complex proteins in PELP1 immunoprecipitates. mTOR-targeting drugs (rapamycin and AZD8055) significantly reduced proliferation of PELP1-overexpressed breast cancer cells in both in vitro and in vivo xenograft tumor models. MCF7 cells that uniquely retain PELP1 in the cytoplasm showed resistance to hormonal therapy and mTOR inhibitors sensitized PELP1cyto cells to hormonal therapy in xenograft assays. Notably, immunohistochemical studies using xenograft tumors derived from PELP1 overexpression model cells showed increased mTOR signaling and inhibition of mTOR rendered PELP1-driven tumors to be highly sensitive to therapeutic inhibition. Collectively, our data identified the PELP1-mTOR axis as a novel component of PELP1 oncogenic functions and suggest that mTOR inhibitor(s) will be effective chemotherapeutic agents for downregulating PELP1 oncogenic functions.


The CDK9 C-helix exhibits conformational plasticity that may explain the selectivity of CAN508.
CDK9 is the kinase of positive transcription elongation factor b and facilitates the transition of paused RNA polymerase II to processive transcription elongation. CDK9 is a validated target for the treatment of cancer, cardiac hypertrophy, and human immunodeficiency virus. Here we analyze different CDK9/cyclin T variants to identify a form of the complex amenable to use in inhibitor design. To demonstrate the utility of this system, we have determined the crystal structures of CDK9/cyclin T and CDK2/cyclin A bound to the CDK9-specific inhibitor CAN508. Comparison of the structures reveals CDK9-specific conformational changes and identifies a CDK9-specific hydrophobic pocket, adjacent to the αC-helix. By comparison with a previously published structure of CDK9/cyclin T/human immunodeficiency virus TAT we find that the CDK9 αC-helix has a degree of conformational variability that has the potential to be exploited for inhibitor design.