Mediterranean-type diet is associated with higher psychological resilience in a general adult population: findings from the Moli-sani study.
Psychological resilience is a measure of stress coping ability and has been associated with favourable health outcomes. While evidence on the relationship of dietary habits with a number of psychosocial conditions is available, there is lack of studies on their association with psychological resilience in a general adult population. Cross-sectional analysis on 10 812 subjects recruited within the cohort of the Moli-sani study (2005-2010). Psychological resilience was measured by the 25-item Connor-Davidson Psychological Resilience Scale. Food intake was recorded by the EPIC food frequency questionnaire and adherence to Mediterranean diet was appraised by both a Greek Mediterranean diet score and an Italian Mediterranean Index. Empirically derived dietary patterns were obtained by principal factor analysis. Multivariable linear regression analysis (95%CI) was used to test the association between dietary scores and psychological resilience. Higher adherence to Mediterranean-type diets or consumption of a vegetable-based dietary pattern (obtained from principal factor analysis) were positively associated with psychological resilience (β=0.43; 95%CI: 0.19-0.66, β=0.92; 0.69-1.16, and β=1.18; 0.93-1.44, for Greek Mediterranean diet score, Italian Mediterranean Index and the 'Olive oil and vegetables pattern', respectively). Dietary polyphenol or antioxidant intakes and greater variety in fruit and vegetable consumption were also positively associated with psychological resilience, while the associations with Western-like diets were weak. In conclusion, Mediterranean diet, vegetable-based dietary patterns and better diet quality were all positively associated with higher psychological resilience, whereas Western-type diets were not.


Hospital outbreaks caused by Pseudomonas aeruginosa: importance of serogroup O11.
Suspected outbreaks caused by pseudomonas aeruginosa in 19 hospitals and two motels were studied. On the basis of epidemiological analysis, serological typing, and antibiotic resistance patterns, 17 were classified as single-strain outbreaks. Six were classified as common-source outbreaks: of these, three were caused by contaminated urological instruments or solutions, two involved bathing in contaminated whirlpools, and one was caused by contaminated lens prostheses implanted during eye surgery. The ability of P. aeruginosa to survive or grow in wet environments was important in each of these six outbreaks. Eight outbreaks were classified as cross-infection. Two involved the urinary tract and were caused by antibiotic-resistant strains. Six involved the respiratory tract, but only one was caused by an antibiotic-resistant strain. In 2 of the 17 single-strain outbreaks, the exact mode of transmission could not be determined. One was an outbreak of pseudobacteremia in which patient blood cultures were contaminated with a single strain, presumably during collection of specimens or culture processing, P. aeruginosa serogroup O11 caused 9 of 17 (53%) single-strain outbreaks, a surprising finding since this serogroup represents only about 8% of endemic hospital isolates of this species. Serotyping was very useful in epidemiological analysis, but antibiotic susceptibility patterns were less useful.


Food-Derived Peptides: Beneficial CNS Effects and Cross-BBB Transmission Strategies.
Food-derived peptides, as dietary supplements, have significant effects on promoting brain health and relieving central nervous system (CNS) diseases. However, the blood-brain barrier (BBB) greatly limits their in-brain bioavailability. Thus, overcoming the BBB to target the CNS is a major challenge for bioactive peptides in the prevention and treatment of CNS diseases. This review discusses improvement in the neuroprotective function of food-derived active peptides in CNS diseases, as well as the source of BBB penetrating peptides (BBB-shuttles) and the mechanism of transmembrane transport. Notably, this review also discusses various peptide modification methods to overcome the low permeability and stability of the BBB. Lipification, glycosylation, introduction of disulfide bonds, and cyclization are effective strategies for improving the penetration efficiency of peptides through the BBB. This review provides a new prospective for improving their neuroprotective function and developing treatments to delay or even prevent CNS diseases.


Lactate dehydrogenase inhibition synergizes with IL-21 to promote CD8+ T cell stemness and antitumor immunity.
Interleukin (IL)-2 and IL-21 dichotomously shape CD8+ T cell differentiation. IL-2 drives terminal differentiation, generating cells that are poorly effective against tumors, whereas IL-21 promotes stem cell memory T cells (TSCM) and antitumor responses. Here we investigated the role of metabolic programming in the developmental differences induced by these cytokines. IL-2 promoted effector-like metabolism and aerobic glycolysis, robustly inducing lactate dehydrogenase (LDH) and lactate production, whereas IL-21 maintained a metabolically quiescent state dependent on oxidative phosphorylation. LDH inhibition rewired IL-2-induced effects, promoting pyruvate entry into the tricarboxylic acid cycle and inhibiting terminal effector and exhaustion programs, including mRNA expression of members of the NR4A family of nuclear receptors, as well as Prdm1 and Xbp1 While deletion of Ldha prevented development of cells with antitumor effector function, transient LDH inhibition enhanced the generation of memory cells capable of triggering robust antitumor responses after adoptive transfer. LDH inhibition did not significantly affect IL-21-induced metabolism but caused major transcriptomic changes, including the suppression of IL-21-induced exhaustion markers LAG3, PD1, 2B4, and TIM3. LDH inhibition combined with IL-21 increased the formation of TSCM cells, resulting in more profound antitumor responses and prolonged host survival. These findings indicate a pivotal role for LDH in modulating cytokine-mediated T cell differentiation and underscore the therapeutic potential of transiently inhibiting LDH during adoptive T cell-based immunotherapy, with an unanticipated cooperative antitumor effect of LDH inhibition and IL-21.


In vitro stimulation of Na-K-ATPase in rat thick ascending limb by dexamethasone.
Dexamethasone has been reported to stimulate Na-K-ATPase activity in the medullary thick ascending limb of adrenalectomized animals within a few hours. The present study was aimed at characterizing the mechanism of this action by investigating the stimulatory effect of the hormone in vitro. Dexamethasone (10(-8) M) added in vitro to segments of the medullary thick ascending limb of Henle's loop, which were microdissected from adrenalectomized rats, restored in a dose-dependent manner the depressed Na-K-ATPase activity within one h of incubation. This stimulation of Na-K-ATPase was inhibited by cycloheximide and actinomycin D. Dexamethasone also stimulated the component of oxidative metabolism coupled to sodium transport. These results, which confirm previous in vivo observations, demonstrate that dexamethasone-induced stimulation of Na-K-ATPase is a direct tubular action of the hormone mediated by protein synthesis. They suggest that this short-term effect of dexamethasone corresponds to the stimulation of sodium reabsorption by the dilution segment.


Microwave induced shift of the main phase transition in phosphatidylcholine membranes.
Numerous experimental evidence show that exposure of biological systems to extremely high frequency microwaves may induce significant effects even at low powers. These effects are thought to occur via nonthermal mechanisms involving primarily the interaction of microwaves with phospholipid membrane structures. However, no conclusive experimental evidence that biomembranes exhibit remarkable sensitivity to this radiation has been provided up to now. Here, deuterium nuclear magnetic resonance spectroscopy is used to study the effects of microwaves on 1,2-Dimyristoyl-sn-glycero-3-phosphatidylcholine/(2)H(2)O multilamellar vesicles that serve as biomimetic membranes. Here we show that, if the membrane is brought into close proximity to the transition point, microwaves induce a reduction of water ordering at the membrane interface, an upward shift of the main phase transition temperature and a broadening of the transition region. A deep dosimetric analysis shows that the above effects are nonthermal, indicating the need for a nonthermal hypothesis to explain them. This study suggests that exposure to high-frequency microwaves can have far reaching consequences on active biological systems.


How does dietary particle size affect carnivore gastrointestinal transit: A dog model.
The effect of dietary particle size on gastrointestinal transit in carnivores has not been studied and might offer more insight into their digestive physiology. This study evaluated the effect of two dietary particle sizes (fine = 7.8 mm vs. coarse = 13 mm) of chunked day-old chicks on transit parameters in dogs. Six beagle dogs were fed both dietary treatments in a crossover design of 7 days with transit testing on the fifth day. Transit parameters were assessed using two markers, that is a wireless motility capsule (IntelliCap® ) and titanium oxide (TiO2 ). Dietary particle size did not affect gastric emptying time (GRT), small bowel transit time (SBTT), colonic transit time (CTT) and total transit time (aTTT) of the capsule (p > .05). There was no effect of dietary particle size on TiO2 mean retention time (MRT) (p > .05). The time of last TiO2 excretion (MaxRT) differed (p = .013) between diets, being later for the coarse diet. Both MRT (R = 0.617, p = .032) and MaxRT (R = 0.814; p = .001) were positively correlated to aTTT. The ratio MRT/aTTT tended towards a difference between diets (p = .059) with the coarse diet exceeding fine diet values. Results show that the difference between capsule measurements and TiO2 is larger for the fine than the coarse diet suggesting that the capsule becomes more accurate when dietary particle size approaches marker size. Dietary particle size might have affected transit parameters but differences are too small to claim major physiological consequences.


Occurrence of Clinically Diagnosed Hypertrophic Cardiomyopathy in the United States.
Hypertrophic cardiomyopathy (HC) is the most common genetic heart disease and an important cause of sudden death and heart failure symptoms. The current prevalence for HC (1:500) is based on echocardiographic population studies in which a substantial proportion of affected subjects have not come to clinical recognition. Therefore, we sought to define the subset of patients with HC who are diagnosed in the US. A proprietary integrated claims database including medical condition International Classification of Diseases, Ninth Revision diagnostic codes for over 160 million individual patients in the US was interrogated for 2013 to identify the prevalence of clinically recognized HC. Patients with ≥1 claim for any of the HC International Classification of Diseases, Ninth Revision diagnosis codes from January to December 2013 were identified. The combined occurrence rate of HC was stratified by age and gender and multiplied by the 2013 United States population in the same age/gender categories to produce the final projected prevalence. The analysis was performed on 169,089,614 patients, of whom 59,009 unique patients were identified with ≥1 claim for HC. The projected estimated occurrence of diagnosed HC in the US in 2013 was 1:3,195 for a total of 98,958 subjects. Average age at HC diagnosis was in the fifth decade of life, with 43% of the cohort composed of women. In conclusion, leveraging a claims-based data analytic technique, about 100,000 patients are diagnosed clinically with HC in the US, an occurrence which is less than the prevalence reported in systematic population studies based on echocardiographic diagnosis. This observation supports the view that many patients with HC are undiagnosed throughout life and enhances our understanding of the burden of this genetic heart disease on the health care system.


Increase in Obesity Among Women of Reproductive Age in Zambia, 2002-2014.
Objective: To describe trends in obesity in Zambian women of reproductive age and to identify factors that may have contributed to changes in trends and nutrition outcomes. Materials and Methods: We obtained data on body mass index and individual factors of women from the Zambia Demographic and Health Survey for the period 2002 to 2014. From these data, we calculated descriptive statistics and examined the extent to which factors link to the odds of obesity over time. We also reviewed primary and secondary data sources, such as government documents, theses, and search engines to identify factors that may have contributed to trends and changes in nutrition outcomes. Results: The proportion of obesity doubled from 2002 (12.5%) to 2014 (22.3%). The odds were higher among educated, currently married and wealthy women, and it increased with age. Rural residence and working in agricultural-related jobs were linked to lower odds for obesity. This disparity varies by province. In addition, despite the presence of many nutrition policies and strategies, the increase in obesity occurred within the past two decades when urbanization and other factors (e.g., sedentary work, a proliferation of fast food restaurants, and advertisements) may have affected changes in nutrition outcomes for women. Conclusions: We identified increasing trends in obesity in women of reproductive age over time. The rapid urbanization and other factors that occurred in Zambia during this period are significant risk factors for obesity in Zambian women. The findings will be of interest to countries that are undergoing a nutrition transition.


Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes.
Vaccinia virus is the prototypic orthopoxvirus and was the vaccine used to eradicate smallpox, yet the expression profiles of many of its genes remain unknown. Using a genome tiling array approach, we simultaneously measured the expression levels of all 223 annotated vaccinia virus genes during infection and determined their kinetics. For 95% of these genes, significant transcript levels were detected. Most remarkably, classification of the genes by their expression profiles revealed 35 genes exhibiting immediate-early expression. Although a similar kinetic class has been described for other virus families, to our knowledge, this is the first demonstration of its existence in orthopoxviruses. Despite expression levels higher than for genes in the other three kinetic classes, the functions of more than half of these remain unknown. Additionally, genes within each kinetic class were spatially grouped together in the genome. This genome-wide picture of transcription alters our understanding of how orthopoxviruses regulate gene expression.


Multi-analyte fluorescence sensing based on a post-synthetically functionalized two-dimensional Zn-MOF nanosheets featuring excited-state proton transfer process.
Covalent post-synthetic modification of metal-organic frameworks (MOFs) represents an underexplored but promising avenue for allowing the addition of specific fluorescent recognition elements to produce the novel MOF-based sensory materials with multiple-analyte detection capability. Here, an excited-state proton transfer (ESPT) active sensor 2D-Zn-NS-P was designed and constructed by covalent post-synthetic incorporation of the excited-state tautomeric 2-hydroxypyridine moiety into the ultrasonically exfoliated amino-tagged 2D Zn-MOF nanosheets (2D-Zn-NS). The water-mediated ESPT process facilitates the highly accessible active sites incorporated on the surface of 2D-Zn-NS-P to specifically respond to the presence of water in common organic solvents via fluorescence turn-on behavior, and accurate quantification of trace amount of water in acetonitrile, acetone and ethanol was established using the as-synthesized nanosheet sensor with the detection sensitivity (<0.01% v/v) superior to the conventional Karl Fischer titration. Upon exposure to Fe3+ or Cr2O72-, the intense blue emission of the aqueous colloidal dispersion of 2D-Zn-NS-P was selectively quenched even in the coexistence of common inorganic interferents. The prohibition of the water-mediated ESPT process and local emission, induced by the coordination of ESPT fluorophore with Fe3+ or by Cr2O72- competitively absorbs the excitation energy, was proposed to responsible for the fluorescence turn-off sensing of the respective analytes. The present study offers the attractive prospect to develop the ESPT-based fluorescent MOF nanosheets by covalent post-synthetic modification strategy as multi-functional sensors for detection of target analytes.


Estradiol-induced regulation of GLUT4 in 3T3-L1 cells: involvement of ESR1 and AKT activation.
Impaired insulin-stimulated glucose uptake involves reduced expression of the GLUT4 (solute carrier family 2 facilitated glucose transporter member 4, SLC2A4 gene). 17β-estradiol (E2) modulates SLC2A4/GLUT4 expression, but the involved mechanisms are unclear. Although E2 exerts biological effects by binding to estrogen receptors 1/2 (ESR1/2), which are nuclear transcriptional factors; extranuclear effects have also been proposed. We hypothesize that E2 regulates GLUT4 through an extranuclear ESR1 mechanism. Thus, we investigated the effects of E2 upon (1) subcellular distribution of ESRs and the proto-oncogene tyrosine-protein kinases (SRC) involvement; (2) serine/threonine-protein kinase (AKT) activation; (3) Slc2a4/GLUT4 expression and (4) GLUT4 subcellular distribution and glucose uptake in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were cultivated or not with E2 for 24 h, and additionally treated or not with ESR1-selective agonist (PPT), ESR1-selective antagonist (MPP) or selective SRC inhibitor (PP2). Subcellular distribution of ESR1, ESR2 and GLUT4 was analyzed by immunocytochemistry; Slc2a4 mRNA and GLUT4 were quantified by qPCR and Western blotting, respectively; plasma membrane GLUT4 translocation and glucose uptake were analyzed under insulin stimulus for 20 min or not. E2 induced (1) translocation of ESR1, but not of ESR2, from nucleus to plasma membrane and AKT phosphorylation, effects mimicked by PPT and blocked by MPP and PP2; (2) increased Slc2a4/GLUT4 expression and (3) increased insulin-stimulated GLUT4 translocation and glucose uptake. In conclusion, E2 treatment promoted a SRC-mediated nucleus-plasma membrane shuttle of ESR1, and increased AKT phosphorylation, Slc2a4/GLUT4 expression and plasma membrane GLUT4 translocation; consequently, improving insulin-stimulated glucose uptake. These results unravel mechanisms through which estrogen improves insulin sensitivity.


Prediction tools and risk stratification in epilepsy surgery.
To conduct external validation of previously published epilepsy surgery prediction tools using a large independent multicenter dataset and to assess whether these tools can stratify patients for being operated and for becoming free of disabling seizures (ILAE 1 and 2). We analyzed a dataset of 1,562 patients, not used for tool development. We applied two scales: Epilepsy Surgery Grading Scale (ESGS) and Seizure Freedom Score (SFS), and two versions of Epilepsy Surgery Nomograms (ESNs): the original version and the modified version which included EEG data. For the ESNs we used calibration curves and concordance indexes. We stratified the patients into three tiers, for assessing the chances of attaining freedom of disabling seizures after surgery: high (ESGS 1, SFS 3-4, ESNs>70%), moderate (ESGS 2, SFS 2, ESNs 40-70%) and low (ESGS 2, SFS 0-1, ESNs<40%). We compared the three tiers as stratified by these tools, concerning the proportion of patients who were operated, and for the proportion of patients who became free of disabling seizures. The concordance indexes for the various versions of the nomograms were between 0.56 and 0.69. Both scales (ESGS, SFS) and nomograms accurately stratified the patients for becoming free of disabling seizures, with significant differences among the three tiers (p<0.05). In addition, ESGS and the modified ESN accurately stratified the patients for having been offered surgery, with significant difference among the three tiers (p<0.05). ESGS and the modified ESN (at thresholds of 40% and 70%) stratify patients undergoing presurgical evaluation into three tiers, with high, moderate and low chance for favorable outcome, with significant differences between the groups concerning having surgery and becoming free of disabling seizures. Stratifying patients for epilepsy surgery has the potential to help select the optimal candidates in underprivileged areas and better allocation of resources in developed countries.


The Great Leap Forward: the anatomic basis for the acquisition of speech and obstructive sleep apnea.
Obstructive sleep apnea is an anatomic illness caused by evolutionary changes in the human upper respiratory tract. These changes include shortening of the maxillary, ethmoid, palatal and mandibular bones, acute oral cavity-skull base angulation, pharyngeal collapse with anterior migration of the foramen magnum, posterior migration of the tongue into the pharynx, descent of the larynx and shortening of the soft palate with loss of the epiglottic-soft palate lock-up. While it is commonly believed that some of these changes had positive selection pressures for bipedalism, binocular vision and locomotion, development of voice, speech and language ultimately became a substantial contributing factor. Here it is shown that these changes are the anatomic basis of obstructive sleep apnea.


Heart-specific autoantibodies can be eluted from the hearts of Coxsackievirus B3-infected mice.
This study was undertaken to determine if immunoglobulin G (IgG) antibodies could be eluted from the hearts of mice with Coxsackievirus B3-induced autoimmune myocarditis and to characterize the immunoreactivity of any elutable autoantibodies. Susceptible (A/J) and resistant (B10.A) mice were administered the virus or the control treatment and killed at various times after treatment. Acid eluates from pooled heart tissue from each treatment group and each time were tested for IgG reactivity with normal heart tissue by immunohistochemistry and with normal heart extracts by Western immunostaining. Eluates from infected A/J mice reacted strongly with syngeneic heart and modestly with syngeneic skeletal muscle tissue. Eluates from infected B10.A or control mice of either strain exhibited little reactivity with either tissue. Tissue reactivity was similar when allogeneic tissue was used as the substrate. Eluates from infected A/J mice recognized the heavy chain of cardiac myosin and several other cardiac antigens by Western immunostaining while eluates from the other treatment groups exhibited little or no reactivity with any normal heart constituents. These results indicate that in vivo IgG deposition occurs in the hearts of mice with post-infectious autoimmune myocarditis and that the specificity of these antibodies is similar to that reported for serum from animals with this disease. The mechanism(s) leading to myocardial IgG deposition and its possible role in pathogenesis remain to be elucidated.


[Effect of removal of the hypothalamus and pituitary gland from rat fetuses on the development of pancreatic B-cell function].
The reactivity of pancreatic B-cells of the decapitated and encephalectomized rat foetuses was determined by changes of insulin secretion in response to glucose introduction in the incubation medium. Upon removal of hypophysis together with hypothalamus in the intact rat females and females with alloxanic diabetes on the 17.5-18.5 day of prenatal development, their pancreas, unlike that of the control foetuses, remained refractory to glucose on the 21.5 day of development. The removal of hypothalamus only upon encephalectomy of the foetuses of the same age led to a similar effect, thus suggesting possible influence of hypothalamus on the regulation of functional maturation of the pancreatic B-cells in the end of prenatal development. Upon the combined incubation of pancreas fragments of the decapitated rat foetuses with adenohypophyses of the adult rats, the effect of decapitation was cut short and the gland responded by the increase of insulin secretion to the rise of glucose content in the medium.


[The electrophoretic properties of gels of high-resolution MS-4 agarose for separating DNA fragments in molecular genetic analysis].
The Hispanlab firm (Spain) offers a new series of Molecular Screen analytical agarose, including MS-4 agarose intended for separation of small fragments of DNA. This low-endosmotic agarose attracts attention as a probable alternative to well-known FMC Bio Products special agarose (USA) due to its useful properties. We characterized a gel-electrophoretic system based on MS-4 agarose (and its correspondence to its passport) and tested its fitness for standard molecular genetic expert investigations. The following parameters were assessed: mechanical properties of gel, its transparency, degree of background fluorescence upon ethidium bromide staining, resolving power, and position distortions in the electrophoregram. Important features of MS-4 agarose gels were detected, not typical of other agarose systems, which should be borne in mind during investigations with this agarose.


The interaction of mitochondrial translational initiation factor 2 with the small ribosomal subunit.
Bovine mitochondrial translational initiation factor 2 (IF-2(mt)) is organized into four domains, an N-terminal domain, a central G-domain and two C-terminal domains. These domains correspond to domains III-VI in the six-domain model of Escherichia coli IF-2. Variants in IF-2(mt) were prepared and tested for their abilities to bind the small (28S) subunit of the mitochondrial ribosome. The binding of wild-type IF-2(mt) was strong (K(d) approximately 10-20 nM) and was not affected by fMet-tRNA. Deletion of the N-terminal domain substantially reduced the binding of IF-2(mt) to 28S subunits. However, the addition of fMet-tRNA stimulated the binding of this variant at least 2-fold demonstrating that contacts between fMet-tRNA and IF-2(mt) can stabilize the binding of this factor to 28S subunits. No binding was observed for IF-2(mt) variants lacking the G-domain which probably plays a critical role in organizing the structure of IF-2(mt). IF-2(mt) contains a 37-amino acid insertion region between domains V and VI that is not found in the prokaryotic factors. Mutations in this region caused a significant reduction in the ability of the factor to promote initiation complex formation and to bind 28S subunits.


[Reference curves for hearing surface scaling in clinical use].
A commercially available system for the purpose of categorial loudness rating (WHF, WESTRA ELECTRONIC GmbH, Wertingen) was applied with 57 subjects with normal hearing to evaluate reference loudness level functions for 1/3-octave bandpass filtered noise and octave bandpass filtered environmental sounds. The procedure can be carried out with high reproducibility without effects of training due to repetition and only slight influence of subject sex, whereas the labeling of input device and temporal structure of the environmental sounds influence results, as does the center frequency. Our results showed deviations compared with the reference functions displayed in the WHF system.


The effect of choline supplementation on hepatic steatosis in the parenterally fed rat.
Information regarding hepatic function during total parenteral nutrition in rats is often extrapolated to the clinical situation, but the steatosis observed in that species may simply reflect choline deficiency and be irrelevant to man. The effect of choline supplementation on hepatic lipid content and triglyceride secretion was examined in parenterally fed rats. Eighty to 90-day-old rats were randomized into three groups; group I received oral Purina Chow ad libitum, groups II and III received identical total parenteral nutrition regimens with the exception that group III received supplemental choline. After 7 days, peripheral triglyceride uptake was inhibited with Triton WR1339, the rate of secretion of 14C-labeled triglyceride measured after a bolus injection of 1-14C-palmitic acid, and total hepatic lipid content was measured. Total hepatic lipid content was elevated in group II (86.3 mg/g) and group III (83.3 mg/g), and both differed significantly from the control group I (35.2 mg/g, p less than 0.01), but the choline supplementation appeared to make no difference. Hepatic secretion of 14C-palmitic acid as 14C-triglyceride was reduced in group II (0.73%/ml plasma), and group III (0.72%/ml plasma) compared to group I (1.06%/ml plasma, p less than 0.05), and was unaffected by choline. The hepatic steatosis produced in the parenterally fed rat did not appear to be due to choline deficiency but to some other factors which may be important in man.


Production of bacteremia and meningitis in infant rats with group B streptococcal serotypes.
Group B streptococcal strains, representing the five major serotypes, were inoculated into infant rats by intranasal, oral, and intraperitoneal routes. Bacteremia regularly followed injection by the intraperitoneal route. Four strains (three of type III) isolated from human cerebrospinal fluid appeared more virulent for 5-day-old rats. Injection of fewer than 10 colony-forming units of one strain, a type III, led to bacteremia and death in 27% of animals. The cumulative bacteremia and mortality rate with this strain was 66% in animals given inocula of less than 10 to 10(3) colony-forming units. Bacteremia developed by 24 to 48 h with concentrations of greater than 10(5) colony-forming units per ml of blood, and death occurred soon afterward. Among bacteremic animals, positive cerebrospinal fluid cultures were found in 97%, and cerebrospinal fluid bacterial concentrations were equal to or exceeded bacterial counts in blood. The susceptibility of infant rats to infection with type Ia, Ic, or III strains was age dependent. Histopathological studies of the brain and meninges in 34 bacteremic animals with culture-positive cerebrospinal fluid revealed that 5- to 10-day-old animals had numerous bacteria distributed in a perivascular pattern but, with one exception, no leukocytic infiltration. In contrast, three of the 11- to 12-day-old and two 15-day-old animals had very thickened meninges infiltrated with polymorphonuclear leukocytes, macrophages, and bacteria.


Medication use and risk of ovarian carcinoma: a prospective study.
Inflammation and gonadotropins are hypothesized to influence ovarian carcinogenesis. In a prospective study, we evaluated ovarian cancer risk associated with self-reported use of medications that influence inflammation or gonadotropin levels. The Breast Cancer Detection Demonstration Project Follow-Up Study enrolled 61,431 women in 1979 and used telephone interviews and 3 mailed questionnaires through 1998 to update risk factor information and identify incident ovarian cancers. The 1992-95 questionnaire ascertained medication use, including duration and frequency of use for aspirin, acetaminophen, other nonsteroidal anti-inflammatory drugs (NSAIDs), tranquilizers and histamine-receptor antagonists. A Poisson regression analysis generated rate ratios (RRs) and 95% confidence intervals (CIs) for the 31,364 women who were at risk of ovarian cancer and responded to the questionnaire that queried regular medication use. One hundred sixteen women developed ovarian cancer during follow-up. None of the anti-inflammatory medications was associated with ovarian cancer, but the RR for more than 1 aspirin per day for 1 year or longer was 0.56 (95% CI 0.20-1.5) and the RR for more than 5 years of regular "other NSAID" use was 2.0 (95% CI 0.95-4.2). Regular tranquilizer use was not associated with ovarian cancer, but histamine-receptor antagonists used regularly for more than 5 years (RR = 3.6, 95% CI 1.4-9.1) or more than once daily (RR = 3.1, 95% CI 1.5-6.5) appeared to increase risk. In our study, neither anti-inflammatory medications nor anti-psychotic medications were associated with ovarian cancer. Potential associations with histamine-receptor antagonists may warrant further study.


Learning Invariant Features in Modulatory Networks through Conflict and Ambiguity.
This work lays the foundation for a framework of cortical learning based on the idea of a competitive column, which is inspired by the functional organization of neurons in the cortex. A column describes a prototypical organization for neurons that gives rise to an ability to learn scale, rotation, and translation-invariant features. This is empowered by a recently developed learning rule, conflict learning, which enables the network to learn over both driving and modulatory feedforward, feedback, and lateral inputs. The framework is further supported by introducing both a notion of neural ambiguity and an adaptive threshold scheme. Ambiguity, which captures the idea that too many decisions lead to indecision, gives the network a dynamic way to resolve locally ambiguous decisions. The adaptive threshold operates over multiple timescales to regulate neural activity under the varied arrival timings of input in a highly interconnected multilayer network with feedforward and feedback. The competitive column architecture is demonstrated on a large-scale (54,000 neurons and 18 million synapses), invariant model of border ownership. The model is trained on four simple, fixed-scale shapes: two squares, one rectangle, and one symmetric L-shape. Tested on 1899 synthetic shapes of varying scale and complexity, the model correctly assigned border ownership with 74% accuracy. The model's abilities were also illustrated on contours of objects taken from natural images. Combined with conflict learning, the competitive column and ambiguity give a better intuitive understanding of how feedback, modulation, and inhibition may interact in the brain to influence activation and learning.


Aggregation and metal-binding properties of mutant forms of the amyloid A beta peptide of Alzheimer's disease.
The fibrillogenic properties of Alzheimer's A beta peptides corresponding to residues 1-40 of the normal human sequence and to two mutant forms containing the replacement Ala21 to Gly or Glu22 to Gln were compared. At pH 7.4 and 37 degrees C the Gln22 peptide was found to aggregate and precipitate from solution faster than the normal A beta, whereas the Gly21 peptide aggregated much more slowly. Electron microscopy showed that the aggregates all had fibrillar structures. Circular dichroism spectra of these peptides revealed that aggregation of the normal and Gln22 sequences was associated with spectral changes consistent with a transformation from random coil to beta sheet, whereas the spectrum of the Gly21 peptide remained almost unchanged during a period in which little or no aggregation occurred. When immobilised by spotting onto nitrocellulose membranes the peptides bound similar amounts of the radioisotope 65Zn2+. Of several competing metal ions, tested at 20x the concentration of Zn2+, Cu2+ displaced > 95% of the radioactivity from all three peptides and Ni2+ produced >50% displacement in each case. Some other metal ions tested caused lesser displacement, but Fe2+ and Al3+ were without effect. In a saturation binding assay, a value of 3.2 microM was obtained for the binding of Zn2+ to A beta but our data provided no evidence for a reported higher affinity site (107 nM). The results suggest that the neuropathology associated with the Gly21 mutation is not due to enhanced fibrillogenic or different metal-binding properties of the peptide and that the binding of zinc to amyloid peptides is not a specific phenomenon.


Palliative care clinical rotations among undergraduate and postgraduate medical trainees in Canada: a descriptive study.
The number of medical undergraduate and postgraduate students completing palliative care clinical rotations in Canadian medical schools is currently unknown. The aim of this study was to assess the proportion of Canadian medical trainees completing clinical rotations in palliative care and to determine whether changes took place between 2008 and 2018. In this descriptive study, all Canadian medical schools (n = 17) were invited to provide data at the undergraduate and postgraduate levels (2007/08-2015/16 and 2007/08-2017/18, respectively). Information collected included the number, type and length of palliative care clinical rotations offered and the total number of medical trainees or residents enrolled at each school. All 17 Canadian medical schools responded to the request for information. At the undergraduate level, palliative care clinical rotations were not offered in 2 schools, mandatory in 2 and optional in 13. Three schools that offered optional rotations were unable to provide complete data and were therefore excluded from further analyses. In 2015/16, only 29.7% of undergraduate medical students completed palliative care clinical rotations, yet this was a significant improvement compared to 2011/12 (13.6%, p = 0.02). At the postgraduate level, on average, 57.9% of family medicine trainees completed such rotations between 2007/08 and 2016/17. During the same period, palliative care clinical rotations were completed by trainees in specialty or subspecialty programs in anesthesiology (34.2%), geriatric medicine (64.4%), internal medicine (30.9%), neurology (28.2%) and psychiatry (64.5%). Between 2008 and 2018, a large proportion of Canadian medical trainees graduated without the benefit of a clinical rotation in palliative care. Without dedicated clinical exposure to palliative care, many physicians will enter practice without vital palliative care competencies.


Loss of CG Methylation in Marchantia polymorpha Causes Disorganization of Cell Division and Reveals Unique DNA Methylation Regulatory Mechanisms of Non-CG Methylation.
DNA methylation is an epigenetic mark that ensures silencing of transposable elements (TEs) and affects gene expression in many organisms. The function of different DNA methylation regulatory pathways has been largely characterized in the model plant Arabidopsis thaliana. However, far less is known about DNA methylation regulation and functions in basal land plants. Here we focus on the liverwort Marchantia polymorpha, an emerging model species that represents a basal lineage of land plants. We identified MpMET, the M. polymorpha ortholog of the METHYLTRANSFERASE 1 (MET1) gene required for maintenance of methylation at CG sites in angiosperms. We generated Mpmet mutants using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein9) system, which showed a significant loss of CG methylation and severe morphological changes and developmental defects. The mutants developed many adventitious shoot-like structures, suggesting that MpMET is required for maintaining differentiated cellular identities in the gametophyte. Even though numerous TEs were up-regulated, non-CG methylation was generally highly increased at TEs in the Mpmet mutants. Closer inspection of CHG methylation revealed features unique to M. polymorpha. Methylation of CCG sites in M. polymorpha does not depend on MET1, unlike in A. thaliana and Physcomitrella patens. Our results highlight the diversity of non-CG methylation regulatory mechanisms in plants.


Effects of medical food leucine content in the management of methylmalonic and propionic acidemias.
The current review highlights the varied effects of medical foods high in leucine (Leu) and devoid of valine (Val) and isoleucine (Ile) in the management of methylmalonic acidemia (MMA) and propionic acidemia and cobalamin C (cblC) deficiency, aiming to advance dietary practices. Leu is a key metabolic regulator with a multitude of effects on different organ systems. Recent observational studies have demonstrated that these effects can have unintended consequences in patients with MMA as a result of liberal use of medical foods. The combination of protein restriction and medical food use in MMA and propionic acidemia results in an imbalanced branched-chain amino acid (BCAA) dietary content with a high Leu-to-Val and/or Ile ratio. This leads to decreased plasma levels of Val and Ile and predicts impaired brain uptake of multiple essential amino acids. Decreased transport of methionine (Met) across the blood-brain barrier due to high circulating Leu levels is of particular concern in cblC deficiency in which endogenous Met synthesis is impaired. Investigations into the optimal composition of medical foods for MMA and propionic acidemia, and potential scenarios in which Leu supplementation may be beneficial are needed. Until then, MMA/propionic acidemia medical foods should be used judiciously in the dietary management of these patients and avoided altogether in cblC deficiency.


On the vestibular labyrinth of Brachiosaurus brancai.
The extensive remains of large sauropods, excavated in the Upper Jurassic layers of the Tendaguru region of Tanzania, East Africa by Janensch [15], include an intact fossil cast of a vestibular labyrinth and an endocast of the large Brachiosaurus brancai. The approximately 150 million year old labyrinth cast demonstrates clearly a form and organisation congruent in detail to those of extant vertebrate species. Besides the near-orthogonal arrangement of semicircular canals (SCCs), the superior and inferior branches of the vestibulo-acoustic nerve, the endolymphatic duct, the oval and round windows, and the cochlea can be identified. The orientation of the labyrinth in the temporal bone is also equivalent to that of many extant vertebrates. Furthermore, the existence of the twelve cranial nerves can be identified from the endocast. The present study was initiated after the photogrammetric measurement of the skeleton volume of B. brancai [13] yielded a realistic estimate of body mass (74.42 metric tons). Dimensional analysis shows that body mass and average SCC dimensions of B. brancai generally fit with the allometric relationship found in previous studies of extant species. However, the anterior SCC is significantly larger than the allometric relationship would predict. This would indicate greater sensitivity, supporting the idea that the behavioural repertoire must have included much slower pitch movements of the head. These slower movements would most likely have involved flexion of the neck, rather than head pitching about the atlas joint. Pursuing the relationship between body mass and SCC dimensions further, the SCC frequency response is estimated by scaling up from the SCC dimensions of the rhesus monkey; this yields a range between 0.008-26 Hz, approximately one octave lower than for humans.


Psychometric properties of the virtual patient version of the Lasater Clinical Judgment Rubric.
A number of studies attest to the effectiveness of virtual patients in fostering and assessing students' development of clinical reasoning. An objective assessment of students' clinical reasoning is, however, challenging. This study focused on determining the psychometric properties of the virtual patient version of the Lasater Clinical Judgment Rubric, a rubric that is aimed at assessing nursing students' clinical reasoning processes when encountering virtual patients. A nonexperimental design was used in which data from 125 students' reflections on solving two different virtual patient scenarios were included in the analysis. First, a deductive content analysis was conducted using the categories of the rubric as a lens. After that, each student's performance was quantified according to the different levels of the rubric. Exploratory factor analysis and test of normality and reliability, including the Kaiser-Meyer-Olkin test, Bartlett's test, the Shapiro-Wilk test, and Cronbach's alpha were used in the analysis. The result suggested three factors: "Understanding the patient", "Care planning" and "Reflecting" that explained 81.8% of the variance. Cronbach's alpha was 0.931. The result showed the rubric to be a valid assessment instrument for assessing nursing students' clinical reasoning when encountering virtual patients.


Analysis of genetic diversity in indian natural populations of drosophila ananassae.
Forty five natural populations of Drosophila ananassae, collected from entire geo-climatic regions of the India were analyzed to determine the distribution of genetic diversity relative to different eco-geographic factors. Quantitative data on the frequencies of three cosmopolitan inversions in the sampled populations were utilized to deduce Nei's gene diversity estimates. Populations were grouped according to the time of collection (years and month); collection-regions like coastal and mainland regions, and collection-seasons. Further, data was subjected to network analysis to detect community structure in the populations and Modularity analysis to quantify the strength in community structure. Gene-diversity statistics revealed the presence of significant variability in the Indian natural populations of D.ananassae. Off all the parameters used to group the populations, geographical attributes seems to have maximum, while the time of collection and seasons have minimum influence on the genetic variability in Indian natural populations of D.ananassae. The results clearly link the association of genetic variability with environmental heterogeneity, elucidating the role of environment specific natural selection. The homogenizing effects could be due to genetic hitchhiking and canalization.


[Aspirin. Pseudo-allergic reactions].
The author reviewed 2000 clinical records of his private allergy patients chosen at random. 197 records (9.7%) labeled "allergic to aspirin" were culled. 41 (2.1%) were acute cases and 153 (7.6%) had a history of "intolerance to aspirin". The main symptoms were angioedema or angioedema and urticaria in the majority of patients. No deaths were recorded. Each clinical record was surveyed and 61% of the acute cases and 72% of the patients with a history of intolerance to aspirin had a personal history of atopy. Family history of atopy was present in 63 and 73%, respectively. There was no history of atopy in 12% of the acute cases and in 7% of those with a history of intolerance to aspirin. 56 to 65% of these groups, respectively, had allergic rhinitis and 17 and 34%, had asthma. 23% of these patients showed allergic reactions simultaneously to Pyrazolones and between 12 and 22% showed allergy to Penicillin, and between 2 and 12%, to Acetominophene. The only food to which statistically significant allergy occurred was pancake, in 12%. 53% of the acute cases and 89% of those with a history of intolerance to aspirin showed a pseudoallergic reaction with products which contain aspirin or non steroidal antiinflamatory agents. 80% of the acute cases were treated with aqueous 1:1000 solution of epinephirine subcutaneously. 100% were given antihistaminics I.M. or orally and 61% were given steroids. Each patient was furnished with a list of the main aspirin containing products and non steroidal antiinflammatory agents, which cross react with aspirin.


Cholestasis caused by panhypopituitarism and acquired cytomegalovirus infection in a 2-month-old male infant: A case report.
Septo-optic dysplasia (SOD) is a rare congenital disorder that may cause jaundice in infants. However, it is usually prone to neglect and misdiagnosis in infants with cholestasis because endocrine disorder such as panhypopituitarism is rare in the cause of infantile cholestasis. We report a case of SOD concurrent with acquired cytomegalovirus (CMV) infection, who presented with prolonged jaundice as the first clinical sign. The patient was a 2-month-old male infant who presented with cholestasis, combined with fever and panhypopituitarism. He was diagnosed with SOD and acquired CMV infection. He was treated with hormone replacement therapy and ganciclovir. After correction of the pituitary hormone deficiency and ganciclovir treatment, significant improvements of cholestasis, retinal lesions, and growth rate were seen in our patient. Although an endocrine disorder such as panhypopituitarism is rare in the cause of neonatal or infantile cholestasis, we must keep this reason in mind.


[Bilateral spontaneous pneumothorax as a setting of Langerhans cell histiocytosis].
Pulmonary Langerhans cell histiocytosis is an interstitial lung disease that results from the accumulation of specific histiocytic cells in the lung. Spontaneous pneumothorax is a recognized feature of pulmonary Langerhans cell histiocytosis and results from destruction of lung parenchyma with associated cystic changes. We report on a 2-year-old boy with recurrent bilateral spontaneous pneumothorax; a computed tomography scan showed marked interstitial changes, fibrosis, cystic spaces and bilateral bullae. The diagnosis was confirmed by the histology and the immunohistochemistry examination of the pulmonary biopsy with CD1 and S100 positive antibodies. The child was treated with prednisone and etoposide, and had a good clinical response and favorable changes in the second thoracic CT scan.


The developmental profile of lactoferrin in mouse epididymis.
A sandwich e.l.i.s.a. method was developed to examine the distribution of lactoferrin in mouse reproductive tract. The lactoferrin concentration was found to be much higher in oviduct, uterus, vagina, vas deferens or epididymis than in serum, but the concentration in ovary, testis, seminal vesicle, prostate or coagulating gland was comparable with that in serum. The existence of lactoferrin in male sexual organs was confirmed by Western-blot analyses for tissue proteins. Lactoferrin in male sexual organs was shown to have a molecular mass similar to that of the deglycosylated form of lactoferrin purified from mouse uterine luminal fluid. Northern-blot analyses for total RNA prepared from male sexual organs indicated that only epididymis contained the lactoferrin mRNA. The lactoferrin mRNA was found in the prepubertal period and increased with the growth of epididymis. The mRNA level in prepubertal epididymis could be stimulated by 17 beta-oestradiol, but was not influenced by testosterone.


Contemporary use of dual antiplatelet therapy for preventing cardiovascular events.
CHARISMA was a landmark randomized clinical trial that failed to demonstrate a benefit of dual antiplatelet therapy (DAPT) over aspirin alone for preventing cardiovascular events. However, subgroup analyses of the trial found fewer major adverse cardiovascular events (MACEs) for patients with established cardiovascular disease but more MACEs for patients with multiple risk factors without established cardiovascular disease. Our objective was to examine DAPT use in contemporary clinical practice after publication of CHARISMA results. Retrospective analysis of a large clinical registry of outpatient cardiovascular visits to over 1000 physicians that collected data on patient clinical history, symptoms, vital signs, and medications. Clinical characteristics and prescription rates of aspirin and clopidogrel were compared for patients with established cardiovascular disease and for patients with only multiple cardiovascular risk factors. Prescription of DAPT by calendar quarter was evaluated from 2008 to 2011 using multivariable Poisson regression models. Of 167,839 patients with established cardiovascular disease, 20.5% were prescribed both aspirin and clopidogrel. Of 20,478 patients with multiple risk factors but no known cardiovascular disease, 3.5% were prescribed both aspirin and clopidogrel. Across 14 calendar quarters, prescription rates of DAPT did not change significantly for patients with established CVD but decreased for patients with multiple risk factors with an incidence rate ratio of 0.77. Use of DAPT is modest in patients with established cardiovascular disease, for whom the CHARISMA trial suggested decreased MACEs, and prescription rates have remained stable over time. Use of DAPT in patients with multiple risk factors only, for whom CHARISMA suggested that DAPT may lead to increased MACE, was low and decreased over time.


[Multiple primaries of the upper aerodigestive tract, esophagus, and lung].
The existence of multiple primary tumors of the upper aerodigestive tract, esophagus, and lung (UADT-E-L) is related with a common etiopathogenic factor (alcohol-tobacco association). In a review of 43 patients, 6.75% with a UADT-E-L tumor developed another neoplasm, 3.25% at the same site. Nine percent (8.59%) of the tumors were synchronic and 10.85% were metachronic. The most frequent association was larynx-lung. Another neoplasm was detected by physical examination and/or radiology in 44.18% of cases, with a time lapse of less than 3 years in 50%. The most frequent treatment was surgery with/without complementary radiotherapy. The most common stage was T1-T2 (62.06%) and N0-N1 (68.96%). The survival rate was 31% at 2 years and 25% at 3 years.


The last challenges and barriers to the development of telemedicine programs.
Over the past several decades the concept of telemedicine has evolved to be more commonplace with many unique applications. These applications have been made possible by overcoming challenges and barriers that have been present for most of telemedicine's development. The application of telemedicine continues to undergo growth and scrutiny. However, the rapid infusion of technology continues to serve as a catalyst for adoption and validation. Looking into the future requires a review of the past. Overcoming barriers and challenges has not been easy. Many have been minimized, in part because of technologies rapid flow and diffusion. Yet there are still significant challenges that must be overcome. In many parts of the world, healthcare and education are limited because of access. This has been driven by many influences, which continue to have an impact, albeit in many cases, minimal. Future generations of telemedicine users will look back at this period as one of transition. The first 30 years or so of telemedicine implementation were fraught with barriers and challenges. The next 30 years will see and entirely different paradigm of practicing medicine, including consumerism and smart systems.


Diagnosis and Management of Anxiety Disorders.
This article provides a synopsis of the current understanding of the pathophysiology of anxiety disorders, the biological and environmental risk factors that contribute to their development and maintenance, a review of the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) diagnostic criteria, and a practical approach to the treatment of anxiety disorders in adults. Despite the ubiquity of anxiety, the evidence is that most individuals with an anxiety disorder are not identified and do not receive guideline-level care. In part, this may be because of the manifold clinical presentations of anxiety disorders and clinicians' lack of confidence in accurately diagnosing and treating these conditions, especially in nonpsychiatric settings. Anxiety disorders represent the complex interplay between biological, psychological, temperamental, and environmental factors. Converging lines of evidence point to dysfunction in regulating activity in the "threat circuit" in the brain as a putative common pathophysiology underlying anxiety disorders. Evidence-based treatments for anxiety disorders, such as cognitive-behavioral therapy and antidepressant medications, have been shown to regulate activity in this circuit, which consists of reciprocal connections between the dorsomedial prefrontal cortex, insula, and amygdala. Anxiety disorders are the most common class of emotional disorders and a leading cause of disability worldwide. A variety of effective treatment strategies are available, which may exert their therapeutic benefits from top-down or bottom-up modulation of the dysfunctional brain activity associated with anxiety disorders.


Extended post-ex vivo lung perfusion cold preservation predicts primary graft dysfunction and mortality: Results from a multicentric study.
Ex vivo lung perfusion (EVLP) allows for a reassessment of lung grafts initially deemed unsuitable for transplantation, increasing the available donor pool; however, this requires a pre- and post-EVLP period of cold ischemic time (CIT). Paucity of data exists on how the sequence of cold normothermic-cold preservations affect outcomes. A total of 110 patients were retrospectively analyzed. Duration of 3 preservation phases was measured: cold pre-EVLP, EVLP, and cold post-EVLP. The donor and recipient clinical data were collected. Primary graft dysfunction (PGD) and survival were monitored. Risk of mortality or PGD was calculated using Cox proportional hazards and logistic regression models to adjust for baseline characteristics. Using the highest quartile, patients were stratified into extended vs non-extended pre-EVLP (<264 vs ≥264 minutes) and post-EVLP (<287 vs ≥287 minutes) CIT. The rates of 1-year mortality (8.4% vs 29.6%, p = 0.013), PGD 2-3 (20.5% vs 52%, p = 0.002), and PGD 3 (8.4% vs 29.6%, p = 0.005) at 72 hours were increased in the extended post-EVLP CIT group. After adjusting for baseline risk factors, the extended group remained an independent predictor of PGD ≥2 (odd ratio: 6.18, 95% CI: 1.88-20.3, p = 0.003) and PGD 3 (odd ratio: 20.4, 95% CI: 2.56-161.9, p = 0.004) at 72 hours and 1-year mortality (hazard ratio: 17.9, 95% CI: 3.36-95.3, p = 0.001). Cold pre-EVLP was not a significant predictor of primary outcomes. Extended cold post-EVLP preservation is associated with a risk for PGD and 1-year mortality. Pre-EVLP CIT does not increase mortality or high-grade PGD. These findings from a multicenter trial should caution on the implementation of extended cold preservation after EVLP.


Orthographic depth and developmental dyslexia: a meta-analytic study.
Cross-cultural studies have suggested that reading deficits in developmental dyslexia (DD) can be moderated by orthographic depth. To further explore this issue and assess the moderating role of orthographic depth in the developmental cognitive trajectories of dyslexic and typical readers, we systematically reviewed 113 studies on DD that were published from 2013 to 2018 and selected 79 in which participants received an official DD diagnosis. Each study was classified according to orthographic depth (deep vs. shallow) and participant age (children vs. adults). We assessed the difference between DD and control groups' performance in reading tasks and in a wide range of cognitive domains associated with reading (phonological awareness (PA), rapid automatized naming (RAN), short-term working memory (WM), and nonverbal reasoning), including age and orthographies as moderators. We found an age-by-orthography interaction effect in word reading accuracy and a significant effect of age in pseudoword reading accuracy, but we found no effect of age and orthographic depth on the fluency parameters. These results suggest that reading speed is a reliable index for discriminating between DD and control groups across European orthographies from childhood to adulthood. A similar pattern of results emerged for PA, RAN, and short-term/WM. Our findings are discussed in relation to their impact on clinical practice while considering the orthographic depth and developmental level.


Synchrotron FTIR microspectroscopy of Escherichia coli at single-cell scale under silver-induced stress conditions.
The present work was focused on elucidating biochemical changes in the model bacterium Escherichia coli exposed to ionic silver mediated stress, at a single-cell scale. In order to achieve this, in situ synchrotron Fourier-transform infrared (sFTIR) microspectroscopy was performed, for the first time, on individual cells by attenuated total reflectance (ATR) combined with the use of zinc-selenide hemisphere for high spatial resolution. In a first part, the potential of the method was evaluated on bacteria subjected to a lethal 100 μM AgNO(3) concentration for 2 h compared to untreated 100 % viable cells. Differences in cell composition were assessed for the C-H stretching and protein spectral regions, indicating that the inhibitory action was targeted against both fatty acids and proteins. Transmission electron microscopy (TEM) confirmed morphological damages of the cell ultrastructure. The relevance of ATR-sFTIR microspectroscopy for highlighting the heterogeneity in Ag(+)-mediated effects within a given bacterial population was also pointed out. In a second part, cells were exposed to sub-lethal Ag(+) concentrations (<10 μM AgNO(3)) tested under "dynamic" growth mode: early addition vs. pulse in the mid-exponential phase, and compared to simultaneously batch-grown untreated bacteria or cells sampled just before the pulse, respectively. sFTIR microspectroscopy and TEM imaging were performed in close relation with growth kinetics characterization. No significant effect of the Ag(+) pulses was detected, in accordance with macrokinetics data. For early-treated cells, effects on fatty acid composition were shown, although no major alteration of protein secondary structure was noticed. These partial effects were consistent with TEM observations and growth kinetics.


Fanconi Anemia Pathway Activation by FOXM1 Is Critical to Bladder Cancer Recurrence and Anticancer Drug Resistance.
Although the 5-year survival rate of patients diagnosed with nonmuscle invasive bladder cancer (NMIBC) has reached 85%, more than 50% of patients suffer from frequent recurrences. To identify molecular targets associated with recurrence of NMIBC, we analyzed gene expression data and found that FOXM1 and FANCD2 were involved in recurrence. Therefore, we investigated how these genes were involved in the mechanism of recurrence and confirmed their usefulness as biomarkers. Investigation have shown that FOXM1 directly regulated the transcription of FANCD2, which is the key gene of the Fanconi anemia (FA) pathway. Depletion of FOXM1 resulted in DNA repair defects in the FA pathway and in decreased resistance to chemotherapy. Thus, the FANCD2-associated FA pathway activated by FOXM1 is an important mechanism involved in chemotherapy-related recurrence. In conclusion, FOXM1 and FANCD2 can be used as prognostic factors that are associated with high risk of recurrence and with anticancer drug resistance properties in NMIBC patients.


Efficient synthesis of gamma-alkylidenetetronic esters by sequential Lewis acid catalyzed.
A new approach for the synthesis of gamma-alkylidenetetronic acids and esters is reported which involves Me3SiOTf-catalyzed, regio- and stereoselective cyclization of 4-alkoxy-1,3-bis(trimethylsilyloxy)-1,3-butadienes with oxalyl chloride. The alpha-hydroxy group of the butenolides is efficiently functionalized by palladium-catalyzed cross-coupling reactions via the corresponding enol triflates.


Structural equivalents of latency for lysosome hydrolases.
1. Structure-linked latency, a trait for most lysosome hydrolase activities, is customarily ascribed to the permeability-barrier function performed by the particle-limiting membrane, which shields enzyme sites from externally added substrates. 2. The influence of various substrate concentrations on the reaction rate has been measured for both free (non-latent) and total (completely unmasked by Triton X-100) hydrolase activities in rat liver cell-free preparations. The substrates were: beta-glycerophosphate, phenolphthalein mono-beta-glucuronide. p-nitrophenyl N-acetyl-beta-D-glucosaminide and p-nitrophenyl beta-D-galactopyranoside. The ratio (free activity/total activity) X 100 is called fractional free activity at any given substrate concentration. 3. The fractional free activity of beta-glucuronidase and beta-N-acetylglucosaminidase were clearly independent of substrate concentration, over the range examined, in both homogenates and lysosome-rich fractions. The fractional free activity of acid phosphatase appeared to be either unaffected (homogenate) or even depressed (lysosome-rich fraction) by increasing the beta-glycerophosphate concentration. The fractional free activity of beta-galactosidase consistently showed a non-linear increase with increasing substrate concentration in both homogenates and lysosome-rich fractions. 4. Procedures such as treatment with digitonin, hypo-osmotic shock and acid autolysis, although effective in causing varying degrees of resolution of the latency of lysosome hydrolase activities, were unable to modify appreciably the pattern of dependence or independence of their fractional free activities on substrate concentration, as compared with that exhibited by control preparations. Ouabain did not affect the free beta-N-acetylglucosaminidase activity of liver homogenates at all. 5. Preincubation of control preparations with beta-glycerophosphate or p-nitrophenyl beta-galactoside did not result in any significant stimulation of the free hydrolytic activity toward these substrates. 6. The results consistently support the view that the membrane of "intact" lysosomes is virtually impermeable to all the substrates tested, except for p-nitrophenyl beta-galactoside, for which the evidence is contradictory. Moreover the progressive unmasking of the hydrolase activities produced by these procedures in vitro reflects the increasing proportion of enzyme sites that are fully accessible to their substrates rather than a graded increase in the permeability of the lysosomal membrane.


Swimming speed alteration of Artemia sp. and Brachionus plicatilis as a sub-lethal behavioural end-point for ecotoxicological surveys.
In this study, we investigated the possibility to improve a new behavioural bioassay (Swimming Speed Alteration test-SSA test) using larvae of marine cyst-forming organisms: e.g. the brine shrimp Artemia sp. and the rotifer Brachionus plicatilis. Swimming speed was investigated as a behavioural end-point for application in ecotoxicology studies. A first experiment to analyse the linear swimming speed of the two organisms was performed to verify the applicability of the video-camera tracking system, here referred to as Swimming Behavioural Recorder (SBR). A second experiment was performed, exposing organisms to different toxic compounds (zinc pyrithione, Macrotrol MT-200, and Eserine). Swimming speed alteration was analyzed together with mortality. The results of the first experiment indicate that SBR is a suitable tool to detect linear swimming speed of the two organisms, since the values have been obtained in accordance with other studies using the same organisms (3.05 mm s(-1) for Artemia sp. and 0.62 mm s(-1) for B. plicatilis). Toxicity test results clearly indicate that swimming speed of Artemia sp. and B. plicatilis is a valid behavioural end-point to detect stress at sub-lethal toxic substance concentrations. Indeed, alterations in swimming speed have been detected at toxic compound concentrations as low as less then 0.1-5% of their LC(50) values. In conclusion, the SSA test with B. plicatilis and Artemia sp. can be a good behavioural integrated output for application in marine ecotoxicology and environmental monitoring programs.


Glutamine induces heat shock protein expression via O-glycosylation and phosphorylation of HSF-1 and Sp1.
Glutamine (GLN) improves outcome in experimental and clinical states of illness and injury. The authors hypothesized GLN-mediated enhancement of O-glycosylation and subsequent phosphorylation of key transcription factors in the HSP70 pathway would lead to increased HSP70 expression following experimental sepsis. Mice underwent cecal ligation and puncture (CLP)-induced sepsis and were treated with GLN (0.75 g/kg) or a saline placebo 30 minutes after CLP. A separate group of mice was treated with mithramycin, an Sp1 inhibitor. Lung tissue was harvested at 1, 2, 6, and 24 hours after CLP and was analyzed for HSF-1 and Sp1 O-GlcNAc modification, alpha-p-threonine modification, and HSP70. GLN increased O-GlcNAc modification of HSF-1 and Sp1 at 1 and 2 hours after sepsis (P < .001 vs saline). Samples immunoprecipitated for Sp1 and probed for subsequent phosphorylation showed a significant increase in nuclear alpha-p-threonine-modified Sp1 at 2 and 6 hours after sepsis (P < .001 vs saline). GLN increased phosphorylated nuclear HSF-1 at 1 and 2 hours after CLP (P < .001). Finally, GLN treatment increased HSP70 4-fold (P < .01), but when treated with mithramycin, this increase was attenuated at 2, 6, and 24 hours (P < .001 vs no mithramycin treatment). These results indicate that GLN induces HSF-1 and Sp1, which is known to lead to their nuclear translocation. The molecular mechanism of GLN-mediated HSP70 expression appears to be dependent on O-GlcNAc pathway activation and subsequent O-glycosylation and phosphorylation of key transcription factors required for HSP70 induction.


Molecular Imaging of Retinoic Acids in Live Cells Using Single-Chain Bioluminescence Probes.
Retinoic acid (RA) is a key metabolite necessary for embryonic development and differentiation in vertebrates. We demonstrate the utility of genetically encoded, ligand-activatable single-chain bioluminescence probes for detecting RAs from different biological sources. We examined 13 different molecular designs to identify an efficient single-chain probe that can quantify RA with significant sensitivity. The optimal probe consisted of four components: the N- and C-terminal fragments of artificial luciferase variant-16 (ALuc16), the ligand binding domain of retinoic acid receptor α (RARα LBD), and an LXXLL interaction motif. This probe showed a 5.2-fold greater bioluminescence intensity in response to RA when compared to the vehicle control in live mammalian cells. The probe was highly selective to all-trans-RA (at-RA), and highly sensitive in determining at-RA levels in cells derived from tumor xenografts created using MDA-MB-231 cells engineered to stably express the probe. We also detected RA levels in serum and cerebrospinal fluid. Using this probe, the detection limit for at-RA was ∼10-9.5 M, with a linear range of two orders. We present a highly useful technique to quantitatively image endogenous at-RA levels in live mammalian cells expressing novel single-chain bioluminescence probes.


Brain micro-ecologies: neural stem cell niches in the adult mammalian brain.
Neurogenesis persists in two germinal regions in the adult mammalian brain, the subventricular zone of the lateral ventricles and the subgranular zone in the hippocampal formation. Within these two neurogenic niches, specialized astrocytes are neural stem cells, capable of self-renewing and generating neurons and glia. Cues within the niche, from cell-cell interactions to diffusible factors, are spatially and temporally coordinated to regulate proliferation and neurogenesis, ultimately affecting stem cell fate choices. Here, we review the components of adult neural stem cell niches and how they act to regulate neurogenesis in these regions.


Aortic reconstruction vs extra-anatomic bypass and angioplasty. Thoughts on evolving a protocol for selection.
One hundred forty-eight patients were evaluated for inflow revascularization and stratified by age, vascular anatomy, medical history, and cardiac functional class into aortic reconstruction (AR), extra-anatomic bypass (EAB), or iliac angioplasty based on a protocol that restricted AR to good-risk patients and liberalized indications for EAB. Fifty-five patients underwent AR with a 1.8% mortality, 1.8% myocardial infarction and 12% morbidity, and cumulative life-table patency of 94% at two years; 69 patients underwent EAB with no mortality and negligible morbidity; cumulative life-table patency was 93% at two years for crossover femoral and 83% at two years for axillofemoral grafts. Operative selection based on a protocol restricting AR to better-risk patients and liberalizing use of EAB may decrease overall patient mortality and morbidity without jeopardizing limb preservation.


The putative nuclear localization signal of the human RAD52 protein is a potential sumoylation site.
RAD52, a key factor in homologous recombination (HR), plays important roles in both RAD51-dependent and -independent HR pathways. Several studies have suggested a link between the functional regulation of RAD52 and the protein modification by a small ubiquitin-like modifier (SUMO). However, the molecular mechanism underlying the regulation of RAD52 by SUMO is unknown. To begin investigating this mechanism, we identified possible target sites for sumoylation in the human RAD52 protein by preparing a RAD52-SUMO complex using an established Escherichia coli sumoylation system. Mass spectrometry and amino acid sequencing of the enzymatically digested fragments of the purified complex revealed that the putative nuclear localization signal located near the C terminus of RAD52 was sumoylated. Biochemical studies of the RAD52-SUMO complex suggested that sumoylation at the identified site has no apparent effect on the DNA binding, D-loop formation, ssDNA annealing and RAD51-binding activities of RAD52. On the other hand, visualization of the GFP-fused RAD52 protein in the human cell that contained mutations at the identified sumoylation sites showed clear differences in the cytosolic and nuclear distributions of the protein. These results suggest the possibility of sumoylation playing an important role in the nuclear transport of RAD52.


Recruiting new principals in general practice.
New principals in general practice who were appointed from 1981 to 1983 by two family practitioner committees, one in an inner city and one in a combination of an inner city and suburban area, were surveyed to find out if they were making improvements to primary medical care in their new practices. Most were not. The highly trained, motivated, young doctors on the whole had joined group practices and practices in health centres, where facilities tended to be good. Older doctors, who may not be as concerned with change, had joined smaller practices, in which it was difficult to make changes owing to, for example, the type of premises and costs.


[Validation of the French version of the Raine Schizotypal Personality Disorder Questionnaire--categorial and dimensional approach to schizotypal personality traits in a normal student population].
Most of existing self-report measures of schizotypal personality assess only few of the nine traits of Schizotypal Personality Disorder (SPD) according to DSM III-R or DSM IV. The Raine's Schizotypal Personality Questionnaire (SPQ) is one of the most widely used questionnaire for SPD diagnosis. This well adapted tool for screening SPD in large samples from general population allows simple and quick evaluations by the mean of 74 items and nine sub-scales exploring DSM IV criteria of the trouble. With the original sample of American students, Raine found SPQ to have high internal reliability (0.91) and reported cutoffs for the top and the bottom ten percents of SPQ scores at respectively 41/74 and 12/74. We reported here the validation study of the French version of the SPQ in a sample of 232 students (males: n = 107; females: n = 125; mean age: 21.17 +/- 1.47; mean educational level: 13.36 +/- 1.06). The French version has high internal reliability (SPQ total: Cronbach's alpha = 0.91; SPQ nine subscales: Cronbach's alpha = 0.57 to 0.76). The ten percent high and low cutoff scores on the distribution of SPQ scores are respectively 40/74 and 7/74 for the total sample. However, gender differences are observed: 38/74 and 9/74 for females; 42/74 and 6/74 for males. A Principal Component Analysis (PCA) confirms the high internal reliability. Moreover, PCA evidences a three-factor model of schizotypy reflecting "positive or cognitive-perceptual", "negative or social-interpersonal", and "disorganization" latent factors. These results replicate previous works on the topic with the same instrument.


Phlegmonous gastritis associated with Kaposi sarcoma: a case report and review of the literature.
We report a case of phlegmonous gastritis associated with Kaposi sarcoma in a 37-year-old, human immunodeficiency virus (HIV)-positive man who presented with an acute abdomen. Computed tomographic scan revealed free fluid in the abdominal cavity and a thickened gastric wall. A partial gastrectomy was performed. The resected portion of stomach had a hemorrhagic, necrotic thickened wall and showed extensive, acute suppurative inflammation, especially in the submucosa, with focal transmural involvement. Beneath an area of healing ulceration, a focus of Kaposi sarcoma was present. Group A beta-hemolytic streptococcus was grown from peritoneal fluid, and treatment with numerous antibiotics was initiated. After a difficult postoperative course that responded to 8 weeks of antibiotic therapy, the patient was medically stable and discharged from the hospital on antiretroviral therapy for HIV. Phlegmonous gastritis is a rare and rapidly progressive bacterial infection of the gastric wall. Kaposi sarcoma is one of the most common malignancies in HIV-positive patients, and gastric involvement is relatively common in those patients with systemic Kaposi sarcoma. To our knowledge, this is the first reported case of phlegmonous gastritis associated with Kaposi sarcoma, and it represents a rare survival following surgical and antibiotic therapy.


A pilot study of interleukin-11 in subjects with chronic hepatitis C and advanced liver disease nonresponsive to antiviral therapy.
To evaluate the effects of recombinant human interleukin (rhIL)-11 on liver histology in patients with chronic hepatitis C virus (HCV) infection and advanced liver disease who had failed antiviral therapy. This was an open-label study of rhIL-11 (Neumega), Wyeth Laboratories, Collegeville, PA) at a dose of 5 microg/kg administered by subcutaneous injection daily for 12 wk. The primary efficacy endpoint was the change in the Knodell Histology Activity Index (HAI) between pre- and posttreatment liver biopsies. Secondary efficacy endpoints included changes in plasma alanine transaminase (ALT) concentrations and in the number of platelets. The Knodell HAI improved in 11 (55%) of the 20 subjects enrolled, with the mean score improving from 7.3 to 5.9 (p= 0.006). Eight subjects (40%) experienced significant improvement as defined by a decrease of at least two points in the HAI. IL-11 treatment was also associated with a decrease in ALT levels from a mean level of 113 IU/L at baseline to 65 IU/L at week 12 (p < 0.001). Platelet levels increased from a mean of 143 x 10(3)/microl at baseline to 198 x 10(3)/mul at week 12 of treatment (p < 0.001). Overall, rhIL-11 was well tolerated and no serious adverse events (AEs) were reported. The most common AE was edema of the lower extremities, which occurred in all subjects. The findings from this pilot study suggest that rhIL-11 may be beneficial for patients with hepatic inflammation and advanced liver disease associated with chronic HCV infection. Larger clinical trials are warranted to further evaluate the long-term antiinflammatory and antifibrotic effects of rhIL-11.


Copper affects the binding of HIF-1α to the critical motifs of its target genes.
Copper (Cu) regulates hypoxia-inducible factor-1 (HIF-1) transcription activity by affecting the selectivity of HIF-1α binding to the promoters of affected genes. In hypoxia, Cu is required for HIF-1α binding to the promoter of BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), but the metal does not affect the binding of HIF-1α to the promoter of insulin-like growth factor 2 (IGF2). Here, we made an effort to provide a comprehensive understanding of Cu regulation of the selectivity of HIF-1α binding across the genome. We used tetraethylenepentamine (TEPA), a Cu selective chelator, to reduce Cu content in the cells. We conducted chromatin immunoprecipitation combined with massively parallel DNA sequencing (ChIP-seq) to globally map the binding sites of HIF-1α, RNA polymerase II (Pol II), and the acetylation at the 27th lysine residue of the histone H3 protein (H3K27ac). We also performed RNA-sequencing (RNA-seq) in EA.hy926 cells under hypoxia (1% O2) with or without Cu depression to determine the profile of mRNA expression. Our analyses identified 3197 HIF-1α binding sites under hypoxia. Cu depression by TEPA reduced the number of binding sites to 1820 from the original 3197, but induced 274 new binding sites. We analysed these binding sites in the promoter and putative enhancer regions, coupled with their mRNA expression profiles, and found 281 Cu-dependent and 10 Cu-independent HIF-1α target genes. We found that the core bases "GGAA" and "TTCC" constituted the critical motifs for the binding sites of Cu-dependent genes. This study thus revealed that Cu, by affecting the binding of HIF-1α to the critical motifs in the promoter and putative enhancer regions of HIF-1 regulated genes, leads to the selectivity of HIF-1 regulated expression of Cu-dependent genes.


Dictionary of immune responses to cytokines at single-cell resolution.
Cytokines mediate cell-cell communication in the immune system and represent important therapeutic targets1-3. A myriad of studies have highlighted their central role in immune function4-13, yet we lack a global view of the cellular responses of each immune cell type to each cytokine. To address this gap, we created the Immune Dictionary, a compendium of single-cell transcriptomic profiles of more than 17 immune cell types in response to each of 86 cytokines (>1,400 cytokine-cell type combinations) in mouse lymph nodes in vivo. A cytokine-centric view of the dictionary revealed that most cytokines induce highly cell-type-specific responses. For example, the inflammatory cytokine interleukin-1β induces distinct gene programmes in almost every cell type. A cell-type-centric view of the dictionary identified more than 66 cytokine-driven cellular polarization states across immune cell types, including previously uncharacterized states such as an interleukin-18-induced polyfunctional natural killer cell state. Based on this dictionary, we developed companion software, Immune Response Enrichment Analysis, for assessing cytokine activities and immune cell polarization from gene expression data, and applied it to reveal cytokine networks in tumours following immune checkpoint blockade therapy. Our dictionary generates new hypotheses for cytokine functions, illuminates pleiotropic effects of cytokines, expands our knowledge of activation states of each immune cell type, and provides a framework to deduce the roles of specific cytokines and cell-cell communication networks in any immune response.


Leg regeneration in Drosophila abridges the normal developmental program.
Regeneration of lost body parts has traditionally been seen as a redeployment of embryonic development. However, whether regeneration and embryonic development are controlled by identical, similar or different genetic programmes has not been fully tested. Here, we analyse proximal-distal regeneration in Drosophila leg imaginal discs using the expression of positional markers, and by cell-lineage experiments, and we compare it with the pattern already known in normal development. During regeneration, the first proximal-distal positional markers reappear in overlapping patterns. As the regenerate expands, they segregate and further markers appear until the normal pattern is produced, following a proximal to distal sequence that is in fact the reverse of normal leg imaginal disc development. The results of lineage tracing support this interpretation and show that regenerated structures derive from cells near the wound edge. Although leg development and leg regeneration are served by a set of identical genes, the ways their proximal-distal patterns are achieved are distinct from each other. Such differences can result from similar developmental gene interactions acting under different starting conditions.


Opening up conversations: Collaborative working across sociomaterial contexts in nursing in London.
To discuss nurses' use of networks to address nursing recruitment and retention in London, UK. Qualitative evaluation of the Capital Nurse programme reporting on 30 narrative interviews with executive, clinical and student nurses in 2019. Executive nurses within the Capital Nurse programme recognized the importance of sociomaterial contexts in the health and social care system in London and worked strategically across these contexts to achieve change. Supported through the Capital Nurse programme, executive nurses from health organizations across London initiated collaborative working to improve recruitment and retention. Primarily by designing and delivering sociomaterial products (organizational and educational) to support nurses to build a career in London. Drawing on ideas from actor network theory, in particular sociomaterial contexts, nurses' actions at all levels to develop and sustain networks to address nursing recruitment and retention across the NHS in London are described. Capital Nurse supported collaborative working both within single organizations and across organizations in London. There is evidence of change in how nurses across the capital work together to improve patient care, improve recruitment and retention. Findings may resonate with nurses in other settings who seek to address the problem of recruitment and retention. They show how nurses coming together in networks to effect changes in practice can work successfully. Nurses' use of networks led to novel models of communication and action to address the problems of recruitment and retention in London. We argue that sociomateriality should be considered outside the clinical practice setting, as part of nurses' professional development and organizational practice, that is how they plan their career, how they address recruitment and retention, how they communicate across organizations about nursing issues. This was an evaluation of a staff development project in London, which sought to elicit nurses' experiences of participation in Capital Nurse.


Reporter gene constructs suggest that the Caenorhabditis elegans avermectin receptor beta-subunit is expressed solely in the pharynx.
Gene promoter/LacZ reporter constructs were made in order to analyse the expression of the beta-subunit of the Caenorhabditis elegans glutamate-gated Cl- channel (Glu-Cl) receptor. Southern blot analysis of the C. elegans cosmid C35E8 identified a 4kbp EcoRI fragment which contained the 5' portion of the Glu-Cl beta coding sequence together with 5' flanking sequences. This was subcloned and used as the template for polymerase chain reaction (PCR) amplification of a DNA fragment encoding the first 24 amino acid residues of Glu-Cl beta together with 1.4 kbp of 5' genomic sequence. The fragment was subcloned into the LacZ expression vector pPD22.11 to form a translational reporter fusion. After injection of the construct into worms, six stably transformed lines were established and assayed for beta-galactosidase activity. Stained nuclei were observed in the pharyngeal metacorpus in adults and in all larval stages, and stained nuclei were seen in many embryos undergoing morphogenesis. Additional stained nuclei towards the terminal bulb of the pharynx were observed in larval stages. These results provide further evidence that the Glu-Cl receptor mediates the glutamatergic inhibition of pharyngeal muscle via the M3 motor neurone and point to inhibition of pharyngeal pumping as a major mode of action for avermectins.


Dissection of immediate-early gene promoters from herpes simplex virus: sequences that respond to the virus transcriptional activators.
The immediate-early promoters of herpes simplex virus give rise to the first series of transcripts after infection. These promoters are composed of compound sequence elements that govern basal level and regulated transcription. The response of three core (truncated) promoters from the herpes simplex virus type 1 IE-4, IE-0, and IE-27 genes to a battery of virus-encoded trans-acting proteins was examined in a short-term transient expression assay system. The results of this study reveal (i) a role for a sequence, 5'---GGGGG---3', flanked by 3 to 5 base pairs of symmetry (the G box), which is present in the upstream region of all immediate-early gene promoters, (ii) a requirement for the consensus sequence protected by ICP4 for autoregulation by this immediate-early gene product, and (iii) an alternative, sequence-independent mechanism for the augmentation of alpha gene expression by the virion-associated transcriptional activator Vmw65, now designated as TIF.


Design, validation, and implementation of a rapid-injection NMR system.
A Rapid Injection NMR (RINMR) apparatus has been designed and constructed to allow the observation of fast chemical reactions in real time by NMR spectroscopy. The instrument was designed to allow the rapid (<2 s) injection and mixing of a metered volume of a reagent into a spinning NMR tube followed by rapid acquisition of the data resulting from the evolution of the chemical process. The various design criteria for this universal system included the ability to deliver any chemical reagent at any temperature and allow for the observation of any nucleus. The various challenges associated with the construction and implementation of this instrument are documented along with the validation of the accuracy of the apparatus with respect to volume and temperature. Finally, the ultimate validation and reproducibility of the technique is presented in the form of three case studies that used the instrument to elucidate various aspects of organic reaction mechanisms. The authors urge interested parties to not embark on the construction of their own instrument and invite those whose research problems might be amenable to this kind of analysis to contact the corresponding author for access to the apparatus described herein.


Bone and cartilage tissue constructs grown using human bone marrow stromal cells, silk scaffolds and rotating bioreactors.
Human bone marrow contains a population of bone marrow stromal cells (hBMSCs) capable of forming several types of mesenchymal tissues, including bone and cartilage. The present study was designed to test whether large cartilaginous and bone-like tissue constructs can be selectively engineered using the same cell population (hBMSCs), the same scaffold type (porous silk) and same hydrodynamic environment (construct settling in rotating bioreactors), by varying the medium composition (chondrogenic vs. osteogenic differentiation factors). The hBMSCs were harvested, expanded and characterized with respect to their differentiation potential and population distribution. Passage two cells were seeded on scaffolds and cultured for 5 weeks in bioreactors using osteogenic, chondrogenic or control medium. The three media yielded constructs with comparable wet weights and compressive moduli ( approximately 25 kPa). Chondrogenic medium yielded constructs with higher amounts of DNA (1.5-fold) and glycosaminoglycans (GAG, 4-fold) per unit wet weight (ww) than control medium. In contrast, osteogenic medium yielded constructs with higher dry weight (1.6-fold), alkaline phosphatase (AP) activity (8-fold) and calcium content (100-fold) per unit ww than control medium. Chondrogenic medium yielded constructs that were weakly positive for GAG by contrast-enhanced MRI and alcian blue stain, whereas osteogenic medium yielded constructs that were highly mineralized by microCT and von Kossa stain. Engineered bone constructs were large (8mm diameter x 2mm thick disks) and resembled trabecular bone with respect to structure and mineralized tissue volume fraction (12%).


Predictors of post-operative cardiovascular events, focused on atrial fibrillation, after valve surgery for primary mitral regurgitation.
Primary mitral regurgitation (PMR) can be considered as a heterogeneous clinical disease. The optimal timing of valve surgery for severe PMR remains unknown. To determine whether unbiased clustering analysis using dense phenotypic data (phenomapping) could identify phenotypically distinct PMR categories of patients. One hundred and twenty-two patients who underwent surgery were analysed, excluding patients with pre-operative permanent atrial fibrillation (AF), were prospectively included before surgery. They were given an extensive echocardiographic evaluation before surgery, and clinical data were collected. These phenotypic variables were grouped in clusters using hierarchical clustering analysis. Then, different groups were created using a dedicated phenomapping algorithm. Post-operative outcomes were compared between the groups. The primary endpoint was post-operative cardiovascular events (PCE), defined as a composite of: deaths, AF, stroke, and rehospitalization. The secondary endpoint was post-operative AF. Data from three phenogroups with different characteristics and prognoses were identified. Phenogroup-1 (67 patients) was the reference group. Phenogroup-2 (33 patients) included intermediate-risk male and smoker patients with heart remodelling. Phenogroup-3 (22 patients) included older female patients with comorbidities (chronic renal failure, paroxysmal AF) and diastolic dysfunction. They had a higher risk of developing both PCE [(hazard ratio) HR = 3.57(1.72-7.44), P < 0.001] and post-operative AF [HR = 4.75(2.03-11.10), P < 0.001]. Pre-operative paroxysmal AF was identified as an independent risk factor for PCE. Classification of PMR can be improved using statistical learning algorithms to define therapeutically homogeneous patient subclasses. High-risk patients can be identified, and these patients should be carefully monitored and may even be treated earlier.


On the mechanism of quinol oxidation at the QP site in the cytochrome bc1 complex: studied using mutants lacking cytochrome bL or bH.
To elucidate the mechanism of bifurcated oxidation of quinol in the cytochrome bc1 complex, Rhodobacter sphaeroides mutants, H198N and H111N, lacking heme bL and heme bH, respectively, were constructed and characterized. Purified mutant complexes have the same subunit composition as that of the wild-type complex, but have only 9-11% of the electron transfer activity, which is sensitive to stigmatellin or myxothiazol. The Em values for hemes bL and bH in the H111N and H198N complexes are -95 and -35 mV, respectively. The pseudo first-order reduction rate constants for hemes bL and bH in H111N and H198N, by ubiquiniol, are 16.3 and 12.4 s(-1), respectively. These indicate that the Qp site in the H111N mutant complex is similar to that in the wild-type complex. Pre-steady state reduction rates of heme c1 by these two mutant complexes decrease to a similar extent of their activity, suggesting that the decrease in electron transfer activity is due to impairment of movement of the head domain of reduced iron-sulfur protein, caused by disruption of electron transfer from heme bL to heme bH. Both mutant complexes produce as much superoxide as does antimycin A-treated wild-type complex. Ascorbate eliminates all superoxide generating activity in the intact or antimycin inhibited wild-type or mutant complexes.


Modified immunoliposome sandwich assay for the detection of Escherichia coli O157:H7 in apple cider.
Detection of Escherichia coli O157:H7 in fruit juices such as apple cider is necessary for diagnosis of infection and epidemiological investigations. However, inhibitors in the apple cider, such as endogenous polyphenols and acids, often decrease the sensitivity of PCR assays and immunoassays, thus routinely requiring laborious cell separation steps to increase the sensitivity. In the current study, polyethylene glycol (PEG)-derivatized liposomes encapsulating sulforhodamine B were tagged with anti-E. coli O157:H7 antibodies and used in an immunoliposome sandwich assay for the detection of E. coli O157:H7 in apple cider. Even without prior separation, this assay can detect E. coli O157:H7 in apple cider samples inoculated with as few as 1 CFU/ml after an 8-h enrichment period. The lower limit of detection in pure cultures without enrichment was 7 x 10(3) CFU/ml (280 CFU/40-microl sample). PEGylated immunoliposomes are suitable as an analytical reagent for the detection of E. coli O157:H7 in fruit juices containing polyphenols.


Assessment of treatment response in cardiac sarcoidosis based on myocardial 18F-FDG uptake.
Immunosuppressive therapy for cardiac sarcoidosis (CS) still largely consists of corticosteroid monotherapy. However, high relapse rates after tapering and insufficient efficacy are significant problems. The objective of this study was to investigate the efficacy and safety of non-biological and biological disease-modifying anti-rheumatic drugs (nb/bDMARDs) considering control of myocardial inflammation assessed by 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) of the heart. We conducted a retrospective analysis of treatment response to nb/bDMARDs of all CS patients seen in the sarcoidosis center of the University Hospital Zurich between January 2016 and December 2020. We identified 50 patients with CS. Forty-five patients with at least one follow-up PET/CT scan were followed up for a mean of 20.5 ± 12.8 months. Most of the patients were treated with prednisone and concomitant nb/bDMARDs. At the first follow-up PET/CT scan after approximately 6.7 ± 3 months, only adalimumab showed a significant reduction in cardiac metabolic activity. Furthermore, comparing all serial follow-up PET/CT scans (143), tumor necrosis factor inhibitor (TNFi)-based therapies showed statistically significant better suppression of myocardial 18F-FDG uptake compared to other treatment regimens. On the last follow-up, most adalimumab-treated patients were inactive (n = 15, 48%) or remitting (n = 11, 35%), and only five patients (16%) were progressive. TNFi was safe even in patients with severely reduced left ventricular ejection fraction (LVEF), and a significant improvement in LVEF under TNFi treatment was observed. TNFi shows better control of myocardial inflammation compared to nbDMARDs and corticosteroid monotherapies in patients with CS. TNFi was efficient and safe even in patients with severely reduced LVEF.