[Analysis of gene mutations and clinic features in 108 patients with myeloproliferative neoplasm].
Objective: To analyze the genetic mutations and clinical features of the subtypes of classical BCR-ABL-negative myeloproliferative neoplasm (MPN) . Methods: Mutations of 108 newly diagnosed BCR-ABL-negative MPN patients [including 55 patients with essential thrombocytopenia (ET) , 24 with polycythemia vera (PV) , and 29 with primary myelofibrosis (PMF) ] were identified using next-generation sequencing with 127-gene panel, and the relationship between gene mutations and clinical features were analyzed. Results: Total 211 mutations in 32 genes were detected in 100 MPN patients (92.59% ) , per capita carried (1.96±1.32) mutations. 85.19% (92/108) patients carried the driver gene (JAK2, CALR, MPL) mutations, 69.56% (64/92) of these patients carried at least 1 additional gene mutation. In descending order of mutation frequency, the highest frequency was for activation signaling pathway genes (42.2% , 89/211) , methylation genes (17.6% , 36/211) , and chromatin-modified genes (16.1% , 34/211) . There was a significant difference in the number of mutations in the activation signaling pathway genes, epigenetic regulatory genes, spliceosomes, and RNA metabolism genes among the three MPN subgroups. The average number of additional mutations in PMF patients was higher than that in ET and PV patients (1.69±1.39, 0.67±0.70, 0.87±1.22, χ(2)=13.445, P=0.001) . MPN-SAF-TSS (MPN 10 score) (P=0.006) and myelofibrosis level (P=0.015) in patients with ≥ 3 mutant genes were higher and the HGB level (P=0.002) was lower than in those with<3 mutations. Twenty-six patients (24.1% ) carried high-risk mutation (HMR) , and patients with HMR had lower PLT (P=0.017) , HGB levels (P<0.001) , and higher myelofibrosis level (P=0.010) and MPN10 score (P<0.001) . The frequency of ASXL1 mutations was higher in PMF than in PV patients (34.5% vs. 4.2% , P=0.005) . PMF patients with ASXL1 had lower levels of PLT and HGB (P=0.029 and 0.019) . Conclusion: 69.56% of MPN patients carry at least one additional mutation, and 24.1% patients had HMR. Each subgroup had different mutation patterns. PMF patients had a higher average number of additional gene mutations, especially a higher frequency of ASXL1 mutation; PLT and HGB levels were lower in ASXL1 mutation PMF patients.


[The development of an organizational socialization process model for new nurses using a system dynamics approach].
The purpose of this study was to examine the problems and relevant variables for effective Organizational Socialization of new nurses, to produce a causal map, to build up a simulation model and to test its validity. The basic data was collected from Sep. 2002 to July 2003. The Organizational Socialization process of new nurses was analyzed through a model simulation. The VENSIM 5.0b DSS program was used to develop the study model. This Model shows interrelation of these result variables: organizational commitment, job satisfaction, job performance, intention of leaving the work setting, decision making ability, and general results of Organizational Socialization. The model's factors are characteristic of organization and individual values, task-related knowledge and skills, and emotion and communication that affects new nurses' socialization process. These elements go through processes of anticipatory socialization, encounter, change and acquisition. The Model was devised to induce effective Organizational Socialization results within 24 months of its implementation. The basic model is the most efficient and will also contribute to the development of knowledge in the body of nursing. This study will provide proper direction for new Nurse's Organizational Socialization. Therefore, developing an Organizational Socialization Process Model is meaningful in a sense that it could provide a framework that could create effective Organizational Socialization for new nurses.


Mortality in mental disorders and global disease burden implications: a systematic review and meta-analysis.
Despite the potential importance of understanding excess mortality among people with mental disorders, no comprehensive meta-analyses have been conducted quantifying mortality across mental disorders. To conduct a systematic review and meta-analysis of mortality among people with mental disorders and examine differences in mortality risks by type of death, diagnosis, and study characteristics. We searched EMBASE, MEDLINE, PsychINFO, and Web of Science from inception through May 7, 2014, including references of eligible articles. Our search strategy included terms for mental disorders (eg, mental disorders, serious mental illness, and severe mental illness), specific diagnoses (eg, schizophrenia, depression, anxiety, and bipolar disorder), and mortality. We also used Google Scholar to identify articles that cited eligible articles. English-language cohort studies that reported a mortality estimate of mental disorders compared with a general population or controls from the same study setting without mental illness were included. Two reviewers independently reviewed the titles, abstracts, and articles. Of 2481 studies identified, 203 articles met the eligibility criteria and represented 29 countries in 6 continents. One reviewer conducted a full abstraction of all data, and 2 reviewers verified accuracy. Mortality estimates (eg, standardized mortality ratios, relative risks, hazard ratios, odds ratios, and years of potential life lost) comparing people with mental disorders and the general population or people without mental disorders. We used random-effects meta-analysis models to pool mortality ratios for all, natural, and unnatural causes of death. We also examined years of potential life lost and estimated the population attributable risk of mortality due to mental disorders. For all-cause mortality, the pooled relative risk of mortality among those with mental disorders (from 148 studies) was 2.22 (95% CI, 2.12-2.33). Of these, 135 studies revealed that mortality was significantly higher among people with mental disorders than among the comparison population. A total of 67.3% of deaths among people with mental disorders were due to natural causes, 17.5% to unnatural causes, and the remainder to other or unknown causes. The median years of potential life lost was 10 years (n = 24 studies). We estimate that 14.3% of deaths worldwide, or approximately 8 million deaths each year, are attributable to mental disorders. These estimates suggest that mental disorders rank among the most substantial causes of death worldwide. Efforts to quantify and address the global burden of illness need to better consider the role of mental disorders in preventable mortality.


Adaptive mechanically controlled lubrication mechanism found in articular joints.
Articular cartilage is a highly efficacious water-based tribological system that is optimized to provide low friction and wear protection at both low and high loads (pressures) and sliding velocities that must last over a lifetime. Although many different lubrication mechanisms have been proposed, it is becoming increasingly apparent that the tribological performance of cartilage cannot be attributed to a single mechanism acting alone but on the synergistic action of multiple "modes" of lubrication that are adapted to provide optimum lubrication as the normal loads, shear stresses, and rates change. Hyaluronic acid (HA) is abundant in cartilage and synovial fluid and widely thought to play a principal role in joint lubrication although this role remains unclear. HA is also known to complex readily with the glycoprotein lubricin (LUB) to form a cross-linked network that has also been shown to be critical to the wear prevention mechanism of joints. Friction experiments on porcine cartilage using the surface forces apparatus, and enzymatic digestion, reveal an "adaptive" role for an HA-LUB complex whereby, under compression, nominally free HA diffusing out of the cartilage becomes mechanically, i.e., physically, trapped at the interface by the increasingly constricted collagen pore network. The mechanically trapped HA-LUB complex now acts as an effective (chemically bound) "boundary lubricant"--reducing the friction force slightly but, more importantly, eliminating wear damage to the rubbing/shearing surfaces. This paper focuses on the contribution of HA in cartilage lubrication; however, the system as a whole requires both HA and LUB to function optimally under all conditions.


Vitamin A facilitates enteric nervous system precursor migration by reducing Pten accumulation.
Hirschsprung disease is a serious disorder of enteric nervous system (ENS) development caused by the failure of ENS precursor migration into the distal bowel. We now demonstrate that retinoic acid (RA) is crucial for GDNF-induced ENS precursor migration, cell polarization and lamellipodia formation, and that vitamin A depletion causes distal bowel aganglionosis in serum retinol-binding-protein-deficient (Rbp4(-/-)) mice. Ret heterozygosity increases the incidence and severity of distal bowel aganglionosis induced by vitamin A deficiency in Rbp4(-/-) animals. Furthermore, RA reduces phosphatase and tensin homolog (Pten) accumulation in migrating cells, whereas Pten overexpression slows ENS precursor migration. Collectively, these data support the hypothesis that vitamin A deficiency is a non-genetic risk factor that increases Hirschsprung disease penetrance and expressivity, suggesting that some cases of Hirschsprung disease might be preventable by optimizing maternal nutrition.


[Pharmacology of troparil].
The physiological action of the tropan derivative troparil (2-beta-methoxycarbonyl-3-beta-phenyltropan or 2-exo-3-exo-2-carbomethoxy-aryltropan was studied. Troparil was demonstrated to be a highly active stimulant of the nervous activity according to a lot of parameters. The drug appeared more powerful than amphetamine but less toxic.


Influence of pectinase treatment on the physicochemical properties of potato flours.
Untreated and pectinase-treated potato flours from Atlantic and Superior cultivars were characterised to identify the effects of pectinase treatment on their physicochemical properties. Steam-cooked potato whole-tissues were treated with and without pectinase to prepare the dehydrated potato flours. Untreated and pectinase-treated potato flours were investigated with respect to morphology, chemical composition, starch leaching, swelling power, gelatinization, and pasting viscosity. Upon viewing with scanning electron microscopy and light microscopy, the pectinase-treated (relative to untreated) potato flours revealed that the retrograded starch materials were present in intact parenchyma cells, apparently exhibiting granular structures. Their protein and ash contents were reduced through pectinase treatment. While starch leachate contents were lower for the pectinase-treated potato flours, the opposite trend in swelling powers was observed. Pectinase-treated potato flours exhibited higher melting temperatures and pasting viscosities than untreated counterparts. Overall, the modification of potato flour morphology by pectinase treatment may result in alteration of physicochemical properties of potato flours.


Impaired control over alcohol use: An under-addressed risk factor for problem drinking in young adults?
Impaired control over alcohol use may be defined as "a breakdown of an intention to limit consumption in a particular situation" (Heather, Tebbutt, Mattick, & Zamir, 1993, p. 701) and has long been considered an important feature of alcohol dependence. Evidence suggests impaired control is highly relevant to young adult problem drinking. In the natural history of problem drinking, impaired control tends to develop early and may predict alcohol-related problems prospectively in undergraduates. Impaired control over alcohol use may be a facet of generalized behavioral undercontrol specifically related to drinking. In particular, impaired control is theoretically and empirically related to impulsivity. The question of whether impaired control represents a facet of impulsivity or a related but separate construct requires further study. However, theoretical arguments and empirical evidence suggest that there are unique qualities to the constructs. Specifically, existing data suggest that self-report measures of impaired control and impulsivity over alcohol use relate distinctly to problem drinking indices in young adults. Several lines of future research concerning impaired control are suggested, using the impulsivity literature as a guide. We conclude that impaired control is a valuable construct to the study of young adult problem drinking and that measures of impaired control should be included in more young adult alcohol studies. The extent to which impaired control over the use of other substances and impaired control over engagement in other addictive behaviors are clinically relevant constructs requires additional study.


Social relationships and physiological determinants of longevity across the human life span.
Two decades of research indicate causal associations between social relationships and mortality, but important questions remain as to how social relationships affect health, when effects emerge, and how long they last. Drawing on data from four nationally representative longitudinal samples of the US population, we implemented an innovative life course design to assess the prospective association of both structural and functional dimensions of social relationships (social integration, social support, and social strain) with objectively measured biomarkers of physical health (C-reactive protein, systolic and diastolic blood pressure, waist circumference, and body mass index) within each life stage, including adolescence and young, middle, and late adulthood, and compare such associations across life stages. We found that a higher degree of social integration was associated with lower risk of physiological dysregulation in a dose-response manner in both early and later life. Conversely, lack of social connections was associated with vastly elevated risk in specific life stages. For example, social isolation increased the risk of inflammation by the same magnitude as physical inactivity in adolescence, and the effect of social isolation on hypertension exceeded that of clinical risk factors such as diabetes in old age. Analyses of multiple dimensions of social relationships within multiple samples across the life course produced consistent and robust associations with health. Physiological impacts of structural and functional dimensions of social relationships emerge uniquely in adolescence and midlife and persist into old age.


Bleeding and thrombosis in a patient with secondary antiphospholipid syndrome.
Antiphospholipid antibodies have been associated with occurrence of arterial and venous thrombotic events and fetal loss, which together constitute the antiphospholipid syndrome (APS). However, bleeding is rare in this syndrome. We report a case of systemic lupus erythematosus (SLE) with APS complicated simultaneously by thrombotic and hemorrhagic events. A 34-year-old woman was a known case of diffuse proliferative lupus nephritis associated with APS, on treatment with corticosteroids, cyclophosphamide and anticoagulants. She presented in February 2004 with severe anemia, menorrhagia, gingival bleeding and acute loss of vision in the left eye. Investigations revealed a hematoma in the psoas muscle with thrombosis of the inferior vena cava and occlusion of the retinal vein. Blood tests revealed a strongly positive lupus anticoagulant, factor XI deficiency (35%) and decrease of free protein S (44%). Factor XI inhibitor, anti-prothrombin, and anti-protein S antibodies were absent. The patient was treated with corticosteroids and six pulses of cyclophosphamide, which resulted in a rapid disappearance of bleeding, reduction of hematoma and normalization of hematological abnormalities. She was maintained on corticosteroids, azathioprine and anticoagulant agents were introduced. After a follow-up of 28 months, there was no recurrence of bleeding, the thrombosis had resolved, and there was a decrease in the levels of circulating anticoagulant as well as anticardiolipin antibodies.


An integrated analysis of host- and tumor-derived markers for predicting high-grade dysplasia and associated invasive carcinoma of intraductal papillary mucinous neoplasms of the pancreas.
To clarify the usefulness of cancer-related inflammation, hypermetabolism, and subsequent host malnutrition biomarkers for predicting the histological grades of intraductal papillary mucinous neoplasms of the pancreas (IPMNs). The systemic immune-inflammation index (SII), prognostic nutritional index (PNI), and maximum standardized uptake value (SUVmax) on fluorodeoxyglucose-positron emission tomography were compared across 171 resected IPMN cases of different histological grades. The diagnostic performance of each marker and of their combinations for predicting IPMN with high-grade dysplasia (HGD)/associated invasive carcinoma (INV) was also tested. Of the 171 IPMNs, the IPMN cases with HGD showed significantly higher values of SII (median 406 vs. 340; P = 0.041) and SUVmax (median 2.5 vs. 2.0; P = 0.001) than those with low-grade dysplasia (LGD). On a multivariate analysis, the SII and SUVmax were both independent markers for predicting HGD/INV. A combination analysis including the tumor- and host-derived markers in combination with imaging findings showed an improved diagnostic performance (area under the curve 0.824; sensitivity 75.9%; specificity 80.0%). The combination of multiple markers of host-derived inflammation and tumor-derived focal hypermetabolism can serve as a predictor for the presence of HGD/INV.


The increasing prevalence of snacking among US children from 1977 to 1996.
To determine snacking trends and changes in nutrient contribution of snacking over time. Nationally representative data from the 1977-78 Nationwide Food Consumption Survey (NFCS77), 1989-91 Continuing Survey of Food Intake by Individuals (CSFII89), and 1994-96 (CSFII96) were used. The sample consisted of 21,236 individuals aged 2 to 18 years. For each survey year, mean numbers of snacks consumed, mean grams consumed per snack, and mean energy intake from snacks were computed, as was contribution of snacking to total energy intake and fat intake. Snacking was self-defined, and a snacking occasion consisted of all snack foods consumed during a 15-minute period. Differences in means between age groups and across survey years were compared. The prevalence of snacking increased in all age groups. The average size of snacks and energy per snack remained relatively constant; however, the number of snacking occasions increased significantly, therefore increasing the average daily energy from snacks. Compared with non-snack eating occasions, the nutrient contribution of snacks decreased in calcium density and increased in energy density and proportion of energy from fat. Snacking is extremely prevalent in our society. Healthy snack food choices should be emphasized over high-energy density convenience snacks for children.


[Superficial haemosiderosis of the central nervous system, neuro otological manifestations and images from a magnetic resonance brain scan: a case report].
Superficial haemosiderosis of the central nervous system (SHCNS) is an infrequent clinical entity; it is produced by the formation of clinically silent haemosiderin deposits in the leptomeninges, the subpial tissue, the cranial nerves and spinal cord, secondary to chronic bleeding in the subarachnoid space. Aetiology is idiopathic in half the cases or secondary to a vascular malformation or other structural abnormalities. At least 100 cases have been reported in the literature, most of which were diagnosed post mortem. In these descriptions there is a predominance of cerebellar ataxia, progressive hypoacusis, nystagmus, recurring headaches, pyramidal signs, an absence of caloric responses and xanthochromic cerebrospinal fluid. Magnetic resonance (MR) brain scanning in T2 revealed hypointensity in the brain stem, the cerebellum and the Sylvian fissure, with atrophy of the cerebellum and the brain stem. A 64 year old male with bilateral tinnitus and progressive neurosensory hipoacusis, who visited because of loss of balance, urinary incontinence and oscillopsia. The patient s personal history included an episode of unbearable headache at the age of 53. The neurological and neuro otological examination was pathological and the MR brain scan confirmed the diagnosis of SHCNS. We wish to highlight how extremely rare this entity is, and especially so when there is a predominance of progressive neuro otological manifestations; we also want to emphasize the importance of carrying out specific imaging studies to enable a diagnosis to be made while the patient is still alive.


Isolation and characterization of a spontaneously immortalized multipotent mesenchymal cell line derived from mouse subcutaneous adipose tissue.
The emerging field of tissue engineering and regenerative medicine is a multidisciplinary science that is based on the combination of a reliable source of stem cells, biomaterial scaffolds, and cytokine growth factors. Adult mesenchymal stem cells are considered important cells for applications in this field, and adipose tissue has revealed to be an excellent source of them. Indeed, adipose-derived stem cells (ASCs) can be easily isolated from the stromal vascular fraction (SVF) of adipose tissue. During the isolation and propagation of murine ASCs, we observed the appearance of a spontaneously immortalized cell clone, named m17.ASC. This clone has been propagated for more than 180 passages and stably expresses a variety of stemness markers, such as Sca-1, c-kit/CD117, CD44, CD106, islet-1, nestin, and nucleostemin. Furthermore, these cells can be induced to differentiate toward osteogenic, chondrogenic, adipogenic, and cardiogenic phenotypes. m17.ASC clone displays a normal karyotype and stable telomeres; it neither proliferates when plated in soft agar nor gives rise to tumors when injected subcutaneously in NOD/SCID-γ (null) mice. The analysis of gene expression highlighted transcriptional traits of SVF cells. m17.ASCs were genetically modified by lentiviral vectors carrying green fluorescent protein (GFP) as a marker transgene and efficiently engrafted in the liver, when injected in the spleen of NOD/SCID-γ (null) monocrotaline-treated mice. These results suggest that this non-tumorigenic spontaneously immortalized ASC line may represent a useful tool (cell model) for studying the differentiation mechanisms involved in tissue repair as well as a model for pharmacological/toxicological studies.


Autism with del15p.11.1: case report with a new cytogenetic finding.
Autism with dell5Sp.11.1: case report with a new cytogenetic finding: Autism is a neurodevelopmental disorder and believed to be mainly genetic in origin, and environmental factors may modulate phenotypic expression. Less than four percent of cases of autism are associated with chromosomal abnormalities. Cytogenetic abnormalities found at the 15q11-q13 locus are reported most frequently in patients with autism. We performed GTG-banding and FISH studies to the present case and his parents and found a maternally inherited deletion on chromosome 15p in this case. With an understanding of the many genetic causes of autism, prenatal screening and counseling may one day become available for affected families as more autism-causing conditions become diagnosable.


[Ultrastructure of the ocular ciliary epithelium of the common frog].
The ultrastructure of the adult frog ciliary epithelium cells has definite regional differences. Cells of ciliary epithelium folds near the iris display morphological features characterizing its barrier and secretory functions which lead to the formation of aqueous humor. These are junctional complexes with tight junctions (zonula occludents) in the apical parts of contacting sides of cells of the inner leaf: a great quantity of mitochondria, ribosomes and various vesicles, well developed endoplasmic reticulum in the cytoplasm, much folded basal surface, gap junctions between cells of external and internal leaflets. In the mammalian inner epithelial layer different cell junctions are known to be arranged in a fixed spatial fashion. Unlike, in the frog's epithelium both zonula adherent and desmosomes may be found in any sequence. Tight junctions are formed during metamorphosis, on the place of focal junctions, whereas gap junctions, referred to earlier as "extended", start functioning between cells just on the very early stages of eye morphogenesis (Dabagyan et al., 1979). The epithelium of the posterior part of the ciliary fold and pars plana of the ciliary body have, in addition, the number of morphological sign indicating the cell involvement in the accomodational function of any eye (i. e. a majority of desmosomes binding all cells together and of zonulae adherentes, well developed intracellular skeleton of tonofilament bundles). These features are characteristic of the whole distal part of ciliary epithelium rather than of the place of attachment of zonula fiber only.


Prognostic role of FDG PET/CT in patients with differentiated thyroid cancer treated with 131-iodine empiric therapy.
To assess the long-term prognostic value of F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) in patients with differentiated thyroid carcinoma (DTC) undergoing empiric radioiodine (RAI) therapy due to raising values of thyroglobulin (Tg). Forty-nine patients with histological diagnosis of DTC (31 with papillary and 18 with follicular carcinoma) follow-up for a mean period of 7.9 ± 5 years after empiric RAI therapy were retrospectively analyzed. FDG-PET/CT was negative in 15 (30.6%) patients and positive in 34 (69.4%), whereas postradioiodine therapy whole body scan (t-WBS) was negative in 16 (32.7%) and positive in 33 (67.3%) patients. FDG-PET/CT and t-WBS were in agreement in 32 patients (7 both negative and 25 both positive); on the contrary, in 17 patients there was disagreement between FDG-PET/CT and t-WBS (P =ns). At short-term follow-up, Tg normalized in 19 (38.8%) patients and was unchanged or increased in 30 (61.2%). Of the 15 patients with negative FDG-PET/CT, 11 (73.3%) showed Tg normalization, whereas of the 34 patients with positive FDG-PET/CT, only 8 (23.5%) had Tg normalization (χ =8.9, P < .005). At multivariate analysis, FDG-PET/CT and Tg normalization at short-term follow-up were independent predictors of disease-free survival (χ =26.3, P < .0001), while Tg normalization was the only variable associated with overall survival χ =7.2, P < .01). FDG-PET/CT in association with Tg normalization at short-term follow-up may be useful for long-term prognostic stratification in DTC patients.


Bronchial hypersecretion in preterm neonates.
During an 18-month period, 11 preterm infants with birthweights between 700 and 1560 g (mean 1.2 kg) developed excessive tracheobronchial secretions during intensive care. No single obstetric factor was incriminated. Copious, viscous, tracheobronchial secretions were noted at about 5 days during mechanical ventilation via endotracheal tube causing recurrent segmental collapse, hypoxia, and hypercapnia (median peak PCO2 13.5 kPa). All infants were treated with frequent bronchial lavages and continued intermittent positive pressure ventilation, together with high concentrations of oxygen. No infant died, but morbidity was high. Tracheostomy was performed on 2 infants (one at age 3 months, because of severe croup) and 2 others had clinical or physiological evidence of upper airways narrowing. Follow-up studies showed that this group had more problems of airways obstruction throughout the first year of life as well as increased lung stiffness. The hypersecretion group showed a higher incidence of chronic lung disease. Likely aetiological factors were sought. Contamination of the mechanical ventilation equipment by detergent and activated glutaraldehyde was found; this could have been a contributory factor.


Impact of increased occlusal contact, interleukin-1 genotype, and periodontitis severity on gingival crevicular fluid IL-1beta levels.
The study of occlusal therapy in humans with periodontitis is problematic due to potential irreversible bone loss in control subjects. The hypothesis of this pilot investigation was that increased interocclusal contact initiated by short-term occlusal splint disuse would increase tooth mobility and the bone-resorptive cytokine interleukin (IL)-1beta in gingival crevicular fluid (GCF), comparable to IL-1-positive genotype and increased periodontitis severity. Nineteen non-smoking chronic periodontitis patients using nocturnal occlusal splints and undergoing periodontal maintenance in a private practice were evaluated at five time points: 24 hours after continuous splint use; 1, 2, and 3 days after no occlusal splint use; and 14 days after resumption of customary nighttime splint use. Subjects were evaluated to confirm that the plaque index and gingival index were < or = 1.0, and to categorize past periodontitis (moderate or severe) and IL-1 genotype (1A +4845 plus IL-1B +3954). Test sites on two anterior teeth vulnerable to occlusal trauma were sampled for mobility, GCF IL-1beta, and IL-1 receptor antagonist (ra). Tooth mobility remained low during the 3-day period when patients were not wearing their occlusal appliance. GCF IL-1beta decreased after not wearing the appliances (P = 0.016), especially at 48 hours. At this time, genotype-positive subjects had higher levels of GCF IL-1beta/IL-1ra than genotype-negative subjects (P = 0.045), and patients who had experienced severe periodontitis had higher IL-1beta levels than moderate periodontitis subjects (P = 0.004). These findings suggest that short-term discontinuation of occlusal splint therapy in non-smoking periodontitis patients undergoing periodontal maintenance does not result in potential signs of early occlusal trauma (increasing mobility or GCF IL-1beta). Longer-term studies may be needed to determine appropriate therapy applications for periodontitis-susceptible patients with definable occlusal discrepancies and/or parafunction.


Corticosteroids for acute severe asthma in hospitalised patients.
Corticosteroids are currently used routinely in the management of acute severe asthma. The optimal dose and route of administration continues to be debated. Some investigators have reported a greater benefit of higher doses of corticosteroids in the management of severe asthma, while others have not. To determine whether higher doses of systemic corticosteroids (oral, intravenous or intramuscular) are more effective than lower doses in the management of patients with acute severe asthma requiring hospital admission. Randomised controlled trials were identified from the Cochrane Airways Group Asthma Register. In addition, primary authors and content experts were contacted to identify eligible studies. Bibliographies from included studies, known reviews and texts were also searched. Studies were selected for inclusion in the review if they met the following broad inclusion criteria: described as randomised controlled trials, included patients with acute severe asthma, compared different doses of corticosteroids (any route) in 2 or more treatment arms, and had a minimum period of follow up of 24 hours. Two reviewers independently assessed the studies for inclusion and disagreement was resolved by third party adjudication. Data were extracted independently by two reviewers if the authors were unable to verify the validity of information. Missing data were obtained from authors or calculated from other data presented in the paper. The data were analysed as weighted mean differences (WMD) for primary pulmonary function outcomes using a fixed effects model. For the purposes of the review, three broad categories of corticosteroid dose (equivalent dose of methylprednisolone in 24 hours) were defined in advance: low dose (< or = 80 mg), medium dose (> 80 mg and < or = 360 mg) and high dose (> 360 mg). There were thus 3 main comparison groups: low versus medium dose, medium versus high dose and low versus high dose. Nine trials were included; a total of 344 adult patients have been studied (96 with low dose, 85 with medium dose and 163 with high dose corticosteroids). Only 6 trials provided sufficient data for the meta-analysis. There were no clinically or statistically significant differences detected in % predicted FEV1 among comparison groups after 24, 48 or 72 hours. At 48 hours, the weighted mean difference was -3.3% predicted (95% confidence interval -12.4 to + 5.8) for the low vs medium dose comparison, -1. 9% predicted (95% CI -8.1 to + 4.3) for the medium vs high dose comparison and + 0.5% predicted (95% CI - 7.8 to + 8.8) for the low vs high dose comparison. There appeared to be no significant differences in side effects or rates of respiratory failure among the varying doses of corticosteroids. No differences were identified among the different doses of corticosteroids in acute asthma requiring hospital admission. Low dose corticosteroids (< or = 80 mg/day of methylprednisolone or < or = 400 mg/day of hydrocortisone) appear to be adequate in the initial management of these adult patients. Higher doses do not appear to offer a therapeutic advantage.


Scandcleft randomized trials of primary surgery for unilateral cleft lip and palate: occlusion in 8-year-olds according to the Modified Huddart and Bodenham index.
The Scandcleft international multicenter study is a prospective clinical trial of the long-term outcome after four different surgical protocols for palatal closure in patients born with unilateral cleft lip and palate (UCLP). This paper is one of a series of follow-up studies in 8-year olds. To evaluate the dental occlusion of 8-year-old patients after four different protocols of primary surgery for UCLP. Ten cleft centres in five countries tested three different surgical procedures for primary palatal repair in three parallel trials (Arms B, C, and D) against a common procedure (Arm A). Initially 448 children born with non-syndromic UCLP were included in the project. At 8 years of age, 428 children remained in the study. Dental casts of 411 patients (270 boys, 141 girls), mean age 8.1 years (range 7.0-10.0) were taken. The casts were blindly assessed with the Modified Huddart and Bodenham (MHB) index by four orthodontists. The main outcome measures were anterior (+2 to -6) and posterior (0 to -8) mean scores. Comparisons were made with previous data in 5-year-olds. The inter- and intra-examiner reliability was good to excellent (0.75-0.90; 0.73-0.97), respectively. The mean total scores varied from -7.09 (Trial 2C) to -10.13 (Trial 3D). The mean anterior scores varied from -1.75 (Trial 2C) to -3.18 (Trial 1A). The mean posterior cleft-side scores varied from -4.32 (Trial 1B) to -5.21 (Trial 3D) and the mean non-cleft-side scores varied from -0.88 (Trial 2C) to -2.40 (Trial 3A). No significant differences were found within the trials. A significant difference was found between Trials 2 and 3 (Arm C/D) for the total score (P = 0.004). There was no evidence of clinically significant differences in occlusion between the two surgical methods in each trial or between the trials. All mean scores showed more negative values in 8-year-olds compared with previously reported values in 5-year-olds. ISRCTN29932826.


Basic Residues at Position 11 of α-Conotoxin LvIA Influence Subtype Selectivity between α3β2 and α3β4 Nicotinic Receptors via an Electrostatic Mechanism.
Understanding the determinants of α-conotoxin (α-CTX) selectivity for different nicotinic acetylcholine receptor (nAChR) subtypes is a prerequisite for the design of tool compounds to study nAChRs. However, selectivity optimization of these small, disulfide-rich peptides is difficult not only because of an absence of α-CTX/nAChR co-structures but also because it is challenging to predict how a mutation to an α-CTX will alter its potency and selectivity. As a prototypical system to investigate selectivity, we employed the α-CTX LvIA that is 25-fold selective for the α3β2 nAChR over the related α3β4 nAChR subtype, which is a target for nicotine addiction. Using two-electrode voltage clamp electrophysiology, we identified LvIA[D11R] that is 2-fold selective for the α3β4 nAChR, reversing the subtype preference. This effect is specifically due to the change in charge and not shape of LvIA[D11R], as substitution of D11 with citrulline retains selectivity for the α3β2 nAChR. Furthermore, LvIA[D11K] shows a stronger reversal, with 4-fold selectivity for the α3β4 nAChR. Motivated by these findings, using site-directed mutagenesis, we found that β2[K79A] (I79 on β4), but not β2[K78A] (N78 on β4), largely restores the potency of basic mutants at position 11. Finally, to understand the structural basis of this effect, we used AlphaFold2 to generate models of LvIA in complex with both nAChR subtypes. Both models confirm the plausibility of an electrostatic mechanism to explain the data and also reproduce a broad range of potency and selectivity structure-activity relationships for LvIA mutants, as measured using free energy perturbation simulations. Our work highlights how electrostatic interactions can drive α-CTX selectivity and may serve as a strategy for optimizing the selectivity of LvIA and other α-CTXs.


Augmentation mammoplasty: results of a patient survey.
A detailed survey on aesthetic augmentation mammoplasty was sent to patients who had undergone this procedure at the Massachusetts General Hospital between July 1973 and July 1991 to determine the incidence of postoperative complications after augmentation mammoplasty, and to qualify the factors related to patient satisfaction (n = 304). Surgical records of respondents were examined. After a mean follow-up of 8.2 years, there were no patients with known breast cancer or autoimmune disease after augmentation. Five implants (2.5%) were replaced due to leakage. Other complications were rare. Overall satisfaction was high and correlated inversely with capsular contracture ratings and elapsed time. Contracture ratings increased with time for silicone implants. Gel implants in the submuscular position were softer than subglandular ones after 5 years elapsed time. Most women are satisfied after augmentation mammoplasty, despite a significant incidence of capsular contracture. The incidence of other adverse effects after augmentation is low. Augmentation mammoplasty does not appear to be an inducer of autoimmune disease or of breast cancer.


Stable Interface between a NaCl-AlCl3 Melt and a Liquid Ga Negative Electrode for a Long-Life Stationary Al-Ion Energy Storage Battery.
Intermediate temperature NaCl-AlCl3-based Al-ion batteries are considered as a promising stationary energy storage system due to their low cost, high safety, etc. However, such a cheap electrolyte has a critical feature, i.e., strong corrosion, which results in the short cycle life of the conventional Al-metal anode and also limits the development of the NaCl-AlCl3-based Al-ion batteries. A noncorrosive electrolyte may be a good choice for addressing the above challenge, while it is difficult to obtain the electrolyte that has advantages of both noncorrosion and low cost. Therefore, here, we report a Ga-metal anode in the affordable NaCl-AlCl3 electrolyte for constructing a long-life stationary Al-ion energy storage system. This featured liquid metal anode shows good alloying and dealloying processes between metallic Ga and Al, as well as renders superior stability of the interface between the electrolyte and the anode (e.g., smoothly running for over 580 h at 2 mA cm-2). No-corrosion and no-pulverization problems appear in this novel liquid/liquid interface. Those advantages demonstrate that the liquid Ga-metal anode has a great promise for the improvement of the NaCl-AlCl3-based Al-ion batteries for large-scale stationary energy storage applications.


Symptoms of neck, shoulder, forearms, and hands: a cohort study among computer office workers in Sudan.
Complaints of the arms, neck, and shoulders in general and computer-related disorders in particular affect many office workers in economically developed countries. However, with the increased use of computer systems in developing countries, the associated musculoskeletal symptoms and risk factors are yet to be investigated. The study investigates the relationship between work-related physical and psychosocial characteristics and symptoms of the arms, neck, and shoulders in an African economically developed worksite. A prospective cohort study with 1-year follow-up was conducted. Data were used from 250 computer office workers in Khartoum, Sudan. Data were collected using the Arabic version of the Maastricht Upper Extremity Questionnaire. Prevalence rates for symptoms were calculated and the categories of risk factors being evaluated consisted of physical, psychological, and individual risk factors. Multivariable logistic regression analysis was used to identify risk factors associated with complaints that were expressed as odds ratios (ORs) with 95% confidence intervals (CIs). The 1-year follow-up prevalence rate was 0.63 (95% CI: 0.58-0.70) for neck symptoms, 0.56 (95% CI: 0.45-0.66) for shoulder symptoms, and 0.46 (95% CI: 0.42-0.59) for symptoms of the forearms/hands. Three main risk factors were significantly associated with the presence of symptoms: (1) time pressure [OR: 1.31 (1.00 to 1.90) P=0.05], (2) task difficulty [OR: 1.85 (1.73 to 1.99) P=0.03], and (3) previous history of symptoms [OR: 4.62 (2.20 to 9.35) P=0.01]. The targeted office workers in Sudan are reporting symptoms in the neck, shoulders, and forearms. Work-related psychological factors predicted more significantly the presence of symptoms in the targeted population.


An outbreak of Sarcocystis calchasi encephalitis in multiple psittacine species within an enclosed zoological aviary.
A total of 5 psittacine birds in an enclosed zoological exhibit, including 2 princess parrots and 3 cockatoos of 2 different species, developed severe central nervous system clinical signs over a 2-3-month period and died or were euthanized. Histologically, all birds had a lymphoplasmacytic and histiocytic encephalitis with intralesional protozoa consistent with a Sarcocystis species in addition to intramuscular tissue sarcocysts. By immunohistochemical staining, merozoites in brain and tissue cysts in muscle did not react with polyclonal antisera against Sarcocystis falcatula, Sarcocystis neurona, Toxoplasma gondii, and Neospora caninum, or with a monoclonal antibody to S. neurona. Transmission electron microscopy on sarcocyst tissue cyst walls from 2 birds was morphologically consistent with Sarcocystis calchasi. Polymerase chain reaction (PCR) amplification and sequencing of partial 18S ribosomal RNA from muscle tissue cysts and brain schizonts from 3 birds was consistent with a clade containing S. calchasi and Sarcocystis columbae but could not distinguish these closely related Sarcocystis species. However, PCR amplification and sequencing of the internal transcribed spacer 1 RNA segment in the brain from 2 birds and muscle from 2 birds specifically identified the isolates as S. calchasi. The current report documents that multiple psittacine species are susceptible intermediate hosts of S. calchasi, and that infection can cause encephalitis resulting in significant morbidity and mortality in psittacine aviaries.


Small-molecule inhibition of HIV-1 Vif.
The HIV-1 protein Vif, essential for in vivo viral replication, targets the human DNA-editing enzyme, APOBEC3G (A3G), which inhibits replication of retroviruses and hepatitis B virus. As Vif has no known cellular homologs, it is an attractive, yet unrealized, target for antiviral intervention. Although zinc chelation inhibits Vif and enhances viral sensitivity to A3G, this effect is unrelated to the interaction of Vif with A3G. We identify a small molecule, RN-18, that antagonizes Vif function and inhibits HIV-1 replication only in the presence of A3G. RN-18 increases cellular A3G levels in a Vif-dependent manner and increases A3G incorporation into virions without inhibiting general proteasome-mediated protein degradation. RN-18 enhances Vif degradation only in the presence of A3G, reduces viral infectivity by increasing A3G incorporation into virions and enhances cytidine deamination of the viral genome. These results demonstrate that the HIV-1 Vif-A3G axis is a valid target for developing small molecule-based new therapies for HIV infection or for enhancing innate immunity against viruses.


Proximity to injury, but neither number of nuclei nor ploidy define pathological adaptation and plasticity in cardiomyocytes.
The adult mammalian heart consists of mononuclear and binuclear cardiomyocytes (CMs) with various ploidies. However, it remains unclear whether a variation in ploidy or number of nuclei is associated with distinct functions and injury responses in CMs, including regeneration. Therefore, we investigated transcriptomes and cellular as well as nuclear features of mononucleated and binucleated CMs in adult mouse hearts with and without injury. To be able to identify the role of ploidy we analyzed control and failing human ventricular CMs because human CMs show a larger and disease-sensitive degree of polyploidization. Using transgenic Myh6-H2BmCh to identify mononucleated and binucleated mouse CMs, we found that cellular volume and RNA content were similar in both. On average nuclei of mononuclear CMs showed a 2-fold higher ploidy, as compared to binuclear CMs indicating that most mononuclear CMs are tetraploid. After myocardial infarction mononucleated and binucleated CMs in the border zone of the lesion responded with hypertrophy and corresponding changes in gene expression, as well as a low level of induction of cell cycle gene expression. Human CMs allowed us to study a wide range of polyploidy spanning from 2n to 16n. Notably, basal as well as pathological gene expression signatures and programs in failing CMs proved to be independent of ploidy. In summary, gene expression profiles were induced in proximity to injury, but independent of number of nuclei or ploidy levels in CMs.


Anti-C5a antibody vilobelimab treatment and the effect on biomarkers of inflammation and coagulation in patients with severe COVID-19: a substudy of the phase 2 PANAMO trial.
We recently reported in the phase 3 PANAMO trial that selectively blocking complement 5a (C5a) with vilobelimab led to improved survival in critically ill COVID-19 patients. C5a is an important contributor to the innate immune system and can also activate the coagulation system. High C5a levels have been reported in severely ill COVID-19 patients and correlate with disease severity and mortality. Previously, we assessed the potential benefit and safety of vilobelimab in severe COVID-19 patients. In the current substudy of the phase 2 PANAMO trial, we aim to explore the effects of vilobelimab on various biomarkers of inflammation and coagulation. Between March 31 and April 24, 2020, 17 patients with severe COVID-19 pneumonia were enrolled in an exploratory, open-label, randomised phase 2 trial. Blood markers of complement, endothelial activation, epithelial barrier disruption, inflammation, neutrophil activation, neutrophil extracellular trap (NET) formation and coagulopathy were measured using enzyme-linked immunosorbent assay (ELISA) or utilizing the Luminex platform. During the first 15 days after inclusion, change in biomarker concentrations between the two groups were modelled with linear mixed-effects models with spatial splines and compared. Eight patients were randomized to vilobelimab treatment plus best supportive care (BSC) and nine patients were randomized to BSC only. A significant decrease over time was seen in the vilobelimab plus BSC group for C5a compared to the BSC only group (p < 0.001). ADAMTS13 levels decreased over time in the BSC only group compared to the vilobelimab plus BSC group (p < 0.01) and interleukin-8 (IL-8) levels were statistically more suppressed in the vilobelimab plus BSC group compared to the BSC group (p = 0.03). Our preliminary results show that C5a inhibition decreases the inflammatory response and hypercoagulability, which likely explains the beneficial effect of vilobelimab in severe COVID-19 patients. Validation of these results in a larger sample size is warranted.


Effect of experimental temperature on the permeation of model diffusants across porcine buccal mucosa.
The influence of experimental temperature on the permeability of model diffusants across porcine buccal mucosa was investigated in vitro. The permeability increased significantly as the experimental temperature was increased in increments of approximately 7°C. It was observed that the apparent permeability and temperature were related by an exponential relationship that conformed to the Arrhenius equation. Diffusants with higher lipophilicities--buspirone and bupivacaine--had lower activation energies for diffusion when compared to hydrophilic diffusants--antipyrine and caffeine. The activation energy for diffusion of the model diffusants decreased linearly with increasing distribution coefficients across porcine buccal mucosa. The results suggested that the buccal mucosa acts as a stronger barrier to the diffusion of hydrophilic diffusants than the lipophilic ones. The log-linear relationship between permeability and temperature indicates that temperature should be carefully controlled in diffusion experiments. These results also point to the possibility of developing heat-generating buccal delivery devices, especially for hydrophobic diffusants.


Resveratrol supplementation reduces pain experience by postmenopausal women.
Pain is a common complaint among postmenopausal women. It has been postulated that vascular dysfunction caused by estrogen decline at menopause plays a key role in the initiation and progression of degradative joint disease, namely age-related osteoarthritis. We evaluated whether supplementation with resveratrol, a phytoestrogen, could improve aspects of well-being such as chronic pain that is commonly experienced by postmenopausal women. A 14-week randomized, double-blind, placebo-controlled intervention with trans-resveratrol (75 mg, twice daily) was conducted in 80 healthy postmenopausal women. Aspects of well-being, including pain, menopausal symptoms, sleep quality, depressive symptoms, mood states, and quality of life were assessed by Short form-36 at baseline and at the end of treatment. Rating scales were averaged to provide a composite score representing overall well-being. Cerebral vasodilator responsiveness to hypercapnia was also assessed as a surrogate marker for cerebrovascular function. Compared with placebo treatment, there was a significant reduction in pain and an improvement in total well-being after resveratrol supplementation. Both benefits, including measures of quality of life, correlated with improvements in cerebrovascular function. Our preliminary findings indicate potential for resveratrol treatment to reduce chronic pain in age-related osteoarthritis. Resveratrol consumption may also boost perceptions of well-being in postmenopausal women. Further investigation to elucidate underlying mechanisms is warranted.


Lifestyle Behaviors and Clinical Outcomes in Idiopathic Pulmonary Fibrosis.
Lifestyle behaviors are not well-characterized in idiopathic pulmonary fibrosis (IPF). To assess the association between lifestyle behaviors and clinical outcomes in patients with IPF. A total of 34 IPF patients (median age 68 years) were assessed for daily sitting and weekly walking times using the International Physical Activity Questionnaire by in-person interview at baseline, and they were followed up for up to 40 months. Cox proportional hazard analysis was conducted for cardiorespiratory-related hospitalizations and mortality as outcomes. Fifty percent of all patients were hospitalized, and 32% died during the follow-up period. Sitting and walking times were associated with hospitalizations and mortality in IPF. Compared to patients who reported a sitting time of <5 h/day, patients who sat 5 to <10 and ≥10 h/day experienced an increased risk of 2.4 and 5.8 (p trend = 0.036) for hospitalization and of 4.6 and 21.2 (p trend = 0.018) for mortality, respectively. Compared to patients walking <100 min/week, patients with a walking time of 100 to <150 and ≥150 min/week were associated with a 49 and 74% reduced risk for hospitalizations (p trend = 0.022) and a 62 and 86% reduced risk for mortality (p trend = 0.018), respectively. The risk for mortality was further reduced with a combination of shorter sitting and extended walking times. Shorter daily sitting and longer weekly walking times were associated with reduced hospitalization and mortality risks in patients with IPF. These findings suggest a clinical importance of assessing lifestyle behaviors in a comprehensive evaluation and prognostication of IPF patients. The results underscore potential clinical benefits of reducing sedentary behaviors among IPF patients; however, this warrants further investigation.


Age-related hearing loss in older adults with cognitive impairment.
Hearing loss can impair effective communication between caregivers and individuals with cognitive impairment. However, hearing loss is not often measured or addressed in care plans for these individuals. The aim of this study is to measure the prevalence of hearing loss and the utilization of hearing aids in a sample of individuals with cognitive impairment in a tertiary care memory clinic. A retrospective review of 133 charts of individuals >50 years who underwent hearing assessment at a tertiary care memory clinic over a 12-month period (June 2014-June 2015) was undertaken. Using descriptive statistics, the prevalence of hearing loss was determined and associations with demographic variables, relevant medical history, cognitive status, and hearing aid utilization were investigated. Results indicate that hearing loss is highly prevalent among this sample of cognitively impaired older adults. Sixty percent of the sample had at least a mild hearing loss in the better hearing ear. Among variables examined, age, MMSE, and medical history of diabetes were strongly associated with hearing impairment. Hearing aid utilization increased in concordance with severity of hearing loss, from 9% to 54% of individuals with a mild or moderate/severe hearing loss, respectively. Hearing loss is highly prevalent among older adults with cognitive impairment. Despite high prevalence of hearing loss, hearing aid utilization remains low. Our study highlights the importance of hearing evaluation and rehabilitation as part of the cognitive assessment and care management plan in this vulnerable population.


Repetition related changes in activation and functional connectivity in hippocampus predict subsequent memory.
Using fMRI, this study examined the relationship between repetition-related changes in the medial temporal lobe (MTL) activation during encoding and subsequent memory for similarity of repetitions. During scanning, subjects classified pictures of objects as natural or man-made. Each object-type was judged twice with presentations of either identical pictures or pictures of different exemplars of the same object. After scanning, a surprise recognition test required subjects to decide whether a probe word corresponded to pictures judged previously. When a subject judged the word as "old," a second judgment was made concerning the physical similarity of the two pictures. Repetition related changes in MTL activation varied depending on whether or not subjects could correctly state that pictures were different. Moreover, psychophysiological interactions analyses showed that accuracy in recalling whether the two pictures were different was predicted by repetition-related changes in the functional connectivity of MTL with frontal regions. Specifically, correct recollection was predicted by increased connectivity between the left posterior hippocampus and the right inferior frontal gyrus, and also by decreased connectivity between the left posterior hippocampus and the left precentral gyrus on the second stimulus presentation. The opposite pattern was found for trials that were incorrectly judged on the nature of the repetition. These results suggest that successful encoding is predicted by a combination of increases and decreases in both the MTL activation and functional connectivity, and not merely by increases in activation and connectivity as suggested previously.


[Possible role of phospholipid molecule reorientation in ion transport through membranes].
A theory of ionic transport through membranes in which the phospholipid molecules play the role of ion-carriers is developed. The transport of ions is accompanied by a membrane asymmetry. Expressions for stationary-state ion-fluxes in the case of one sort of ions transport, and in the case of coupled transport of ions of two sorts, are obtained. In the latter case near equilibrium the cross-coefficients are equal in accordance with Onsager's law. The possible role of phospholipids as ioncarriers in native membranes is discussed.


Increased blood pressure after abrupt cessation of daily cannabis use.
Cannabis is the most widely used illicit drug. Acute cannabis administration increases blood pressure (BP) and heart rate and tolerance develops to these effects with heavy use. A valid and reliable withdrawal syndrome occurs in most daily users, but few studies have assessed the cardiovascular effects of withdrawal. The objective of this report is to describe unexpected changes in cardiovascular function during brief periods of supervised cannabis use and abstinence in daily cannabis users. A within-subjects ABAC crossover study in which inpatient volunteers smoked cannabis ad libitum (A), and abstained from cannabis (B/C). Vital signs were obtained 3 times daily during 11 inpatient days for 13 daily cannabis users (11 men, 8 African American). BP increased significantly during periods of cannabis abstinence compared with periods of cannabis use. The magnitude of increase was substantial in a subset (N = 6) of participants, with mean increases of up to 22.8 mm Hg systolic and 12.3 mm Hg diastolic BP observed. A main effect of heart rate was not observed. Secondary analysis limited to morning assessments suggested that resting heart rate increased during abstinence, but the magnitude of this effect was not clinically significant. Abrupt cessation of heavy cannabis use may cause clinically significant increases in BP in a subset of users. BP should be monitored among those attempting to reduce or quit frequent cannabis use, particularly those with preexisting hypertension. The time course of this effect is currently unknown and requires further study.


Biliary stent or surgical bypass in unresectable pancreatic cancer with obstructive jaundice.
To investigate the effects of surgical and non-surgical palliation of jaundice in unresectable pancreatic carcinoma this retrospective study was performed. Between 1980 and 1983 90 patients were treated of whom 54 (69%) were jaundiced. Of these 36 were treated with biliary bypass (67%), four underwent resection (7%), five were treated by percutaneous drainage (9%) and nine (17%) were in such poor general condition that no treatment for jaundice was possible. Ninety-eight patients were treated between 1984 and 1987 when the initial approach to palliation of jaundice was endoscopic stenting. Transhepatic drainage was used only if stenting failed, and operation only if both non-surgical methods failed. Seventy-two of the 98 patients (73%) were jaundiced, of whom 18 (25%) received a stent placed endoscopically, 11 (15%) underwent transhepatic drainage, 27 (38%) underwent biliary bypass, and 14 (19%) underwent pancreatic resection. Significantly fewer patients in the second group could not be treated because of their poor general condition (n = 2, 3%, p less than 0.02). There were no differences among the methods in overall and 30 day complication rates, or the length of hospital stay, but the late complication rate was 1/63 (2%) for biliary bypass compared with 7/29 (24%) for biliary stenting (p less than 0.001). The difference was because of the high incidence of blockage of the stents causing recurrent jaundice, but the stents could easily be replaced. There was no difference in mortality between the two periods. We conclude that stenting is an acceptable alternative to biliary decompression in the treatment of obstructive jaundice in unresectable pancreatic cancer.


A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting.
Molecular biology critically depends upon the isolation of desired DNA sequences. Flow cytometry, with its capacity to interrogate and sort more than 50,000 cells/s, shows great potential to expedite clone characterization and isolation. Intrinsic heterogeneity of protein expression levels in cells limits the utility of single fluorescent reporters for cell-sorting. Here, we report a novel dual-fluorescence strategy that overcomes the inherent limitations of single reporter systems by controlling for expression variability. We demonstrate a dual-reporter system using the green fluorescent protein (GFP) gene fused to the Discosoma red fluorescent protein (DsRed) gene. The system reports the successful insertion of foreign DNA with the loss of DsRed fluorescence and the maintenance of GFP fluorescence. Single cells containing inserts are readily recognized by their altered ratios of green to red fluorescence and separated using a high-speed cell-sorter for further processing. This novel reporter system and vector were successfully validated by shotgun library construction, cloned sequence isolation, PCR amplification and DNA sequencing of cloned inserts from bacteria after cell-sorting. This simple, robust system can also be adapted for diverse biosensor assays and is amenable to miniaturization. We demonstrated that dual-fluorescence reporting coupled with high-speed cell-sorting provides a more efficient alternative to traditional methods of clone isolation.


Construction and application of riboswitch-based sensors that detect metabolites within bacterial cells.
A riboswitch is an RNA element that detects the level of a specific metabolite within the cell and regulates the expression of co-transcribed genes. By fusing a riboswitch to a reporter protein in a carefully designed and tested construct, this ability can be exploited to create an intracellular sensor that detects the level of a particular small molecule within live bacterial cells. There is a great deal of flexibility in the design of such a sensor and factors such as the molecule to be detected and the downstream experiments in which the sensor will be applied should guide the specific blueprint of the final construct. The completed sensor plasmid needs to be rigorously tested with appropriate controls to ensure that its dynamic range, signal strength, sensitivity and specificity are suitable for its intended applications. In this chapter, methods for the design, assessment and use of riboswitch sensors are provided along with those for one example application for which riboswitch sensors are ideally suited.


Molecular recognition with DNA nanoswitches: effects of single base mutations on structure.
This paper investigates the properties of a simple DNA-based nanodevice capable of detecting single base mutations in unlabeled nucleic acid target sequences. Detection is achieved by a two-stage process combining first complementary-base hybridization of a target and then a conformational change as molecular recognition criteria. A probe molecule is constructed from a single DNA strand designed to adopt a partial cruciform structure with a pair of exposed (unhybridized) strands. Upon target binding, a switchable cruciform construct (similar to a Holliday junction) is formed which can adopt open and closed junction conformations. Switching between these forms occurs by junction folding in the presence of divalent ions. It has been shown from the steady-state fluorescence of judiciously labeled constructs that there are differences between the fluorescence resonance energy transfer (FRET) efficiencies of closed forms, dependent on the target sequence near the branch point, where the arms of the cruciform cross. This difference in FRET efficiency is attributed to structural variations between these folded junctions with their different branch point sequences arising from the single base mutations. This provides a robust means for the discrimination of single nucleotide mismatches in a specific region of the target. In this paper, these structural differences are analyzed by fitting observed time-resolved donor fluorescence decay data to a Gaussian distribution of donor-acceptor separations. This shows the closest mean separation (approximately 40 A) for the perfectly matched case, whereas larger separations (up to 50 A) are found for the single point mutations. These differences therefore indicate a structural basis for the observed FRET differences in the closed configuration which underpins the operation of these devices as biosensors capable of resolving single base mutations.


A multicenter double-blind study of controlled-release physostigmine for the treatment of symptoms secondary to Alzheimer's disease. Physostigmine Study Group.
A multicenter trial to evaluate the efficacy of controlled-release physostigmine salicylate, a cholinesterase inhibitor, was conducted in 1,111 mild-to-moderate Alzheimer's disease (AD) subjects. During dose titration, subjects received 18, 24, or 30 mg of physostigmine or placebo daily. After a 2-week washout period, 366 subjects with putative improvement were randomized to receive either placebo or their best dose of physostigmine in a 6-week double-blind trial. Nonresponding patients (439) were randomized to receive in a separate double-blind trial either placebo or their highest tolerated dose of physostigmine. The primary efficacy measures included the cognitive subscale of the Alzheimer Disease Assessment Scale (ADAS) and a Clinical Global Impression of Change (CGIC). Secondary measures included the Mini-Mental State Examination and two activities-of-daily-living scales. At the end of the 6-week double-blind phase, physostigmine-treated patients scored 1.75 points higher than placebo-treated patients on the ADAS (p = 0.003) and 0.26 points higher on the CGIC (p = 0.012) in the intent-to-treat analysis. There was no significant improvement on the secondary outcome measures. Patients failing to respond to physostigmine during the dose titration phase failed to respond on any of the outcome measures during the double-blind period of re-exposure. Common adverse events included nausea, vomiting, diarrhea, and anorexia. There were no significant changes in liver function tests. This study demonstrated statistically significant differences between physostigmine and placebo on both a performance-based cognitive functioning instrument and a clinician's global evaluation. The magnitude of the effect size was small and occurred only in the subset of patients who responded in the initial dose titration study period. Nevertheless, the results suggest that in a subset of patients, physostigmine can induce a degree of cognitive improvement over 6 weeks of treatment.


Fast-forward breeding for a food-secure world.
Crop production systems need to expand their outputs sustainably to feed a burgeoning human population. Advances in genome sequencing technologies combined with efficient trait mapping procedures accelerate the availability of beneficial alleles for breeding and research. Enhanced interoperability between different omics and phenotyping platforms, leveraged by evolving machine learning tools, will help provide mechanistic explanations for complex plant traits. Targeted and rapid assembly of beneficial alleles using optimized breeding strategies and precise genome editing techniques could deliver ideal crops for the future. Realizing desired productivity gains in the field is imperative for securing an adequate future food supply for 10 billion people.


Compartmentalization and spatiotemporal organization of macromolecules in bacteria.
For many years, the bacterial cells were regarded as tiny vessels lacking internal organization. This view, which stemmed from the scarcity of membrane-bounded organelles, has changed considerably in recent years, mainly due to advancements in imaging capabilities. Consequently, despite the rareness of conventional organelles, bacteria are now known to have an intricate internal organization, which is vital for many cellular processes. The list of bacterial macromolecules reported to have distinct localization patterns is rapidly growing. Moreover, time-lapse imaging revealed the spatiotemporal dynamics of various bacterial macromolecules. Although the regulatory mechanisms that underlie macromolecules localization in bacterial cells are largely unknown, certain strategies elucidated thus far include the establishment of cell polarity, the employment of cytoskeletal proteins, and the use of the membrane properties, that is, curvature, electric potential, and composition, as localization signals. The most surprising mechanism discovered thus far is targeting of certain mRNAs to the subcellular domains where their protein products are required. This mechanism relies on localization features in the mRNA itself and does not depend on translation. Localization of other mRNAs near their genetic loci suggests that the bacterial chromosome is involved in organizing gene expression. Taken together, the deep-rooted separation between cells with nucleus and without is currently changing, highlighting bacteria as suitable models for studying universal mechanisms underlying cell architecture.


Nitrate in public water supplies and the risk of colon and rectum cancers.
Nitrate is a widespread contaminant of drinking water, but its potential health effects are unclear. In the body, nitrate is reduced to nitrite, which can react with amines and amides by nitrosation to form N-nitroso compounds, known animal carcinogens. N-nitroso compound formation is inhibited by certain nutrients, such as vitamin C, and increased by meat intake. We investigated the association of nitrate in public water supplies with incident colon and rectum cancers in a case-control study conducted in Iowa from 1986 to 1989. Nitrate levels in Iowa towns were linked to the participants' water source histories. We focused our analyses on the period from 1960 onward, during which nitrate measurements were more frequent, and we restricted analyses to those persons with public water supplies that had nitrate data (actual or imputed) for greater than 70% of this time period (376 colon cancer cases, 338 rectum cancer cases, and 1244 controls). There were negligible overall associations of colon or rectum cancers with measures of nitrate in public water supplies, including average nitrate and the number of years with elevated average nitrate levels. For more than 10 years with average nitrate greater than 5 mg/L, the odds ratio (OR) for colon cancer was 1.2 (95% confidence interval [CI] = 0.9-1.6) and for rectum the OR was 1.1 (CI = 0.7-1.5). However, nitrate exposure (>10 years with average nitrate >5 mg/L) was associated with increased colon cancer risk among subgroups with low vitamin C intake (OR = 2.0; CI = 1.2-3.3) and high meat intake (OR = 2.2; CI = 1.4-3.6). These patterns were not observed for rectum cancer. Our analyses suggest that any increased risk of colon cancer associated with nitrate in public water supplies might occur only among susceptible subpopulations.


Glucocorticoids plus N-acetylcysteine in severe alcoholic hepatitis.
Mortality among patients with severe acute alcoholic hepatitis is high, even among those treated with glucocorticoids. We investigated whether combination therapy with glucocorticoids plus N-acetylcysteine would improve survival. We randomly assigned 174 patients to receive prednisolone plus N-acetylcysteine (85 patients) or only prednisolone (89 patients). All patients received 4 weeks of prednisolone. The prednisolone-N-acetylcysteine group received intravenous N-acetylcysteine on day 1 (at a dose of 150, 50, and 100 mg per kilogram of body weight in 250, 500, and 1000 ml of 5% glucose solution over a period of 30 minutes, 4 hours, and 16 hours, respectively) and on days 2 through 5 (100 mg per kilogram per day in 1000 ml of 5% glucose solution). The prednisolone-only group received an infusion in 1000 ml of 5% glucose solution per day on days 1 through 5. The primary outcome was 6-month survival. Secondary outcomes included survival at 1 and 3 months, hepatitis complications, adverse events related to N-acetylcysteine use, and changes in bilirubin levels on days 7 and 14. Mortality was not significantly lower in the prednisolone-N-acetylcysteine group than in the prednisolone-only group at 6 months (27% vs. 38%, P = 0.07). Mortality was significantly lower at 1 month (8% vs. 24%, P = 0.006) but not at 3 months (22% vs. 34%, P = 0.06). Death due to the hepatorenal syndrome was less frequent in the prednisolone-N-acetylcysteine group than in the prednisolone-only group at 6 months (9% vs. 22%, P = 0.02). In a multivariate analysis, factors associated with 6-month survival were a younger age (P<0.001), a shorter prothrombin time (P<0.001), a lower level of bilirubin at baseline (P<0.001), and a decrease in bilirubin on day 14 (P<0.001). Infections were less frequent in the prednisolone-N-acetylcysteine group than in the prednisolone-only group (P = 0.001); other side effects were similar in the two groups. Although combination therapy with prednisolone plus N-acetylcysteine increased 1-month survival among patients with severe acute alcoholic hepatitis, 6-month survival, the primary outcome, was not improved. (Funded by Programme Hospitalier de Recherche Clinique; AAH-NAC ClinicalTrials.gov number, NCT00863785 .).


Linking the genomic signatures of human beat synchronization and learned song in birds.
The development of rhythmicity is foundational to communicative and social behaviours in humans and many other species, and mechanisms of synchrony could be conserved across species. The goal of the current paper is to explore evolutionary hypotheses linking vocal learning and beat synchronization through genomic approaches, testing the prediction that genetic underpinnings of birdsong also contribute to the aetiology of human interactions with musical beat structure. We combined state-of-the-art-genomic datasets that account for underlying polygenicity of these traits: birdsong genome-wide transcriptomics linked to singing in zebra finches, and a human genome-wide association study of beat synchronization. Results of competitive gene set analysis revealed that the genetic architecture of human beat synchronization is significantly enriched for birdsong genes expressed in songbird Area X (a key nucleus for vocal learning, and homologous to human basal ganglia). These findings complement ethological and neural evidence of the relationship between vocal learning and beat synchronization, supporting a framework of some degree of common genomic substrates underlying rhythm-related behaviours in two clades, humans and songbirds (the largest evolutionary radiation of vocal learners). Future cross-species approaches investigating the genetic underpinnings of beat synchronization in a broad evolutionary context are discussed. This article is part of the theme issue 'Synchrony and rhythm interaction: from the brain to behavioural ecology'.


Reducing the overuse of βhCG measurements in the emergency gynaecology clinic.
Serial βhCG testing can be a helpful tool in deciding how to manage pregnancy of unknown location. Its use in emergency gynaecology clinics can prevent unnecessary admission and intervention. However, despite NICE Guidelines on when it is safe to opt for conservative management, it was identified that there was a problem with over-testing of βhCG when patients could be discharged with instructions to repeat a urinary pregnancy test in two weeks. Two PDSA cycles were undertaken to improve the awareness of NICE guidelines: the first involved formal and informal educational sessions and the second involved the inclusion of a guideline summary on the front of patients' notes when they were having serial βhCG tests for doctors to refer to. Case notes were reviewed for 157 women who had βhCG tests at baseline and 48 hours. Of these, 139 were suitable for serial βhCG testing, and 83 of these were suitable for discharge after 48 hours. Of the 83 patients that were eligible for discharge, there were 31 unnecessary βhCG tests done, 23 of which were prior to intervention. A significant improvement was noted, with between 4-10 unnecessary βhCG tests per fortnight prior to intervention, 0-3 following the first intervention, and 0-2 following the second. Reduction in unnecessary βhCG testing has positive implications for patients, who do not have to take unnecessary time off work, prolong an already very distressing period, and have unnecessary blood tests. There are also cost and time saving implications for the hospital.


Complex regional pain syndrome: are the IASP diagnostic criteria valid and sufficiently comprehensive?
This is a multisite study examining the internal validity and comprehensiveness of the International Association for the Study of Pain (IASP) diagnostic criteria for Complex Regional Pain Syndrome (CRPS). A standardized sign/symptom checklist was used in patient evaluations to obtain data on CRPS-related signs and symptoms in a series of 123 patients meeting IASP criteria for CRPS. Principal components factor analysis (PCA) was used to detect statistical groupings of signs/symptoms (factors). CRPS signs and symptoms grouped together statistically in a manner somewhat different than in current IASP/CRPS criteria. As in current criteria, a separate pain/sensation criterion was supported. However, unlike in current criteria, PCA indicated that vasomotor symptoms form a factor distinct from a sudomotor/edema factor. Changes in range of motion, motor dysfunction, and trophic changes, which are not included in the IASP criteria, formed a distinct fourth factor. Scores on the pain/sensation factor correlated positively with pain duration (P<0. 001), but there was a negative correlation between the sudomotor/edema factor scores and pain duration (P<0.05). The motor/trophic factor predicted positive responses to sympathetic block (P<0.05). These results suggest that the internal validity of the IASP/CRPS criteria could be improved by separating vasomotor signs/symptoms (e.g. temperature and skin color asymmetry) from those reflecting sudomotor dysfunction (e.g. sweating changes) and edema. Results also indicate motor and trophic changes may be an important and distinct component of CRPS which is not currently incorporated in the IASP criteria. An experimental revision of CRPS diagnostic criteria for research purposes is proposed. Implications for diagnostic sensitivity and specificity are discussed.


Differential stabilization of cytochrome P-450 isoenzymes in primary cultures of adult rat liver parenchymal cells.
Cytochrome P-450 dependent hydroxylation of testosterone was measured in 7-day-old cultures of primary rat liver parenchymal cells. Determinations were carried out in monocultures of parenchymal cells and co-cultures of parenchymal cells with rat liver nonparenchymal epithelial cells, or mouse embryo fibroblasts. In the monoculture system, testosterone metabolism was drastically reduced and hardly measurable after 7 days in culture. In the co-culture systems, individual P-450 isoenzymes were stabilized on different levels. P-450s p and presumably c were well preserved, P-450 a was reduced but clearly measurable, P-450 h was totally lost whereas P-450s b and e were not measurable after 7 days (the activities of these isoenzymes however were already low in freshly isolated parenchymal cells). The results were independent of the cell line used for co-cultivation and of the method of parenchymal cell isolation, that is whether collagenase or EDTA was used as the agent for dissociating the cells from the liver. The results showed that the co-cultivation of liver parenchymal cells with other nonparenchymal cells significantly improved the differentiated status of the former. In this cell culture system however, not every parameter was equally well stabilized.


Implementing stakeholder-informed research in the substance abuse treatment sector: strategies used by Connections, a Canadian knowledge translation and exchange project.
Researcher-stakeholder collaboration has been identified as critical to bridging research and health system change. While collaboration models vary, meaningful stakeholder involvement over time ("integrated knowledge translation") is advocated to improve the relevance of research to knowledge users. This short report describes the integrated knowledge translation efforts of Connections, a knowledge translation and exchange project to improve services for women with substance abuse problems and their children, and implementation barriers and facilitators. Strategies of varying intensities were used to engage diverse stakeholders, including policy makers and people with lived experience, and executive directors, program managers, and service providers from Canadian addiction agencies serving women. Barriers to participation included individual (e.g., interest), organizational (e.g., funding), and system level (e.g., lack of centralized stakeholder database) barriers. Similarly, facilitators included individual (e.g., perceived relevance) and organizational (e.g., support) facilitators, as well as initiative characteristics (e.g., multiple involvement opportunities). Despite barriers, Connections' stakeholder-informed research efforts proved essential for developing clinically relevant and feasible processes, measures, and implementation strategies. Stakeholder-researcher collaboration is possible and robust integrated knowledge translation efforts can be productive. Future work should emphasize developing and evaluating a range of strategies to address stakeholders' knowledge translation needs and to facilitate sustained and meaningful involvement in research.


Congenital Heart Surgery Nomenclature and Database Project: atrial septal defect.
The extant nomenclature for atrial septal defect (ASD) is reviewed for the purpose of establishing a unified reporting system. The subject was debated and reviewed by members of the STS-Congenital Heart Surgery Database Committee and representatives from the European Association for Cardiothoracic Surgery. All efforts were made to include all relevant nomenclature categories using synonyms where appropriate. A comprehensive database set is presented that is based on a hierarchical scheme. Data are entered at various levels of complexity and detail that can be determined by the clinician. These data can lay the foundation for comprehensive risk stratification analyses. A minimum database set is also presented that will allow for data sharing and would lend itself to basic interpretation of trends. Outcome tables relating diagnoses, procedures, and various risk factors are presented.


Autonomic reactivity to cognitive and emotional stress of low, medium, and high hypnotizable healthy subjects: testing predictions from the high risk model of threat perception.
This study tested hypotheses derived from Wickramasekera's High Risk Model of Threat Perception (HRMTP) by comparing autonomic and affective responses to a cognitive and an emotional stress task in high, medium, and low hypnotizables. Electrodermal activity (EDA) was used as a measure of sympathetic activity, and the high frequency (HF) spectral component of heart rate variability as a measure of parasympathetic activity. High hypnotizables exhibited greater EDA at baseline and slower EDA recovery following both tasks than did medium and lows. Medium hypnotizables responded with greater decreases in normalized HF power than did highs and lows during the emotional stress task. The results suggest diminished EDA variability in high hypnotizables and the potential for HF power as an indicator of autonomic dysregulation in low and high hypnotizables, compared to mediums. The results are discussed in relation to predictions based on the HRMTP.


Laboratory animals and respiratory allergies: the prevalence of allergies among laboratory animal workers and the need for prophylaxis.
Subjects exposed to laboratory animals are at a heightened risk of developing respiratory and allergic diseases. These diseases can be prevented by simple measures such as the use of personal protective equipment. We report here the primary findings of the Laboratory Animals and Respiratory Allergies Study regarding the prevalence of allergic diseases among laboratory animal workers, the routine use of preventive measures in laboratories and animal facilities, and the need for prevention programs. Animal handlers and non-animal handlers from 2 Brazilian universities (University of São Paulo and State University of Campinas) answered specific questionnaires to assess work conditions and symptoms. These subjects also underwent spirometry, a bronchial challenge test with mannitol, and skin prick tests for 11 common allergens and 5 occupational allergens (rat, mouse, guinea pig, hamster, and rabbit). Four hundred fifty-five animal handlers (32±10 years old [mean±SD], 209 men) and 387 non-animal handlers (33±11 years old, 121 men) were evaluated. Sensitization to occupational allergens was higher among animal handlers (16%) than non-animal handlers (3%, p<0.01). Accessibility to personal protective equipment was measured at 85% (median, considering 73 workplaces of the animal handler group). Nineteen percent of the animal handlers indicated that they wear a respirator at all times while handling animals or working in the animal room, and only 25% of the animal handlers had received an orientation about animal-induced allergies, asthma, or rhinitis. In conclusion, our data indicate that preventive programs are necessary. We suggest providing individual advice to workers associated with institutional programs to promote a safer work environment.


Varenicline enhances dopamine release facilitation more than nicotine after long-term nicotine treatment and withdrawal.
An important factor contributing to the high relapse rates among smokers is nicotine withdrawal symptoms. Multiple studies suggest that decreased dopamine release in nucleus accumbens plays a key role in withdrawal. However, recent reports showed that long-term nicotine exposure itself also decreases accumbal dopamine release, suggesting that additional mechanisms are involved in withdrawal. Here, we used real-time cyclic voltammetry in brain slices containing the nucleus accumbens to further elucidate the changes in dopamine release linked to nicotine withdrawal. Rats received vehicle or nicotine via the drinking water for 2-3 months. Studies assessing the expression of somatic signs in vehicle-treated, nicotine-treated, and 24-h nicotine withdrawn rats showed that nicotine withdrawal led to a significant increase in somatic signs. Subsequent voltammetry studies showed that long-term nicotine decreased single-pulse-stimulated dopamine release via an interaction at α6β2* receptors. Nicotine withdrawal led to a partial recovery in α6β2* receptor-mediated release. In addition, long-term nicotine treatment alone increased dopamine release paired-pulse ratios and this was partially reversed with nicotine removal. We then evaluated the effect of bath-applied nicotine and varenicline on dopamine release. Nicotine and varenicline both decreased single-pulse-stimulated release in vehicle-treated, nicotine-treated, and nicotine withdrawn rats. However, bath-applied varenicline increased paired-pulse ratios to a greater extent than nicotine during long-term nicotine treatment and after its withdrawal. Altogether these data suggest that nicotine withdrawal is associated with a partial restoration of dopamine release measures to control levels and that varenicline's differential modulation of dopamine release may contribute to its mechanism of action.


Triiodothyronine down-regulates thyrotropin-releasing hormone (TRH) synthesis and decreases pTRH-(160-169) and insulin releases from fetal rat islets in culture.
TRH is localized with insulin in beta-cells. It is synthesized as a prohormone containing five copies of TRH and seven cryptic peptides, including pro (p)-TRH-(160-169). Thyroid hormone regulates the expression of several genes encoding peptide hormones. We found that circulating T3 concentrations were inversely correlated with TRH levels in two physiopathological situations. There are low concentrations of circulating thyroid hormone and very high concentrations of TRH and pTRH-(160-169) during development, and experimental hypothyroidism results in higher concentrations of prepro (pp)-TRH messenger RNA (mRNA) and TRH content in the adult rat pancreas than are present in the euthyroid pancreas. The present study was carried out to investigate the interaction between T3 and pancreatic TRH during the functional maturation of islets in culture and to validate the data obtained in vivo. T3 decreases ppTRH mRNA in islets in a dose-dependent manner. The primary impact of T3 on islet function may be mediated by ppTRH mRNA, as short term T3 treatment had no effect. Long term T3 treatment reduced the islet TRH content and the amounts of pTRH-(160-169) and insulin released. This secretory pattern and coordinated regulation of pTRH-(160-169) and insulin suggests that pTRH-(160-169) plays a specific role in the regulation of insulin secretion.


No impact of polymorphism in the phosphodiesterase 5A gene in Cavalier King Charles Spaniels on pimobendan-induced inhibition of platelet aggregation response.
A variant in the canine phosphodiesterase (PDE) 5A gene (PDE5A:E90K) is associated with decreased concentrations of circulating cyclic guanosine monophosphate (cGMP) and response to PDE5 inhibitor treatment. Pimobendan is a PDE inhibitor recommended for medical treatment of certain stages of myxomatous mitral valve disease (MMVD) in dogs. PDE5A:E90K polymorphism attenuates the inhibitory effect of pimobendan on in vitro platelet aggregation and increases basal platelet aggregation in Cavalier King Charles Spaniels (CKCS). Selected clinical variables (MMVD severity, sex, age, hematocrit, platelet count in platelet-rich plasma [PRP], and echocardiographic left ventricular fractional shortening [LV FS]) will not show an association with results. Fifty-two privately owned CKCS with no or preclinical MMVD. Using blood samples, we prospectively assessed PDE5A genotype using Sanger sequencing and adenosine diphosphate-induced platelet aggregation response (area under the curve [AUC], maximal aggregation [MaxA], and velocity [Vel]) with and without pimobendan using light transmission aggregometry. Dogs also underwent echocardiography. Pimobendan inhibited platelet function as measured by AUC, MaxA, and Vel at a concentration of 10 μM (P < .0001) and Vel at 0.03 μM (P < .001). PDE5A:E90K polymorphism did not influence the inhibitory effect of pimobendan or basal platelet aggregation response. The PDE5A:E90K polymorphism did not influence in vitro basal platelet aggregation response or the inhibitory effect of pimobendan on platelet aggregation in CKCS. Dogs with the PDE5A:E90K polymorphism did not appear to have altered platelet function or response to pimobendan treatment.


Conserved regions of the timeless (tim) clock gene in Drosophila analyzed through phylogenetic and functional studies.
Circadian (approximately 24-hr) rhythms in Drosophila melanogaster depend upon cyclic expression of the period (per) and timeless (tim) genes, which encode interacting components of the endogenous clock. The per gene has been isolated from other insects and, more recently, a per ortholog was found in mammals where its expression oscillates in a circadian fashion. We report here the complete sequence of a tim gene from another species, Drosophila virilis. TIM is better conserved than the PER protein is between these two species (76 vs. 54% overall amino acid identity), and putative functional domains, such as the PER interaction domains and the nuclear localization signal, are highly conserved. The acidic domain and the cytoplasmic localization domain, however, are within the least conserved regions. In addition, the initiating methionine in the D. virilis gene lies downstream of the proposed translation start for the original D. melanogaster tim cDNA and corresponds to the one used by D. simulans and D. yakuba. Among the most conserved parts of TIM is a region of unknown function near the N terminus. We show here that deletion of a 32 amino acid segment within this region affects rescue of rhythms in arrhythmic tim01 flies. Flies carrying a full-length tim transgene displayed rhythms with approximately 24-hr periods, indicating that a fully functional clock can be restored in tim01 flies through expression of a tim transgene. Deletion of the segment mentioned above resulted in very long activity rhythms with periods ranging from 30.5 to 48 hr.


Sympathetic sprouting in the dorsal root ganglia of the injured peripheral nerve in a rat neuropathic pain model.
The extent of the sprouting of sympathetic postganglionic fibers in the dorsal root ganglion (DRG) and the peripheral nerves was examined in neuropathic rats at different postoperative times. After the L5 and L6 spinal nerves were ligated on one side, three different pain behavior tests (representing mechanical allodynia, cold allodynia, ongoing pain exacerbated by cold stress) were performed at various time intervals. The sympathetic postganglionic fibers were visualized by immunostaining with antibodies to tyrosine hydroxylase (TH). In the neuropathic rats, all three pain behaviors were fully developed within 3 days after the surgery, maintained up to 2 weeks, and then started to decline gradually afterward. At 20 weeks after neuropathic surgery, pain behaviors were reduced significantly compared to the peak response, but were still higher than the presurgery levels. Sympathectomy, performed 4 days after neuropathic surgery, almost completely abolished the signs of mechanical allodynia and ongoing pain behaviors, and it reduced the behaviors of cold allodynia to approximately half. The numerical density of sympathetic fibers in the DRG of an injured segment was significantly higher at 1, 4, and 20 weeks after neuropathic surgery as compared to the normal, suggesting that there is sprouting of sympathetic fibers in the DRG after peripheral nerve injury. Sprouting of sympathetic fibers in the DRG was extensive as early as 2 days after the spinal nerve ligation, and the sprouted fibers were almost completely eliminated after sympathectomy. The data suggest that sympathetic innervation of the DRG may play an important role in the development and maintenance of sympathetically maintained neuropathic pain.


Differential analgetic actions of amphetamine enantiomers in the mouse: a drug-drug interaction study.
A series of agents were tested for their ability to interact with the analgetic actions of either d-amphetamine (d-AMP) or l-amphetamine (l-AMP), or morphine in rats using the hot plate procedure. The analgetic action of l-AMP was potentiated by morphine and slightly antagonized by naloxone; it was antagonized by clonidine, alpha-MT, yohimbine, fluoxetine and metergoline, but enhanced by PCPA. The analgetic action of morphine was antagonized by both naloxone and PCPA. The analgetic actions of l-AMP and morphine appear to involve different biogenic amine systems.


Intermittent hypoxia exposure in a hypobaric chamber and erythropoietin abuse interpretation.
The aim of this study was to assess the effect of intermittent hypoxia exposure on direct and indirect methods used to evaluate recombinant human erythropoietin (rhEPO) misuse. Sixteen male triathletes were randomly assigned to either the intermittent hypoxia exposure group (experimental group) or the control normoxic group (control group). The members of the experimental group were exposed to simulated altitude (from 4000 to 5500 m) in a hypobaric chamber for 3 h per day, 5 days a week, for 4 weeks. Blood and urine samples were collected before and after the first and the final exposures, and again 2 weeks after the final exposure. While serum EPO significantly increased after the first [from a mean 8.3 IU x l(-1) (s = 3.2) to 16.6 IU x l(-1) (s = 4.7)] and final exposures [from 4.6 IU x l(-1) (s = 1.4) to 24.8 IU x l(-1) (s = 9.3)], haemoglobin, percentage of reticulocytes, and soluble transferrin receptor were not elevated. Second-generation ON/OFF models (indirect rhEPO misuse detection) were insensitive to intermittent hypoxia exposure. The distribution of the urinary EPO isoelectric profiles (direct rhEPO misuse detection) was altered after intermittent hypoxia exposure with a slight shift towards more basic isoforms. However, those shifts never resulted in misinterpretation of results. The intermittent hypoxia exposure protocol studied did not produce any false-positive result for indirect or direct detection of rhEPO misuse in spite of the changes in EPO serum concentrations and urinary EPO isoelectric profiles, respectively.


[Formation and action of interferon in experimental respiratory syncytial infection].
Respiratory syncytial virus multiplying activity in the lungs of newborn and 6-day-old cotton rats, induced production of considerable amounts of interferon. Interferon production correlated with virus multiplication. Lower interferon titers were observed in the lungs of newborn cotton rats than in the lungs of 6-day-old animals. RS virus also induced production of serum interferon upon intraperitoneal inoculation, its titers being 2 times as low as after inoculation of equal doses of Newcastle disease virus. Poly I: poly C was shown to be a quite active interferon inducer in cotton rats and to inhibit RS virus reproduction in their lungs.