Carcinoma in situ of the urinary bladder: review of clinicopathologic characteristics with an emphasis on aspects related to molecular diagnostic techniques and prognosis.
Carcinoma in situ (CIS) of the urinary bladder is defined as a flat lesion comprising of cytologically malignant cells which may involve either full or partial thickness of the urothelium. De novo CIS constitutes less than 3% of all urothelial neoplasms; however, CIS detected concurrently or secondarily during follow-up of urothelial carcinoma constitutes 45% and 90%, respectively, of bladder cancer. CIS is noted predominantly in male smokers in the sixth or seventh decade. Patients may present with dysuria, nocturia, and urinary frequency and urgency with microscopic hematuria. Cystoscopic findings may range from unremarkable to erythema or edema. Urine cytology is an important diagnostic tool. Cellular anaplasia, loss of polarity, discohesion, nuclear enlargement, hyperchromasia, pleomorphism, and atypical mitoses are the histopathologic hallmarks of CIS. Extensive denudation of the urothelium, monomorphic appearance of the neoplastic cells, inflammatory atypia, radiation induced nuclear smudging, multinucleation, and pagetoid spread of CIS may cause diagnostic difficulties. Together with clinical and morphologic correlation, immunostaining with CK 20, p53 (full thickness), and CD44 (absence of staining) may help accurately diagnose CIS. Fluorescent in situ hybridization analysis of voided urine for amplification of chromosomes 3, 7, and 17 and deletion of 9p has high sensitivity and specificity for diagnosing CIS in surveillance cases. Several other molecular markers, such as NMP 22 and BTA, are under evaluation or used variably in clinical pathology. Intravesical bacillus Calmette-Guerin (BCG) instillation is considered the preferred treatment, with radical cystectomy being offered to refractory cases. Chemotherapy, alpha-interferon, and photodynamic therapy are other modalities that can be considered in BCG-refractory cases. Multifocality, involvement of prostatic urethra, and response to BCG remain the most important prognostic factors, although newer molecular markers are being evaluated for this entity. Patient outcome varies based on whether it is de novo development or diagnosed secondary to prior or concomitant papillary bladder cancer. From a clinical perspective, the principal determinants of outcome are extent of disease, involvement of prostatic urethra, response to therapy, and time to recurrence.


Mössbauer characterization of an unusual high-spin side-on peroxo-Fe3+ species in the active site of superoxide reductase from Desulfoarculus Baarsii. Density functional calculations on related models.
Superoxide reductase (SOR) is an Fe protein that catalyzes the reduction of superoxide to give H(2)O(2). Recently, the mutation of the Glu47 residue into alanine (E47A) in the active site of SOR from Desulfoarculus baarsii has allowed the stabilization of an iron-peroxo species when quickly reacted with H(2)O(2) [Mathé et al. (2002) J. Am. Chem. Soc. 124, 4966-4967]. To further investigate this non-heme peroxo-iron species, we have carried out a Mössbauer study of the (57)Fe-enriched E47A SOR from D. baarsii reacted quickly with H(2)O(2). Considering the Mössbauer data, we conclude, in conjunction with the other spectroscopic data available and with the results of density functional calculations on related models, that this species corresponds to a high-spin side-on peroxo-Fe(3+) complex. This is one of the first examples of such a species in a biological system for which Mössbauer parameters are now available: delta(/Fe) = 0.54 (1) mm/s, DeltaE(Q) = -0.80 (5) mm/s, and the asymmetry parameter eta = 0.60 (5) mm/s. The Mössbauer and spin Hamiltonian parameters have been evaluated on a model from the side-on peroxo complex (model 2) issued from the oxidized iron center in SOR from Pyrococcus furiosus, for which structural data are available in the literature [Yeh et al. (2000) Biochemistry 39, 2499-2508]. For comparison, similar calculations have been carried out on a model derived from 2 (model 3), where the [CH(3)-S](1)(-) group has been replaced by the neutral [NH(3)](0) group [Neese and Solomon (1998) J. Am. Chem. Soc. 120, 12829-12848]. Both models 2 and 3 contain a formally high-spin Fe(3+) ion (i.e., with empty minority spin orbitals). We found, however, a significant fraction ( approximately 0.6 for 2, approximately 0.8 for 3) of spin (equivalently charge) spread over two occupied (minority spin) orbitals. The quadrupole splitting value for 2 is found to be negative and matches quite well the experimental value. The computed quadrupole tensors are rhombic in the case of 2 and axial in the case of 3. This difference originates directly from the presence of the thiolate ligand in 2. A correlation between experimental isomer shifts for Fe(3+) mononuclear complexes with computed electron densities at the iron nucleus has been built and used to evaluate the isomer shift values for 2 and 3 (0.56 and 0.63 mm/s, respectively). A significant increase of isomer shift value is found upon going from a methylthiolate to a nitrogen ligand for the Fe(3+) ion, consistent with covalency effects due to the presence of the axial thiolate ligand. Considering that the isomer shift value for 3 is likely to be in the 0.61-0.65 mm/s range [Horner et al. (2002) Eur. J. Inorg. Chem., 3278-3283], the isomer shift value for a high-spin eta(2)-O(2) Fe(3+) complex with an axial thiolate group can be estimated to be in the 0.54-0.58 mm/s range. The occurrence of a side-on peroxo intermediate in SOR is discussed in relation to the recent data published for a side-on peroxo-Fe(3+) species in another biological system [Karlsson et al. (2003) Science 299, 1039-1042].


Clinical safety-in-use study of a new tampon design.
To confirm the safety of a new experimental Tampax tampon and applicator compared with that of a currently marketed Tampax tampon and applicator using comprehensive gynecological and microbiological assessments. A 2-month, single-blind, randomized, crossover study was conducted in which each subject served as her own control. Safety was evaluated by comparing potential product-related irritation (using colposcopic examination and subject diary data), assessment of vaginal discharge, vaginal pH, and effects on selected microorganisms (yeast, Escherichia coli, Staphylococcus aureus and group B streptococci) obtained by vaginal swab cultures after normal menstrual use in the experimental and control groups. In total, 110 women completed the study. There were no significant differences between the groups that used either the experimental or control tampon with regard to prevalence or mean cell density for the selected microorganisms. No differences were observed in the incidence or severity of erythema, in abrasion or ulceration of the cervix, vagina, introitus, vulva or perineum, or in mean vaginal pH and discharge assessments. There were equivalent low incidences of reported symptoms such as discomfort during insertion, wear or removal, and a similar low incidence of burning, stinging or itching during use of either the control or experimental tampon. There was a more favorable overall product rating for the experimental tampon (p = 0.003). This approach provides a combination of gynecological, microbiological and self-reported (diary recall) methodologies in order to assess tampon safety during use more thoroughly than has previously been reported, and it supports a comparable safety profile for the experimental tampon and a currently marketed tampon.


Predictive Factors of In-Hospital Mortality in Older Adults with Community-Acquired Bloodstream Infection.
To assess the prevalence of intra-hospital mortality and associated risk factors in older people aged 75+, admitted with blood stream infections (BSI). Single center retrospective study performed in an 850-bed of the academic hospital of the Université Libre de Bruxelles. From January 2015 to December 2017, all inpatients over 75 years old admitted with BSI were included. Demographical, clinical and microbiological data were collected. 212 patients were included: median age was 82 [79-85] years and 60 % were female. The in-hospital mortality rate was 19%. The majority of microorganisms were Gram-negative strains, of which Escherichia coli was the most common, and urinary tract infection was the most common origin of BSI. Compared to patients who survived, the non-survivor group had a higher SOFA score (6 versus 3, p<0.0001), a higher comorbidity score (5 versus 4, p<0.0001), more respiratory tract infections (28 vs 6 %, p < 0.0001) and fungal infections (5 vs 1 %, p = 0.033), bedridden status (60 vs 25 %, p < 0.0001), and healthcare related infections (60 vs 40 %, p = 0.019). Using Cox multivariable regression analysis, only SOFA score was independently associated with mortality (HR 1.75 [95%IC 1.52-2.03], p<0.0001). BSI in older people are severe infections associated with a significant in-hospital mortality. Severity of clinical presentation at onset remains the most important predictor of mortality for BSI in older people. BSI originating from respiratory source and bedridden patients are at greater risk of intra-hospital mortality. Further prospective studies are needed to confirm these results.


Immunization with mutant p53- and K-ras-derived peptides in cancer patients: immune response and clinical outcome.
To determine the ability to induce tumor-specific immunity with individual mutant K-ras-or p53-derived peptides and to monitor clinical outcome. Patients in varying stages of disease underwent genetic analysis for mutations in K-ras and p53. Thirty-nine patients were enrolled. Seventeen-mer peptides were custom synthesized to the corresponding mutation. Baseline immunity was assessed for cytotoxic T-lymphocyte (CTL) response and interferon gamma (IFN-gamma) release from mutant peptide-primed lymphocytes. Patients' peripheral-blood mononuclear cells were pulsed with the corresponding peptide, irradiated, and applied intravenously. Patients were observed for CTL, IFN-gamma, interleukin (IL) -2, IL-5, and granulocyte-macrophage colony-stimulating factor responses, for treatment-related toxicity, and for tumor response. No toxicity was observed. Ten (26%) of 38 patients had detectable CTL against mutant p53 or K-ras, and two patients were positive for CTL at baseline. Positive IFN-gamma responses occurred in 16 patients (42%) after vaccination, whereas four patients had positive IFN-gamma reaction before vaccination. Of 29 patients with evident disease, five experienced a period of stable disease. Favorable prognostic markers were detectable CTL activity and a positive IFN-gamma reaction but not IL-5 release. Median survival times of 393 v 98 days for a positive versus negative CTL response (P = .04), respectively, and of 470 v 88 days for a positive versus negative IFN-gamma response (P = .02), respectively, were detected. Custom-made peptide vaccination is feasible without any toxicity. CTL and cytokine responses specific to a given mutation can be induced or enhanced with peptide vaccines. Cellular immunity to mutant p53 and K-ras oncopeptides is associated with longer survival.


Dietary resistant starch upregulates total GLP-1 and PYY in a sustained day-long manner through fermentation in rodents.
Glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are anti-diabetes/obesity hormones secreted from the gut after meal ingestion. We have shown that dietary-resistant starch (RS) increased GLP-1 and PYY secretion, but the mechanism remains unknown. RS is a fermentable fiber that lowers the glycemic index of the diet and liberates short-chain fatty acids (SCFAs) through fermentation in the gut. This study investigates the two possible mechanisms by which RS stimulates GLP-1 and PYY secretion: the effect of a meal or glycemic index, and the effect of fermentation. Because GLP-1 and PYY secretions are stimulated by nutrient availability in the gut, the timing of blood sample collections could influence the outcome when two diets with different glycemic indexes are compared. Thus we examined GLP-1 and PYY plasma levels at various time points over a 24-h period in RS-fed rats. In addition, we tested proglucagon (a precursor to GLP-1) and PYY gene expression patterns in specific areas of the gut of RS-fed rats and in an enteroendocrine cell line following exposure to SCFAs in vitro. Our findings are as follows. 1) RS stimulates GLP-1 and PYY secretion in a substantial day-long manner, independent of meal effect or changes in dietary glycemia. 2) Fermentation and the liberation of SCFAs in the lower gut are associated with increased proglucagon and PYY gene expression. 3) Glucose tolerance, an indicator of increased active forms of GLP-1 and PYY, was improved in RS-fed diabetic mice. We conclude that fermentation of RS is most likely the primary mechanism for increased endogenous secretions of total GLP-1 and PYY in rodents. Thus any factor that affects fermentation should be considered when dietary fermentable fiber is used to stimulate GLP-1 and PYY secretion.


Adrenergic blood pressure responses in the shark.
Both nurse and lemon sharks recovered slowly from the pressor effect of epinephrine and from the depressor effect of isoproterenol. The recovery time increased with the dose of epinephrine. Grading of dosage and the use of dibenzyline revealed that these species exhibit alpha and beta adrenergic vascular responses in a manner qualitatively similar but quantitatively dissimilar to that for mammals.


Structure and dynamics of γ-secretase with presenilin 2 compared to presenilin 1.
Severe early-onset familial Alzheimer's disease (FAD) is caused by more than 200 different mutations in the genes coding for presenilin, the catalytic subunit of the 4-subunit protease complex γ-secretase, which cleaves the C99 fragment of the amyloid precursor protein (APP) to produce Aβ peptides. γ-Secretase exists with either of two homologues, PS1 and PS2. All cryo-electron microscopic structures and computational work has so far focused on γ-secretase with PS1, yet PS2 mutations also cause FAD. A central question is thus whether there are structural and dynamic differences between PS1 and PS2. To address this question, we use the cryo-electron microscopic data for PS1 to develop the first structural and dynamic model of PS2-γ-secretase in the catalytically relevant mature membrane-bound state at ambient temperature, equilibrated by three independent 500 ns molecular dynamics simulations. We find that the characteristic nicastrin extra-cellular domain breathing mode and major movements in the cytosolic loop between TM6 and TM7 occur in both PS2- and PS1-γ-secretase. The overall structures and conformational states are similar, suggesting similar catalytic activities. However, at the sequence level, charge-controlled membrane-anchoring is extracellular for PS1 and intracellular for PS2, which suggests different subcellular locations. The tilt angles of the TM2, TM6, TM7 and TM9 helices differ in the two forms of γ-secretase, suggesting that the two proteins have somewhat different substrate processing and channel sizes. Our MD simulations consistently indicated that PS2 retains several water molecules near the catalytic site at the bilayer, as required for catalysis. The possible reasons for the differences of PS1 and PS2 are discussed in relation to their location and function.


Somatization in Family Practice: Comparing 5 Methods of Classification.
BACKGROUND: It has been suggested that patients with somatoform disorders are high utilizers of medical care, yet interpretation of studies has been difficult because of variant methods of diagnosis. The goal of this study was to compare 5 different methods of classification on the same group of subjects and to examine prevalence rates of somatoform disorders and medical utilization. METHOD: Subjects completed a demographic questionnaire in the physician's office and the somatization section of the Diagnostic Interview Schedule (DIS) by telephone. Subsequently, their medical charts were examined. Using the 5 methods of diagnosis, somatizers were compared with nonsomatizers for level of utilization of medical services. The setting was a medical school-based family practice residency training center. Participants were 119 patients waiting to see their family doctors. The main outcome measures were prevalence of somatization symptoms and chart information (utilization, number of health problems). RESULTS: One subject met the Diagnostic and Statistical Manual (DSM-IV)-based DIS criteria for somatization disorder. With the Bucholz modification for scoring the DIS, 10 subjects were diagnosed with somatization disorder. With the abridged Escobar criteria of 6 symptoms, 7 subjects met diagnostic criteria, while 28 met the criteria for multisomatoform disorder of 3 symptoms, as suggested by Kroenke. The diagnosis of undifferentiated somatoform disorder, requiring only 1 unexplained symptom, was obtained by 94 of the subjects. CONCLUSION: Findings from this study revealed widely divergent prevalence rates of somatoform disorders, depending on methods of diagnosis used. Correlation with rates of medical utilization is suggested as an external criterion for validating diagnostic methods.


Prefrontal gray matter volume predicts metacognitive accuracy following traumatic brain injury.
To examine metacognitive ability (MC) following moderate to severe traumatic brain injury (TBI) using an empirical assessment approach and to determine the relationship between alterations in gray matter volume (GMV) and MC. A sample of 62 individuals (TBI n = 34; healthy control [HC] n = 28) were included in the study. Neuroimaging and neuropsychological data were collected for all participants during the same visit. MC was quantified using an approach borrowed from signal detection theory (Type II area under the receiver operating characteristic curve calculation) to evaluate judgments during a modified version of the 3rd edition of the Wechsler Adult Intelligence Scale's Matrix Reasoning subtest where half of the items were presented randomly and half were presented in the order of increasing difficulty. Retrospective confidence judgments were collected on an item-by-item basis. Brain volumetric analyses were conducted using FreeSurfer software. Analyses of the modified Matrix Reasoning task data demonstrated that HCs significantly outperformed TBIs (ordered: d = .63; random: d = .58). There was a significant difference between groups for MC for the randomly presented stimuli (d = .54) but not the ordered stimuli. There was an association between GMV and MC in the TBI group between the right orbital region and MC (R2 = .11). In the HC group, there were associations between the left posterior (R2 = .17), left orbital (R2 = .29), and left dorsolateral (R2 = .21) regions and MC. These results are consistent with those of previous research on MC in the cognitive neurosciences, but this study demonstrates that injury may moderate the regional contributions to MC. (PsycINFO Database Record


Community-based psychosocial interventions for people with schizophrenia in low and middle-income countries: systematic review and meta-analysis.
There is consensus that the treatment of schizophrenia should combine anti-psychotic medication and psychosocial interventions in order to address complex social, economic and health needs. It is recommended that family therapy or support; community-based rehabilitation; and/or self-help and support groups should be provided for people with schizophrenia in low and middle-income countries. The effectiveness of community-based psychosocial interventions in these settings is unclear. Studies evaluating community-based psychosocial interventions for people with schizophrenia were identified through database searching up to April 2016. Randomised controlled trials were included if they compared the intervention group with a control group receiving treatment as usual including medication. Only studies set in low and middle-income countries were included. Random effects meta-analyses were performed separately for each intervention type. Eleven randomised controlled trials in five middle-income countries were identified, with a total of 1580 participants. The content of included interventions varied from single-faceted psychoeducational interventions, to multi-component rehabilitation-focused interventions, to case management interventions. A third of the included studies did not incorporate any community involvement in the intervention. The quality of evidence was often low. Amongst the seven studies that reported on symptom severity up to 18 months post intervention, the pooled standardised mean difference (SMD) across all intervention types was 0.95 (95% CI 0.28, 1.61; P 0.005; I 2 = 95%; n = 862), representing a strong effect. A strong effect on symptom severity remained after excluding two studies with a high risk of bias (SMD 0.80; 95% CI 0.07, 1.53; P 0.03; I 2 = 94%; n = 676). Community-based psychosocial interventions may also have beneficial impacts on functioning (SMD 1.12; 95% CI 0.25, 2.00; P 0.01; I 2 = 94%; n = 511) and reducing hospital readmissions (SMD 0.68; 95% CI 0.27, 1.09; P 0.001; I2 = 33%; n = 167). The limited evidence from low and middle-income countries supports the feasibility and effectiveness of community-based psychosocial interventions for schizophrenia, even in the absence of community mobilisation. Community-based psychosocial interventions should therefore be provided in these settings as an adjuvant service in addition to facility-based care for people with schizophrenia.


Capuchin monkey research priorities and urgent issues.
The "Capuchin research community roundtable: working together towards a comparative biology of Cebus and Sapajus" was held at the International Primatological Society Congress in Cancún, Mexico, August 2012. Goals of the roundtable were to strengthen interactions among the capuchin research community, and to prioritize and coordinate research and training in a more systematic and interactive way in light of increasing conservation urgency. New phylogenetic and biogeographic evidence highlights the distinct evolutionary histories of the two radiations of capuchin monkeys, Cebus (untufted or gracile capuchins) and Sapajus (tufted or robust capuchins), that were formerly lumped under Cebus, and points to a higher number of species, or Evolutionarily Significant Units, in each compared to past capuchin taxonomies. Many of the lesser-known species face increasing fragmentation and destruction of habitat, and most populations of still non-threatened species face encroachment from human settlements. Here, we present capuchin research priorities and urgent issues based on the discussion by capuchin researchers in the roundtable. These include a call for the immediate end to the use of the name Cebus apella and the employment of the term Sapajus spp. instead for captive robust capuchins of unknown origin; for the implementation of rapid assessments for previously unstudied capuchin species or populations in biomes of interest; for the development of standardized methods to allow for comparative analyses across capuchin field sites; and for the creation and maintenance of an open-access website for capuchin monkey data. Finally, we planned the creation of an international Capuchin Action Network, to help disseminate research information; to work as a research community in a more efficient, collaborative manner; to help prioritize research and conservation goals as a community of experts; and to strengthen our political voice.


The efficacy and safety of pefloxacin in the treatment of typhoid fever in Algeria.
The efficacy and safety of pefloxacin 400 mg twice daily for seven days in the treatment of typhoid fever was investigated in an open, non-comparative study involving 37 patients infected with Salmonella typhi. Between day 5 and the end of treatment, all patients showed clinical improvement. The clinical cure rate at the end of the treatment period was 89.2%. One month after the end of treatment, the final assessment showed a clinical cure rate of 94.6% with relapses occurring in only two patients. Both these patients belonged to the group with negative blood cultures but positive serodiagnostic tests. Bacteriological tests confirmed eradication of the pathogen in 94.6% of patients and the relapses in the two serologically positive patients. Temperature became normal in a mean of 5.14 days. Clinical adverse reactions were reported in 5.9% (3/51) of patients (vomiting in two, transient pruritic rash in one). The results of this study indicate that a seven-day course of pefloxacin 400 mg twice daily provides a suitable alternative in the treatment of typhoid fever.


Helicobacter pylori cholesteryl α-glucosides contribute to its pathogenicity and immune response by natural killer T cells.
Approximately 10-15% of individuals infected with Helicobacter pylori will develop ulcer disease (gastric or duodenal ulcer), while most people infected with H. pylori will be asymptomatic. The majority of infected individuals remain asymptomatic partly due to the inhibition of synthesis of cholesteryl α-glucosides in H. pylori cell wall by α1,4-GlcNAc-capped mucin O-glycans, which are expressed in the deeper portion of gastric mucosa. However, it has not been determined how cholesteryl α-glucosyltransferase (αCgT), which forms cholesteryl α-glucosides, functions in the pathogenesis of H. pylori infection. Here, we show that the activity of αCgT from H. pylori clinical isolates is highly correlated with the degree of gastric atrophy. We investigated the role of cholesteryl α-glucosides in various aspects of the immune response. Phagocytosis and activation of dendritic cells were observed at similar degrees in the presence of wild-type H. pylori or variants harboring mutant forms of αCgT showing a range of enzymatic activity. However, cholesteryl α-glucosides were recognized by invariant natural killer T (iNKT) cells, eliciting an immune response in vitro and in vivo. Following inoculation of H. pylori harboring highly active αCgT into iNKT cell-deficient (Jα18(-/-)) or wild-type mice, bacterial recovery significantly increased in Jα18(-/-) compared to wild-type mice. Moreover, cytokine production characteristic of Th1 and Th2 cells dramatically decreased in Jα18(-/-) compared to wild-type mice. These findings demonstrate that cholesteryl α-glucosides play critical roles in H. pylori-mediated gastric inflammation and precancerous atrophic gastritis.


Adaptation of cough reflex with different types of stimulation.
Inhalation cough challenge has become an accepted method of investigating antitussive agents. It is, therefore, important to examine the degree of tachyphylaxis seen with repeated cough challenge. In addition, different types of challenge may reveal important differences in the neuronal pathways involved in the cough reflex. Citric acid, distilled water and capsaicin were examined to determine adaptation of the cough response during acute and long-term inhalation studies in healthy subjects. To study acute tachyphylaxis two separate one minute continuous inhalation challenges (n = 13) were performed. Long-term tachyphylaxis (n = 10) was examined using citric acid and capsaicin inhalation at 10 min intervals for 40 min, and at 4 and 6 h. Cross-tachyphylaxis to citric acid and capsaicin was examined in a separate randomized crossover study (n = 10). Highly significant adaptation occurred between the first and last 10 s of the one minute challenge with citric acid (90-100%) and distilled water (74-84%), but was less pronounced with capsaicin (37-49%, at 2 microM). Cough during the whole of the second test was significantly reduced for citric acid (50%). During long-term challenge cough was attenuated over 40 min both with citric acid (100 mM, p less than 0.05; 300 mM, p less than 0.001; 1 M, p less than 0.001) and with capsaicin (3 microM, p less than 0.01; 10, 30, 100 microM, p less than 0.001 each). With higher doses, tachyphylaxis was still present at 180 min with both citric acid (300 mM, p less than 0.05) and capsaicin (100 microM, p less than 0.008).(ABSTRACT TRUNCATED AT 250 WORDS)


The identification of chromosome abnormalities associated with the invasive phenotype of uveal melanoma in vitro.
Tumour cell cultures are often highly heterogeneous, containing sub-populations of cells with differing characteristics. To identify chromosome abnormalities that are associated with the invasive phenotype, we isolated highly invasive uveal melanoma cell populations using the Transwell assay. Using this invasion assay, invasive sub-populations of primary uveal melanoma short-term cultures, and an established cell line, were specifically isolated. A series of sequential assays were undertaken to enrich the invasive population, and the enhanced invasive ability was confirmed by Transwell invasion assay. Chromosome abnormalities in invasive and parental cells were identified by karyotyping and confirmed by comparative genome hybridisation. Invasive sub-populations of uveal melanoma cells were isolated from 3 uveal melanoma short term cultures and a uveal melanoma cell line. In all cases, invasive sub-populations had either acquired additional chromosome abnormalities to those present in the parental cell line, or other abnormalities present in the parental lines were lost. In the established cell line (SOM 157), invasive cells were characterised by widespread chromosomal instability, frequent telomere associations and additional copies of chromosome 20. The invasive phenotype of SOM 196 associated with the presence of a derivative chromosome 5, der(5)t(5;11)(q35;q12) whilst a translocation t(17;20)(q12;q13) was predominant amongst non-invasive cells. In two additional cultures, deletions on chromosome 6q were associated with reduced invasive ability. In conclusion, highly invasive populations of uveal melanoma cells demonstrate chromosomal abnormalities that differ from non-invasive cells. These include chromosome instability and abnormalities of chromosome 20, observations echoing those seen in metastatic uveal melanoma.


Risk and protective factors associated with domestic abuse among older Chinese in the People's Republic of China.
To identify risk and protective factors associated with elder abuse among older Chinese with cognitive and physical impairment in the People's Republic of China (PRC). 1002 dyads of care recipients and family caregivers were interviewed. The mutually reported rates were 9.7%, 0.8%, 33.2%, and 39.7% for psychological abuse, physical abuse, financial exploitation, and caregiver neglect respectively. Caregiver burden, care recipient neuropsychiatric symptoms, and cognitive impairments are prominent risk factors, while protective factors associated with multiple abuse subtypes include caregiver's use of emotion-focused and solution-focused coping, perception of familism, and premorbid relationship rewards. Distinct factors were observed for respective forms of abuse. Psychological abuse with family poverty; Caregiver neglect with caregiver's substance use and poor physical health; Financial exploitation with caregiver's neurotic personality, as well as care recipient's younger age, absence of chronic illness, and co-residence between the caregiver and care recipient. Both similar and distinct factors were observed for different forms of abuse, potentially useful for designing prevention and intervention programs.


Meckel diverticulum: the Mayo Clinic experience with 1476 patients (1950-2002).
Through a comprehensive review of the Mayo Clinic experience with patients who had Meckel diverticulum, we sought to determine which diverticula should be removed when discovered incidentally during abdominal surgery. Meckel diverticula occur so infrequently that most articles have reported either small series or isolated cases. From these limited series, various conclusions have been reported without clearly indicating which incidental diverticula should be removed. Medical records were reviewed of 1476 patients found to have a Meckel diverticulum during surgery from 1950 to 2002. Preoperative diagnosis; age; sex; date of surgery; and intraoperative, macroscopic, and microscopic findings from operative and pathology reports were recorded. Logistic regression analysis was used to determine which clinical or histologic features were associated with symptomatic Meckel diverticulum. The features analyzed were age; sex; length, base width, and ratio of length to base width of the diverticulum; and the presence of ectopic tissue or abnormal tissue (inflammation or enteroliths). Among the 1476 patients, 16% of the Meckel diverticula were symptomatic. The most common clinical presentation in adults was bleeding; in children, obstruction. Among patients with a symptomatic Meckel diverticulum, the male-female ratio was approximately 3:1. Clinical or histologic features most commonly associated with symptomatic Meckel diverticula were patient age younger than 50 years (odds ratio [OR], 3.5; 95% confidence interval [CI], 2.6-4.8; P < 0.001), male sex (OR, 1.8; 95% CI, 1.3-2.4; P < 0.001); diverticulum length greater than 2 cm (OR, 2.2; 95% CI, 1.1-4.4; P = 0.02), and the presence of histologically abnormal tissue (OR, 13.9; 95% CI, 9.9-19.6; P < 0.001). After analyzing our data, we neither support nor reject the recommendation that all Meckel diverticula found incidentally should be removed, although the procedure today has little risk. If a selective approach is taken, we recommend removing all incidental Meckel diverticula that have any of the 4 features most commonly associated with symptomatic Meckel diverticulum.


Potentially Curable Pancreatic Adenocarcinoma: ASCO Clinical Practice Guideline Update.
The purpose of this guideline update is to incorporate recently reported practice-changing evidence into ASCO's recommendations on potentially curable pancreatic adenocarcinoma. ASCO convened an Expert Panel to evaluate data from PRODIGE 24/CCTG PA.6, a phase III, multicenter, randomized clinical trial of postoperative leucovorin calcium, fluorouracil, irinotecan hydrochloride, and oxaliplatin (FOLFIRINOX) versus gemcitabine alone, presented at the 2018 ASCO Annual Meeting. In addition, PubMed was searched for additional papers that may influence the existing recommendations. The Expert Panel only updated Recommendation 4.1 as a result of the practice-changing data. Recommendation 4.1 states that all patients with resected pancreatic adenocarcinoma who did not receive preoperative therapy should be offered 6 months of adjuvant chemotherapy in the absence of medical or surgical contraindications. The modified combination regimen of 5-fluorouracil, oxaliplatin, and irinotecan (mFOLFIRINOX; oxaliplatin 85 mg/m2, leucovorin 400 mg/m2, irinotecan 150 mg/m2 D1, and 5-fluorouracil 2.4 g/m2 over 46 hours every 14 days for 12 cycles) is now preferred in the absence of concerns for toxicity or tolerance; alternatively, doublet therapy with gemcitabine and capecitabine or monotherapy with gemcitabine alone or fluorouracil plus folinic acid alone can be offered.Additional information can be found at www.asco.org/gastrointestinal-cancer-guidelines.


A comparative study between sterile freeze-dried and sterile pre-hydrated acellular dermal matrix in tissue expander/implant breast reconstruction.
In implant-based breast reconstruction, acellular dermal matrix (ADM) is essential for supporting the inferolateral pole. Recent studies have compared non-sterilized freeze-dried ADM and sterilized pre-hydrated ADM, but have not assessed whether differences were attributable to factors related to sterile processing or packaging. This study was conducted to compare the clinical outcomes of breast reconstruction using two types of sterile-processed ADMs. Through a retrospective chart review, we analyzed 77 consecutive patients (85 breasts) who underwent tissue expander/implant breast reconstruction with either freezedried ADM (35 breasts) or pre-hydrated ADM (50 breasts) from March 2016 to February 2018. Demographic variables, postoperative outcomes, and operative parameters were compared between freeze-dried and pre-hydrated ADM. Biopsy specimens were obtained for histologic analysis. We obtained results after adjusting for variables found to be significant in univariate analyses. The total complication rate for freeze-dried and pre-hydrated ADMs was 25.7% and 22.0%, respectively. Skin necrosis was significantly more frequent in the freeze-dried group than in the pre-hydrated group (8.6% vs. 4.0%, P=0.038). All other complications and operative parameters showed no significant differences. In the histologic analysis, collagen density, inflammation, and vascularity were higher in the pre-hydrated ADM group (P=0.042, P=0.006, P=0.005, respectively). There are limited data comparing the outcomes of tissue expander/implant breast reconstruction using two types of sterile-processed ADMs. In this study, we found that using pre-hydrated ADM resulted in less skin necrosis and better integration into host tissue. Pre-hydrated ADM may therefore be preferable to freeze-dried ADM in terms of convenience and safety.


Plasma membrane Ca2+ pumps and Na+/Ca2+ exchangers.
Membrane-intrinsic transport systems play an essential role in intracellular Ca2+ homeostasis. ATP-driven Ca2+ pumps and carrier-mediated Na+/Ca2+ exchangers are the two specific Ca2+ transporting systems mainly responsible for Ca2+ extrusion across the plasma membrane. Ca2+ pumps operate in all eukaryotic cell types and are characterized by their high Ca2+ affinity and their specific regulation by direct interaction with Ca2+/calmodulin. Na+/Ca2+ exchangers are particularly abundant in excitable tissues and are responsible for the bulk Ca2+ efflux in these tissues. Recent success in the molecular characterization of the pumps has led to the determination of complete amino acid sequences for several isoforms and has allowed the identification and topological assignment of important functional and regulatory domains. Genetic evidence indicates that mammalian Ca2+ pump diversity is generated from a multigene family and via alternative RNA splicing. Different isoforms may vary in their regulatory properties, presumably reflecting different physiological requirements of the tissues of their expression. Although the molecular characterization of Na+/Ca2+ exchangers is not as far advanced as that of the pumps, recent studies have established detailed kinetic, stoichiometric and regulatory properties of these systems. Together with advances in expression cloning methods these studies promise to result in a rapid improvement of our knowledge of the functional properties of these ion transporters on a molecular level.


Severe hypoxia and multiple infarctions resembling Creutzfeldt-Jakob disease.
Although neuropathological examination is still required for the definite diagnosis of Creutzfeldt-Jakob disease (CJD), specialised clinical assessment predicts probable CJD. Here we present a 73-year-old female patient presenting with rapid cognitive decline, visual, acoustic and cerebellar disturbances, ataxia and EEG changes compatible with early CJD stages. MRI revealed hyperintensities within the thalami, hypothalami, corpora mammillaria, the tectum and the cortex. Initial neuropathological examination showed severe cortical and subcortical spongiosis. However, both immunohistochemistry and Western blotting showed no pathological prion protein. Finally, small infarctions affecting the tectum, tegmentum, corpora mammillaria and global hypoxic-ischaemic changes could be identified as the probable reason for the changes interpreted as CJD-related pathology. Hypoxic-ischaemic CNS alterations mainly affecting the supply area of the basilar artery should be ruled out in case of probable CJD. In addition, severe spongiosis can be misleading in the histological examination, suggesting the diagnosis of a prion-induced spongiform encephalopathy.


Clinical outcomes of rotational atherectomy in severely calcified in-stent restenosis: a single-center, retrospective study.
Neointimal calcification after stent implantation has been reported as one of the forms of neoatherosclerosis. There are a few patients with in-stent restenosis (ISR) and an undilatable calcified neointima who require rotational atherectomy to achieve sufficient acute gain in lumen diameter. However, the clinical outcomes of rotational atherectomy for undilatable calcified ISR have not been fully elucidated. Therefore, we investigated the safety and efficacy of rotational atherectomy for treating calcified ISR. This retrospective study included 17 patients (20 lesions) who had undergone percutaneous coronary intervention including rotational atherectomy to treat ISR with severely calcified neointima. Kaplan-Meier analysis was used to analyze the data. The mean age of the enrolled patients was 67±18 years, and 71% were men. The patients had highly atherogenic characteristics: 65% had diabetes mellitus and 53% were receiving hemodialysis. Procedural success was obtained in 19 (95%) patients, and the acute gain in lumen diameter was acceptable (1.7±0.6 mm). However, during a median follow-up of 571 days, the incidences of major adverse cardiac and cerebrovascular events per patient and clinical-driven target lesion revascularizations per lesion were relatively high. There were no differences in clinical outcomes according to the baseline characteristics, type of restenotic stents, and therapeutic strategy. In conclusion, clinical outcomes of rotational atherectomy for severely calcified ISR were unfavorable despite a high success rate and acceptable acute gain in lumen diameter.


Coseasonal sublingual immunotherapy reduces the development of asthma in children with allergic rhinoconjunctivitis.
We wondered whether short-term coseasonal sublingual immunotherapy (SLIT) can reduce the development of asthma in children with hay fever in an open randomized study. We sought to determine whether SLIT is as effective as subcutaneous immunotherapy in reducing hay fever symptoms and the development of asthma in children with hay fever. One hundred thirteen children aged 5 to 14 years (mean age, 7.7 years) with hay fever limited to grass pollen and no other clinically important allergies were randomized in an open study involving 6 Italian pediatric allergy centers to receive specific SLIT for 3 years or standard symptomatic therapy. All of the subjects had hay fever symptoms, but at the time of study entry, none reported seasonal asthma with more than 3 episodes per season. Symptomatic treatment was limited to cetirizine, loratadine, nasal budesonide, and salbutamol on demand. The hay fever and asthma symptoms were quantified clinically. The actively treated children used less medication in the second and third years of therapy, and their symptom scores tended to be lower. From the second year of immunotherapy, subjective evaluation of overall allergy symptoms was favorable in the actively treated children. Development of asthma after 3 years was 3.8 times more frequent (95% confidence limits, 1.5-10.0) in the control subjects. Three years of coseasonal SLIT improves seasonal allergic rhinitis symptoms and reduces the development of seasonal asthma in children with hay fever.


Pseudo-direct bandgap transitions in silicon nanocrystals: effects on optoelectronics and thermoelectrics.
While silicon nanostructures are extensively used in electronics, the indirect bandgap of silicon poses challenges for optoelectronic applications like photovoltaics and light emitting diodes (LEDs). Here, we show that size-dependent pseudo-direct bandgap transitions in silicon nanocrystals dominate the interactions between (photoexcited) charge carriers and phonons, and hence the optoelectronic properties of silicon nanocrystals. Direct measurements of the electronic density of states (DOS) for different sized silicon nanocrystals reveal that these pseudo-direct transitions, likely arising from the nanocrystal surface, can couple with the quantum-confined silicon states. Moreover, we demonstrate that since these transitions determine the interactions of charge carriers with phonons, they change the light emission, absorption, charge carrier diffusion and phonon drag (Seebeck coefficient) in nanoscaled silicon semiconductors. Therefore, these results can have important implications for the design of optoelectronics and thermoelectric devices based on nanostructured silicon.


Depressive symptoms across adolescence and young adulthood in men: predictions from parental and contextual risk factors.
The current study examined the course of depressive symptoms of young men from early adolescence through young adulthood using a growth model approach and the influence of early parental (i.e.,depressive symptoms, antisocial behaviors, substance use) and contextual (i.e., family income and parental marital transitions) risk factors on both the level of depressive symptoms in early adolescence and on changes over a 10-year period, controlling for young men's early antisocial behavior. On average, depressive symptoms of the youths tended to decrease slightly as they reached young adulthood. Among the parental and contextual risk factors, parent's marital transitions was the most significant predictor for the level of depressive symptoms in early adolescence. Parent's marital transitions and family income were also significantly related to changes in depressive symptoms. As hypothesized, there was a significant interaction effect of paternal and maternal depressive symptoms on depressive symptoms in early adolescence and on changes over time. Maternal depressive symptoms were related to the son's depressive symptoms only when the father's depressive symptoms were also high. The results also suggested that parenting practices did not mediate the effects of the parental and contextual risk factors on the development of depressive symptoms among young men.


The interpretation of forensic conclusions by criminal justice professionals: The same evidence interpreted differently.
Forensic reports use various types of conclusions, such as a categorical (CAT) conclusion or a likelihood ratio (LR). In order to correctly assess the evidence, users of forensic reports need to understand the conclusion and its evidential strength. The aim of this paper is to study the interpretation of the evidential strength of forensic conclusions by criminal justice professionals. In an online questionnaire 269 professionals assessed 768 reports on fingerprint examination and answered questions that measured self-proclaimed and actual understanding of the reports and conclusions. The reports entailed CAT, verbal LR and numerical LR conclusions with low or high evidential strength and were assessed by crime scene investigators, police detectives, public prosecutors, criminal lawyers, and judges. The results show that about a quarter of all questions measuring actual understanding of the reports were answered incorrectly. The CAT conclusion was best understood for the weak conclusions, the three strong conclusions were all assessed similarly. The weak CAT conclusion correctly emphasizes the uncertainty of any conclusion type used. However, most participants underestimated the strength of this weak CAT conclusion compared to the other weak conclusion types. Looking at the self-proclaimed understanding of all professionals, they in general overestimated their actual understanding of all conclusion types.


Goal striving, goal attainment, and well-being: adapting and testing the self-concordance model in sport.
Grounded in self-determination theory (Deci & Ryan, 1985) and the self-concordance model (Sheldon & Elliot, 1999), this study examined the motivational processes underlying goal striving in sport as well as the role of perceived coach autonomy support in the goal process. Structural equation modeling with a sample of 210 British athletes showed that autonomous goal motives positively predicted effort, which, in turn, predicted goal attainment. Goal attainment was positively linked to need satisfaction, which, in turn, predicted psychological well-being. Effort and need satisfaction were found to mediate the associations between autonomous motives and goal attainment and between attainment and well-being, respectively. Controlled motives negatively predicted well-being, and coach autonomy support positively predicted both autonomous motives and need satisfaction. Associations of autonomous motives with effort were not reducible to goal difficulty, goal specificity, or goal efficacy. These findings support the self-concordance model as a framework for further research on goal setting in sport.


Metanx in type 2 diabetes with peripheral neuropathy: a randomized trial.
To determine whether a combination of L-methylfolate, methylcobalamin, and pyridoxal-5'-phosphate (LMF-MC-PLP [Metanx; Pamlab LLC, Covington, La]) improves sensory neuropathy. This multicenter, randomized, double-blind, placebo-controlled trial involved 214 patients with type 2 diabetes and neuropathy (baseline vibration perception threshold [VPT]: 25-45 volts), who were randomly assigned to 24 weeks of treatment with either L-methylfolate calcium 3 mg, methylcobalamin 2 mg, and pyridoxal-5'-phosphate 35 mg or placebo. The primary end point was effect on VPT. Secondary end points included Neuropathy Total Symptom Score (NTSS-6) and Short Form 36 (SF-36), as well as plasma levels of folate, vitamins B(6) and B(12), methylmalonic acid (MMA), and homocysteine. There was no significant effect on VPT. However, patients receiving LMF-MC-PLP consistently reported symptomatic relief, with clinically significant improvement in NTSS-6 scores at week 16 (P=.013 vs placebo) and week 24 (P=.033). Improvement in NTSS scores was related to baseline MMA and inversely related to baseline PLP and metformin use. Quality-of-life measures also improved. Homocysteine decreased by 2.7±3.0 μmol/L with LMF-MC-PLP versus an increase of 0.5±2.4 μmol/L with placebo (P=.0001). Adverse events were infrequent, with no single event occurring in ≥2% of subjects. LMF-MC-PLP appears to be a safe and effective therapy for alleviation of peripheral neuropathy symptoms, at least in the short term. Additional long-term studies should be conducted, as the trial duration may have been too short to show an effect on VPT. In addition, further research on the effects in patients with cobalamin deficiency would be useful.


Direct detection of Coccidioides from Arizona soils using CocciENV, a highly sensitive and specific real-time PCR assay.
Coccidioides immitis and Coccidioides posadasii are soil fungi endemic to desert regions of the southwestern United States, and the causative agents of valley fever, or coccidioidomycosis. Studies have shown that the distribution of Coccidioides in soils is sporadic and cannot be explained by soil characteristics alone, suggesting that biotic and other abiotic factors should be examined. However, tools to reliably and robustly screen the large number of soils needed to investigate these potential associations have not been available. Thus, we developed a real-time polymerase chain reaction (PCR) assay for testing environmental samples by modifying CocciDx, an assay validated for testing clinical specimens to facilitate coccidioidomycosis diagnosis. For this study, we collected soil samples from previously established locations of C. posadasii in Arizona and new locations in fall 2013 and spring 2014, and screened the extracted DNA with the new assay known as CocciEnv. To verify the presence of Coccidioides in soil using an alternate method, we employed next generation amplicon sequencing targeting the ITS2 region. Results show our modified assay, CocciEnv, is a rapid and robust method for detecting Coccidioides DNA in complex environmental samples. The ability to test a large number of soils for the presence of Coccidioides is a much-needed tool in the understanding of the ecology of the organism and epidemiology of the disease and will greatly improve our understanding of this human pathogen.


The influence of rural clinical school experiences on medical students' levels of interest in rural careers.
Australian Rural Clinical School (RCS) programmes have been designed to create experiences that positively influence graduates to choose rural medical careers. Rural career intent is a categorical evaluation measure and has been used to assess the Australian RCS model. Predictors for rural medical career intent have been associated with extrinsic values such as students with a rural background. Intrinsic values such as personal interest have not been assessed with respect to rural career intent. In psychology, a predictor of the motivation or emotion for a specific career or career location is the level of interest. Our primary aims are to model over one year of Australian RCS training, change in self-reported interest for future rural career intent. Secondary aims are to model student factors associated with rural career intent while attending an RCS. The study participants were medical students enrolled in a RCS in the year 2013 at the University of New South Wales (UNSW) and who completed the newly developed self-administered UNSW Undergraduate Destinations Study (UDS) questionnaire. Data were collected at baseline and after one year of RCS training on preferred location for internship, work and intended specialty. Interest for graduate practice location (career intent) was assessed on a five-variable Likert scale at both baseline and at follow-up. A total of 165 students completed the UDS at baseline and 150 students after 1 year of follow-up. Factors associated with intent to practise in a rural location were rural background (χ2 = 28.4, P < 0.001), two or more previous years at an RCS (χ2 = 9.0, P = 0.003), and preference for a rural internship (χ2 = 17.8, P < 0.001). At follow-up, 41% of participants who originally intended to work in a metropolitan location at baseline changed their preference and indicated a preference for a rural location. The level of interest in intended practice location was significantly higher for those intending to work in a rural area than those with intention to work in a metropolitan (urban area) location (t = -3.1, P = 0.002). Initial rural career location intention was associated with increased interest levels after 1 year of follow-up (paired t = -2.3, P = 0.02). When evaluating the success of RCS outcomes with respect to rural workforce destination, both rural practice intentions and level of interest are key factors related to projected career destination. RCS experience can positively influence practice intent (toward rural practice) and interest levels (toward greater interest in rural practice).


Detection of hearing loss using 2f2-f1 and 2f1-f2 distortion-product otoacoustic emissions.
Although many distortion-product otoacoustic emissions (DPOAEs) may be measured in the ear canal in response to 2 pure tone stimuli, the majority of clinical studies have focused exclusively on the DPOAE at the frequency 2f1-f2. This study investigated another DPOAE, 2f2-f1, in an attempt to determine the following: (a) the optimal stimulus parameters for its clinical measurement and (b) its utility in differentiating between normal-hearing and hearing-impaired ears at low-to-mid frequencies (<or=2,000 Hz) when measured either alone or in conjunction with the 2f1-f2 DPOAE. Two experiments were conducted. In Experiment 1, the effects of primary level, level separation, and frequency separation (f2/f1) on 2f2-f1 DPOAE level were evaluated in normal-hearing ears for low-to-mid f2 frequencies (700-2,000 Hz). Moderately high-level primaries (60-70 dB SPL) presented at equal levels or with f2 slightly higher than f1 produced the highest 2f2-f1 DPOAE levels. When the f2/f1 ratio that produced the highest 2f2-f1 DPOAE levels was examined across participants, the mean optimal f2/f1 ratio across f2 frequencies and primary level separations was 1.08. In Experiment 2, the accuracy with which DPOAE level or signal-to-noise ratio identified hearing status at the f2 frequency as normal or impaired was evaluated using clinical decision analysis. The 2f2-f1 and 2f1-f2 DPOAEs were measured from both normal-hearing and hearing-impaired ears using 2 sets of stimulus parameters: (a) the traditional parameters for measuring the 2f1-f2 DPOAE (f2/f1 = 1.22; L1, L2 = 65, 55 dB SPL) and (b) the new parameters that were deemed optimal for the 2f2-f1 DPOAE in Experiment 1 (f2/f1 = 1.073, L1 and L2 = 65 dB SPL). Identification of hearing status using 2f2-f1 DPOAE level and signal-to-noise ratio was more accurate when the new stimulus parameters were used compared with the results achieved when the 2f2-f1 DPOAE was recorded using the traditional parameters. However, identification of hearing status was less accurate for the 2f2-f1 DPOAE measured using the new parameters than for the 2f1-f2 DPOAE measured using the traditional parameters. No statistically significant improvements in test performance were achieved when the information from the 2 DPOAEs was combined, either by summing the DPOAE levels or by using logistic regression analysis.


The MKK7 gene encodes a group of c-Jun NH2-terminal kinase kinases.
The c-Jun NH2-terminal protein kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) group and is an essential component of a signaling cascade that is activated by exposure of cells to environmental stress. JNK activation is regulated by phosphorylation on both Thr and Tyr residues by a dual-specificity MAPK kinase (MAPKK). Two MAPKKs, MKK4 and MKK7, have been identified as JNK activators. Genetic studies demonstrate that MKK4 and MKK7 serve nonredundant functions as activators of JNK in vivo. We report here the molecular cloning of the gene that encodes MKK7 and demonstrate that six isoforms are created by alternative splicing to generate a group of protein kinases with three different NH2 termini (alpha, beta, and gamma isoforms) and two different COOH termini (1 and 2 isoforms). The MKK7alpha isoforms lack an NH2-terminal extension that is present in the other MKK7 isoforms. This NH2-terminal extension binds directly to the MKK7 substrate JNK. Comparison of the activities of the MKK7 isoforms demonstrates that the MKK7alpha isoforms exhibit lower activity, but a higher level of inducible fold activation, than the corresponding MKK7beta and MKK7gamma isoforms. Immunofluorescence analysis demonstrates that these MKK7 isoforms are detected in both cytoplasmic and nuclear compartments of cultured cells. The presence of MKK7 in the nucleus was not, however, required for JNK activation in vivo. These data establish that the MKK4 and MKK7 genes encode a group of protein kinases with different biochemical properties that mediate activation of JNK in response to extracellular stimuli.


Fast signals and slow marks: the dynamics of histone modifications.
Most multi-cellular organisms adopt a specific gene expression pattern during cellular differentiation. Once established, this pattern is frequently maintained over several cell divisions despite the fact that the initiating signal is no longer present. Differential packaging into chromatin is one such mechanism that allows fixation of transcriptional activity. Recent genome-wide studies demonstrate that actively transcribed regions are characterized by a specific modification pattern of histones, the main protein component of chromatin. These findings support the hypothesis that a histone code uses histone post-translational modifications to stably inscribe particular chromatin structures into the genome. Experiments on the dynamics of histone modifications reveal a striking kinetic difference between methylation, phosphorylation and acetylation, suggesting different roles of these modifications in epigenetically fixing specific gene expression patterns.


Role of translocator protein density, a marker of neuroinflammation, in the brain during major depressive episodes.
The neuroinflammatory hypothesis of major depressive disorder is supported by several main findings. First, in humans and animals, activation of the immune system causes sickness behaviors that present during a major depressive episode (MDE), such as low mood, anhedonia, anorexia, and weight loss. Second, peripheral markers of inflammation are frequently reported in major depressive disorder. Third, neuroinflammatory illnesses are associated with high rates of MDEs. However, a fundamental limitation of the neuroinflammatory hypothesis is a paucity of evidence of brain inflammation during MDE. Translocator protein density measured by distribution volume (TSPO VT) is increased in activated microglia, an important aspect of neuroinflammation. To determine whether TSPO VT is elevated in the prefrontal cortex, anterior cingulate cortex (ACC), and insula in patients with MDE secondary to major depressive disorder. Case-control study in a tertiary care psychiatric hospital from May 1, 2010, through February 1, 2014. Twenty patients with MDE secondary to major depressive disorder and 20 healthy control participants underwent positron emission tomography with fluorine F 18-labeled N-(2-(2-fluoroethoxy)benzyl)-N-(4-phenoxypyridin-3-yl)acetamide ([18F]FEPPA). Patients with MDE were medication free for at least 6 weeks. All participants were otherwise healthy and nonsmokers. Values of TSPO VT in the prefrontal cortex, ACC, and insula. In MDE, TSPO VT was significantly elevated in all brain regions examined (multivariate analysis of variance, F15,23 = 4.5 [P = .001]). The magnitude of TSPO VT elevation was 26% in the prefrontal cortex (mean [SD] TSPO VT, 12.5 [3.6] in patients with MDE and 10.0 [2.4] in controls), 32% in the ACC (mean [SD] TSPO VT, 12.3 [3.5] in patients with MDE and 9.3 [2.2] in controls), and 33% in the insula (mean [SD] TSPO VT, 12.9 [3.7] in patients with MDE and 9.7 [2.3] in controls). In MDE, greater TSPO VT in the ACC correlated with greater depression severity (r = 0.63 [P = .005]). This finding provides the most compelling evidence to date of brain inflammation, and more specifically microglial activation, in MDE. This finding is important for improving treatment because it implies that therapeutics that reduce microglial activation should be promising for MDE. The correlation between higher ACC TSPO VT and the severity of MDE is consistent with the concept that neuroinflammation in specific regions may contribute to sickness behaviors that overlap with the symptoms of MDE.


Does Time with Dad in Childhood Pay Off in Adolescence?
We aim to understand the association between father involvement in middle childhood and adolescent behaviors and whether the relationship differs by father residence. Internalizing and externalizing behaviors in adolescence can trigger a cascade of negative outcomes later in life, including lower educational attainment, criminal justice involvement, and future psychological distress. Evidence, largely focusing on nonresidential fathers and older cohort, suggests that father involvement-particularly closeness and engagement-may reduce adolescents' internalizing and externalizing behaviors. We use data six waves of the Fragile Families and Child Wellbeing Study, a birth cohort survey representative of births in large U.S. cities between 1998 and 2000, to estimate OLS regression models examining (a) whether father involvement in middle childhood is associated with fewer problem behaviors at Age 15, (b) if the salience of father involvement differs depending on whether the father was present in the home (i.e., was married to or living with his child's mother) in middle childhood, and (c) whether father involvement matters differently based on the child's sex. We find protective associations between father involvement and adolescent behavioural outcomes that persist even among children who were not living with their fathers. In models stratified by the child's sex, father involvement matters for both boys and girls. In all models, father presence alone, apart from active involvement, is not significantly associated with behavioral outcomes. Father involvement protects against negative adolescent behaviors even among children with nonresidential fathers and for both boys and girls. These results suggest that policies that promote greater father involvement and father-child bonds, rather than other options such as promoting marriage, may be more effective in reducing behavioral problems among adolescents.


Incidence, clinical course, and predictors of prolonged recovery time following sport-related concussion in high school and college athletes.
Sport-related concussion (SRC) is typically followed by clinical recovery within days, but reports of prolonged symptoms are common. We investigated the incidence of prolonged recovery in a large cohort (n = 18,531) of athlete seasons over a 10-year period. A total of 570 athletes with concussion (3.1%) and 166 controls who underwent pre-injury baseline assessments of symptoms, neurocognitive functioning and balance were re-assessed immediately, 3 hr, and 1, 2, 3, 5, 7, and 45 or 90 days after concussion. Concussed athletes were stratified into typical (within 7 days) or prolonged (> 7 days) recovery groups based on symptom recovery time. Ten percent of athletes (n = 57) had a prolonged symptom recovery, which was also associated with lengthier recovery on neurocognitive testing (p < .001). At 45-90 days post-injury, the prolonged recovery group reported elevated symptoms, without deficits on cognitive or balance testing. Prolonged recovery was associated with unconsciousness [odds ratio (OR), 4.15; 95% confidence interval (CI) 2.12-8.15], posttraumatic amnesia (OR, 1.81; 95% CI, 1.00-3.28), and more severe acute symptoms (p < .0001). These results suggest that a small percentage of athletes may experience symptoms and functional impairments beyond the typical window of recovery after SRC, and that prolonged recovery is associated with acute indicators of more severe injury.


Genetic Architecture and Candidate Genes for Deep-Sowing Tolerance in Rice Revealed by Non-syn GWAS.
Dry direct-seeding of rice is rapidly increasing in China, but variable planting depth associated with machine sowing can lead to low seedling emergence rates. Phenotype analysis of 621 rice accessions showed that mesocotyl length (ML) was induced by deep soil covering and was important in deep-sowing tolerance in the field. Here, we performed and compared GWAS using three types of SNPs (non-synonymous SNP, non-synonymous SNPs and SNPs within promoters and 3 million randomly selected SNPs from the entire set of SNPs) and found that Non-Syn GWAS (GWAS using non-synonyomous SNP) decreased computation time and eliminated confounding by other loci relative to GWAS using randomly selected SNPs. Thirteen QTLs were finally detected, and two new major-effect genes, named OsML1 and OsML2, were identified by an integrated analysis. There were 2 and 7 non-synonymous SNPs in OsML1 and OsML2, respectively, from which 3 and 4 haplotypes were detected in cultivated rice. Combinations of superior haplotypes of OsML1 and OsML2 increased ML by up to 4 cm, representing high emergence rate (85%) in the field with 10 cm of soil cover. The studies provide key loci and naturally occurring alleles of ML that can be used in improving tolerance to dry direct-seeding.


What influences general practitioners' use of exercise for patients with chronic knee pain? Results from a national survey.
Exercise is a recommended 'core' treatment for chronic knee pain (CKP), however it appears to be underused by general practitioners (GPs). While behavioural theories suggest that attitudes and beliefs influence behaviours, no single theory reliably predicts GPs' behaviours. A theoretical analysis framework, developed from sociocognitive theories, was used to underpin investigation of the key influences associated with GPs' use of exercise for patients with CKP, to inform future interventions to optimise GPs' use of exercise. A cross-sectional postal questionnaire survey investigated UK GPs' reported use of exercise based on a patient case vignette. Factors influencing GPs' exercise use (behaviour) were examined using attitude statements, free-text questions and multiple response option questions related to factors within the analysis framework. Unadjusted logistic regression analyses explored the associations between GPs' attitudes/beliefs and behaviour. From a total sample of 5000 GPs, 835 (17%) returned a questionnaire. Most respondents (n = 729, 87%) reported that they would use exercise. Factors significantly associated with exercise use (OR (95% CI)) included GPs' beliefs about their role (belief that GPs should give information on type, duration and frequency of exercise (30.71 (5.02,188.01)), beliefs about consequences (agreement that knee problems are improved by local (3.23 (1.94,5.39)) and general exercise (2.63 (1.38,5.02))), moral norm (agreement that GPs should prescribe all patients local (3.08 (1.96,4.83)) and general exercise (2.63 (1.45,4.76))), and GP-related beliefs about capabilities (prior experience of insufficient expertise to give detailed exercise information (0.50 (0.33,0.76)). Whilst perceived time limitations were not associated with exercise use (1.00 (0.33,3.01)), GPs who disagreed that they experienced time limitations were more likely to suggest general (2.17 (1.04,4.55)), or demonstrate local (2.16 (1.06,4.42)), exercises. GPs' attitudes and beliefs are associated with their use of exercise for patients with CKP, particularly beliefs about role, responsibilities and skills in initiating exercise, and about the efficacy of exercise. Although the low response risks response bias, these results can inform future interventions to optimise GPs' behaviour. The role of GP uncertainty and influences on clinical decision-making need further exploration, thus an amended analysis framework is suggested, which should be tested in future research.


Qualitative insights into promotion of pharmaceutical products in Bangladesh: how ethical are the practices?
The pharmaceutical market in Bangladesh is highly concentrated (top ten control around 70 % of the market). Due to high competition aggressive marketing strategies are adopted for greater market share, which sometimes cross limit. There is lack of data on this aspect in Bangladesh. This exploratory study aimed to fill this gap by investigating current promotional practices of the pharmaceutical companies including the role of their medical representatives (MR). This qualitative study was conducted as part of a larger study to explore the status of governance in health sector in 2009. Data were collected from Dhaka, Chittagong and Bogra districts through in-depth interview (healthcare providers and MRs), observation (physician-MR interaction), and round table discussion (chief executives and top management of the pharmaceutical companies). Findings reveal a highly structured system geared to generate prescriptions and ensure market share instituted by the pharmaceuticals. A comprehensive training curriculum for the MRs prepares the newly recruited science graduates for generating enough prescriptions by catering to the identified needs and demands of the physicians expressed or otherwise, and thus grab higher market-share for the companies they represent. Approaches such as inducements, persuasion, emotional blackmail, serving family members, etc. are used. The type, quantity and quality of inducements offered to the physicians depend upon his/her capacity to produce prescriptions. The popular physicians are cultivated meticulously by the MRs to establish brand loyalty and fulfill individual and company targets. The physicians, willingly or unwillingly, become part of the system with few exceptions. Neither the regulatory authority nor the professional or consumer rights bodies has any role to control or ractify the process. The aggressive marketing of the pharmaceutical companies compel their MRs, programmed to maximize market share, to adopt unethical means if and when necessary. When medicines are prescribed and dispensed more for financial interests than for needs of the patients, it reflects system's failed ability to hold individuals and entities accountable for adhering to basic professional ethics, code of conduct, and statutory laws.


Cigarette Smoking and Estrogen-Related Cancer.
Cigarette smoking is a known cause of many cancers, yet epidemiologic studies have found protective associations with the risk of four "estrogen-related" malignancies: endometrial cancer, endometrioid and clear cell ovarian cancers, and thyroid cancer. This review considers epidemiologic and biological aspects of these associations, focusing particularly on estrogen signaling, and contrasts them with those for breast cancer, another estrogen-related malignancy. The observational findings regarding the inverse associations are consistent and remain after adjustment for possible confounding factors. In general, women who smoke do not have lower circulating estrogen levels than nonsmokers, eliminating one possible explanation for reduced risks of these malignancies. For endometrial and endometrioid ovarian cancer, the negative associations could plausibly be explained by interference with signaling through the estrogen receptor α. However, this is unlikely to explain the lower risks of thyroid and clear cell ovarian cancers. For thyroid cancer, an anti-inflammatory effect of nicotine and reduced TSH levels from smoking have been proposed explanations for the inverse association, but both lack convincing evidence. While the overall impact of cigarette smoking is overwhelmingly negative, protective associations such as those discussed here can provide potential clues to disease etiology, treatment, and prevention.


The iScore predicts total healthcare costs early after hospitalization for an acute ischemic stroke.
The ischemic Stroke risk score is a validated prognostic score which can be used by clinicians to estimate patient outcomes after the occurrence of an acute ischemic stroke. In this study, we examined the association between the ischemic Stroke risk score and patients' 30-day, one-year, and two-year healthcare costs from the perspective of a third party healthcare payer. Patients who had an acute ischemic stroke were identified from the Registry of Canadian Stroke Network. The 30-day ischemic Stroke risk score prognostic score was determined for each patient. Direct healthcare costs at each time point were determined using administrative databases in the province of Ontario. Unadjusted mean and the impact of a 10-point increase ischemic Stroke risk score and a patient's risk of death or disability on total cost were determined. There were 12,686 patients eligible for the study. Total unadjusted mean costs were greatest among patients at high risk. When adjusting for patient characteristics, a 10-point increase in the ischemic Stroke risk score was associated with 8%, 7%, and 4% increase in total costs at 30 days, one-year, and two-years. The same increase was found to impact patients at low, medium, and high risk differently. When adjusting for patient characteristics, patients in the high-risk group had the highest total costs at 30 days, while patients at medium risk had the highest costs at both one and two-years. The ischemic Stroke risk score can be useful as a predictor of healthcare utilization and costs early after hospitalization for an acute ischemic stroke.


On-the-fly cross flow laser guided separation of aerosol particles based on size, refractive index and density-theoretical analysis.
Laser separation of particles is achieved using forces resulting from the momentum exchange between particles and photons constituting the laser radiation. Particles can experience different optical forces depending on their size and/or optical properties, such as refractive index. Thus, particles can move at different speeds in the presence of an optical force, leading to spatial separations. In this paper, we present a theoretical analysis on laser separation of non-absorbing aerosol particles moving at speeds (1-10 cm/sec) which are several orders of magnitude greater than typical particle speeds used in previous studies in liquid medium. The calculations are presented for particle deflection by a loosely focused Gaussian 1064 nm laser, which simultaneously holds and deflects particles entrained in flow perpendicular to their direction of travel. The gradient force holds the particles against the viscous drag for a short period of time. The scattering force simultaneously pushes the particles, perpendicular to the flow, during this period. Our calculations show particle deflections of over 2500 µm for 15 µm aerosol particles, and a separation of over 1500 µm between 5 µm and 10 µm particles when the laser is operated at 10 W. We show that a separation of about 421 µm can be achieved between two particles of the same size (10 µm) but having a refractive index difference of 0.1. Density based separations are also possible. Two 10 µm particles with a density difference of 600 kg/m3 can be separated by 193 µm. Examples are shown for separation distances between polystyrene, poly(methylmethacrylate), silica and water particles. These large laser guided deflections represent a novel achievement for optical separation in the gas phase.


Direct visualization of the arterial wall water permeability barrier using CARS microscopy.
The artery wall is equipped with a water permeation barrier that allows blood to flow at high pressure without significant water leak. The precise location of this barrier is unknown despite its importance in vascular function and its contribution to many vascular complications when it is compromised. Herein we map the water permeability in intact arteries, using coherent anti-Stokes Raman scattering (CARS) microscopy and isotopic perfusion experiments. Generation of the CARS signal is optimized for water imaging with broadband excitation. We identify the water permeation barrier as the endothelial basolateral membrane and show that the apical membrane is highly permeable. This is confirmed by the distribution of the AQP1 water channel within endothelial membranes. These results indicate that arterial pressure equilibrates within the endothelium and is transmitted to the supporting basement membrane and internal elastic lamina macromolecules with minimal deformation of the sensitive endothelial cell. Disruption of this pressure transmission could contribute to endothelial cell dysfunction in various pathologies.


Molecular diagnosis of amoebiasis.
Amoebiasis is an intestinal parasitosis caused by the protozoan Entamoeba histolytica that represents the third leading cause of mortality due to parasitosis. It is a prevalent disease in tropical climate regions with poor or absent sanitary services. Microscopy and antigen detection techniques are routinely used to diagnose amoebiasis because of their low cost and ease of application. However, these techniques do not differentiate E. histolytica infections and other potentially pathogenic species such as Entamoeba moshkovskii or Entamoeba bangladeshi. Therefore, in the last decades, molecular tests that allow correct identification of the causal agent of amoebiasis and the establishment of the prevalence of the infecting species have been developed. Techniques based on nucleic acids, such as conventional, multiplex, or real-time polymerase chain reaction (PCR), are being seriously considered in clinical laboratories, because they detect the etiologic agent directly from the sample without the need for previous prolonged culture, thus reducing diagnostic time. Also, the nested PCR test and the sequencing of ribosomal markers have allowed the identification of new parasitic species in humans, such as E. moshkovskii and E. bangladeshi, and an improved characterization of the known infecting species. The application of multiplex platforms allows the simultaneous identification of infecting species, increasing the sensitivity and specificity of these techniques. Therefore, the molecular diagnosis of amoebiasis is projected as an innovative tool in the fight against this parasitosis.


Development and Testing of a Cognitive Behavioral Therapy Resource for Children's Dental Anxiety.
Cognitive Behavioral Therapy (CBT) is an evidence-based treatment for dental anxiety; however, access to therapy is limited. The current study aimed to develop a self-help CBT resource for reducing dental anxiety in children, and to assess the feasibility of conducting a trial to evaluate the treatment efficacy and cost-effectiveness of such an intervention. A mixed methods design was employed. Within phase 1, a qualitative "person-based" approach informed the development of the self-help CBT resource. This also employed guidelines for the development and evaluation of complex interventions. Within phase 2, children, aged between 9 and 16 y, who had elevated self-reported dental anxiety and were attending a community dental service or dental hospital, were invited to use the CBT resource. Children completed questionnaires, which assessed their dental anxiety and health-related quality of life (HRQoL) prior to and following their use of the resource. Recruitment and completion rates were recorded. Acceptability of the CBT resource was explored using interviews and focus groups with children, parents/carers and dental professionals. For this analysis, the authors adhered to the Mixed Methods Appraisal Tool criteria. There were 24 families and 25 dental professionals participating in the development and qualitative evaluation of the CBT resource for children with dental anxiety. A total of 56 children agreed to trial the CBT resource (66% response rate) and 48 of these children completed the study (86% completion rate). There was a significant reduction in dental anxiety (mean score difference = 7.7, t = 7.9, df = 45, P < 0.001, Cohen's d ES = 1.2) and an increase in HRQoL following the use of the CBT resource (mean score difference = -0.03, t = 2.14, df = 46, P < 0.05, Cohen's d ES = 0.3). The self-help approach had high levels of acceptability to stakeholders. These findings provide preliminary evidence for the effectiveness and acceptability of the resource in reducing dental anxiety in children and support the further evaluation of this approach in a randomized control trial. Knowledge Transfer Statement: This study details the development of a guided self-help Cognitive Behavioral Therapy resource for the management of dental anxiety in children and provides preliminary evidence for the feasibility and acceptability of this approach with children aged between 9 and 16 y. The results of this study will inform the design of a definitive trial to examine the treatment- and cost-effectiveness of the resource for reducing dental anxiety in children.


A fluorescence spectroscopy study on the interactions of the TAT-PTD peptide with model lipid membranes.
Protein-transduction domains (PTDs) have been shown to translocate into and through the living cells in a rapid manner by an as yet unknown mechanism. Regardless of the mechanism of translocation, the first necessary step must be binding of the PTD peptide to the surface of the lipid membrane. We used fluorescence spectroscopy to study the interaction between PTD of the HIV-1 Tat protein (TAT-PTD; residues 47-60 of Tat, fluorescently labeled with tryptophan) and the lipid bilayer labeled with various fluorescence membrane probes. The TAT-PTD tryptophan exhibited a decrease in fluorescence intensity and an increase in anisotropy upon interaction with lipid bilayers. The fluorescence changes were linearly proportional to the density of negative charge in the membrane. Kinetic analysis of the interaction showed two apparent dissociation constants. The value of one dissociation constant (Kd1 = 2.6 +/- 0.6 microM), which accounted for 24% of the interaction, was found to be independent of the negative charge density, suggesting its nonelectrostatic nature. The value of the second dissociation constant (Kd2), which accounted for 76% of the interaction, decreased linearly from 610 +/- 150 to 130 +/- 30 microM with an increase in negative charge density from 0 to 25 mol %, suggesting this interaction is electrostatic in nature. Even though the binding was predominantly electrostatic, it could not be reversed by high salt, indicating the presence of a second, irreversible, step in the interaction with lipid. When TAT-PTD was bound to lipid vesicles labeled with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), fluorescence resonance energy transfer between the tryptophan and the probe occurred at a distance of 3.4 nm. No change in fluorescence anisotropy of either TMA-DPH or DPH was observed upon the interaction with TAT-PTD, indicating no significant disruption or perturbation of the lipid bilayer by the peptide. TAT-PTD did not cause dissipation of membrane potential (165 mV, negative inside). Inclusion of 3% pyrene-labeled phosphatidylglycerol (pyrene-PG) in the membrane revealed that TAT-PTD preferentially bound to the membrane in the liquid state. We conclude that membrane fluidity is an important physicochemical parameter, which may regulate binding of TAT-PTD to the membrane.


Fibronectin adsorption on tantalum: the influence of nanoroughness.
The complex mechanisms of protein adsorption at the solid-liquid interface is of great importance in many research areas, including protein purification, biocompatibility of medical implants, biosensing, and biofouling. The protein adsorption process depends crucially on both the nanoscale chemistry and topography of the interface. Here, we investigate the adsorption of the cell-binding protein fibronectin on flat and nanometer scale rough tantalum oxide surfaces using ellipsometry and quartz crystal microbalance with dissipation (QCM-D). On the flat tantalum oxide surfaces, the interfacial protein spreading causes an increase in the rigidity and a decrease in the thickness of the adsorbed fibronectin layer with decreasing bulk protein concentration. For the tantalum oxide surfaces with well-controlled, stochastic nanometer scale roughness, similar concentration effects are observed for the rigidity of the fibronectin layer and saturated fibronectin uptake. However, we find that the nanorough tantalum oxide surfaces promote additional protein conformational changes, an effect especially apparent from the QCM-D signals, interpreted as an additional stiffening of the formed fibronectin layers.


Smokers at higher risk for undetected antibody for oncogenic human papillomavirus type 16 infection.
To determine the association between tobacco smoking and serologic evidence of human papillomavirus type 16 (HPV16)-specific antibodies among HPV16 DNA-positive women. Baseline health history, physical examination, and laboratory data for 205 HPV16 DNA-positive women with no prior cytologic evidence of squamous intraepithelial lesions who were enrolled subsequently in a randomized clinical trial. HPV16-L1 antibody (anti-HPV16 antibody) detected from serum using RIA or ELISA. Eighty-seven percent (179 of 205) of women tested positive for HPV16 DNA using cervicovaginal swabs or lavage specimens, and 26 women showed similar results using swab specimens of external genitalia alone. HPV16-infected women who reported increasingly greater levels of daily cigarette smoking were less likely to test positive for anti-HPV16 antibodies than nonsmoking women (P = 0.02). Smokers were twice as likely as nonsmokers to test negative for anti-HPV16 antibodies, even after controlling for the effects of other covariates in the analyses (adjusted odds ratio, 0.5; 95% confidence limits, 0.2-0.9). Although Papanicolaou test findings and smoking characteristics were poorly correlated (r(2) = 0.01), women who showed atypical cells of unknown significance or squamous intraepithelial lesion were twice as likely to test anti-HPV16 antibody positive as women who showed normal Papanicolaou tests (adjusted odds ratio, 2.0; 95% confidence limits, 1.1-3.7). These data suggest that smoking may influence the long-term risk for cancer by perturbing early immune responses to the virus and may increase the likelihood of persistent infection. Patient education messages should alert women to this additional risk of smoking. A clinical trial of smoking cessation should be explored as a therapeutic intervention for primary HPV16 infection.


An alternative gonadotropin-releasing hormone (GnRH) RNA splicing product found in cultured GnRH neurons and mouse hypothalamus.
Gonadotropin-releasing hormone (GnRH) is encoded by the proGnRH gene which contains four exons and three introns. In this study, two immortalized GnRH-expressing cell lines (Gn11 and NLT) were characterized. The NLT and Gn11 cells, derived from a same brain tumor in a transgenic mouse, display neuronal morphology and neuron-specific markers. However, NLT cells secrete much higher levels of GnRH than Gn11 cells. To delineate the mechanism underlying this difference, reverse transcriptase-polymerase chain reaction and RNase protection assays were performed to examine proGnRH gene expression. While the mature proGnRH mRNA was predominately expressed in NLT cells, Gn11 cells express an abundant short transcript. Sequence analysis revealed that this short transcript contains exons 1, 3, and 4, but not exon 2, which encodes the GnRH decapeptide. RNase protection assays demonstrated that NLT cells express much higher levels of mature proGnRH mRNA than Gn11 cells. The lower level of GnRH secreting capacity in Gn11 cells is due, in part, to decreased expression of mature proGnRH mRNA. When proGnRH gene expression in the mouse brain was examined, the same short splicing variant was observed in the olfactory area and preoptic area-anterior hypothalamus. But the prevalent transcript in these regions was the mature proGnRH mRNA. In contrast, only the mature proGnRH mRNA was found in the caudal hypothalamus. These results suggest that alternative splicing may be one of the mechanisms regulating proGnRH gene expression in the animal brain.


Sieving of ionic constituents across moving boundaries in gel electrophoresis.
The representative beta-hydroxyethylmorpholinium-chloride-bicinate moving boundary with a trailing ion net mobility relative to Na+ of 0.41, detected by precipitation of chloride with silver nitrate, exhibits a decreasing chloride mobility at increasing polyacrylamide gel concentrations from 3.5 to 45%T, 5%CBis. This decrease, largely due to an increase of field strength at constant current, is described by a convex* plot of log (mobility) vs. %T (Ferguson plot) and signifies that chloride/bicinate are sieved by the gel. In agarose gels, the same plot of mobility vs. gel concentration is constant below 7% gel concentration, since in those gels field strength and migration rate remain the same within that gel concentration range. Both in polyacrylamide and in agarose gels the displacement rate of the chloride-bicinate boundary as a function of the time of electrophoresis or distance migrated remains invariant within 15%. The plot of log (mobility) vs. gel concentration extrapolated to 0%T is 5.85 and 5.41 (10(-5) cm2s-1V-1) for polyacrylamide and for agarose (SeaKem HGT-P,FMC) gels, respectively. The slightly decreased mobility intercept at 0%T for agarose is presumably due either to the electroendosmotic properties of agarose HGT-P and/or failure to Sufficiently take into account the flattening of the Ferguson plot in the polyacrylamide concentration range below 3% in which a transition from a gel to a fluid (sol) medium takes place.


Combining recombineering and ends-out homologous recombination to systematically characterize Drosophila gene families: Rab GTPases as a case study.
Evaluating how an individual gene contributes to a particular biological process benefits greatly from a comprehensive understanding of all members of its gene family. Such knowledge is ideally obtained using multicellular model organisms, which provide rapid and decisive platforms for determining gene function. We recently established a novel transgenesis platform in Drosophila to systematically knock out all members of the Rab small GTPase family of membrane regulators. This platform combines BAC transgenesis/recombineering with ends-out homologous recombinations and Gateway(TM) technologies and provides a new rapid and scalable method that eases the manipulation of endogenous loci. This method not only allows for the generation of molecularly defined lesions, but also the precise replacement or tagging of genes in their endogenous loci. Using this method, we found that up to half of all Rab GTPases exhibit enriched expression at synapses in the nervous system. Here we provide critical details about the underlying recombineering and transgenesis method, new cassettes for tagging endogenous loci and information on important parameters that will allow Drosophila researchers to target members of other gene families.


Identification of regulatory factors for mesenchymal stem cell-derived salivary epithelial cells in a co-culture system.
Patients with Sjögren's syndrome or head and neck cancer patients who have undergone radiation therapy suffer from severe dry mouth (xerostomia) due to salivary exocrine cell death. Regeneration of the salivary glands requires a better understanding of regulatory mechanisms by which stem cells differentiate into exocrine cells. In our study, bone marrow-derived mesenchymal stem cells were co-cultured with primary salivary epithelial cells from C57BL/6 mice. Co-cultured bone marrow-derived mesenchymal stem cells clearly resembled salivary epithelial cells, as confirmed by strong expression of salivary gland epithelial cell-specific markers, such as alpha-amylase, muscarinic type 3 receptor, aquaporin-5, and cytokeratin 19. To identify regulatory factors involved in this differentiation, transdifferentiated mesenchymal stem cells were analyzed temporarily by two-dimensional-gel-electrophoresis, which detected 58 protein spots (>1.5 fold change, p<0.05) that were further categorized into 12 temporal expression patterns. Of those proteins only induced in differentiated mesenchymal stem cells, ankryin-repeat-domain-containing-protein 56, high-mobility-group-protein 20B, and transcription factor E2a were selected as putative regulatory factors for mesenchymal stem cell transdifferentiation based on putative roles in salivary gland development. Induction of these molecules was confirmed by RT-PCR and western blotting on separate sets of co-cultured mesenchymal stem cells. In conclusion, our study is the first to identify differentially expressed proteins that are implicated in mesenchymal stem cell differentiation into salivary gland epithelial cells. Further investigation to elucidate regulatory roles of these three transcription factors in mesenchymal stem cell reprogramming will provide a critical foundation for a novel cell-based regenerative therapy for patients with xerostomia.


A Randomized, Triple-Blind, Comparator-Controlled Parallel Study Investigating the Pharmacokinetics of Cannabidiol and Tetrahydrocannabinol in a Novel Delivery System, Solutech, in Association with Cannabis Use History.
Background: An oral route of administration for tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) eliminates the harmful effects of smoking and has potential for efficacious cannabis delivery for therapeutic and recreational applications. We investigated the pharmacokinetics of CBD, Δ9-THC, 11-OH-THC, and 11-nor-9-carboxy-Δ9-THC (THC-COOH) in a novel oral delivery system, Solutech™, compared to medium-chain triglyceride-diluted cannabis oil (MCT-oil) in a healthy population. Materials and Methods: Thirty-two participants were randomized and divided into two study arms employing a comparator-controlled, parallel-study design. To evaluate the pharmacokinetics of Δ9-THC, CBD, 11-OH-THC, and THC-COOH, blood was collected at pre-dose (t=0) and 10, 20, 30, and 45, min and 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, 24, and 48 h post-dose after a single dose of Solutech (10.0 mg Δ9-THC, 9.76 mg CBD) or MCT (10.0 mg Δ9-THC, 9.92 mg CBD). Heart rate and blood pressure were measured at 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 h. Relationships between cannabis use history, body mass index, sex, and pharmacokinetic parameters were investigated. Safety was assessed before and at 48 h post-acute dose. Results: Acute consumption of Solutech provided a significantly greater maximum concentration (Cmax), larger elimination and absorption rate constants, faster time to Cmax and lag time, and half-life for all analytes compared to MCT-oil (p<0.001). In addition, cannabis use history had a significant influence on the pharmacokinetic parameters of CBD, Δ9-THC, 11-OH-THC, and THC-COOH. On average, participants with later age of first use had higher Δ9-THC, CBD, and THC-COOH Cmax and later time-to-Cmax and half-life for Δ9-THC, CBD, THC-COOH, and 11-OH-THC than those with earlier age of first use (p≤0.032). Those with more years of recreational cannabis use had higher area under the curve for Δ9-THC and CBD, Cmax for CBD, and longer 11-OH-THC half-life than those with less (p≤0.048). Conclusion: This study demonstrated that consumption of Solutech enhanced most pharmacokinetics parameters measured compared to MCT-oil. Participant's cannabis use history, including their age of first use and number of years using cannabis significantly impacted pharmacokinetic parameters investigated. Acute consumption of both products was found to be safe and well tolerated. The results suggest that Solutech may optimize bioavailability from cannabis formulations.