Altered calcium handling in cardiomyocytes from arginine-glycine amidinotransferase-knockout mice is rescued by creatine.
The creatine kinase system facilitates energy transfer between mitochondria and the major ATPases in the heart. Creatine-deficient mice, which lack arginine-glycine amidinotransferase (AGAT) to synthesize creatine and homoarginine, exhibit reduced cardiac contractility. We studied how the absence of a functional CK system influences calcium handling in isolated cardiomyocytes from AGAT-knockouts and wild-type littermates as well as in AGAT-knockout mice receiving lifelong creatine supplementation via the food. Using a combination of whole cell patch clamp and fluorescence microscopy, we demonstrate that the L-type calcium channel (LTCC) current amplitude and voltage range of activation were significantly lower in AGAT-knockout compared with wild-type littermates. Additionally, the inactivation of LTCC and the calcium transient decay were significantly slower. According to our modeling results, these changes can be reproduced by reducing three parameters in knockout mice when compared with wild-type: LTCC conductance, the exchange constant of Ca2+ transfer between subspace and cytosol, and SERCA activity. Because tissue expression of LTCC and SERCA protein were not significantly different between genotypes, this suggests the involvement of posttranslational regulatory mechanisms or structural reorganization. The AGAT-knockout phenotype of calcium handling was fully reversed by dietary creatine supplementation throughout life. Our results indicate reduced calcium cycling in cardiomyocytes from AGAT-knockouts and suggest that the creatine kinase system is important for the development of calcium handling in the heart.NEW & NOTEWORTHY Creatine-deficient mice lacking arginine-glycine amidinotransferase exhibit compromised cardiac function. Here, we show that this is at least partially due to an overall slowing of calcium dynamics. Calcium influx into the cytosol via the L-type calcium current (LTCC) is diminished, and the rate of the sarcoendoplasmic reticulum calcium ATPase (SERCA) pumping calcium back into the sarcoplasmic reticulum is slower. The expression of LTCC and SERCA did not change, suggesting that the changes are regulatory.


Population structure of mixed Mycobacterium tuberculosis infection is strain genotype and culture medium dependent.
Molecular genotyping methods have shown infection with more than one Mycobacterium tuberculosis strain genotype in a single sputum culture, indicating mixed infection. This study aimed to develop a PCR-based genotyping tool to determine the population structure of M. tuberculosis strain genotypes in primary Mycobacterial Growth Indicator Tubes (MGIT) and Löwenstein-Jensen (LJ) cultures to identify mixed infections and to establish whether the growth media influenced the recovery of certain strain genotypes. A convenience sample of 206 paired MGIT and LJ M. tuberculosis cultures from pulmonary tuberculosis patients resident in Khayelitsha, South Africa were genotyped using an in-house PCR-based method to detect defined M. tuberculosis strain genotypes. The sensitivity and specificity of the PCR-based method for detecting Beijing, Haarlem, S-family, and LAM genotypes was 100%, and 75% and 50% for detecting the Low Copy Clade, respectively. Thirty-one (15%) of the 206 cases showed the presence of more than one M. tuberculosis strain genotype. Strains of the Beijing and Haarlem genotypes were significantly more associated with a mixed infection (on both media) when compared to infections with a single strain (Beijing MGIT p = 0.02; LJ, p<0.01) and (Haarlem: MGIT p<0.01; LJ, p = 0.01). Strains with the Beijing genotype were less likely to be with "other genotype" strains (p<0.01) while LAM, Haarlem, S-family and LCC occurred independently with the Beijing genotype. The PCR-based method was able to identify mixed infection in at least 15% of the cases. LJ media was more sensitive in detecting mixed infections than MGIT media, implying that the growth characteristics of M. tuberculosis on different media may influence our ability to detect mixed infections. The Beijing and Haarlem genotypes were more likely to occur in a mixed infection than any of the other genotypes tested suggesting pathogen-pathogen compatibility.


Morphologic and hemodynamic analysis of paraclinoid aneurysms: ruptured versus unruptured.
In order to determine the risk factors related to aneurysm rupture, we studied the aneurysms at the paraclinoid segment of the internal carotid artery by applying morphologic and hemodynamic numerical analyzes. 107 patients with 110 paraclinoid aneurysms (26 ruptured, 84 unruptured) were analyzed using computational fluid dynamics based on patient-specific three-dimensional geometrical models. A series of morphologic and hemodynamic parameters were evaluated to find the potential indicators of aneurysm rupture. Aneurysms with an irregular shape accounted for 23.1% of the ruptured group and only 8.3% of the unruptured group. The difference was statistically significant (p=0.042, χ(2) test). Ruptured paraclinoid aneurysms were found to be significantly smaller than unruptured aneurysms (p=0.041), which is different from the results of most previous studies. Energy loss (EL) and inflow concentration showed a level of statistical significance to assess the risk of rupture in paraclinoid aneurysms. By multivariate logistic regression analysis, aneurysm shape (regular or irregular), EL and inflow concentration were retained as independently significant parameters. The odds of rupture were increased by 1.65 times for a 10% increase in EL, by 4.88 times for an aneurysm with an irregular shape and by 2.91 times for an aneurysm with concentrated inflow jet. Irregular shape, larger EL and concentrated inflow jet were independently associated with the rupture status of paraclinoid aneurysms. These findings need to be further confirmed based on large multicenter and multipopulation data.


Clinical Science Editorial 2020 - a year gone by and the year ahead.
As this extraordinary year, blemished by COVID-19, comes to an end, I look back as Editor-in-Chief to the many great successes and new initiatives of Clinical Science. Despite the challenges we all faced during 2020, our journal has remained strong and vibrant. While we have all adapted to new working conditions, with life very different to what it was pre-COVID-19, the one thing that remains intact and secure is the communication of scientific discoveries through peer-reviewed journals. I am delighted to share with you some of the many achievements of our journal over the past year and to highlight some exciting new activities planned for 2021.


Environmental factors influencing older adults' walking for transportation: a study using walk-along interviews.
Current knowledge on the relationship between the physical environment and walking for transportation among older adults (≥ 65 years) is limited. Qualitative research can provide valuable information and inform further research. However, qualitative studies are scarce and fail to include neighborhood outings necessary to study participants' experiences and perceptions while interacting with and interpreting the local social and physical environment. The current study sought to uncover the perceived environmental influences on Flemish older adults' walking for transportation. To get detailed and context-sensitive environmental information, it used walk-along interviews. Purposeful convenience sampling was used to recruit 57 older adults residing in urban or semi-urban areas. Walk-along interviews to and from a destination (e.g. a shop) located within a 15 minutes' walk from the participants' home were conducted. Content analysis was performed using NVivo 9 software (QSR International). An inductive approach was used to derive categories and subcategories from the data. Data were categorized in the following categories and subcategories: access to facilities (shops & services, public transit, connectivity), walking facilities (sidewalk quality, crossings, legibility, benches), traffic safety (busy traffic, behavior of other road users), familiarity, safety from crime (physical factors, other persons), social contacts, aesthetics (buildings, natural elements, noise & smell, openness, decay) and weather. The findings indicate that to promote walking for transportation a neighborhood should provide good access to shops and services, well-maintained walking facilities, aesthetically appealing places, streets with little traffic and places for social interaction. In addition, the neighborhood environment should evoke feelings of familiarity and safety from crime. Future quantitative studies should investigate if (changes in) these environmental factors relate to (changes in) older adults' walking for transportation.


Relationship between physical activity and chronic musculoskeletal pain among community-dwelling Japanese adults.
Both little and excessive physical activity (PA) may relate to chronic musculoskeletal pain. The primary objective of this study was to characterize the relationship of PA levels with chronic low back pain (CLBP) and chronic knee pain (CKP). We evaluated 4559 adults aged 40-79 years in a community-based cross-sectional survey conducted in 2009 in Shimane, Japan. We used self-administered questionnaires to assess sociodemographics and health status: PA was assessed by the International Physical Activity Questionnaire, and CLBP and CKP were assessed by a modified version of the Knee Pain Screening Tool. We examined relationships of PA with prevalence of CLBP and CKP using Poisson regression, controlling for potential confounders. CLBP and CKP were both prevalent (14.1% and 10.7%, respectively) and associated with history of injury, medication use, and consultation with physicians. PA was not significantly related to CLBP or CKP (P > 0.05) before or after adjustment for potential confounders. For example, compared with adults reporting moderate PA (8.25-23.0 MET-hours/week), prevalence ratios for CKP adjusted for sex, age, education years, self-rated health, depressive symptom, smoking, chronic disease history, and body-mass index were 1.12 (95% confidential interval [CI] 0.84-1.50) among those with the lowest PA and 1.26 (95% CI 0.93-1.70) among those with the highest PA (P quadratic = 0.08). The prevalence ratios were further attenuated toward the null after additional adjustment for history of injury, medication use, and consultation (P quadratic = 0.17). This cross-sectional study showed that there were no significant linear or quadratic relationships of self-reported PA with CLBP and CKP. Future longitudinal study with objective measurements is needed.


Dead-time distortion in fluorescence correlation measurements.
The dead time of the detector significantly distorts the fluorescence correlation function of fluorescent particles in solution. This distortion of the correlation function is similar to the saturation effect of the correlation function in a high-power excitation region. The correlation amplitude is significantly reduced by the dead time. The deviations in the number of molecules and the diffusion time are empirically given by the deviation of the fluorescence intensity linearity. The empirical curves of the deviations can be applied to the systematic error estimation of the parameters. The proportionality of the number of molecules to the concentration of fluorophores is no longer maintained with a large dead time, although almost all of the proportionality of the diffusion time to the inverse diffusion constant remains. This fact makes the dead-time effect different from the saturation effect, which is due to photokinetics. In practice, these distortions can be reduced by use of a smaller excitation power in which the proportionality of the fluorescence intensity is maintained.


Mental Health and Psychosocial Needs of Patients Being Treated for Opioid Use Disorder in a Primary Care Residency Clinic.
Purpose: Primary care is an ideal setting to deliver efficacious treatments for opioid use disorder (OUD). Primary care providers need to be aware of other concerns patients with OUD might have in order to provide comprehensive care. This study describes the prevalence of mental health, comorbid substance use, and psychosocial concerns of patients seeking treatment for OUD in primary care and their relation to 6-month treatment retention. Methods: Patients (N = 100; M age = 34.9 years (SD = 10.8), 74% white, 46% female) with OUD who were starting treatment with buprenorphine at an academic family medicine residency clinic completed surveys of mental health concerns (depression, anxiety, trauma), psychosocial needs (food insecurity, income, transportation, employment), and demographic variables. Chart reviews were conducted to gather information on comorbid substance use, mental health diagnoses, and 6-month treatment retention. Results: Mental health symptoms were highly prevalent in this sample (44% screened positive for anxiety, 31% for depression, and 52% for posttraumatic stress disorder). Three-quarters reported use of illicit substances other than opioids. Many patients also had significant psychosocial concerns, including unemployment (54%), low income (75%), food insecurity (51%), and lacking reliable transportation (64%). Two-thirds (67%) of the sample were retained at 6 months; patients who previously used intravenous opioids were more likely to discontinue treatment (P = .003). Conclusions: Many patients receiving treatment for OUD have significant mental health problems, comorbid substance use, and psychosocial concerns; interestingly, none of these factors predicted treatment retention at 6 months. Primary care clinics would benefit from having appropriate resources, interventions, and referrals for these comorbid issues in order to enhance overall patient well-being and promote recovery.


May omega-3 fatty acid dietary supplementation help reduce severe complications in Covid-19 patients?
In around 10% of SARS-CoV-2 infected patients, coronavirus disease-2019 (Covid-19) symptoms are complicated with a severe lung damage called Acute Respiratory Distress Syndrome (ARDS), which is often lethal. ARDS is mainly associated with an uncontrolled overproduction of immune cells and cytokines, called "cytokine storm syndrome"; it appears 7-15 days following the onset of symptoms, leading to systemic inflammation and multiple organ failure. Because they are well-known metabolic precursors of specialized pro-resolving lipid mediators (SPMs), omega-3 long-chain polyunsaturated fatty acids (omega-3 LC-PUFAs) could help improve the resolution of the inflammatory balance, limiting therefore the level and duration of the critical inflammatory period. Omega-3 LC-PUFAs may also interact at different stages of the viral infection, notably on the virus entry and replication. In the absence of demonstrated treatment and while waiting for vaccine possibility, the use of omega-3 LC-PUFAs deserve therefore to be considered, based on previous clinical studies suggesting that omega-3 supplementation could improve clinical outcomes of critically ill patients at the acute phase of ARDS. In this context, it is crucial to remind that the omega-3 PUFA dietary intake levels in Western countries remains largely below the current recommendations, considering both the omega-3 precursor α-linolenic acid (ALA) and long chain derivatives such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). An optimized omega-3 PUFAs status could be helpful to prevent infectious diseases, including Covid-19.


Breast cancer prevention and control programs in Malaysia.
Breast cancer is the most common cancer in Malaysian females. The National Cancer Registry in 2003 and 2006 reported that the age standardized incidence of breast cancer was 46.2 and 39.3 per 100,000 populations, respectively. With the cumulative risk at 5.0; a woman in Malaysia had a 1 in 20 chance of developing breast cancer in her lifetime. The incidence of cancer in general, and for breast cancer specifically was highest in the Chinese, followed by Indians and Malays. Most of the patients with breast cancers presented at late stages (stage I: 15.45%, stage II: 46.9%, stage III: 22.2% and stage IV: 15.5%). The Healthy Lifestyles Campaign which started in the early nineties had created awareness on breast cancer and after a decade the effort was enhanced with the Breast Health Awareness program to promote breast self examination (BSE) to all women, to perform annual clinical breast examination (CBE) on women above 40 and mammogram on women above 50. The National Health Morbidity Survey in 2006 showed that the prevalence rate of 70.35% by any of three methods of breast screening; 57.1% by BSE, 51.8% by CBE and 7.6% by mammogram. The current screening policy for breast cancer focuses on CBE whereby all women at the age of 20 years and above must undergo breast examination by trained health care providers every 3 years for age between 20-39 years, and annually for age 40 and above. Several breast cancer preventive programs had been developed by various ministries in Malaysia; among which are the RM50 subsidy for mammogram by the Ministry of Women, Family and Community Development and the SIPPS program (a call-recall system for women to do PAP smear and CBE) by the Ministry of Health. Measures to increase uptake of breast cancer screening and factors as to why women with breast cancer present late should be studied to assist in more development of policy on the prevention of breast cancer in Malaysia.


Nuclear export and import of human hepatitis B virus capsid protein and particles.
It remains unclear what determines the subcellular localization of hepatitis B virus (HBV) core protein (HBc) and particles. To address this fundamental issue, we have identified four distinct HBc localization signals in the arginine rich domain (ARD) of HBc, using immunofluorescence confocal microscopy and fractionation/Western blot analysis. ARD consists of four tight clustering arginine-rich subdomains. ARD-I and ARD-III are associated with two co-dependent nuclear localization signals (NLS), while ARD-II and ARD-IV behave like two independent nuclear export signals (NES). This conclusion is based on five independent lines of experimental evidence: i) Using an HBV replication system in hepatoma cells, we demonstrated in a double-blind manner that only the HBc of mutant ARD-II+IV, among a total of 15 ARD mutants, can predominantly localize to the nucleus. ii) These results were confirmed using a chimera reporter system by placing mutant or wild type HBc trafficking signals in the heterologous context of SV40 large T antigen (LT). iii) By a heterokaryon or homokaryon analysis, the fusion protein of SV40 LT-HBc ARD appeared to transport from nuclei of transfected donor cells to nuclei of recipient cells, suggesting the existence of an NES in HBc ARD. This putative NES is leptomycin B resistant. iv) We demonstrated by co-immunoprecipitation that HBc ARD can physically interact with a cellular factor TAP/NXF1 (Tip-associated protein/nuclear export factor-1), which is known to be important for nuclear export of mRNA and proteins. Treatment with a TAP-specific siRNA strikingly shifted cytoplasmic HBc to nucleus, and led to a near 7-fold reduction of viral replication, and a near 10-fold reduction in HBsAg secretion. v) HBc of mutant ARD-II+IV was accumulated predominantly in the nucleus in a mouse model by hydrodynamic delivery. In addition to the revised map of NLS, our results suggest that HBc could shuttle rapidly between nucleus and cytoplasm via a novel TAP-dependent NES.


Green tea catechin, epigallocatechin-3-gallate (EGCG): mechanisms, perspectives and clinical applications.
An expanding body of preclinical evidence suggests EGCG, the major catechin found in green tea (Camellia sinensis), has the potential to impact a variety of human diseases. Apparently, EGCG functions as a powerful antioxidant, preventing oxidative damage in healthy cells, but also as an antiangiogenic and antitumor agent and as a modulator of tumor cell response to chemotherapy. Much of the cancer chemopreventive properties of green tea are mediated by EGCG that induces apoptosis and promotes cell growth arrest by altering the expression of cell cycle regulatory proteins, activating killer caspases, and suppressing oncogenic transcription factors and pluripotency maintain factors. In vitro studies have demonstrated that EGCG blocks carcinogenesis by affecting a wide array of signal transduction pathways including JAK/STAT, MAPK, PI3K/AKT, Wnt and Notch. EGCG stimulates telomere fragmentation through inhibiting telomerase activity. Various clinical studies have revealed that treatment by EGCG inhibits tumor incidence and multiplicity in different organ sites such as liver, stomach, skin, lung, mammary gland and colon. Recent work demonstrated that EGCG reduced DNMTs, proteases, and DHFR activities, which would affect transcription of TSGs and protein synthesis. EGCG has great potential in cancer prevention because of its safety, low cost and bioavailability. In this review, we discuss its cancer preventive properties and its mechanism of action at numerous points regulating cancer cell growth, survival, angiogenesis and metastasis. Therefore, non-toxic natural agent could be useful either alone or in combination with conventional therapeutics for the prevention of tumor progression and/or treatment of human malignancies.


Long-Term Premorbid Blood Pressure and Cerebral Small Vessel Disease Burden on Imaging in Transient Ischemic Attack and Ischemic Stroke.
Background and Purpose- Studies of causes of cerebral small vessel disease (SVD) should fully adjust for blood pressure (BP), but most etiological studies use a single BP measurement or history of hypertension, which might underestimate the role of hypertension. In patients with transient ischemic attack and ischemic stroke, we therefore compared the associations of baseline and long-term premorbid BP with measures of SVD on magnetic resonance imaging brain. Methods- We studied 1009 transient ischemic attack/ischemic stroke patients who had a brain magnetic resonance imaging, in the population-based OXVASC (Oxford Vascular Study), and related baseline and 20-year premorbid BP (median: 15 readings/patient) to the total SVD score on imaging. Results- SVD score was associated with increasing mean baseline systolic BP (SBP; odds ratio of top versus bottom BP quartile: 2.28; [95% CI, 1.62-3.21]; P<0.0001) and with prior hypertension (2.53; [95% CI, 2.01-3.20]; P<0.0001), but the association was much stronger with mean premorbid SBP (6.09; [95% CI, 4.34-8.55]; P<0.0001). Mean diastolic BP at baseline was negatively associated with SVD score (0.71; [95% CI, 0.51-1.00]; P=0.050), and a positive association was only evident for diastolic BP 10 to 20 years previously (3.35; [95% CI, 2.33-4.84]; both P<0.0001). Relationships between overall mean premorbid BP and SVD burden were strongest in patients age <70 (SBP: 6.99; 4.11-11.86; diastolic BP: 3.13; 1.95-5.07; both P<0.0001) versus ≥70 years (2.37; 1.42-3.94; P=0.001; and 1.16; 0.74-1.84; P=0.52). Conclusions- Mean premorbid SBP is more strongly associated with SVD burden than baseline SBP or history of hypertension, and baseline diastolic BP yields a misleading estimate of the likely etiological importance of midlife hypertension for the subsequent development of SVD. Studies of novel potential etiological factors for SVD should aim to adjust for long-term prior BP, and trials of BP lowering with only a few years of follow-up may underestimate the overall impact on SVD.


Abnormal refraction of microwave in ferrite/wire metamaterials.
We report the experimentally observed abnormal refraction in metamaterials (MMs) consisting of ferrite rods and metallic wires with two kinds of configurations. Negative refraction (NR) and positive refraction (PR) are demonstrated in an MM constructed with parallel-arranged rods and wires. The frequencies of both NR and PR can be adjusted dynamically and together by an applied magnetic field and the PR occurs at frequencies slightly lower than that of the NR. The NR is attributed to simultaneously negative effective permittivity and permeability, and the PR is resulted from positive effective permittivity and permeability with the positive effective permittivity originating from electromagnetic coupling between the closest rod and wire. By making the rod cross the wire to reduce the coupling, we observed sole NR in an MM consisting of the cross-arranged rods and wires. Theoretical analysis explained qualitatively the abnormal refraction behaviors of microwave for the two kinds of MMs and it is supported by the retrieved effective parameters and field distributions.


Over 20 Years of Progress in Radiation Oncology: Hodgkin's Disease.
The Patterns of Care Study (PCS) surveys for Hodgkin's disease have documented important correlations between treatment processes and patient outcome. Nationwide improvements in radiotherapy for Hodgkin's disease since 1973, such as routine use of extended fields (subtotal lymphoid irradiation) in patients with early stage disease, individualized blocking, and treatment using linear accelerators, have resulted in greater freedom from relapse and overall survival for patients with stage I/II disease treated with radiotherapy alone. In addition, the introduction of computed tomography along with increased use of chemotherapy in high-risk patients has reduced the use of routine laparotomy and has improved outcome for patients with stage III disease. Overall survival for Hodgkin's disease in the national practice is excellent, reflecting the dissemination of complex treatment programs and radiation therapy technology to the oncologic community at large. Future studies of the national practice will be important in assessing the impact of managed care on workup and other facility practices, as well as evaluating the transfer of new approaches aimed at reducing treatment toxicities.


Diagnostic value of computed tomography in dogs with chronic nasal disease.
Computed tomographic (CT) studies of 80 dogs with chronic nasal disease (nasal neoplasia (n = 19), nasal aspergillosis (n = 46), nonspecific rhinitis (n = 11), and foreign body rhinitis (n = 4)) were reviewed retrospectively by two independent observers. Each observer filled out a custom-designed list to record his or her interpretation of the CT signs and selected a diagnosis. Accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for the diagnosis of each disease. The agreement between observers was evaluated. The CT signs corresponded to those previously described in the literature. CT had an accuracy greater than 90% for each observer in all disease processes. The sensitivity, specificity, PPV, and NPV were greater than 80% in all dogs with the exception of the PPV of foreign body rhinitis (80% for observer A and 44% for observer B). There was a substantial, to almost perfect, agreement between the two observers regarding the CT signs and diagnosis. This study indicates a high accuracy of CT for diagnosis of dogs with chronic nasal disease. The differentiation between nasal aspergillosis restricted to the nasal passages and foreign body rhinitis may be difficult when the foreign body is not visible.


A distal single nucleotide polymorphism alters long-range regulation of the PU.1 gene in acute myeloid leukemia.
Targeted disruption of a highly conserved distal enhancer reduces expression of the PU.1 transcription factor by 80% and leads to acute myeloid leukemia (AML) with frequent cytogenetic aberrations in mice. Here we identify a SNP within this element in humans that is more frequent in AML with a complex karyotype, leads to decreased enhancer activity, and reduces PU.1 expression in myeloid progenitors in a development-dependent manner. This SNP inhibits binding of the chromatin-remodeling transcriptional regulator special AT-rich sequence binding protein 1 (SATB1). Overexpression of SATB1 increased PU.1 expression, and siRNA inhibition of SATB1 downregulated PU.1 expression. Targeted disruption of the distal enhancer led to a loss of regulation of PU.1 by SATB1. Interestingly, disruption of SATB1 in mice led to a selective decrease of PU.1 RNA in specific progenitor types (granulocyte-macrophage and megakaryocyte-erythrocyte progenitors) and a similar effect was observed in AML samples harboring this SNP. Thus we have identified a SNP within a distal enhancer that is associated with a subtype of leukemia and exerts a deleterious effect through remote transcriptional dysregulation in specific progenitor subtypes.


The effects of antireflux therapy on pulmonary function in patients with severe gastroesophageal reflux disease. Department of Veterans Affairs Gastroesophageal Reflux Disease Study Group.
Gastroesophageal reflux can induce bronchospasm, and antireflux therapy has been shown to improve pulmonary function in patients who have gastroesophageal reflux disease (GERD) associated with asthma. Our objective was to study the pulmonary effects of antireflux therapy in patients who had severe GERD without clinically apparent lung disease. In a Department of Veterans Affairs Cooperative Study, patients who had complicated GERD without important lung disease were randomly assigned to receive one of three types of antireflux treatment, including two kinds of medical therapy and a surgical therapy. Patients had pulmonary function tests (PFTs), including total lung capacity, residual volume, forced vital capacity, forced expiratory volume in 1 s, maximal midexpiratory flow, and diffusing capacity for carbon monoxide. Two hundred forty-seven patients (243 men, four women; mean age 58 yr) entered the randomized trial, and 151 returned for PFTs at 1 yr. For the entire study group and for all three treatment groups, mean values for PFTs at 1 yr did not differ significantly from those at baseline. Even in subgroups of patients whose baseline PFTs were abnormal and whose esophagitis had healed completely, there were no significant changes in results of PFTs. For veteran patients with severe GERD and no obvious lung disease, 1 yr of antireflux therapy had no important effect on pulmonary function. These findings suggest that GERD is not commonly associated with inapparent, reversible pulmonary dysfunction.


Light and electron microscope study of cell walls of brown and red algae.
A survey of the structure of the cell walls of green, brown, and red algae, as seen under light and electron microscopes is in progress. In this report a comparison of the cell wall structure of a brown alga, Dictyota flabellata, and a red alga, Helminthocladia californica, is presented. In Dictyota, typicalofthe brown algae, the microfibrillar pattern in the apical cells and in the adjacent cells of the thallus tip is reticulate. In mature cells the microfibrils are dominantly parallel in orientation. Pits, fields ofclosely set pores, are distinctive. The microfibrils in the pit areas are masked by nonfibrillar material. Helminthocladia, with a cell wall characteristic of the red algae, differs from Dictyota in that the microfibrillar pattern is reticulate throughout the thallus. In the pit areas the microfibrils are not masked by amorphous material.


Use of the word schizophrenia in Portuguese newspapers.
Stigmatizing references to schizophrenia have a negative impact on self-esteem, deter treatment seeking and diminish the effectiveness of treatment. To analyze the reporting of schizophrenia in Portuguese newspapers. We analyzed five high circulation Portuguese newspapers between 2007 and 2013. We selected all news containing the word "esquizofrenia" (schizophrenia). Several variables were collected. About 1058 news items contained the word schizophrenia. Schizophrenia was mentioned metaphorically in 40% of the cases and in the context of Crime in 22%. When used in a Criminal context, schizophrenia was mostly attributed to people who were the perpetrators of the crime (93%). When used metaphorically, schizophrenia had a negative connotation in 90% of cases. We found an increasing reporting of schizophrenia in the criminal news and serious crimes. Our results suggest the media has an active role promoting stigma, as well as passively broadcasting and thus passing on prejudices.


Cytosine methylation and mammalian development.
Programmed methylation and demethylation of regulatory sequences has been proposed to play a central role in vertebrate development. We report here that the methylation status of the 5' regions of a panel of tissue-specific genes could not be correlated with expression in tissues of fetal and newborn mice. Genes reported to be regulated by reversible methylation were not expressed ectopically or precociously in Dnmt1-deficient mouse embryos under conditions where demethylation caused biallelic expression of imprinted genes and activated transcription of endogenous retroviruses of the IAP class. These and other data suggest that the numerous published expression-methylation correlations may have described not a cause but a consequence of transcriptional activation. A model is proposed under which cytosine methylation represents a biochemical specialization of large genomes that participates in specialized biological functions such as allele-specific gene expression and the heritable transcriptional silencing of parasitic sequence elements, whereas cellular differentiation is controlled by conserved regulatory networks that do not depend on covalent modification of the genome.


Factors that influence health-seeking behaviors of patients experiencing acute stroke.
Stroke is the third leading cause of death in the United States and the leading cause of serious, long-term disability. Patients who sustain a sudden ischemic stroke can benefit from the administration of intravenous recombinant tissue plasminogen activator (rt-PA). rt-PA may reduce disability, yet few patients arrive in emergency departments in time to be eligible for rt-PA administration. The purpose of this study was to discover factors that influence patients' decision-making process when seeking treatment for acute-stroke symptoms. Qualitative inquiry using grounded-theory methods was used to elicit descriptions of the decision-making process. The primary purpose of grounded theory is to generate explanatory models of human behavior that are grounded in the data. A purposive sample of 10 clinically stable patients or their caregivers were interviewed after acute ischemic stroke. Data were analyzed and broken down into descriptive themes following the grounded-theory approach. Distinct themes emerged for patients who presented for treatment early and late. Five themes emerged for patients who presented late for treatment: lack of knowledge, fear of hospitals, denial, living alone, and preexisting health concerns. The theme that emerged from the group that presented early for treatment was previous history or knowledge of stroke. The most significant implication for practice identified in this study is that nurses must take a leading role in educating the public regarding symptoms, risk factors, and available treatments for stroke.


Inhibin as a marker for granulosa-cell tumors.
Inhibin is a peptide hormone normally produced by ovarian granulosa cells. It reaches a peak of 772 +/- 38 U per liter in the follicular phase of the menstrual cycle and is undetectable in the serum of menopausal women. To determine whether measurements of serum inhibin levels would provide a biochemical marker of the presence or progression of ovarian granulosa-cell tumors and their metastases, we measured the serum immunoreactive inhibin concentrations in six women with such tumors. Three women had been treated by hysterectomy and bilateral salpingo-oophorectomy. In the two women with residual or recurrent disease, the serum inhibin levels were abnormally elevated 5 and 20 months before the clinical manifestations of recurrence became evident. The maximal concentrations approached 3000 U per liter. The serum inhibin level remained undetectable in one patient who was disease-free for 11 years. Serum inhibin concentrations were also elevated in three women with amenorrhea and infertility that resulted from small granulosa-cell tumors. After the removal of the tumors, the serum inhibin levels in these women became normal, and fertility returned. There was a significant negative correlation between the serum concentrations of inhibin and follicle-stimulating hormone, in a manner consistent with the autonomous production of inhibin by granulosa-cell tumors. We conclude that granulosa-cell tumors produce inhibin. Since serum inhibin levels reflect the size of the tumor, measurements of inhibin can be used as a marker for primary as well as recurrent disease.


African midwifery students' self-assessed confidence in antenatal care: a multi-country study.
Background: Evidence-based antenatal care is one cornerstone in Safe Motherhood and educated and confident midwives remain to be optimal caregivers in Africa. Confidence in antenatal midwifery skills is important and could differ depending on the provision of education among the training institutions across Africa.Objective: The aim of the study was to describe and compare midwifery students' confidence in basic antenatal skills, in relation to age, sex, program type and level of program.Methods: A survey in seven sub-Saharan African countries was conducted. Enrolled midwifery students from selected midwifery institutions in each country presented selfreported data on confidence to provide antenatal care. Data were collected using a selfadministered questionnaire. The questionnaire consisted of 22 antenatal skills based on the competency framework from the International Confederation of Midwives. The skills were grouped into three domains; Identify fetal and maternal risk factors and educate parents; Manage and document emergent complications and Physical assessment and nutrition.Results: In total, 1407 midwifery students from seven Sub-Saharan countries responded. Almost one third (25-32%) of the students reported high levels of confidence in all three domains. Direct entry programs were associated with higher levels of confidence in all three domains, compared to post-nursing and double degree programs. Students enrolled at education with diploma level presented with high levels of confidence in two out of three domains.Conclusions: A significant proportion of student midwives rated themselves low on confidence to provide ANC. Midwifery students enrolled in direct entry programs reported higher levels of confidence in all domains. It is important that local governments develop education standards, based on recommendations from the International Confederation of midwives. Further research is needed for the evaluation of actual competence.


Expanding the phenotype of craniofrontonasal syndrome: two unrelated boys with EFNB1 mutations and congenital diaphragmatic hernia.
Craniofrontonasal syndrome (CFNS, MIM 304110) is an X-linked craniofacial disorder that shows paradoxically greater severity in heterozygous females than in hemizygous males. Mutations have been identified in the EFNB1 gene that encodes a member of the ephrin-B family of transmembrane ligands for Eph receptor tyrosine kinases. Here, we describe two unrelated families, in both of which a mother and her son have proven mutations in EFNB1. The mothers have classical features of CFNS; although the sons have no major craniofacial features other than telecanthus, both had a congenital diaphragmatic hernia (CDH). Our cases represent the first in which CDH has been confirmed in males with mutations in EFNB1, highlighting an important role for signalling by ephrin-B1 in the development of the diaphragm.


The neglected bee trees: European beech forests as a home for feral honey bee colonies.
It is a common belief that feral honey bee colonies (Apis mellifera L.) were eradicated in Europe through the loss of habitats, domestication by man and spread of pathogens and parasites. Interestingly, no scientific data are available, neither about the past nor the present status of naturally nesting honeybee colonies. We expected near-natural beech (Fagus sylvatica L.) forests to provide enough suitable nest sites to be a home for feral honey bee colonies in Europe. Here, we made a first assessment of their occurrence and density in two German woodland areas based on two methods, the tracing of nest sites based on forager flight routes (beelining technique), and the direct inspection of potential cavity trees. Further, we established experimental swarms at forest edges and decoded dances for nest sites performed by scout bees in order to study how far swarms from beekeeper-managed hives would potentially move into a forest. We found that feral honey bee colonies regularly inhabit tree cavities in near-natural beech forests at densities of at least 0.11-0.14 colonies/km2. Colonies were not confined to the forest edges; they were also living deep inside the forests. We estimated a median distance of 2,600 m from the bee trees to the next apiaries, while scout bees in experimental swarms communicated nest sites in close distances (median: 470 m). We extrapolate that there are several thousand feral honey bee colonies in German woodlands. These have to be taken in account when assessing the role of forest areas in providing pollination services to the surrounding land, and their occurrence has implications for the species' perception among researchers, beekeepers and conservationists. This study provides a starting point for investigating the life-histories and the ecological interactions of honey bees in temperate European forest environments.


Quantitative phase restoration by direct inversion using the optical transfer function.
Quantitative phase recovery of phase objects is achieved by a direct inversion using the defocused weak object transfer function. The presented method is noniterative and is based on partially coherent principles. It also takes into account the optical properties of the system and gives the phase of the object directly. The proposed method is especially suitable for application to weak phase objects, such as live and unstained biological samples but, surprisingly, has also been shown to work with comparatively strong phase objects.


Evaluation of a Swedish version of the Watts Sexual Function Questionnaire (WSFQ) in persons with heart disease: a pilot study.
As part of preparation for a Swedish multicentre study, exploring sexual and married life in patients with myocardial infarction and their partners, a Swedish validated instrument was required. The aim of this pilot study was to evaluate the validity and reliability of a Swedish version of the Watts Sexual Function Questionnaire (WSFQ) among persons with a heart disease. A convenience sample of 79 persons (47 men and 32 women) living with a heart disease was recruited from the members of the National Association of Heart and Lung Patients. They completed a Swedish version of the WSFQ on two occasions. Two separate factor analyses each revealed a two-factor structure on both occasions: "Sexual appetite" and "Sexual expectations" with gender-neutral questions and "Sexual sensitiveness" and "Sexual ability" with gender-specific questions. Cronbach's alpha coefficients ranged from 0.48 to 0.86 and test-retest values for all but one question exceeded 0.70. The Swedish version of the WSFQ showed good validity and stability and acceptable internal homogeneity. Extended evaluations of the questionnaire are recommended.


CCD camera response to diffraction patterns simulating particle images.
We present a statistical study of CCD (or CMOS) camera response to small images. Diffraction patterns simulating particle images of a size around 2-3 pixels were experimentally generated and characterized using three-point Gaussian peak fitting, currently used in particle image velocimetry (PIV) for accurate location estimation. Based on this peak-fitting technique, the bias and RMS error between locations of simulated and real images were accurately calculated by using a homemade program. The influence of the intensity variation of the simulated particle images on the response of the CCD camera was studied. The experimental results show that the accuracy of the position determination is very good and brings attention to superresolution PIV algorithms. Some tracks are proposed in the conclusion to enlarge and improve the study.


Downregulation of Human DAB2IP Gene Expression in Renal Cell Carcinoma Results in Resistance to Ionizing Radiation.
Renal cell carcinoma (RCC) is known to be highly radioresistant but the mechanisms associated with radioresistance have remained elusive. We found DOC-2/DAB2 interactive protein (DAB2IP) frequently downregulated in RCC, is associated with radioresistance. In this study, we investigated the underlying mechanism regulating radioresistance by DAB2IP and developed appropriate treatment. Several RCC lines with or without DAB2IP expression were irradiated with ionizing radiation (IR) for determining their radiosensitivities based on colony formation assay. To investigate the underlying regulatory mechanism of DAB2IP, immunoprecipitation-mass spectrometry was performed to identify DAB2IP-interactive proteins. PARP-1 expression and enzymatic activity were determined using qRT-PCR, Western blot analysis, and ELISA. In vivo ubiquitination assay was used to test PARP-1 degradation. Furthermore, in vivo mice xenograft model and patient-derived xenograft (PDX) model were used to determine the effect of combination therapy to sensitizing tumors to IR. We notice that DAB2IP-deficient RCC cells acquire IR-resistance. Mechanistically, DAB2IP can form a complex with PARP-1 and E3 ligases that is responsible for degrading PARP-1. Indeed, elevated PARP-1 levels are associated with the IR resistance in RCC cells. Furthermore, PARP-1 inhibitor can enhance the IR response of either RCC xenograft model or PDX model. In this study, we unveil that loss of DAB2IP resulted in elevated PARP-1 protein is associated with IR-resistance in RCC. These results provide a new targeting strategy to improve the efficacy of radiotherapy of RCC.


Inactivation of glutathione S-transferases by nitric oxide-derived oxidants: exploring a role for tyrosine nitration.
Reactive intermediates derived from nitric oxide ((*)NO) are thought to play a contributing role in disease states associated with inflammation and infection. We show here that glutathione S-transferases (GSTs), principal enzymes responsible for detoxification of endogenous and exogenous electrophiles, are susceptible to inactivation by reactive nitrogen species (RNS). Treatment of isolated GSTs or rat liver homogenates with either peroxynitrite, the myeloperoxidase/hydrogen peroxide/nitrite system, or tetranitromethane, resulted in loss of GST activity with a concomitant increase in the formation of protein-associated 3-nitrotyrosine (NO(2)Tyr). This inactivation was only partially (<25%) reversible by dithiothreitol, and exposure of GSTs to hydrogen peroxide or S-nitrosoglutathione was only partially inhibitory (<25%) and did not result in protein nitration. Thus, irreversible modifications such as tyrosine nitration may have contributed to GST inactivation by RNS. Since all GSTs contain a critical, highly conserved, active-site tyrosine residue, we postulated that this Tyr residue might present a primary target for nitration by RNS, thus leading to enzyme inactivation. To directly investigate this possibility, we analyzed purified mouse liver GST-mu, following nitration by several RNS, by trypsin digestion, HPLC separation, and matrix-assisted laser desorption/ionization-time of flight analysis, to determine the degree of tyrosine nitration of individual Tyr residues. Indeed, nitration was found to occur preferentially on several tyrosine residues located in and around the GST active site. However, RNS concentrations that resulted in near complete GST inactivation only caused up to 25% nitration of even preferentially targeted tyrosine residues. Hence, nitration of active-site tyrosine residues may contribute to GST inactivation by RNS, but is unlikely to fully account for enzyme inactivation. Overall, our studies illustrate a potential mechanism by which RNS may promote (oxidative) injury by environmental pollutants in association with inflammation.


The inter-tidal zone is the pathway of input of enterococci to a subtropical recreational marine beach.
Efforts were made to evaluate the source of enterococci levels at a recreational beach. Four monitoring efforts were implemented which included tidal studies, hourly sampling, runoff sampling, and spatially intensive sediment sampling. Spatially intensive sediment sampling indicated that enterococci concentrations consistently decreased away from the inter-tidal zone, both seaward and landward. During dry conditions, the highest concentrations in the water were observed during high tide (71±48 CFU/100 mL) and lower concentrations were observed during low tide (4±3 CFU/100 mL). Runoff was characterised by very high levels (11,700 CFU/100 mL). Results from these monitoring efforts collectively showed that the source of enterococci to the study beach is geographically located within the inter-tidal zone. Wash-in from the inter-tidal zone through tidal action and runoff plays a major role in controlling enterococci levels within the water column. Such results are significant in identifying the source and transport mechanisms of enterococci, which can subsequently be used as part of a modelling effort aimed at predicting enterococci levels at recreational beaches.


Young patients with suspected uncomplicated renal colic are unlikely to have dangerous alternative diagnoses or need emergent intervention.
In the United States there is debate regarding the appropriate first test for new-onset renal colic, with non-contrast helical computed tomography (CT) receiving the highest ratings from both Agency for Healthcare Research and Quality and the American Urological Association. This is based not only on its accuracy for the diagnosis of renal colic, but also its ability to diagnose other surgical emergencies, which have been thought to occur in 10-15% of patients with suspected renal colic, based on previous studies. In younger patients, it may be reasonable to attempt to avoid immediate CT if concern for dangerous alternative diagnosis is low, based on the risks of radiation from CTs, and particularly in light of evidence that patients with renal colic have a very high likelihood of having multiple CTs in their lifetimes. The objective is to determine the proportion of patients with a dangerous alternative diagnosis in adult patients age 50 and under presenting with uncomplicated (non-infected) suspected renal colic, and also to determine what proportion of these patients undergo emergent urologic intervention. Retrospective chart review of 12 months of patients age 18-50 presenting with "flank pain," excluding patients with end stage renal disease, urinary tract infection, pregnancy and trauma. Dangerous alternative diagnosis was determined by CT. Two hundred and ninety-one patients met inclusion criteria. One hundred and fifteen patients had renal protocol CTs, and zero alternative emergent or urgent diagnoses were identified (one-sided 95% CI [0-2.7%]). Of the 291 encounters, there were 7 urologic procedures performed upon first admission (2.4%, 95% CI [1.0-4.9%]). The prevalence of kidney stone by final diagnosis was 58.8%. This small sample suggests that in younger patients with uncomplicated renal colic, the benefit of immediate CT for suspected renal colic should be questioned. Further studies are needed to determine which patients benefit from immediate CT for suspected renal colic, and which patients could undergo alternate imaging such as ultrasound.


Safety and efficacy of CMX001 as salvage therapy for severe adenovirus infections in immunocompromised patients.
No therapeutic agent has yet been established as the definitive therapy for adenovirus infections. We describe the clinical experience of 13 immunocompromised patients who received CMX001 (hexadecyloxypropyl cidofovir), an orally bioavailable lipid conjugate of cidofovir, for adenovirus disease. We retrospectively analyzed 13 patients with adenovirus disease and viremia treated with CMX001; data were available for ≥ 4 weeks after initiation of CMX001 therapy. Virologic response (VR) was defined as a 99% drop from baseline or undetectable adenovirus DNA in serum. The median age of the group was 6 years (range, 0.92-66 years). One patient had severe combined immunodeficiency, 1 patient was a small bowel transplant recipient, and 11 were allogeneic stem cell transplant recipients. Adenovirus disease was diagnosed at a median of 75 days (range, 15-720 days) after transplantation. All patients received i.v. cidofovir for a median of 21 days (range, 5-90 days) before CMX001 therapy. The median absolute lymphocyte count at CMX001 initiation was 300 cells/μL (range, 7-1500 cells/μL). Eight patients (61.5%) had a ≥ 1 log10 drop in viral load after the first week of therapy. By week 8, 9 patients (69.2%) demonstrated a VR, with a median time to achieve VR of 7 days (range, 3-35 days). The change in absolute lymphocyte count was inversely correlated with the change in log10 viral load only at week 6 (r = -0.74; P = .03). Patients with VR had longer survival than those without VR (median 196 days versus 54.5 days; P = .04). No serious adverse events were attributed to CMX001 during therapy. CMX001 may be a promising therapeutic option for the treatment of severe adenovirus disease in immunocompromised patients.


Induction and regulation of conoid extrusion in Toxoplasma gondii.
Cell invasion by the intracellular parasite Toxoplasma gondii occurs through an active process that involves dynamic events, such as gliding motility and conoid extrusion, followed by a sequential secretion from specialized secretory organelles. Increase of intracellular Ca(2+) by ionophores induces conoid extrusion, although in an irreversible way, thus limiting the characterization of the regulatory pathways. In this report we studied the effect of different activating conoid conditions to characterize the regulatory mechanisms involved. Exposure of tachyzoites to ethanol, a well-known activator of microneme secretion through the increase of intracellular Ca(2+), induced conoid extrusion without affecting parasite viability nor its in vitro invasive capability, in a process that could be completely reverted and repeatedly reactivated. A temporal relationship between conoid extrusion and microneme secretion was here studied. Under this condition, signal transduction pathways and the precise role of the parasite cytoskeleton were characterized. Our results indicate that phospholipase C, Ca(2+) released through channels sensitive to inositol-3-phosphate and ryanodine, as well as myosin together with actin filaments, but not microtubules, all participate in conoid extrusion. Specific inhibitors for serine-threonine kinases blocked conoid extrusion; in contrast, calmodulin inhibitors did not affect the induction. A regulatory model for conoid activation is here proposed.


Chemokine gene expression in lung CD8 T cells correlates with protective immunity in mice immunized intra-nasally with Adenovirus-85A.
Immunization of BALB/c mice with a recombinant adenovirus expressing Mycobacterium tuberculosis (M. tuberculosis) antigen 85A (Ad85A) protects against aerosol challenge with M. tuberculosis only when it is administered intra-nasally (i.n.). Immunization with Ad85A induces a lung-resident population of activated CD8 T cells that is antigen dependent, highly activated and mediates protection by early inhibition of M. tuberculosis growth. In order to determine why the i.n. route is so effective compared to parenteral immunization, we used microarray analysis to compare gene expression profiles of pulmonary and splenic CD8 T cells after i.n. or intra-dermal (i.d.) immunization. Total RNA from CD8 T cells was isolated from lungs or spleens of mice immunized with Ad85A by the i.n. or i.d. route. The gene profiles generated from each condition were compared. Statistically significant (p ≤ 0.05) differentially expressed genes were analyzed to determine if they mapped to particular molecular functions, biological processes or pathways using Gene Ontology and Panther DB mapping tools. CD8 T cells from lungs of i.n. immunized mice expressed a large number of chemokines chemotactic for resting and activated T cells as well as activation and survival genes. Lung lymphocytes from i.n. immunized mice also express the chemokine receptor gene Cxcr6, which is thought to aid long-term retention of antigen-responding T cells in the lungs. Expression of CXCR6 on CD8 T cells was confirmed by flow cytometry. Our microarray analysis represents the first ex vivo study comparing gene expression profiles of CD8 T cells isolated from distinct sites after immunization with an adenoviral vector by different routes. It confirms earlier phenotypic data indicating that lung i.n. cells are more activated than lung i.d. CD8 T cells. The sustained expression of chemokines and activation genes enables CD8 T cells to remain in the lungs for extended periods after i.n. immunization. This may account for the early inhibition of M. tuberculosis growth observed in Ad85A i.n. immunized mice and explain the effectiveness of i.n. compared to parenteral immunization with this viral vector.


erbB-2 is a potent oncogene when overexpressed in NIH/3T3 cells.
A wide variety of human tumors contain an amplified or overexpressed erbB-2 gene, which encodes a growth factor receptor-like protein. When erbB-2 complementary DNA was expressed in NIH/3T3 cells under the control of the SV40 promoter, the gene lacked transforming activity despite expression of detectable levels of the erbB-2 protein. A further five- to tenfold increase in its expression under influence of the long terminal repeat of Moloney murine leukemia virus was associated with activation of erbB-2 as a potent oncogene. The high levels of the erbB-2 product associated with malignant transformation of NIH/3T3 cells were observed in human mammary tumor cells that overexpressed this gene. These findings demonstrate a new mechanism for acquisition of oncogenic properties by genes encoding growth factor receptor-like proteins and provide a functional basis for the role of their overexpression in the development of human malignancies.


Role of STAT4 polymorphisms in systemic lupus erythematosus in a Japanese population: a case-control association study of the STAT1-STAT4 region.
Recent studies identified STAT4 (signal transducers and activators of transcription-4) as a susceptibility gene for systemic lupus erythematosus (SLE). STAT1 is encoded adjacently to STAT4 on 2q32.2-q32.3, upregulated in peripheral blood mononuclear cells from SLE patients, and functionally relevant to SLE. This study was conducted to test whether STAT4 is associated with SLE in a Japanese population also, to identify the risk haplotype, and to examine the potential genetic contribution of STAT1. To accomplish these aims, we carried out a comprehensive association analysis of 52 tag single nucleotide polymorphisms (SNPs) encompassing the STAT1-STAT4 region. In the first screening, 52 tag SNPs were selected based on HapMap Phase II JPT (Japanese in Tokyo, Japan) data, and case-control association analysis was carried out on 105 Japanese female patients with SLE and 102 female controls. For associated SNPs, additional cases and controls were genotyped and association was analyzed using 308 SLE patients and 306 controls. Estimation of haplotype frequencies and an association study using the permutation test were performed with Haploview version 4.0 software. Population attributable risk percentage was estimated to compare the epidemiological significance of the risk genotype among populations. In the first screening, rs7574865, rs11889341, and rs10168266 in STAT4 were most significantly associated (P < 0.01). Significant association was not observed for STAT1. Subsequent association studies of the three SNPs using 308 SLE patients and 306 controls confirmed a strong association of the rs7574865T allele (SLE patients: 46.3%, controls: 33.5%, P = 4.9 x 10(-6), odds ratio 1.71) as well as TTT haplotype (rs10168266/rs11889341/rs7574865) (P = 1.5 x 10(-6)). The association was stronger in subgroups of SLE with nephritis and anti-double-stranded DNA antibodies. Population attributable risk percentage was estimated to be higher in the Japanese population (40.2%) than in Americans of European descent (19.5%). The same STAT4 risk allele is associated with SLE in Caucasian and Japanese populations. Evidence for a role of STAT1 in genetic susceptibility to SLE was not detected. The contribution of STAT4 for the genetic background of SLE may be greater in the Japanese population than in Americans of European descent.


Induction of c-Fos and DeltaFosB immunoreactivity in rat brain by Vagal nerve stimulation.
Vagus nerve stimulation (VNS) is used as therapy for treatment-resistant depression or epilepsy. This study used immunohistochemistry for biomarkers of short-term (c-Fos) and long-term (DeltaFosB) neuronal activation to map regions in brain that are activated by acute (2 h) or chronic (3 weeks) VNS in conscious Sprague-Dawley rats. Electrodes (Cyberonics Inc.) were implanted on the left vagus nerve and 1 week after surgery, stimulation began using parameters employed clinically (one burst of 20 Hz, 250 micros pulse width, 0.25 mA stimulation for 30 s every 5 min). Radio telemetry transmitters were used for monitoring blood pressure, heart rate, activity, and respiratory rate during VNS; neither acute nor chronic VNS significantly affected these parameters. Acute VNS significantly increased c-Fos staining in the nucleus of the solitary tract, paraventricular nucleus of the hypothalamus, parabrachial nucleus, ventral bed nucleus of the stria terminalis, and locus coeruleus but not in the cingulate cortex or dorsal raphe nucleus (DRN). Acute VNS did not affect DeltaFosB staining in any region. Chronic VNS significantly increased DeltaFosB and c-Fos staining bilaterally in each region affected by acute VNS as well as in the cingulate cortex and DRN. Using these stimulation parameters, VNS was tested for antidepressant-like activity using the forced swim test (FST). Both VNS and desipramine significantly decreased immobility in the FST; whereas desipramine decreased immobility by increasing climbing behavior, VNS did so by increasing swimming behavior. This study, then, identified potential sites in brain where VNS may produce its clinical effects.


Analgesia, pruritus, and ventilation exhibit a dose-response relationship in parturients receiving intrathecal fentanyl during labor.
Several studies have characterized the 50% and 95% effective doses (ED50 and ED95, respectively) of intrathecal sufentanil for labor analgesia. Few have investigated these same criteria for the less expensive alternative, fentanyl. In addition, the ventilatory effects of intrathecal fentanyl at clinically relevant doses are unclear. We performed this study to establish the dose-response relationship of intrathecal fentanyl for both analgesia and ventilatory depression. Ninety parturients in active early labor (< or = 5 cm dilation) received intrathecal fentanyl 5, 7.5, 10, 15, 20, or 25 micrograms in a double-blinded, randomized fashion (n = 15 patients in each group). Parturients were monitored for degree of pain (measured using a 100-mm visual analog pain scale), blood pressure, arterial oxygen saturation (SaO2), respiratory rate, ETCO2, and fetal heart rate 0, 1, 5, 10, 15, 20, 25, and 30 min after the administration of intrathecal fentanyl. An absolute visual analog pain scale score < or = 25 mm was defined a priori as analgesic success. The percentage of parturients who achieved analgesic success was used to construct quantal dose-response curves, from which the ED50 and ED95 values were derived for the total population (mixed parity) and the nulliparous and multiparous subpopulations separately. Overall ED50 and ED95 values (95% CI) were 5.5 (3.4-7.2) and 17.4 (13.8-27.1) micrograms, respectively. Nulliparous values were lower (5.3 and 15.9 micrograms, respectively) than multiparous values (6.9 and 26.0 micrograms, respectively) but were within the 95% CIs of the total population. Pruritus incidence in parturients with analgesic success displayed a dose-response relationship identical to that seen for analgesia. ETCO2 displayed a dose-related increase, particularly at doses > or = 15 micrograms, without concomitant changes in respiratory rate or SaO2, which suggests a decrease in tidal volume. Even in the absence of overt signs or symptoms of somnolence, intrathecal fentanyl at doses within the effective analgesic range induced a change in ventilation that may last longer than the 30-min period we studied. Intrathecal fentanyl induces rapid and satisfying dose-dependent analgesia in early labor; however, it also produces dose-related decreases in ventilation in the absence of overt somnolence.


Multiple sclerosis: risk factors, prodromes, and potential causal pathways.
Multiple sclerosis (MS) is a common, complex neurological disease. The precise aetiology of MS is not yet known, although epidemiological data indicate that both genetic and environmental factors are important. The evidence that the environment acts long before MS becomes clinically evident is well established and suggests the existence of a prodromal phase for the disease. The increasing incidence of MS emphasises the need for strategies to prevent this chronic disorder, and the possibility of a prodrome indicates a window of opportunity to potentially reverse early disease processes before clinical disease becomes evident. Studying a prodrome requires techniques other than clinical observation such as monitoring endophenotypes that result from associated risk factors. However, our current knowledge of causal pathways and endophenotypes in MS is limited. Identifying and studying individuals with a high risk of developing the disease provides a powerful opportunity to understand the MS causal cascade and is highly relevant to strategies that are aimed at preventing this debilitating disease.


Chronic myelogenous leukemia shapes host immunity by selective deletion of high-avidity leukemia-specific T cells.
We have shown that cytotoxic T lymphocytes specific for PR1, an HLA-A2-restricted nonopeptide derived from proteinase 3, kill leukemia cells and may contribute to the elimination of chronic myelogenous leukemia (CML) after treatment with IFN or allogeneic bone marrow transplant. Some patients with persistent disease also have circulating PR1-specific T cells, however, suggesting the likelihood of immune tolerance. Here we show that both high- and low-avidity PR1-specific T cells from the peripheral blood of healthy donors can be identified and selectively expanded in vitro. Although high-avidity PR1-specific T cells killed CML more effectively than low-avidity T cells, only high-avidity T cells underwent apoptosis when stimulated with high PR1 peptide concentration or when exposed to leukemia that overexpressed proteinase 3. No high-avidity PR1-specific T cells could be identified or expanded from newly diagnosed leukemia patients, whereas low-avidity T cells were readily expanded. Circulating high-avidity PR1-specific T cells were identified in IFN-sensitive patients in cytogenetic remission, however. These results provide evidence that CML shapes the host immune response and that leukemia outgrowth may result in part from leukemia-induced selective deletion of high-avidity PR1-specific T cells.


Molecular basis of fast inactivation in voltage and Ca2+-activated K+ channels: a transmembrane beta-subunit homolog.
Voltage-dependent and calcium-sensitive K+ (MaxiK) channels are key regulators of neuronal excitability, secretion, and vascular tone because of their ability to sense transmembrane voltage and intracellular Ca2+. In most tissues, their stimulation results in a noninactivating hyperpolarizing K+ current that reduces excitability. In addition to noninactivating MaxiK currents, an inactivating MaxiK channel phenotype is found in cells like chromaffin cells and hippocampal neurons. The molecular determinants underlying inactivating MaxiK channels remain unknown. Herein, we report a transmembrane beta subunit (beta2) that yields inactivating MaxiK currents on coexpression with the pore-forming alpha subunit of MaxiK channels. Intracellular application of trypsin as well as deletion of 19 N-terminal amino acids of the beta2 subunit abolished inactivation of the alpha subunit. Conversely, fusion of these N-terminal amino acids to the noninactivating smooth muscle beta1 subunit leads to an inactivating phenotype of MaxiK channels. Furthermore, addition of a synthetic N-terminal peptide of the beta2 subunit causes inactivation of the MaxiK channel alpha subunit by occluding its K+-conducting pore resembling the inactivation caused by the "ball" peptide in voltage-dependent K+ channels. Thus, the inactivating phenotype of MaxiK channels in native tissues can result from the association with different beta subunits.


AHNAK: the giant jack of all trades.
The nucleoprotein AHNAK is an unusual and somewhat mysterious scaffolding protein characterised by its large size of approximately 700 kDa. Several aspects of this protein remain uncertain, including its exact molecular function and regulation on both the gene and protein levels. Various studies have attempted to annotate AHNAK and, notably, protein interaction and expression analyses have contributed greatly to our current understanding of the protein. The implicated biological processes are, however, very diverse, ranging from a role in the formation of the blood-brain barrier, cell architecture and migration, to the regulation of cardiac calcium channels and muscle membrane repair. In addition, recent evidence suggests that AHNAK might be yet another accomplice in the development of tumour metastasis. This review will discuss the different functional roles of AHNAK, highlighting recent advancements that have added foundation to the proposed roles while identifying ties between them. Implications for related fields of research are noted and suggestions for future research that will assist in unravelling the function of AHNAK are offered.


Activation of P2Y1 and P2Y2 receptors induces chloride secretion via calcium-activated chloride channels in kidney inner medullary collecting duct cells.
Dysregulation of urinary sodium chloride (NaCl) excretion can result in extracellular fluid (ECF) volume expansion and hypertension. Recent studies demonstrated that urinary nucleotide excretion increases in mice ingesting a high-salt diet and that these increases in extracellular nucleotides can signal through P2Y(2) receptors in the kidney collecting duct to inhibit epithelial Na(+) channels (ENaC). However, under conditions of ECF volume expansion brought about by high-dietary salt intake, ENaC activity should already be suppressed. We hypothesized that alternative pathways exist by which extracellular nucleotides control renal NaCl excretion. We used an inner medullary collecting duct (mIMCD-K2) cell line in an Ussing chamber system as a model to study additional ion transport pathways that are regulated by extracellular nucleotides. When ENaC was inhibited, the addition of adenosine triphosphate (ATP) to the basal side of cell sheets activated both P2Y(1) and P2Y(2) receptors, inducing a transient increase in short-circuit current (I(sc)); addition of ATP to the apical side activated only P2Y(2) receptors, inducing first a transient and then a sustained increase in I(sc). The ATP-induced increases in I(sc) were blocked by pretreatment with a phospholipase C (PLC) inhibitor, a calcium (Ca(2+)) chelator, or Ca(2+)-activated Cl(-) channel (CACC) inhibitors, suggesting that ATP signals through both PLC and intracellular Ca(2+) to activate CACC. We propose that P2Y(1) and P2Y(2) receptors operate in tandem in IMCD cells to provide an adaptive mechanism for enhancing urinary NaCl excretion in the setting of high-dietary NaCl intake.


[Single-blind study of the efficacy of pirozadil versus alufibrate in the treatment of primary hyperlipoproteinemias].
Pirozadil and alufibrate are compared by a single-blind study over two groups 20 patients with primary hyperlipoproteinemia. Pirozadil seems more effective as a cholesterol-lowering agent. There were no adverse reactions. We conclude that pirozadil can be a valuable agent in preventing atherosclerotic disease.


Genomic analysis of the function of the transcription factor gata3 during development of the mammalian inner ear.
We have studied the function of the zinc finger transcription factor gata3 in auditory system development by analysing temporal profiles of gene expression during differentiation of conditionally immortal cell lines derived to model specific auditory cell types and developmental stages. We tested and applied a novel probabilistic method called the gamma Model for Oligonucleotide Signals to analyse hybridization signals from Affymetrix oligonucleotide arrays. Expression levels estimated by this method correlated closely (p<0.0001) across a 10-fold range with those measured by quantitative RT-PCR for a sample of 61 different genes. In an unbiased list of 26 genes whose temporal profiles clustered most closely with that of gata3 in all cell lines, 10 were linked to Insulin-like Growth Factor signalling, including the serine/threonine kinase Akt/PKB. Knock-down of gata3 in vitro was associated with a decrease in expression of genes linked to IGF-signalling, including IGF1, IGF2 and several IGF-binding proteins. It also led to a small decrease in protein levels of the serine-threonine kinase Akt2/PKBbeta, a dramatic increase in Akt1/PKBalpha protein and relocation of Akt1/PKBalpha from the nucleus to the cytoplasm. The cyclin-dependent kinase inhibitor p27(kip1), a known target of PKB/Akt, simultaneously decreased. In heterozygous gata3 null mice the expression of gata3 correlated with high levels of activated Akt/PKB. This functional relationship could explain the diverse function of gata3 during development, the hearing loss associated with gata3 heterozygous null mice and the broader symptoms of human patients with Hearing-Deafness-Renal anomaly syndrome.


In utero exposure to maternal tobacco smoke and subsequent obesity, hypertension, and gestational diabetes among women in the MoBa cohort.
Environmental factors influencing the developmental origins of health and disease need to be identified and investigated. In utero exposure to tobacco smoke has been associated with obesity and a small increase in blood pressure in children; however, whether there is a corresponding increased risk of conditions such as diabetes and hypertension during adulthood remains unclear. Our goal was to assess the association of self-reported in utero exposure to tobacco smoke with the prevalence of obesity, hypertension, type 2 diabetes mellitus (T2DM), and gestational diabetes mellitus (GDM) in women 14-47 years of age. We conducted a cross-sectional analysis of the Norwegian Mother and Child Cohort Study, which enrolled pregnant women in Norway from 1999 thorough 2008. Exposure to tobacco smoke in utero (yes vs. no) was ascertained on the baseline questionnaire (obtained at ~ 17 weeks' gestation); the outcomes were ascertained from the Medical Birth Registry of Norway and the questionnaire. Our analysis included 74,023 women. Women exposed to tobacco smoke in utero had 1.53 times the odds of obesity [95% confidence interval (CI): 1.45, 1.61] relative to those unexposed, after adjusting for age, education, and personal smoking. After further adjustment for body mass index, the odds ratio for hypertension was 1.68 (95% CI: 1.19, 2.39); for T2DM 1.14 (95% CI: 0.79, 1.65); and for GDM 1.32 (95% CI: 1.10, 1.58) among exposed compared with unexposed. Exposure to tobacco smoke in utero was associated with obesity, hypertension, and GDM in adult women. The possibility that the associations were attributable to unmeasured confounding cannot be excluded.


Cerebrospinal fluid markers including trefoil factor 3 are associated with neurodegeneration in amyloid-positive individuals.
We aimed to identify cerebrospinal fluid (CSF) biomarkers associated with neurodegeneration in individuals with and without CSF evidence of Alzheimer pathology. We investigated 287 Alzheimer's Disease Neuroimaging Initiative (ADNI) subjects (age=74.9±6.9; 22/48/30% with Alzheimer's disease/mild cognitive impairment/controls) with CSF multiplex analyte data and serial volumetric MRI. We calculated brain and hippocampal atrophy rates, ventricular expansion and Mini Mental State Examination decline. We used false discovery rate corrected regression analyses to assess associations between CSF variables and atrophy rates in individuals with and without amyloid pathology, adjusting in stages for tau, baseline volume, p-tau, age, sex, ApoE4 status and diagnosis. Analytes showing statistically significant independent relationships were entered into reverse stepwise analyses. Adjusting for tau, baseline volume, p-tau, age, sex and ApoE4, 4/83 analytes were significantly independently associated with brain atrophy rate, 1/83 with ventricular expansion and 2/83 with hippocampal atrophy. The strongest CSF predictor for the three atrophy measures was low trefoil factor 3 (TFF3). High cystatin C (CysC) was associated with higher whole brain atrophy and hippocampal atrophy rates. Lower levels of vascular endothelial growth factor and chromogranin A (CrA) were associated with higher whole brain atrophy. In exploratory reverse stepwise analyses, lower TFF3 was associated with higher rates of whole brain, hippocampal atrophy and ventricular expansion. Lower levels of CrA were associated with higher whole brain atrophy rate. The relationship between low TFF3 and increased hippocampal atrophy rate remained after adjustment for diagnosis. We identified a series of CSF markers that are independently associated with rate of neurodegeneration in amyloid-positive individuals. TFF3, a substrate for NOTCH processing may be an important biomarker of neurodegeneration across the Alzheimer spectrum.


Growing Ultra-flat Organic Films on Graphene with a Face-on Stacking via Moderate Molecule-Substrate Interaction.
The electronic properties of small molecule organic crystals depend heavily on the molecular orientation. For multi-layer organic photovoltaics, it is desirable for the molecules to have a face-on orientation in order to enhance the out-of-plane transport properties. However, it is challenging to grow well-ordered and smooth films with a face-on stacking on conventional substrates such as metals and oxides. In this work, metal-phthalocyanine molecules is used as a model system to demonstrate that two-dimensional crystals such as graphene can serve as a template for growing high quality, ultra-flat organic films with a face-on orientation. Furthermore, the molecule-substrate interaction is varied systematically from strong to weak interaction regime with the interaction strength characterized by ultrafast electron transfer measurements. We find that in order to achieve the optimum orientation and morphology, the molecule-substrate interaction needs to be strong enough to ensure a face-on stacking while it needs to be weak enough to avoid film roughening.


Sawtooth pacing by real-time auxiliary power control in a tokamak plasma.
In the standard scenario of tokamak plasma operation, sawtooth crashes are the main perturbations that can trigger performance-degrading, and potentially disruption-generating, neoclassical tearing modes. This Letter demonstrates sawtooth pacing by real-time control of the auxiliary power. It is shown that the sawtooth crash takes place in a reproducible manner shortly after the removal of that power, and this can be used to precisely prescribe, i.e., pace, the individual sawteeth. In combination with preemptive stabilization of the neoclassical tearing modes, sawtooth pacing provides a new sawtooth control paradigm for improved performance in burning plasmas.


Health-related quality of life and disease specific symptoms in long-term thyroid cancer survivors: a study from the population-based PROFILES registry.
Given the longevity of thyroid cancer patients, any impairment in health-related quality of life (HRQoL) during the follow-up period is of considerable concern. Therefore, the first aim of this study was to assess (thyroid cancer specific) HRQoL among long-term thyroid cancer survivors and to compare this with the HRQoL of an age- and sex-matched normative population. Secondly, our aim was to investigate which clinical and socio-demographic characteristics and thyroid cancer specific problems were associated with HRQoL. All patients diagnosed with thyroid cancer between 1990 and 2008, as registered in the Eindhoven Cancer Registry, received a survey on HRQoL (EORTC QLQ-C30) and disease-specific symptoms (THYCA-QoL). The scores were compared with age- and sex-matched cancer free controls (n = 800). A series of multiple linear regression analyses were conducted to investigate the independent associations between clinical, socio-demographic and thyroid cancer specific factors with HRQoL. A total of 306 patients (86%) responded to the invitation. Thyroid cancer survivors had significantly lower scores on physical, role, emotional, cognitive and social functioning (p < 0.001) compared to the normative population after adjusting for comorbidities. Sympathetic problems [feeling chilly (52%), hot flushes (40%)], neuromuscular problems [cramp legs (43%) and pain joints/muscles (64%)] and abrupt attacks of fatigue (50%) were the most often reported thyroid cancer specific complaints. Thyroid cancer specific neuromuscular, concentration, sympathetic and psychological problems explained 41-58% of the variance in HRQoL. Clinical and socio-demographic factors explained a small part of the variance in (thyroid cancer specific) HRQoL (1-27%). Long-term thyroid cancer survivors experience more symptoms and deteriorated HRQoL compared to the normative population. Thyroid cancer specific neuromuscular, sympathetic, concentration and psychological symptoms are stronger associated with HRQoL than clinical and socio-demographic factors alone. Awareness of these specific determinants of HRQoL could help health care practitioners to provide better supportive care.


Differential modulation of doxorubicin toxicity to multidrug and intrinsically drug resistant cell lines by anti-oestrogens and their major metabolites.
The ability of the anti-oestrogens tamoxifen, toremifene and their 4-hydroxy and N-desmethyl metabolites to modify doxorubicin (dox) toxicity to intrinsically resistant and multidrug resistant cell lines was compared, using human breast and lung cancer, and Chinese hamster ovary cell lines. The anti-oestrogens significantly enhanced dox toxicity to multidrug resistant, P-glycoprotein-positive cell lines, but did not affect toxicity to intrinsically resistant, P-glycoprotein-negative cells. Modification was observed at clinically achievable anti-oestrogen concentrations. Toremifene and tamoxifen would therefore appear to be good candidates for in vivo studies as MDR modulating agents in selected patients with P-glycoprotein-positive tumours.


Psychosocial Adjustment and Perceived Risk Among Adolescent Girls From Families With BRCA1/2 or Breast Cancer History.
To evaluate the impact of breast cancer family history and maternal BRCA1/2 mutation on the psychosocial adjustment and perceived risk in girls age 11 to 19 years old. Girls age 11 to 19 years old with one or more relatives with breast cancer or a familial BRCA1/2 mutation (breast cancer family history [BCFH] positive, n = 208; n = 69 with BRCA1/2-positive mother), peers (BCFH negative, n = 112), and their mothers completed assessments of psychosocial adjustment, breast cancer-specific distress, and perceived risk of breast cancer. General psychosocial adjustment did not differ significantly between BCFH-positive and BCFH-negative girls, either by self-report or mother report, except for higher self-esteem among BCFH-positive girls (P = .01). BCFH-positive girls had higher breast cancer-specific distress than BCFH-negative girls (P < .001), but girls from BRCA1/2-positive families did not differ from other BCFH-positive peers. BCFH-positive girls were more likely to report themselves at increased self-risk for breast cancer in adulthood than BCFH-negative peers (74% v 33%, respectively; P ≤ .001). Girls from BRCA1/2-positive families were more likely than other BCFH-positive and BCFH-negative peers to report themselves at increased risk (P < .001). In all groups, perceived risk of breast cancer was associated with older age. Higher breast cancer-specific distress among adolescent girls was associated with higher self-perceived risk of breast cancer and higher maternal breast cancer-specific distress. Adolescent girls from BRCA1/2-positive and breast cancer families have higher self-esteem and do not have poorer psychosocial adjustment than peers. However, they do experience greater breast cancer-specific distress and perceived risk of breast cancer, particularly among older girls. Understanding the impact is important to optimize responses to growing up in families at familial and genetic risk for breast cancer, particularly given the debate over the genetic testing of children for cancer susceptibility in adulthood.


Lineage-Specific Changes in Biomarkers in Great Apes and Humans.
Although human biomedical and physiological information is readily available, such information for great apes is limited. We analyzed clinical chemical biomarkers in serum samples from 277 wild- and captive-born great apes and from 312 healthy human volunteers as well as from 20 rhesus macaques. For each individual, we determined a maximum of 33 markers of heart, liver, kidney, thyroid and pancreas function, hemoglobin and lipid metabolism and one marker of inflammation. We identified biomarkers that show differences between humans and the great apes in their average level or activity. Using the rhesus macaques as an outgroup, we identified human-specific differences in the levels of bilirubin, cholinesterase and lactate dehydrogenase, and bonobo-specific differences in the level of apolipoprotein A-I. For the remaining twenty-nine biomarkers there was no evidence for lineage-specific differences. In fact, we find that many biomarkers show differences between individuals of the same species in different environments. Of the four lineage-specific biomarkers, only bilirubin showed no differences between wild- and captive-born great apes. We show that the major factor explaining the human-specific difference in bilirubin levels may be genetic. There are human-specific changes in the sequence of the promoter and the protein-coding sequence of uridine diphosphoglucuronosyltransferase 1 (UGT1A1), the enzyme that transforms bilirubin and toxic plant compounds into water-soluble, excretable metabolites. Experimental evidence that UGT1A1 is down-regulated in the human liver suggests that changes in the promoter may be responsible for the human-specific increase in bilirubin. We speculate that since cooking reduces toxic plant compounds, consumption of cooked foods, which is specific to humans, may have resulted in relaxed constraint on UGT1A1 which has in turn led to higher serum levels of bilirubin in humans.


Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells.
It is here reported that mesenchymal stem cells known to give rise to limb-bud mesoderm can, at the single-cell level, also differentiate into cells of visceral mesoderm and can be expanded extensively by means of clinically applicable methods. These cells were named mesodermal progenitor cells (MPCs). MPCs were selected by depleting bone marrow mononuclear cells from more than 30 healthy human donors of CD45(+)/glycophorin-A (GlyA)(+) cells. Cells were cultured on fibronectin with epidermal growth factor and platelet-derived growth factor BB and 2% or less fetal calf serum. It was found that 1/5 x 10(3) CD45(-)GlyA(-) cells, or 1/10(6) bone marrow mononuclear cells, gave rise to clusters of small adherent cells. Cell-doubling time was 48 to 72 hours, and cells have been expanded in culture for more than 60 cell doublings. MPCs are CD34(-), CD44(low), CD45(-), CD117 (cKit)(-), class I-HLA(-), and HLA-DR(-). MPCs differentiated into cells of limb-bud mesoderm (osteoblasts, chondrocytes, adipocytes, stroma cells, and skeletal myoblasts) as well as visceral mesoderm (endothelial cells). Retroviral marking was used to definitively prove that single MPCs can differentiate into cells of limb bud and visceral mesoderm. Thus, MPCs that proliferate without obvious senescence under clinically applicable conditions and differentiate at the single-cell level not only into mesenchymal cells but also cells of visceral mesoderm may be an ideal source of stem cells for treatment of genetic or degenerative disorders affecting cells of mesodermal origin.


Radiosensitization of glioma cells by modulation of Met signalling with the hepatocyte growth factor neutralizing antibody, AMG102.
The hepatocyte growth factor (HGF)/Met signalling pathway is up-regulated in many cancers, with downstream mediators playing a role in DNA double strand break repair. Previous studies have shown increased radiosensitization of tumours through modulation of Met signalling by genetic methods. We investigated the effects of the anti-HGF monoclonal antibody, AMG102, on the response to ionizing radiation in a model of glioblastoma multiforme in vitro and in vivo. Radiosensitivity was evaluated in vitro in the U-87 MG human glioma cell line. Met activation was measured by Western blot, and the effect on survival following radiation was evaluated by clonogenic assay. Mechanism of cell death was evaluated by apoptosis and mitotic catastrophe assays. DNA damage was quantitated by γH2AX foci and neutral comet assay. Growth kinetics of subcutaneous tumours was used to assess the effects of AMG102 on in vivo tumour radiosensitivity. AMG102 inhibited Met activation after irradiation. An enhancement of radiation cell killing was shown with no toxicity using drug alone. Retention of γH2AX foci at 6 and 24 hrs following the drug/radiation combination indicated an inhibition of DNA repair following radiation, and comet assay confirmed DNA damage persisting over the same duration. At 48 and 72 hrs following radiation, a significant increase of cells undergoing mitotic catastrophe was seen in the drug/radiation treated cells. Growth of subcutaneous tumours was slowed in combination treated mice, with an effect that was greater than additive for each modality individually. Modulation of Met signalling with AMG102 may prove a novel radiation sensitizing strategy. Our data indicate that DNA repair processes downstream of Met are impaired leading to increased cell death through mitotic catastrophe.


Regulatory T lymphocytes from ALS mice suppress microglia and effector T lymphocytes through different cytokine-mediated mechanisms.
Activated microglia and infiltrating lymphocytes are neuropathological hallmarks of amyotrophic lateral sclerosis (ALS), a fatal motoneuron disease. Although both cell types play pivotal roles in the ALS pathogenic process, the interactions between microglia and lymphocytes, specifically regulatory CD4+CD25High T lymphocytes (Tregs) and cytotoxic CD4+CD25- T lymphocytes (Teffs), have not been addressed. When co-cultured with mSOD1 adult microglia, mSOD1 Tregs suppressed the cytotoxic microglial factors NOX2 and iNOS through an IL-4-mediated mechanism, whereas Teffs were only minimally effective; IL-4 inhibitory antibodies blocked the suppressive function of mSOD1 Tregs, and conditioned media from mSOD1 Tregs or the addition of IL-4 reduced microglial NOX2 expression. During the stable disease phase, the total number of Tregs, specifically the numbers of CD4+CD25HighIL-4+, CD4+CD25HighIL-10+ and CD4+CD25HighTGF-β+ Tregs, were increased in ALS mice compared with WT mice; Tregs isolated during this phase reduced Teff proliferation. In contrast, during the rapidly progressing phase, the number of mSOD1 Tregs decreased while the proliferation of mSOD1 Teffs increased. The combination of IL-4, IL-10, and TGF-β was required to inhibit the proliferation of mSOD1 Teffs by mSOD1 Tregs that were isolated during the slow phase, while inhibition of mSOD1 Teffs by mSOD1 Tregs during the rapid phase, as well as WT Teffs, was not dependent on these factors. Thus, mSOD1 Tregs at the slow phase suppressed microglial toxicity and SOD1 Teff proliferation through different mechanisms; microglial activation was suppressed through IL-4 whereas mSOD1 Teffs were suppressed by IL-4, IL-10 and TGF-β. These data suggest that mSOD1 Tregs contribute to the slowly progressing phase in ALS mice and may offer a novel therapeutic option for ALS patients.


Cell parameter measurement of a twisted nematic liquid crystal device using interferometric polarimeter under normal incidence.
Five cell parameters of a twisted nematic liquid crystal device (TNLCD), namely, cell gap, pretilt angle, twisted angle, rubbing angle, and phase retardation are precisely measured by the developed amplitude-sensitive heterodyne polarimeter (ASHP) simultaneously integrated with Yeh and Gu's transfer matrix and Lien's transfer matrix. This proposed method can characterize the five cell parameters under the arrangement of a single wavelength at normal incidence. In contrast to the conventional methods on cell parameter detection either by adopting a multiple wavelength laser beam at normal incidence or by using a single wavelength laser beam under oblique incident to TNLCD, this method presents the advantage of not only having a simple setup but also the possibility to measure simultaneously five cell parameters on the characterization of TNLCD at high speed.


Genome-wide association scan identifies a risk locus for preeclampsia on 2q14, near the inhibin, beta B gene.
Elucidating the genetic architecture of preeclampsia is a major goal in obstetric medicine. We have performed a genome-wide association study (GWAS) for preeclampsia in unrelated Australian individuals of Caucasian ancestry using the Illumina OmniExpress-12 BeadChip to successfully genotype 648,175 SNPs in 538 preeclampsia cases and 540 normal pregnancy controls. Two SNP associations (rs7579169, p = 3.58×10(-7), OR = 1.57; rs12711941, p = 4.26×10(-7), OR = 1.56) satisfied our genome-wide significance threshold (modified Bonferroni p<5.11×10(-7)). These SNPs reside in an intergenic region less than 15 kb downstream from the 3' terminus of the Inhibin, beta B (INHBB) gene on 2q14.2. They are in linkage disequilibrium (LD) with each other (r(2) = 0.92), but not (r(2)<0.80) with any other genotyped SNP ±250 kb. DNA re-sequencing in and around the INHBB structural gene identified an additional 25 variants. Of the 21 variants that we successfully genotyped back in the case-control cohort the most significant association observed was for a third intergenic SNP (rs7576192, p = 1.48×10(-7), OR = 1.59) in strong LD with the two significant GWAS SNPs (r(2)>0.92). We attempted to provide evidence of a putative regulatory role for these SNPs using bioinformatic analyses and found that they all reside within regions of low sequence conservation and/or low complexity, suggesting functional importance is low. We also explored the mRNA expression in decidua of genes ±500 kb of INHBB and found a nominally significant correlation between a transcript encoded by the EPB41L5 gene, ∼250 kb centromeric to INHBB, and preeclampsia (p = 0.03). We were unable to replicate the associations shown by the significant GWAS SNPs in case-control cohorts from Norway and Finland, leading us to conclude that it is more likely that these SNPs are in LD with as yet unidentified causal variant(s).


Prefrontal Cortical Activation With Open and Closed-Loop Tactile Cueing When Walking and Turning in Parkinson Disease: A Pilot Study.
Gait and turning impairments are common in people with Parkinson disease (PwPD). Tactile cues delivered in open- or closed-loop modalities may improve gait and turning in PwPD, but underlying mechanisms are unclear. Attention stemming from the prefrontal cortex (PFC) may play a role in cue response, but PFC contributions to specific cue modalities are unclear. Examining how open- and closed-loop cueing influences PFC activity during walking and turning in PwPD may elucidate mechanisms involved in cue response, which could advance development of effective therapeutics. We examined PFC activity during walking and turning in response to open- and closed-loop cueing in PwPD, and explored relationships between PFC activity and behavioral measures. A mobile functional near-infrared spectroscopy device measured PFC activity during walking and turning in 25 PwPD (n = 13 freezers, n = 12 nonfreezers). Participants performed 180° and 360° turns while walking, and a 2-minute walk under single- and dual-task (AX-CPT) conditions with and without an open- (metronome-like vibration) or closed-loop (biofeedback vibration) tactile cue. PFC activity did not change when walking or turning in PwPD; freezing status or task demands did not influence PFC activity. With both open- and closed-loop cueing dual-task cost of gait significantly improved, whereas turning slowed. Our preliminary results indicate that both open- and closed-loop cueing can improve gait without additional burden to the PFC beyond usual walking. However, turning while walking slowed with cueing with no PFC activity change. Further investigations are necessary to establish these findings in a larger cohort.Video Abstract available for more insights from the authors (see Supplemental Digital Content 1, the Video, available at: http://links.lww.com/JNPT/A280).