eQTL mapping of rare variant associations using RNA-seq data: An evaluation of approaches.
Expression quantitative trait loci (eQTL) provide insight on transcription regulation and illuminate the molecular basis of phenotypic outcomes. High-throughput RNA sequencing (RNA-seq) is becoming a popular technique to measure gene expression abundance. Traditional eQTL mapping methods for microarray expression data often assume the expression data follow a normal distribution. As a result, for RNA-seq data, total read count measurements can be normalized by normal quantile transformation in order to fit the data using a linear regression. Other approaches model the total read counts using a negative binomial regression. While these methods work well for common variants (minor allele frequencies > 5% or 1%), an extension of existing methodology is needed to accommodate a collection of rare variants in RNA-seq data. Here, we examine 2 approaches that are direct applications of existing methodology and apply these approaches to RNAseq studies: 1) collapsing the rare variants in the region and using either negative binomial regression or Poisson regression and 2) using the normalized read counts with the Sequence Kernel Association Test (SKAT), the burden test for SKAT (SKAT-Burden), or an optimal combination of these two tests (SKAT-O). We evaluated these approaches via simulation studies under numerous scenarios and applied these approaches to the 1,000 Genomes Project.


Optimization of Bicomponent Electrospun Fibers for Therapeutic Use: Post-Treatments to Improve Chemical and Biological Stability.
Bicomponent electrospun nanofibers based on the combination of synthetic (i.e., aliphatic polyesters such as polycaprolactone (PCL)) and natural proteins (i.e., gelatin) have been extensively investigated as temporary platforms to instruct cells by the release of molecular/pharmaceutical signals for the regeneration of several tissues. Here, water soluble proteins (i.e., gelatin), strictly embedded to PCL, act as carriers of bioactive molecules, thus improving bioavailability and supporting cell activities during in vitro regeneration. However, these proteins are rapidly digested by enzymes, locally produced by many different cell types, both in vitro and in vivo, with significant drawbacks in the control of molecular release. Hence, we have investigated three post-processing strategies based on the use of different crosslinking agents-(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) (EDC), glyceraldehyde (GC), and 1,4-butanediol diglycidyl ether (BDDGE)-to delay the dissolution time of gelatin macromolecules from bicomponent fibers. All of the qualitative (i.e., SEM, TGA) and quantitative (i.e., Trinitrobenzene sulfonate (TNBS) and bicinchoninic acid (BCA) assays) morphological/chemical analyses as well as biocompatibility assays indicate that EDC crosslinking improves the chemical stability of bicomponent fibers at 37 °C and provides a more efficient encapsulation and controlled sustained release of drug, thus resulting in the best post-treatment to design bio-inspired fibrous platforms for the extended in vitro release of drugs.


An ultra-sensitive LC-MS/MS method to determine midazolam levels in human plasma: development, validation and application to a clinical study.
Midazolam is a commonly used marker substrate for the in vivo assessment of CYP3A activity. Reliable pharmacokinetic assessment at sub-pharmacological doses of midazolam requires an ultra-sensitive analytical method. A new, ultra-sensitive LC-MS/MS method for the determination of midazolam in human plasma using SPE was developed and fully validated. The lowest limit of quantitation is 0.1 pg/ml with a sample volume of 500 μl. The following parameters were validated: sensitivity, assay accuracy and precision, linearity, selectivity, and stability of midazolam at pertinent analytical and storage conditions. The validated method was utilized successfully for the sample assay during a midazolam microdosing study for the evaluation of CYP3A4 activity of a clinical candidate.


Clinical outcomes according to time to treatment.
Twenty years ago, pathophysiologic studies showed that myocardial salvage in acute myocardial infarction depends on early intervention. A meta-analysis of the largest thrombolytic trials showed 1.6 lives saved/1000 treated for each hour closer to symptom onset, and that patients treated in the first hour have a much higher survival rate. The MITI trial found a seven-fold decrease in mortality in patients treated within 70 min of symptom onset. The GUSTO study showed similar results, except that during the first hour mortality was actually higher than in the second hour. In contrast, studies have found that while little survival benefit accrues from treatment after a 12 h delay, significant benefit is achieved by treatment between 6 and 12 h from symptom onset. Thus, mechanisms other than myocardial salvage are at work. A GUSTO substudy demonstrated considerable in-hospital delays in attaining electrocardiographic readings, in deciding on the course of therapy, and most of all in the time to infuse the drug. Other delays in both presentation and treatment are related to patient characteristics such as age, gender, diabetes, and Killip class. The medical community cannot control delays in presentation, but it has been able to reduce in-hospital delays, with resultant benefits in morbidity as well as mortality. At the end of the day, reducing delay outweighs the choice of thrombolytic agent.


Intake of dietary phylloquinone and menaquinones and risk of stroke.
Dietary vitamin K intake is thought to decrease the risk of cardiovascular disease (CVD) by reducing vascular calcification, although vitamin K is also involved in coagulation. Studies investigating the association between phylloquinone intake and risk of stroke are scarce, and the relation with menaquinones has not been investigated to date. We investigated the association between intake of phylloquinone and menaquinones and stroke in a prospective cohort of 35,476 healthy subjects. Information on occurrence of stroke was obtained by linkage to national registries, and stroke was further specified into ischemic and hemorrhagic stroke. Vitamin K intake was estimated using a validated food-frequency questionnaire. Multivariate Cox proportional hazards models adjusted for cardiovascular risk factors, lifestyle, and other dietary factors were used to estimate the associations. During a follow-up of 12.1 ± 2.1 years, 580 incident cases of stroke were identified, 163 of which were hemorrhagic and 324 were ischemic. Phylloquinone intake was not associated with risk of stroke with a hazard ratio (HR) of 1.09 (95% CI: 0.85 to 1.40, P(trend) 0.41) for the highest versus lowest quartile. For intake of menaquinones similar results were found, with an HR(Q4 versus Q1) of 0.99 (95% CI: 0.75 to 1.29, P(trend) 0.82). When specifying hemorrhagic and ischemic stroke or menaquinone subtypes, no significant associations were detected. In our study, neither dietary phylloquinone nor dietary menaquinones intake were associated with stroke risk.


Telehealth in palliative care in the UK: a review of the evidence.
We reviewed telehealth applications which were being used in palliative care settings in the UK. Electronic database searches (Medline, CINAHL, PsychInfo and Embase), searches of the grey literature and cited author searches were conducted. In total, 111 papers were identified and 21 documents were included in the review. Telehealth was being used by a range of health professionals in oncology care settings that included specialist palliative care, hospices, primary care settings, nursing homes and hospitals as well as patients and carers. The most common applications were: out-of-hours telephone support, advice services for palliative care patients, carers and health professionals, videoconferencing for interactive case discussions, consultations and assessments, and training and education of palliative care and other health-care staff. The review suggests that current technology is usable and acceptable to patients and health professionals in palliative care settings. However, there are several challenges in integrating telehealth into routine practice.


Network reconstruction and systems analysis of plant cell wall deconstruction by Neurospora crassa.
Plant biomass degradation by fungal-derived enzymes is rapidly expanding in economic importance as a clean and efficient source for biofuels. The ability to rationally engineer filamentous fungi would facilitate biotechnological applications for degradation of plant cell wall polysaccharides. However, incomplete knowledge of biomolecular networks responsible for plant cell wall deconstruction impedes experimental efforts in this direction. To expand this knowledge base, a detailed network of reactions important for deconstruction of plant cell wall polysaccharides into simple sugars was constructed for the filamentous fungus Neurospora crassa. To reconstruct this network, information was integrated from five heterogeneous data types: functional genomics, transcriptomics, proteomics, genetics, and biochemical characterizations. The combined information was encapsulated into a feature matrix and the evidence weighted to assign annotation confidence scores for each gene within the network. Comparative analyses of RNA-seq and ChIP-seq data shed light on the regulation of the plant cell wall degradation network, leading to a novel hypothesis for degradation of the hemicellulose mannan. The transcription factor CLR-2 was subsequently experimentally shown to play a key role in the mannan degradation pathway of N. crassa. Here we built a network that serves as a scaffold for integration of diverse experimental datasets. This approach led to the elucidation of regulatory design principles for plant cell wall deconstruction by filamentous fungi and a novel function for the transcription factor CLR-2. This expanding network will aid in efforts to rationally engineer industrially relevant hyper-production strains.


Acute stress facilitates long-lasting changes in cholinergic gene expression.
Acute traumatic stress may lead to post-traumatic stress disorder (PTSD), which is characterized by delayed neuropsychiatric symptoms including depression, irritability, and impaired cognitive performance. Curiously, inhibitors of the acetylcholine-hydrolysing enzyme acetylcholinesterase may induce psychopathologies that are reminiscent of PTSD. It is unknown how a single stressful event mediates long-term neuronal plasticity. Moreover, no mechanism has been proposed to explain the convergent neuropsychological outcomes of stress and of acetylcholinesterase inhibition. However, acute stress elicits a transient increase in the amounts released of the neurotransmitter acetylcholine and a phase of enhanced neuronal excitability. Inhibitors of acetylcholinesterase also promote enhanced electrical brain activity, presumably by increasing the survival of acetylcholine at the synapse. Here we report that there is similar bidirectional modulation of genes that regulate acetylcholine availability after stress and blockade of acetylcholinesterase. These calcium-dependent changes in gene expression coincide with phases of rapid enhancement and delayed depression of neuronal excitability. Both of these phases are mediated by muscarinic acetylcholine receptors. Our results suggest a model in which robust cholinergic stimulation triggers rapid induction of the gene encoding the transcription factor c-Fos. This protein then mediates selective regulatory effects on the long-lasting activities of genes involved in acetylcholine metabolism.


Lichen Nitidus of the Eyelids.
Lichen nitidus is a rare, idiopathic inflammatory condition that typically presents as small, flat-topped papules on the chest, abdomen, and upper extremities. The lesions are benign and often asymptomatic and self-resolve. The pediatric population is most often affected. The authors report a case of lichen nitidus presenting as isolated bilateral eyelid lesions increasing in number for several years. Eventual excision and antibiotic/steroid ointment prompted regression.


Direct and indirect effects of dispersion interactions on the electric properties of weakly bound complexes.
Direct (electronic) and indirect (geometrical) modifications of the molecular properties of weakly interacting complexes between the push-pull p-aminobenzoic acid (pABA) molecule and the nonpolar benzene (Bz) have been studied with a large panel of wave function (WF) and density functional theory (DFT) based methods using carefully selected atomic basis sets. For pABA, both the canonical (pABA-c) and zwitterionic (pABA-z) forms have been investigated. Owing to strongly distinct charge distributions, the two forms of pABA enable us to mimic different interaction modes with Bz. In this work, we assessed the performances of dispersion-corrected DFT methods, as well as of long-range corrected exchange-correlation functionals. It follows from the SAPT analysis that both the structure and the interaction energy of the first complex (pABA-c···Bz) is mainly controlled by dispersion interactions whereas, in the second complex (pABA-z···Bz), electrostatic and induction forces play also an important role. Our results suggest that the (non)linear electric properties of push-pull and zwitterionic molecules can be significantly reduced by the presence of a nonpolar compound. We also show that even for a complex with stability strongly determined by dispersion forces, the direct dispersion contributions to its electric properties can be small. Nevertheless, the intersystem distance is influenced by dispersion forces, which, in turn indirectly tune the induced properties. The zwitterionic derivative appears to be more challenging in the context of molecular properties.


A preliminary trial of ascorbic acid as supplemental therapy for autism.
1. This study presents the results of a 30-week double-blind, placebo-controlled trial exploring the effectiveness of ascorbic acid (8g/70kg/day) as a supplemental pharmacological treatment for autistic children in residential treatment. 2. Residential school children (N = 18) were randomly assigned to either ascorbate-ascorbate-placebo treatment order group or ascorbate-placebo-ascorbate treatment order group. Each treatment phase lasted 10 weeks and behaviors were rated weekly using the Ritvo-Freeman scale. 3. Significant group by phase interactions were found for total scores and also sensory motor scores indicating a reduction in symptom severity associated with the ascorbic acid treatment. 4. These results were consistent with a hypothesized dopaminergic mechanism of action of ascorbic acid.


Expression, localization, and function of the thioredoxin system in diabetic nephropathy.
Excessive reactive oxygen species play a key role in the pathogenesis of diabetic nephropathy, but to what extent these result from increased generation, impaired antioxidant systems, or both is incompletely understood. Here, we report the expression, localization, and activity of the antioxidant thioredoxin and its endogenous inhibitor thioredoxin interacting protein (TxnIP) in vivo and in vitro. In normal human and rat kidneys, expression of TxnIP mRNA and protein was most abundant in the glomeruli and distal nephron (distal convoluted tubule and collecting ducts). In contrast, thioredoxin mRNA and protein localized to the renal cortex, particularly within the proximal tubules and to a lesser extent in the distal nephron. Induction of diabetes in rats increased expression of TxnIP but not thioredoxin mRNA. Kidneys from patients with diabetic nephropathy had significantly higher levels of TxnIP than control kidneys, but thioredoxin expression did not differ. In vitro, high glucose increased TxnIP expression in mesangial, NRK (proximal tubule), and MDCK (distal tubule/collecting duct) cells, and decreased the expression of thioredoxin in mesangial and MDCK cells. Knockdown of TxnIP with small interference RNA suggested that TxnIP mediates the glucose-induced impairment of thioredoxin activity. Knockdown of TxnIP also abrogated both glucose-induced 3H-proline incorporation (a marker of collagen production) and oxidative stress. Taken together, these findings suggest that impaired thiol reductive capacity contributes to the generation of reactive oxygen species in diabetes in a site- and cell-specific manner.


The Association of Prenatal Vitamin D Status With Pregnancy and Neonatal Outcomes.
Vitamin D inadequacy is globally prevalent among pregnant women; however, its impact on pregnancy remains inconclusive. This study aims to explore the associations of maternal and umbilical cord serum 25-hydroxyvitamin D (25(OH)D) levels with pregnancy and neonatal outcomes. We used archived serum samples from the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study participants in the Hong Kong center and assayed maternal 25(OH)D levels at midgestation and umbilical cord 25(OH)D at birth using liquid chromatography-tandem mass spectroscopy. Data regarding pregnancy and perinatal outcomes were extracted from the HAPO study dataset and the hospital computerized medical system. Only 247 (16.4%) mothers and 66 (5.0%) neonates met the criteria for vitamin D sufficiency (ie, 25(OH)D ≥ 75 nmol/L). The ratio of umbilical cord to maternal vitamin D levels was positively associated with maternal age and ambient solar radiation at the month of delivery, while negatively associated with maternal serum total 25(OH)D at midgestation (all P < .001). Umbilical cord serum 25(OH)D was independently associated with a lower primary cesarean section rate (OR 0.990, 95% CI 0.982-0.999; P = .032). There were no associations of maternal and umbilical cord 25(OH)D levels with other adverse pregnancy and neonatal outcomes. Placental vitamin D transfer was found to be higher with a lower maternal vitamin D level, older maternal age, and higher ambient solar radiation at the time of the delivery. The protective effect of sufficient vitamin D in a cesarean section will require further studies.


Linkage-specific sialic acid derivatization for MALDI-TOF-MS profiling of IgG glycopeptides.
Glycosylation is a common co- and post-translational protein modification, having a large influence on protein properties like conformation and solubility. Furthermore, glycosylation is an important determinant of efficacy and clearance of biopharmaceuticals such as immunoglobulin G (IgG). Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) shows potential for the site-specific glycosylation analysis of IgG at the glycopeptide level. With this approach, however, important information about glycopeptide sialylation is not duly covered because of in-source and metastable decay of the sialylated species. Here, we present a highly repeatable sialic acid derivatization method to allow subclass-specific MALDI-TOF-MS analysis of tryptic IgG glycopeptides. The method, employing dimethylamidation with the carboxylic acid activator 1-ethyl-3-(3-dimethylamino)propyl)carbodiimide (EDC) and the catalyst 1-hydroxybenzotriazole (HOBt), results in different masses for the functionally divergent α2,3- and α2,6-linked sialic acids. Respective lactonization and dimethylamidation leads to their direct discrimination in MS and importantly, both glycan and peptide moieties reacted in a controlled manner. In addition, stabilization allowed the acquisition of fragmentation spectra informative with respect to glycosylation and peptide sequence. This was in contrast to fragmentation spectra of underivatized samples, which were dominated by sialic acid loss. The method allowed the facile discrimination and relative quantitation of IgG Fc sialylation in therapeutic IgG samples. The method has considerable potential for future site- and sialic acid linkage-specific glycosylation profiling of therapeutic antibodies, as well as for subclass-specific biomarker discovery in clinical IgG samples derived from plasma.


Quality assurance framework for rapid automatic analysis deployment in medical imaging.
Automatic image analysis algorithms have an increasing role in clinical quality assurance (QA) in medical imaging. Although the implementation of QA calculation algorithms may be straightforward at the development level, actual deployment of a new method to clinical routine may require substantial additional effort from supporting services. We sought to develop a multimodal system that enables rapid implementation of new QA analysis methods in clinical practice. The QA system was built using freely available open-source software libraries. The included features were results database, database interface, interactive user interface, e-mail error dispatcher, data processing backend, and DICOM server. An in-house database interface was built, providing the developers of analyses with simple access to the results database. An open-source DICOM server was used for image traffic and automatic initiation of modality-specific QA image analyses. The QA framework enabled rapid adaptation of new analysis methods to automatic image processing workflows. The system provided online data review via an easily accessible user interface. In case of deviations, the system supported simultaneous review of the results for the user and QA expert to trigger corrective actions. In particular, embedded error thresholds, trend analyses, and error-feedback channels were provided to facilitate continuous monitoring and to enable pre-emptive corrective actions. An effective and novel QA framework incorporating easy adaptation and scalability to automated image analysis methods was developed. The framework provides an efficient and responsive web-based tool to manage the normal operation, trends, errors, and abnormalities in medical image quality.


Immunotherapy of bladder cancer using autologous dendritic cells pulsed with human lymphocyte antigen-A24-specific MAGE-3 peptide.
Recent investigations have demonstrated the efficacy of autologous dendritic cells (DCs) pulsed with tumor antigens to generate tumor-specific CTLs against cancer cells. Melanoma antigens (MAGE) are a family of tumor-specific antigens shown to be expressed in various tumors, including bladder cancers and melanoma, but not in normal tissues except for the testis. Because invasive bladder cancers are frequently reported to express MAGE, we explored the possibility of establishing a new immunotherapeutic modality against advanced bladder cancer using autologous DCs pulsed with one of the MAGE-3 epitope peptides (IMPKAGLLI), which is synthesized to bind specifically to HLA-A24. A MAGE-3-expressing bladder cancer cell line, FY, was newly established from a lymph node metastasis of bladder cancer in a HLA-A24+ patient. The FY cell-specific CTL response was significantly higher when CTL was induced by autologous DCs pulsed with IMPKAGLLI than by FY cells alone or by nonpulsed DCs in vitro. A total of four HLA-A24+ patients with advanced MAGE-3+ bladder cancers were treated with s.c. injections of autologous DCs pulsed with IMPKAGLLI every 2 weeks for a minimum of 6 and a maximum of 18 times. Three of four patients showed significant reductions in the size of lymph node metastases and/or liver metastasis. No significant untoward side effects were noted in these patients. This study indicated that, at sometime in the future, tumor-specific DC-based cancer immunotherapy may be useful as an additional treatment modality against advanced bladder cancer.


Astrocyte regulation of cerebral vascular tone.
Cerebral blood flow is controlled by two crucial processes, cerebral autoregulation (CA) and neurovascular coupling (NVC) or functional hyperemia. Whereas CA ensures constant blood flow over a wide range of systemic pressures, NVC ensures rapid spatial and temporal increases in cerebral blood flow in response to neuronal activation. The focus of this review is to discuss the cellular mechanisms by which astrocytes contribute to the regulation of vascular tone in terms of their participation in NVC and, to a lesser extent, CA. We discuss evidence for the various signaling modalities by which astrocytic activation leads to vasodilation and vasoconstriction of parenchymal arterioles. Moreover, we provide a rationale for the contribution of astrocytes to pressure-induced increases in vascular tone via the vasoconstrictor 20-HETE (a downstream metabolite of arachidonic acid). Along these lines, we highlight the importance of the transient receptor potential channel of the vanilloid family (TRPV4) as a key molecular determinant in the regulation of vascular tone in cerebral arterioles. Finally, we discuss current advances in the technical tools available to study NVC mechanisms in the brain as it relates to the participation of astrocytes.


Psychosocial characteristics and pain burden of patients with suspected sphincter of Oddi dysfunction in the EPISOD multicenter trial.
Patients with several painful functional gastrointestinal disorders (FGIDs) are reported to have a high prevalence of psychosocial disturbance. These aspects have not been studied extensively in patients with suspected Sphincter of Oddi dysfunction (SOD). A total of 214 patients with post-cholecystectomy pain and suspected SOD were enrolled in seven US centers in a multicenter-randomized trial (Evaluating Predictors and Interventions in Sphincter of Oddi Dysfunction). Baseline assessments included pain descriptors and burden, structured psychosocial assessments of anxiety/depression, coping, trauma, and health-related quality of life. Patients with high levels of depression, suicidal ideation, or psychosis were excluded. The study population (92% female, mean age 38) reported anxiety (9%), depression (8%), past sexual trauma (18%), and physical abuse (10%). Of the total screened population (n=1460), 3.9% of the patients were excluded because of the presence of defined severe psychological problems. The mean medical outcomes study short-form-36 (SF-36) physical and mental composite scores were 38.70 (s.d.=7.89) and 48.74 (s.d.=9.60), respectively. Most subjects reported symptoms of other FGIDs. There were no correlations between the extent of the pain burden in the 3 months before enrollment and the baseline anxiety scores or victimization history. However, those with greater pain burden were significantly more depressed. There were no meaningful differences in the psychosocial parameters in subjects with or without irritable bowel, and those who had cholecystectomy for stones or functional gallbladder disease. Those declining randomization were comparable to those randomized. Psychosocial comorbidity in SOD is high. However, it does not appear to differ significantly from that reported in surveys of age- and gender-matched general populations, and may be lower than reported with other FGIDs.


The Short- and Long-Term Clinical, Radiological and Functional Consequences of COVID-19.
As with the rapid increase of the number of patients who have recovered from COVID-19 globally, there needs to be a major shift of the focus from rapid pathogen detection, treatment and prevention to the promotion of better recovery. Notwithstanding the scarcity of our understandings, recent studies have unraveled a plethora of pulmonary and systemic consequences which require medical attention. These consequences remained as of the end of follow-up which ranged from 1 month to 1 year. Here, we review the consequences of COVID-19 in terms of the residual symptoms, radiological and functional manifestations, and identify the potential risk factors that contribute to a prolonged recovery. We also summarize the benefits of clinical interventions (particularly the pulmonary rehabilitation program), and address several undetermined concerns and key future research directions.


MRI of the lung: state of the art.
Magnetic resonance imaging (MRI) of the lung is technically challenging due to the low proton density and fast signal decay of the lung parenchyma itself. Additional challenges consist of tissue loss, hyperinflation, and hypoxic hypoperfusion, e.g., in emphysema, a so-called "minus-pathology". However, pathological changes resulting in an increase of tissue ("plus-pathology"), such as atelectases, nodules, infiltrates, mucus, or pleural effusion, are easily depicted with high diagnostic accuracy. Although MRI is inferior or at best equal to multi-detector computed tomography (MDCT) for the detection of subtle morphological features, MRI now offers an increasing spectrum of functional imaging techniques such as perfusion assessment and measurement of ventilation and respiratory mechanics that are superior to what is possible with MDCT. Without putting patients at risk with ionizing radiation, repeated examinations allow for the evaluation of the course of lung disease and monitoring of the therapeutic response through quantitative imaging, providing a level of functional detail that cannot be obtained by any other single imaging modality. As such, MRI will likely be used for clinical applications beyond morphological imaging for many lung diseases. In this article, we review the technical aspects and protocol suggestions for chest MRI and discuss the role of MRI in the evaluation of nodules and masses, airway disease, respiratory mechanics, ventilation, perfusion and hemodynamics, and pulmonary vasculature.


Panax ginseng enhances cognitive performance in Alzheimer disease.
Recent experimental evidences suggest protective and trophic effects of ginseng in the memory function of Alzheimer disease (AD). Thus, we investigated the clinical efficacy of Panax ginseng in the cognitive performance of AD patients in an open-label study. Consecutive AD patients were randomly assigned to the ginseng (n=58) or the control group (n=39), and the ginseng group was treated with Panax ginseng powder (4.5 g/d) for 12 weeks. Cognitive performances were monitored using the mini-mental state examination (MMSE) and Alzheimer disease assessment scale (ADAS) during 12 weeks of the ginseng treatment and at 12 weeks after the ginseng discontinuation. MMSE and ADAS scales showed no baseline difference between the groups. After ginseng treatment, the cognitive subscale of ADAS and the MMSE score began to show improvements and continued up to 12 weeks (P=0.029 and P=0.009 vs. baseline, respectively). After discontinuing ginseng, the improved ADAS and MMSE scores declined to the levels of the control group. These results suggest that Panax ginseng is clinically effective in the cognitive performance of AD patients.


Cardiac mortality in limited-stage small cell lung cancer.
Life expectancy of patients with limited-stage small cell lung cancer (LS-SCLC) continues to rise; thus, characterization of long-term toxicities is essential. Although there are emerging data linking cardiac irradiation doses with survival for non-small cell lung cancer, there are currently minimal data on cardiac-specific mortality (CSM) in LS-SCLC. The goal of this investigation was to evaluate CSM between left- and right-sided cases. The Surveillance, Epidemiology, and End Results database was queried for stage I-III primary SCLC patients receiving radiotherapy; CSM was compared between left- and right-sided diseases. Accounting for mortality from other causes, Gray's test compared cumulative incidences of CSM between both groups. Multiple multivariate models examined the independent effect of laterality on CSM, including the Fine and Gray competing risk model and the Cox proportional hazards model. Of 19,692 patients, 7991 (41%) were left-sided and 11,701 (59%) were right-sided. Left-sided patients experienced significantly higher CSM overall (3.3% vs. 2.6%, p = 0.004). Laterality was an independent predictor of CSM in the overall population in the Fine and Gray competing risk model (p = 0.006) as well as the Cox proportional hazards model (p = 0.007). The overall hazard ratio for CSM by disease laterality was 1.27 (95% confidence interval, 1.08-1.50). Laterality had no statistical association with non-cardiac mortality in the Fine and Gray competing risk model (p = 0.130). Although causation between radiotherapy and CSM in LS-SCLC cannot be stated based on these data, we encourage clinical attentiveness to cardiac-sparing radiotherapy for LS-SCLC, along with further investigation evaluating dosimetric correlates for cardiotoxicity.


Natural killer cell activation and interferon production by peripheral blood lymphocytes after exposure to bacteria.
We have previously shown that peripheral blood natural killer (NK) cells have significant levels of cytotoxic activity against Shigella flexneri-infected HeLa cells. In this report, we show that NK cell activity against K562 tumor cells and Shigella flexneri-infected HeLa cells can be greatly enhanced by preincubating peripheral blood lymphocytes (PBL) for 18 h with kanamycin-treated Shigella flexneri or Salmonella typhimurium. Cell-free supernatants obtained from PBL-bacteria cultures contained high levels of interferon (IFN) activity, which was characterized as a mixture of IFN-gamma and IFN-alpha. Cytotoxic activity associated with PBL precultured with shigellae was associated with predominantly CD16+ (Leu-11+) and CD2+ (OKT-11+) cells. Further, IFN production was dependent upon the presence of CD16+ and CD2+ cells at culture initiation. Enhancement of cytotoxic activity associated with PBL-bacteria cultures did not, however, appear to be dependent upon IFN production, since low numbers of bacteria which failed to stimulate IFN production induced high levels of NK cell activity. Lipopolysaccharide appeared not to be involved in bacteria-induced IFN production or enhanced NK cell activity, since Salmonella lipopolysaccharide failed to induce IFN production or enhance NK cell activity. These results suggest that IFN production by NK cells and the killing of bacteria-infected cells play an important role in host defense against facultative intracellular bacterial infections.


Assessment of T regulatory cells and expanded profiling of autoantibodies may offer novel biomarkers for the clinical management of systemic sclerosis and undifferentiated connective tissue disease.
In order to identify disease biomarkers for the clinical and therapeutic management of autoimmune diseases such as systemic sclerosis (SSc) and undifferentiated connective tissue disease (UCTD), we have explored the setting of peripheral T regulatory (T reg) cells and assessed an expanded profile of autoantibodies in patients with SSc, including either limited (lcSSc) or diffuse (dcSSc) disease, and in patients presenting with clinical signs and symptoms of UCTD. A large panel of serum antibodies directed towards nuclear, nucleolar, and cytoplasmic antigens, including well-recognized molecules as well as less frequently tested antigens, was assessed in order to determine whether different antibody profiles might be associated with distinct clinical settings. Beside the well-recognized association between lcSSc and anti-centromeric or dcSSC and anti-topoisomerase-I antibodies, we found a significative association between dcSSc and anti-SRP or anti-PL-7/12 antibodies. In addition, two distinct groups emerged on the basis of anti-RNP or anti-PM-Scl 75/100 antibody production among UCTD patients. The levels of T reg cells were significantly lower in patients with SSc as compared to patients with UCTD or to healthy controls; in patients with lcSSc, T reg cells were inversely correlated to disease duration, suggesting that their levels may represent a marker of disease progression.


[Successful repair of complete pancreatic rupture and subtotal duodenal avulsion after blunt abdominal trauma in childhood--a case report].
We report the case of a 12-year-old boy who presented after a bicycle accident with handlebar injury of the epigastrium with clinical signs of hollow visceral injury and elevated pancreatic enzymes. Upon emergency laparotomy, a complete rupture of the pancreatic isthmus and a nearly total transsection of the postpyloric duodenum were encountered (Grade IV according to Lucas). Reconstruction consisted of reanastomosis of the proximal duodenum, closure of the distal end of the pancreatic head and internal drainage of the left pancreas into a Roux-en-Y jejunal loop. The postoperative course was uneventful. For rare traumatic pancreaticoduodenal lesions only individual surgical concepts are promising depending on the extent of damage. However, effort should be made to preserve as much pancreatic tissue as possible.


Neuritic regeneration and synaptic reconstruction induced by withanolide A.
We investigated whether withanolide A (WL-A), isolated from the Indian herbal drug Ashwagandha (root of Withania somnifera), could regenerate neurites and reconstruct synapses in severely damaged neurons. We also investigated the effect of WL-A on memory-deficient mice showing neuronal atrophy and synaptic loss in the brain. Axons, dendrites, presynapses, and postsynapses were visualized by immunostaining for phosphorylated neurofilament-H (NF-H), microtubule-associated protein 2 (MAP2), synaptophysin, and postsynaptic density-95 (PSD-95), respectively. Treatment with A beta(25-35) (10 microM) induced axonal and dendritic atrophy, and pre- and postsynaptic loss in cultured rat cortical neurons. Subsequent treatment with WL-A (1 microM) induced significant regeneration of both axons and dendrites, in addition to the reconstruction of pre- and postsynapses in the neurons. WL-A (10 micromol kg(-1) day(-1), for 13 days, p.o.) recovered A beta(25-35)-induced memory deficit in mice. At that time, the decline of axons, dendrites, and synapses in the cerebral cortex and hippocampus was almost recovered. WL-A is therefore an important candidate for the therapeutic treatment of neurodegenerative diseases, as it is able to reconstruct neuronal networks.


Tetrahydrobiopterin biosynthesis, utilization and pharmacological effects.
Tetrahydrobiopterin (H4-biopterin) is an essential cofactor of a set of enzymes that are of central metabolic importance, i.e. the hydroxylases of the three aromatic amino acids phenylalanine, tyrosine, and tryptophan, of ether lipid oxidase, and of the three nitric oxide synthase (NOS) isoenzymes. As a consequence, H4-biopterin plays a key role in a vast number of biological processes and pathological states associated with neurotransmitter formation, vasorelaxation, and immune response. In mammals, its biosynthesis is controlled by hormones, cytokines and certain immune stimuli. This review aims to summarize recent developments concerning regulation of H4-biopterin biosynthetic and regulatory enzymes and pharmacological effects of H4-biopterin in various conditions, e.g. endothelial dysfunction or apoptosis of neuronal cells. Also, approaches towards gene therapy of diseases like the different forms of phenylketonuria or of Parkinson's disease are reviewed. Additional emphasis is given to H4-biopterin biosynthesis and function in non-mammalian species such as fruit fly, zebra fish, fungi, slime molds, the bacterium Nocardia as well as to the parasitic protozoan genus of Leishmania that is not capable of pteridine biosynthesis but has evolved a sophisticated salvage network for scavenging various pteridine compounds, notably folate and biopterin.


Serum lipid levels and thromboembolic complications during estrogen therapy of prostatic cancer.
The effect of diethylstilboestrol diphosphate (DES) and polyestradiol phosphate (PES) on the serum lipid levels of 40 prostatic cancer patients was investigated during a long-term follow-up. In patients treated with DES serum triglyceride levels elevated significantly whereas changes in serum cholesterol and total lipid contents were not significant. In patients treated with PES no significant changes in serum triglyceride, cholesterol and total lipid levels were found. Nineteen out of 40 (48%) of patients developed cardiovasculat complications during the trial; 11 had cerebrovascular attacks, 5 coronary occlusion and 3 thromboembolism in the leg. There was a significant increase in serum triglyceride levels in all patients who developed vascular disorders, whereas the changes in serum cholesterol and total lipids were not significant. The results suggest a possible association between the increase of serum triglycerides and the development of thromboembolic complications.


Linking the genetic architecture of cytosine modifications with human complex traits.
Interindividual variation in cytosine modifications could contribute to heterogeneity in disease risks and other complex traits. We assessed the genetic architecture of cytosine modifications at 283,540 CpG sites in lymphoblastoid cell lines (LCLs) derived from independent samples of European and African descent. Our study suggests that cytosine modification variation was primarily controlled in local by single major modification quantitative trait locus (mQTL) and additional minor loci. Local genetic epistasis was detectable for a small proportion of CpG sites, which were enriched by more than 9-fold for CpG sites mapped to population-specific mQTL. Genetically dependent CpG sites whose modification levels negatively (repressive sites) or positively (facilitative sites) correlated with gene expression levels significantly co-localized with transcription factor binding, with the repressive sites predominantly associated with active promoters whereas the facilitative sites rarely at active promoters. Genetically independent repressive or facilitative sites preferentially modulated gene expression variation by influencing local chromatin accessibility, with the facilitative sites primarily antagonizing H3K27me3 and H3K9me3 deposition. In comparison with expression quantitative trait loci (eQTL), mQTL detected from LCLs were enriched in associations for a broader range of disease categories including chronic inflammatory, autoimmune and psychiatric disorders, suggesting that cytosine modification variation, while possesses a degree of cell linage specificity, is more stably inherited over development than gene expression variation. About 11% of unique single-nucleotide polymorphisms reported in the Genome-Wide Association Study Catalog were annotated, 78% as mQTL and 31% as eQTL in LCLs, which covered 37% of the investigated diseases/traits and provided insights to the biological mechanisms.


[Primary hyperoxaluria: case report and therapeutic perspectives].
Primary hyperoxaluria (PH) is a rare genetic disorder with autosomal recessive transmission, characterized by high endogenous production and markedly excessive urinary excretion of oxalate (Ox). It causes the accumulation of calcium oxide crystals in organs and tissues including bones, heart, arteries, skin and kidneys, where it may cause oxalo-calcic nephrolithiasis, nephrocalcinosis and chronic renal failure. Some forms are secondary to enteric diseases, drugs or dietetic substances, while three primitive forms, caused by various enzymatic defects, are currently known: PH1, PH2 and PH3. An early diagnosis, with the aid of biochemical and genetic investigations, helps prevent complications and establish a therapeutic strategy that often includes liver and liver-kidney transplantation, improving the prognosis of these patients. In this work we describe the clinical case of a patient with PH1 undergoing extracorporeal hemodialysis treatment and we report the latest research results that could change the life of patients with PH.


Occupation as a risk identifier for breast cancer.
Breast cancer mortality may be reduced if the disease is detected early through targeted screening programs. Current screening guidelines are based solely on a woman's age. Because working populations are accessible for intervention, occupational identification may be a way of helping to define and locate risk groups and target prevention. We used a database consisting of 2.9 million occupationally coded death certificates collected from 23 states between 1979 and 1987 to calculate age-adjusted, race-specific proportionate mortality ratios for breast cancer according to occupation. We performed case-control analyses on occupational groups and on stratifications within the teaching profession. We found a number of significant associations between occupation and frequency of breast cancer. For example, white female professional, managerial, and clerical workers all had high proportions of breast cancer death. High rates of breast cancer in teachers were found in both proportionate mortality ratio and case-control analyses. These findings may serve as in an aid in the effective targeting of work-site health promotion programs. They suggest that occupationally coded mortality data can be a useful adjunct in the difficult task of identifying groups at risk of preventable disease.


Evaluation of a system of structured, pro-active care for chronic depression in primary care: a randomised controlled trial.
People with chronic depression are frequently lost from effective care, with resulting psychological, physical and social morbidity and considerable social and financial societal costs. This randomised controlled trial will evaluate whether regular structured practice nurse reviews lead to better mental health and social outcomes for these patients and will assess the cost-effectiveness of the structured reviews compared to usual care.The hypothesis is that structured, pro-active care of patients with chronic depression in primary care will lead to a cost-effective improvement in medical and social outcomes when compared with usual general practitioner (GP) care. Participants were recruited from 42 general practices throughout the United Kingdom. Eligible participants had to have a history of chronic major depression, recurrent major depression or chronic dsythymia confirmed using the Composite International Diagnostic Interview (CIDI). They also needed to score 14 or above on the Beck Depression Inventory (BDI-II) at recruitment.Once consented, participants were randomised to treatment as usual from their general practice (controls) or the practice nurse led intervention. The intervention includes a specially prepared education booklet and a comprehensive baseline assessment of participants' mood and any associated physical and psycho-social factors, followed by regular 3 monthly reviews by the nurse over the 2 year study period. At these appointments intervention participants' mood will be reviewed, together with their current pharmacological and psychological treatments and any relevant social factors, with the nurse suggesting possible amendments according to evidence based guidelines. This is a chronic disease management model, similar to that used for other long-term conditions in primary care.The primary outcome is the BDI-II, measured at baseline and 6 monthly by self-complete postal questionnaire. Secondary outcomes collected by self-complete questionnaire at baseline and 2 years include social functioning, quality of life and data for the economic analyses. Health service data will be collected from GP notes for the 24 months before recruitment and the 24 months of the study. 558 participants were recruited, 282 to the intervention and 276 to the control arm. The majority were recruited via practice database searches using relevant READ codes. ISRCTN36610074.


C4 anatomy can evolve via a single developmental change.
C4 photosynthesis is a complex trait that boosts productivity in warm environments. Paradoxically, it evolved independently in numerous plant lineages, despite requiring specialised leaf anatomy. The anatomical modifications underlying C4 evolution have previously been evaluated through interspecific comparisons, which capture numerous changes besides those needed for C4 functionality. Here, we quantify the anatomical changes accompanying the transition between non-C4 and C4 phenotypes by sampling widely across the continuum of leaf anatomical traits in the grass Alloteropsis semialata. Within this species, the only trait that is shared among and specific to C4 individuals is an increase in vein density, driven specifically by minor vein development that yields multiple secondary effects facilitating C4 function. For species with the necessary anatomical preconditions, developmental proliferation of veins can therefore be sufficient to produce a functional C4 leaf anatomy, creating an evolutionary entry point to complex C4 syndromes that can become more specialised.


Why and how do journals retract articles? An analysis of Medline retractions 1988-2008.
Journal editors are responsible for what they publish and therefore have a duty to correct the record if published work is found to be unreliable. One method for such correction is retraction of an article. Anecdotal evidence suggested a lack of consistency in journal policies and practices regarding retraction. In order to develop guidelines, we reviewed retractions in Medline to discover how and why articles were retracted. We retrieved all available Medline retractions from 2005 to 2008 and a one-in-three random selection of those from 1988 to 2004. This yielded 312 retractions (from a total of 870). Details of the retraction including the reason for retraction were recorded by two investigators. Medline retractions have increased sharply since 1980 and currently represent 0.02% of included articles. Retractions were issued by authors (63%), editors (21%), journals (6%), publishers (2%) and institutions (1%). Reasons for retraction included honest error or non-replicable findings (40%), research misconduct (28%), redundant publication (17%) and unstated/unclear (5%). Some of the stated reasons might have been addressed by corrections. Journals' retraction practices are not uniform. Some retractions fail to state the reason, and therefore fail to distinguish error from misconduct. We have used our findings to inform guidelines on retractions.


Integration of gene expression, clinical, and epidemiologic data to characterize Chronic Fatigue Syndrome.
BACKGROUND: Chronic fatigue syndrome (CFS) has no diagnostic clinical signs or diagnostic laboratory abnormalities and it is unclear if it represents a single illness. The CFS research case definition recommends stratifying subjects by co-morbid conditions, fatigue level and duration, or functional impairment. But to date, this analysis approach has not yielded any further insight into CFS pathogenesis. This study used the integration of peripheral blood gene expression results with epidemiologic and clinical data to determine whether CFS is a single or heterogeneous illness. RESULTS: CFS subjects were grouped by several clinical and epidemiological variables thought to be important in defining the illness. Statistical tests and cluster analysis were used to distinguish CFS subjects and identify differentially expressed genes. These genes were identified only when CFS subjects were grouped according to illness onset and the majority of genes were involved in pathways of purine and pyrimidine metabolism, glycolysis, oxidative phosphorylation, and glucose metabolism. CONCLUSION: These results provide a physiologic basis that suggests CFS is a heterogeneous illness. The differentially expressed genes imply fundamental metabolic perturbations that will be further investigated and illustrates the power of microarray technology for furthering our understanding CFS.


AVAPS-NIV treatment in hypercapnic respiratory failure with insufficient response to fixed-level PS-NIV.
Noninvasive ventilation (NIV) for acute hypercapnic respiratory failure (AHRF) is an established treatment modality. Current evidence does not conclude any superiority between fixed pressure support (PS) and average volume-assured pressure support (AVAPS) modes. However, given the ability of rapid PaCO2 decline in AVAPS mode, we hypothesized that COPD patients with AHRF who did not show the desired reduction in PaCO2 with fixed-level PS-NIV might benefit from the AVAPS mode. Patients admitted to the non-ICU pulmonary ward with acute exacerbation of COPD (AECOPD) and AHRF were included consecutively in this observational study. Patients with hypercapnic respiratory failure due to obesity-hypoventilation, neurological diseases, or chest wall deformities were excluded. All patients started NIV treatment with fixed pressure support (PS) and patients who did not reach clinical and laboratory stability under PS-NIV treatment were switched to the average volume-assured pressure support (AVAPS) mode of NIV. Thirty-five COPD patients with hypercapnic respiratory failure were included. Under PS-NIV treatment, 14 (40%) patients showed a 17.9 (-0.0-29.2) percent change in terms of PaCO2, meaning no improvement or worsening. Therefore, these patients were treated with AVAPS mode. Arterial PaCO2 and pH levels significantly improved after AVAPS-NIV administration. AVAPS-NIV treatment created a significantly better PaCO2 change rate than using PS-NIV [-11.4 (-22.0 - -0.5) vs 8.2 (-5.3-19.5), p= 0.02]. Independent predictors of AVAPS mode requirement were higher Charlson Comorbidity Index [OR= 1.74 (95% CI= 1.02-2.97)] and higher PaCO2 upon admission [OR= 1.18 (95% CI= 1.03-1.35)]. Thirteen (92.8%) patients reaching significant clinical stability with AVAPS-NIV were able to return to fixed-level PS-NIV and maintain acceptable PaCO2 levels. Our study demonstrated that patients can benefit from AVAPSNIV despite insufficient response to fixed-level PS-NIV.


Exploring the proposed DSM-5 criteria in a clinical sample.
The proposed DSM-5 criteria for Autism Spectrum Disorder (ASD) depart substantially from the previous DSM-IV criteria. In this file review study of 131 children aged 2-12, previously diagnosed with either Autistic Disorder or Pervasive Developmental Disorder-Not Otherwise Specified (PDD-NOS), 63 % met the new DSM-5 ASD criteria, including 81 % previously diagnosed with Autistic Disorder and only 17 % of those with PDD-NOS. The proportion of children meeting DSM-5 differed by IQ grouping as well, with higher rates in lower IQ groups. Children who did meet criteria for ASD had significantly lower levels of cognitive and adaptive skills and greater autism severity but were similar in age. These findings raise concerns that the new DSM-5 criteria may miss a number of children who would currently receive a diagnosis.


Phosphorylated derivatives of myo-inositol.
The structures of myo-inositol and its isomers are reviewed, together with those of the phosphoinositides, and of the inositol phosphates resulting from phosphodiesteratic cleavage. Inositol trisphosphate and related derivatives are considered with regard to their second messenger function. Strategies for possible disruption of enzymatic steps leading to the formation and degradation of these substances are discussed.


A history of mitochondrial diseases.
This articles reviews the development of mitochondrial medicine from the premolecular era (1962-1988), when mitochondrial diseases were defined on the basis of clinical examination, muscle biopsy, and biochemical criteria, through the molecular era, when the full complexity of these disorders became evident. In a chronological order, I have followed the introduction of new pathogenic concepts that have shaped a rational genetic classification of these clinically heterogeneous disorders. Thus, mitochondrial DNA (mtDNA)-related diseases can be divided into two main groups: those that impair mitochondrial protein synthesis in toto, and those that affect specific respiratory chain proteins. Mutations in nuclear DNA can affect components of respiratory chain complexes (direct hits) or assembly proteins (indirect hits), but they can also impair mtDNA integrity (multiple mtDNA mutations), replication (mtDNA depletion), or mtDNA translation. Besides these disorders that affect the respiratory chain directly, defects in other mitochondrial functions may also affect oxidative phosphorylation, including problems in mitochondrial protein import, alterations of the inner mitochondrial membrane lipid composition, and defects of mitochondrial dynamics. The enormous and still ongoing progress in our understanding of mitochondrial medicine was made possible by the intense collaboration of an international cadre of "mitochondriacs." Having published my first paper on a patient with mitochondrial myopathy 37 years ago (DiMauro et al., 1973), I feel qualified to write a history of the mitochondrial diseases, a fascinating, still evolving, and continuously puzzling area of medicine. In each section, I follow a chronological order of the salient discoveries and I show only the portraits of distinguished deceased mitochondriacs and those whose names became eponyms of mitochondrial diseases.


Feigned psychiatric symptoms in the emergency room.
Psychiatrists providing emergency services at an urban general hospital completed questionnaires on 227 patients evaluated over a two-month period to assess whether they suspected the patient of malingering or of having secondary gains, and whether the patient was confronted about the suspicions. Thirteen percent of patients were strongly or definitely suspected of feigning symptoms; none received a primary diagnosis of malingering, and less than half were confronted. Suspected secondary gains included food and shelter, medications, financial gains, and avoidance of jail, work, or family responsibilities.


The intricate triangular interaction between protective microbe, pathogen and host determines fitness of the metaorganism.
The microbiota shapes host biology in numerous ways. One example is protection against pathogens, which is likely critical for host fitness in consideration of the ubiquity of pathogens. The host itself can affect abundance of microbiota or pathogens, which has usually been characterized in separate studies. To date, however, it is unclear how the host influences the interaction with both simultaneously and how this triangular interaction determines fitness of the host-microbe assemblage, the so-called metaorganism. To address this current knowledge gap, we focused on a triangular model interaction, consisting of the nematode Caenorhabditis elegans, its protective symbiont Pseudomonas lurida MYb11 and its pathogen Bacillus thuringiensis Bt679. We combined the two microbes with C. elegans mutants with altered immunity and/or microbial colonization, and found that (i) under pathogen stress, immunocompetence has a larger influence on metaorganism fitness than colonization with the protective microbe; (ii) in almost all cases, MYb11 still improves fitness; and (iii) disruption of p38 MAPK signalling, which contributes centrally to immunity against Bt679, completely reverses the protective effect of MYb11, which further reduces nematode survival and fitness upon infection with Bt679. Our study highlights the complex interplay between host, protective microbe and pathogen in shaping metaorganism biology.


Creatine monohydrate in resistant depression: a preliminary study.
Creatine plays a pivotal role in brain energy homeostasis, and altered cerebral energy metabolism may be involved in the pathophysiology of depression. Oral creatine supplementation may modify brain high-energy phosphate metabolism in depressed subjects. Eight unipolar and two bipolar patients with treatment-resistant depression were treated for four weeks with 3-5 g/day of creatine monohydrate in an open add-on design. Outcome measures were the Hamilton Depression Rating Scale, Hamilton Anxiety Scale, and Clinical Global Impression scores, recorded at baseline and at weeks 1, 2, 3 and 4. One patient improved considerably after one week and withdrew. Both bipolar patients developed hypomania/mania. For the remaining seven patients, all scale scores significantly improved. Adverse reactions were mild and transitory. This small, preliminary, open study of creatine monohydrate suggests a beneficial effect of creatine augmentation in unipolar depression, but possible precipitation of a manic switch in bipolar depression.


Self-administered topical 5% imiquimod cream for external anogenital warts. HPV Study Group. Human PapillomaVirus.
To compare the safety and effectiveness of 5% and 1% imiquimod cream with vehicle cream in the treatment of external anogenital warts. Randomized, double-blind, placebo-controlled comparison that evaluated patients for total clearance of their warts. Patients who experienced total clearance were evaluated for recurrence in a 12-week follow-up. Eleven ambulatory offices, including both private physician offices and referral medical centers. Three hundred eleven healthy men and women aged 18 years or older with 2 to 50 external anogenital warts were recruited from the practices of investigators, referring physicians, and advertisements. Eighty-two additional patients were screened but did not qualify. Four patients discontinued use of the medication because of adverse effects. Five percent imiquimod (Aldara) cream, 1% imiquimod cream, or vehicle cream was applied to all external warts overnight 3 times each week for 16 weeks, or until all treated warts disappeared, whichever occurred first. The number of patients experiencing the elimination of all baseline warts and the recurrence rate of these warts. In addition, the reduction in baseline wart area the duration of therapy required to eliminate warts, and the frequency and severity of adverse reactions were principal measurements. In the intent-to-treat analysis, 54 (50%) of 109 patients who received 5% imiquimod cream, 21 (21%) of 102 of those who received 1% imiquimod cream, and 11 (11%) of 100 patients treated with vehicle cream experienced eradication of all treated baseline warts. The difference between the effectiveness of 5% imiquimod cream and the vehicle cream was statistically significant (P < .001). Of those patients whose warts cleared during therapy, 13% of patients who received 5% imiquimod experienced a recurrence of at least 1 wart. Recurrences occurred in none of the patients who used 1% imiquimod cream and in 10% of patients who used the vehicle cream. Local erythema was the most common adverse reaction, but the majority of patients in each group experienced no or only mild local inflammatory reactions. There were no differences in incidences of flulike symptoms among treatment groups. Five percent imiquimod cream is an effective and safe self-administered therapy for external anogenital warts when applied 3 times a week overnight for up to 16 weeks. The recurrence rate is low.


What's New to You? Preschoolers' Partner-Specific Online Processing of Disfluency.
Speech disfluencies (e.g., "Point to thee um turtle") can signal that a speaker is about to refer to something difficult to name. In two experiments, we found evidence that 4-year-olds, like adults, flexibly interpret a particular partner's disfluency based on their estimate of that partner's knowledge, derived from the preceding conversation. In entrainment trials, children established partner-specific shared knowledge of names for tangram pictures with one or two adult interlocutors. In each test trial, an adult named one of two visible tangrams either fluently or disfluently while children's eye-movements were monitored. We manipulated speaker knowledge in the test trials. In Experiment 1, the test-trial speaker was the same speaker from entrainment or a naïve experimenter; in Experiment 2, the test-trial speaker had been one of the child's partners in entrainment and had seen half of the tangrams (either animal or vehicle tangrams). When hearing disfluent expressions, children looked more at a tangram that was unfamiliar from the speaker's perspective; this systematic disfluency effect disappeared in Experiment 1 when the speaker was entirely naïve, and depended on each speaker's entrainment experience in Experiment 2. These findings show that 4-year-olds can keep track of two different partners' knowledge states, and use this information to determine what should be difficult for a particular partner to name, doing so efficiently enough to guide online interpretation of disfluent speech.


Developmental changes in renal renin mRNA half-life and responses to stimulation in fetal lambs.
In the perinatal period there is increased renin gene expression in the kidney compared with other stages of development. This may be related to changes in responsiveness of the renin gene to stimulation and/or differences in renin mRNA stability as development progresses. To ascertain if either responsiveness or stability changes in fetal life, we studied renin mRNA levels in primary cultures of renal cortical cells obtained from fetal lamb kidneys at two stages (0.7 and 0.9) of gestation after stimulation with isoproterenol, forskolin, or isobutyl methylxanthine and after inhibition of transcription with actinomycin D. Forskolin and isobutyl methylxanthine rapidly increased renin mRNA by at least twofold in the cultured cells from fetuses of both ages, with the sensitivity to stimulation higher in the cells from the mature fetal kidneys. Isoproterenol was effective only in mature fetal cells. In addition, the decay of renin mRNA after cessation of transcription was slower in mature cells compared with immature cells, the half-life being 11.6 +/- 0.8 h in mature cells and 6.6 +/- 0.6 h in immature cells (P < 0.05). The data suggest that increases in both renin mRNA sensitivity to stimulation and in stability can contribute to the enhanced renin expression in the perinatal period.


Effect of stent graft fixation types on pararenal aortic diameter and renal function after endovascular aortic repair.
This study aims to identify pararenal aortic diameter changes following endovascular aortic repair and to investigate the effects of suprarenal fixation on renal function. Between May 2006 and January 2019, a total of 168 patients (135 males, 33 females; mean age: 75.2±8.4 years; range, 48 to 93 years) who underwent endovascular aortic repair were retrospectively analyzed. To compare the effects of graft types, we measured the change in pararenal aortic diameter at four levels: below the celiac artery, below the superior mesenteric artery, at the lowest renal artery, and 1 cm below the lowest renal artery. To evaluate the effect of suprarenal fixation on renal function, serial measurements of serum creatinine, glomerular filtration rate, creatinine clearance rate, and estimated glomerular filtration rate were made. We examined the factors related to post-procedural aortic diameter. The mean change in the aortic diameter was statistically significant, being 12.9±10% in the suprarenal fixation group and 6.19±6.9% in the infrarenal fixation group only at the lowest renal artery level (p=0.001). There was no significant difference in the rate of type I endoleak (p=0.330) or renal function and adverse events (p>0.107) between the groups. The formula for calculating post-procedural aortic diameter was as follows: post-procedural aortic diameter (renal artery level, mm) =1.845 (in case of suprarenal fixation) + 1.012 × pre-procedural aortic diameter (renal artery level, mm) + 0.029 × follow-up time (months) + 0.039 × oversizing (%) (R2=0.773, p<0.001). Suprarenal fixation affects the infrarenal aortic diameter at the lowest renal artery level without significant type I endoleak. However, it does not result in significant renal dysfunction. Pre-procedural aortic diameter, computed tomography follow-up time, suprarenal fixation type, and stent oversizing are the only definite independent correlation factors for postprocedural aortic diameter. This formula can predict the change of aortic diameter after endovascular aortic repair.


Screening Regulatory Element Function with CRISPR/Cas9-based Epigenome Editing.
Genomic regulatory elements that control gene expression play an important role in many traits and diseases. Identifying the regulatory elements associated with each gene or phenotype and understanding the function of that element remain a significant challenge. To address this technological need, we developed CRISPR/Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context. This protocol includes detailed instructions for design and cloning of gRNA libraries, construction of endogenous reporter cell lines via CRISPR/Cas9-mediated knock-in of fluorescent proteins, overall screen design, and recovery of the gRNA library for enrichment analysis. This protocol will be generally useful for implementing genome engineering technologies for high-throughput functional annotation of putative regulatory elements in their native chromosomal context.


Heavy metals testing in active pharmaceutical ingredients: an alternate approach.
The principle of the pharmacopoeial heavy metals test is detection and estimation of the metallic impurities colored by sulfide ion by comparison against lead standard. The test suffers from a loss of analytes upon ashing and from having varied responses for various metals. An inductively coupled plasma-optical emission spectroscopy (ICP-OES) for estimating 23 metals in active pharmaceutical ingredients is being proposed. The method covers the metals listed in USP, Ph. Eur and EMEA guidance on "Residues of Metal Catalysts or Metal Reagents".


Vascular NRP2 triggers PNET angiogenesis by activating the SSH1-cofilin axis.
Angiogenesis is a critical step in the growth of pancreatic neuroendocrine tumors (PNETs) and may be a selective target for PNET therapy. However, PNETs are robustly resistant to current anti-angiogenic therapies that primarily target the VEGFR pathway. Thus, the mechanism of PNET angiogenesis urgently needs to be clarified. Dataset analysis was used to identify angiogenesis-related genes in PNETs. Immunohistochemistry was performed to determine the relationship among Neuropilin 2 (NRP2), VEGFR2 and CD31. Cell proliferation, wound-healing and tube formation assays were performed to clarify the function of NRP2 in angiogenesis. The mechanism involved in NRP2-induced angiogenesis was detected by constructing plasmids with mutant variants and performing Western blot, and immunofluorescence assays. A mouse model was used to evaluate the effect of the NRP2 antibody in vivo, and clinical data were collected from patient records to verify the association between NRP2 and patient prognosis. NRP2, a VEGFR2 co-receptor, was positively correlated with vascularity but not with VEGFR2 in PNET tissues. NRP2 promoted the migration of human umbilical vein endothelial cells (HUVECs) cultured in the presence of conditioned medium PNET cells via a VEGF/VEGFR2-independent pathway. Moreover, NRP2 induced F-actin polymerization by activating the actin-binding protein cofilin. Cofilin phosphatase slingshot-1 (SSH1) was highly expressed in NRP2-activating cofilin, and silencing SSH1 ameliorated NRP2-activated HUVEC migration and F-actin polymerization. Furthermore, blocking NRP2 in vivo suppressed PNET angiogenesis and tumor growth. Finally, elevated NRP2 expression was associated with poor prognosis in PNET patients. Vascular NRP2 promotes PNET angiogenesis by activating the SSH1/cofilin/actin axis. Our findings demonstrate that NRP2 is an important regulator of angiogenesis and a potential therapeutic target of anti-angiogenesis therapy for PNET.


Neopolyploidy and diversification in Heuchera grossulariifolia.
Newly formed polyploid lineages must contend with several obstacles to avoid extinction, including minority cytotype exclusion, competition, and inbreeding depression. If polyploidization results in immediate divergence of phenotypic characters these hurdles may be reduced and establishment made more likely. In addition, if polyploidization alters the phenotypic and genotypic associations between traits, that is, the P and G matrices, polyploids may be able to explore novel evolutionary paths, facilitating their divergence and successful establishment. Here, we report results from a study of the perennial plant Heuchera grossulariifolia in which the phenotypic divergence and changes in phenotypic and genotypic covariance matrices caused by neopolyploidization have been estimated. Our results reveal that polyploidization causes immediate divergence for traits relevant to establishment and results in significant changes in the structure of the phenotypic covariance matrix. In contrast, our results do not provide evidence that polyploidization results in immediate and substantial shifts in the genetic covariance matrix.


Inhibitors of TGFβR1/ALK4/JNK3/Flt1 Kinases in Cynomolgus Macaques Lead to the Rapid Induction of Renal Epithelial Tumors.
Two young cynomolgus macaques (Macaca fascicularis) given a small molecule kinase inhibitor ((S)-4-((2-(5-chloro-2-fluorophenyl)-5-isopropylpyrimidin-4-yl)amino)-N-(2-hydroxypropyl)nicotinamide [SCIO-120]) via nasogastric intubation gavage, once-daily for 21 days at 400 mg/kg/day, developed an unusual epithelial proliferative process in the renal parenchyma. Morphological and immunohistochemical characterization of the lesions confirmed an invasive malignant epithelial neoplasm (carcinoma). A similar renal neoplasm was seen in a third macaque after a 14-day exposure to a second kinase inhibitor in the same chemical series ((S) 4-((2-(5-chloro-2-fluorophenyl)-5-methoxypyrimidin-4-yl)amino)-N-cyclopropylnicotinamide [SCIO-974]). Despite remarkably short latency periods, exposure to these kinase inhibitors was likely causally associated with the induction of the renal tumors, as renal carcinomas are exceedingly rare spontaneously in macaques. Both SCIO-120 and SCIO-974 were designed as potent TGFβR1 inhibitors (IC50s 37 and 39 nM, respectively). SCIO-120 and SCIO-974 inhibited additional kinases, most notably closely related ALK4 (IC50 = 34 and 20 nM, respectively), c-Jun n-Terminal kinase 3 (JNK3, IC50 = 10 and 20 nM, respectively), and Fms-related tyrosine kinase 1 (29 and 76 nM, respectively). TGFβR1 has been specifically implicated in epithelial proliferative disorders, including neoplasia. Neither SCIO-120 nor SCIO-974 was genotoxic based on bacterial reverse mutation and/or clastogenicity screening assays. The rapid appearance of renal carcinomas in primates following short-term treatment with nongenotoxic kinase inhibitors is remarkable and suggests that the compounds had noteworthy tumor-enhancing effects, hypothetically linked to their TGFβR1 inhibition activity. These observations have implications for mechanisms of carcinogenesis and TGFβR1 biology.


The Relationship Between Procedural Volume, Hospital Quality, and Postoperative Mortality in Pediatric Neurosurgery: Review of the Literature.
Studies of neurosurgical pediatric patients associate treatment at low-volume hospitals and by low-volume surgeons with increased odds of adverse outcomes. Although these associations suggest that increased centralization of care could be considered, we evaluate whether confounding endogenous factors mitigate against the proposed outcome benefits. Literature review of English-language articles from 1999-2021. We included articles that assessed volume-outcome effects in pediatric neurosurgical patients. Twelve papers were included from 1999-2021. Primary outcomes included mortality (9), length of stay (LOS) (6), complications (4), and shunt revision/failure rates (3). Volume was measured at the hospital level (8) and at the surgeon level (6). Four papers found that higher volume hospitals had lower odds of mortality. Two papers found that hospitals with higher volume had fewer complications. Two papers found that higher volume surgeons had decreased mortality (OR 0.09-0.3). One paper found that high-volume surgeons had fewer complications (-2.4%; P=0.006). After controlling for hospital factors, two out of seven analyses remained significant. Five analyses did not control for hospital factors. The literature consistently demonstrates a relationship between higher hospital and surgeon volume and better outcomes for pediatric neurosurgical patients. Of the seven articles that assessed hospital factors, only two analyses found that surgical volume remained associated with better outcomes. No reports assessed the degree of centralization already present. The call for centralization of pediatric care should be tempered until variables such as hospital factors, distribution of cases, and clinical thresholds can be defined and studied.


Randomized, double-blind, placebo-controlled trial of acetaminophen for preventing mood and memory effects of prednisone bursts.
Corticosteroids are commonly associated with changes in mood, memory, and the hippocampus. Declarative memory decline occurs rapidly after corticosteroid administration. Minimal research has focused on interventions to prevent or reverse corticosteroid effects on the human brain and associated adverse psychiatric effects. Acetaminophen has neuroprotective properties in animal models. We examined acetaminophen add-on therapy in patients prescribed corticosteroids. Thirty outpatients prescribed oral high-dose prednisone therapy for asthma (n = 28) or allergic rhinitis (n = 2) were randomized to approximately 7 days of acetaminophen (4000 mg/day) or placebo in a double-blind fashion at the same time as prednisone. Mood was assessed with the Hamilton Rating Scale for Depression, Young Mania Rating Scale, and Activation subscale of the Internal State Scale. Memory was assessed with the Rey Auditory Learning Test and asthma symptoms with the Asthma Control Questionnaire. Between-group differences were assessed using mixed ANCOVAs and within-group changes were examined with paired t-tests. Baseline mean depression scores were elevated. In the total sample, depressive and asthma symptoms improved significantly, while declarative memory worsened during prednisone therapy. No between treatment-group differences were found in mood or memory measures. Change in asthma symptoms with receiving prednisone was not related to change in mood or memory. Prednisone therapy was associated with a reduction in depressive symptom severity and decline in declarative memory that was not related to changes in asthma symptoms. This is consistent with prior research suggesting that prednisone impairs memory and may have antidepressant properties. Acetaminophen did not attenuate corticosteroid-induced mood or memory changes.


Bioinspired titanium coatings: self-assembly of collagen-alginate films for enhanced osseointegration.
Achieving long term osseointegration is fundamental to the development of successful bone implants. A key aspect for improving long term osseointegration on titania surfaces is to gain control of nano- and microscale features. The so called biological approach is applied here to modify the surface of titania by coating it with a self-assembled and chemically crosslinked biopolymer film made of alginate and collagen. The biofilm coated titania closely mimics the bone extracellular matrix in bio-morphology and mechanical properties. Biofilms are prepared using the layer by layer technique combined with carbodiimide chemistry to achieve a stable and compact structure. Alginate-collagen coatings display fibrillar morphology with an apparent fiber diameter of ∼50 nm and lengths ranging from a few hundred nanometers to ∼3 μm, mimicking therefore the extracellular matrix of the bone in fiber length and extent. Osteoblast MC3T3-E1 cells showed enhanced adhesion on the coated surface compared to the bare titania and a superior biological activity of the alginate-terminated coating that interfaces the cells in biological fluids.


Mechanism of neutrophil activation by an unopsonized inflammatory particulate. Monosodium urate crystals induce pertussis toxin-insensitive hydrolysis of phosphatidylinositol 4,5-bisphosphate.
Monosodium urate crystals are believed to trigger acute inflammation via the direct stimulation of leukocytes. Unopsonized urate crystals activate neutrophil (PMN) membrane G proteins in a pertussis toxin (PT)-sensitive manner, but induce PT-insensitive cytosolic [Ca2+]i elevation. Thus, we have further defined the mechanism of PMN responsiveness to urate crystals in this study. Though urate crystals can increase membrane permeability by lytic effects, we observed elevation of PMN cytosolic [Ca2+]i in the absence of extracellular [Ca2+]i. In addition, the early, crystal-induced cytosolic [Ca2+]i transient was buffered in cells loaded with a [Ca2+]i-chelator. This suggested mobilization of internal [Ca2+]i stores, which was supported by demonstrating rapid phosphatidylinositol bisphosphate (PIP2) hydrolysis, and the formation of inositol (1,4,5) trisphosphate (as well as phosphatidic acid) in a PT-insensitive manner. Importantly, PMN activation by urate crystals was discriminatory, as evidenced by the absence of phosphatidylinositol trisphosphate formation, a PT-sensitive event triggered by chemotactic factors. Urate crystal-induced PIP2 hydrolysis was not a nonspecific consequence of the early cytosolic [Ca2+]i transient itself, and it did not require phagocytosis. However, crystal-induced O2- release was markedly inhibited by buffering of the early cytosolic [Ca2+]i transient under conditions where crystal phagocytosis and PMA-induced O2- release were unaffected. We conclude that urate crystals activate PT-insensitive PIP2 hydrolysis, resulting in IP3 generation, and early urate crystal-induced mobilization of cytosolic [Ca2+]i. This pathway appears to modulate crystal-induced O2- release.


Primary Aldosteronism in the Elderly.
The clinical spectrum and knowledge of the molecular mechanisms underlying primary aldosteronism (PA), the most frequent form of endocrine hypertension, has evolved over recent years. In accordance with the Endocrine Society guidelines and in light of the growing evidence showing adverse cardiovascular outcomes, it is expected that a progressively wider population of patients affected by hypertension will be screened for PA, including the elderly. A systematic search of PubMed was undertaken for studies related to the renin-angiotensin-aldosterone system (RAAS), PA, and adrenal histopathology in the elderly population. Several studies showed an age-dependent decrease in the activity of RAAS, together with a progressive decrease of the aldosterone response to sodium intake, particularly after the sixth decade of life. The positive correlation between age and serum aldosterone during liberal sodium intake over serum aldosterone during sodium restriction is paralleled by histological changes in adrenal aldosterone synthase (CYP11B2) expression patterns. Immunohistochemical studies showed a progressive loss of the continuous expression of CYP11B2 in the adrenal zona glomerulosa with aging and a concomitant increase of aldosterone-producing cell clusters, which might be responsible for relatively autonomous aldosterone production. Additionally, following PA confirmation and subtype diagnosis, older age is correlated with a lower benefit after adrenalectomy for unilateral PA. Accumulating evidence suggests that RAAS physiology and regulation show age-related changes. Further studies may investigate to what extent these variations might affect the diagnostic workup of patients affected by PA.


Molecular diagnosis of dermatofibrosarcoma protuberans: a comparison between reverse transcriptase-polymerase chain reaction and fluorescence in situ hybridization methodologies.
Dermatofibrosarcoma protuberans (DFSP) is characterized by the presence of the t(17;22)(q22;q13) that leads to the fusion of the COL1A1 and PDGFB genes. This translocation can be detected by multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) or fluorescence in situ hybridization (FISH) techniques. We have evaluated the usefulness of a dual color dual fusion FISH probe strategy for COL1A1/PDGFB detection in a series of 103 archival DFSPs and compared the obtained results with RT-PCR analyses. FISH and RT-PCR were carried out on paraffin embedded tissue samples. Regarding the RT-PCR approach, all COL1A1 exons and exon 2 of PDGFB were evaluated. Sensitivity, specificity, positive and negative predictive values were assessed considering the histological diagnosis as the gold standard. We also analyzed the relationship between the genetic findings and the clinicopathological variables of the tumors. The COL1A1/PDGFB translocation was detected in 93% of DFSP. Both techniques showed a similar specificity (100%), but FISH was more sensitive than RT-PCR (90% vs. 72%). Regarding, clinicopathological features, a higher percentage of positive cells detected by FISH was significantly associated with the fibrosarcomatous DFSP variant (P < 0.001). Interestingly, all CD34 negative DFSP (n = 5) were positive for COL1A1/PDGFB translocation by both techniques. In conclusion, the majority of DFSP harbor the COL1A1/PDGFB translocation and FISH technique should be recommended as a routine diagnostic tool, especially in cases showing unusual histopathological subtypes and/or immunohistochemical features.


Standardisation of cardiac troponin I measurement: past and present.
The laboratory measurement of cardiac troponin (cTn) concentration is a critical tool in the diagnosis of acute myocardial infarction (MI). Current cTnI assays produce different absolute troponin numbers and use different clinical cut-off values; hence cTnI values cannot be interchanged, with consequent confusion for clinicians. A recent Australian study compared patient results for seven cTnI assays and showed that between-method variation was approximately 2- to 5-fold. A major reason for poor method agreement is the lack of a suitable common reference material for the calibration of cTnI assays by manufacturers. Purified complexed troponin material lacks adequate commutability for all assays; hence a serum-based secondary reference material is required for cTnI with value assignment by a higher order reference measurement procedure. There is considerable debate about how best to achieve comparability of results for heterogeneous analytes such as cTnI, whether it should be via the harmonisation or the standardisation process. Whereas harmonisation depends upon consensus value assignment and uses those commercial methods which give the closest agreement at the time, standardisation comes closer to the true value through a reference measurement system that is based upon long-term calibration traceability. The current paper describes standardisation efforts by the International Federation of Clinical Chemistry and Laboratory Medicine Working Group on Standardization of cTnI (IFCC WG-TNI) to establish a reference immunoassay measurement procedure for cTnI of a higher order than current commercial immunoassay methods and a commutable secondary reference material for cTnI to which companies can reference their calibration materials.


Risk reduction strategies for variant Creutzfeldt-Jakob disease transmission by UK plasma products and their impact on patients with inherited bleeding disorders.
The appearance and rapid evolution of BSE in UK cattle in the mid 1980s, with compelling data supporting variant Creutzfeldt-Jakob disease (vCJD) as its human manifestation, pose a potentially severe threat to public health. Three clinical cases and one asymptomatic case of vCJD infection have been reported in UK recipients of non-leucodepleted red cell transfusions from donors subsequently diagnosed with vCJD. Plasma from both these and other donors who later developed vCJD has contributed towards plasma pools used to manufacture clotting factor concentrate. The United Kingdom Haemophilia Centre Doctors' Organisation (UKHCDO) Surveillance Study has detected asymptomatic vCJD postmortem in a haemophilic patient treated with UK plasma products including two batches of clotting factor linked to a donor who subsequently developed vCJD. Over 4000 bleeding disorder patients treated with UK plasma products are recorded on the UKHCDO National Haemophilia Database. The risk of vCJD transmission by plasma products is not known. However, public health precautions have been implemented since 2004 in all UK inherited bleeding disorder patients who received UK-sourced plasma products between 1980 and 2001 to minimize the possible risk of onward vCJD transmission. We evaluate vCJD surveillance and risk management measures taken for UK inherited bleeding disorder patients, report current data and discuss resultant challenges and future directions.


Alpha 2-adrenoceptors do not regulate catecholamine secretion by bovine adrenal medullary cells: a study with clonidine.
Experiments have been performed with perfused bovine adrenal glands, with freshly isolated chromaffin cells, and with chromaffin cells maintained in tissue culture to investigate the suggestion that there are alpha-adrenoceptors present which regulate catecholamine secretion. Only one set of observations has lent support to this suggestion: the rather specific alpha 2-adrenoceptor agonist clonidine inhibits catecholamine secretion evoked by the physiological secretagogue, acetylcholine, and by the related nicotinic agonists, carbachol and nicotine. All other observations detract from the suggestion. Other alpha-adrenoceptor agonists (noradrenaline, adrenaline, tramazoline, phenylephrine, and alpha-methyl-noradrenaline) are virtually ineffective at inhibiting secretion evoked by carbachol. In addition, the alpha-adrenoceptor antagonists phentolamine, phenoxybenzamine, and yohimbine not only fail to enhance the secretion of catecholamines evoked by carbachol but also fail to offset the inhibitory action of clonidine. The data suggest that functional alpha 2-adrenoceptors of the classical type are not present upon bovine chromaffin cells and that, in this tissue, clonidine must act in some other way. In the bovine adrenal medullary chromaffin cell clonidine probably acts at the nicotinic receptor because it does not reduce catecholamine secretion evoked by depolarizing concentrations of potassium or veratridine but does reduce the carbachol-evoked influx of 22Na that can be measured in the presence of tetrodotoxin and ouabain and which probably reflects entry through the nicotinic channel. Furthermore, clonidine can abolish, in reversible fashion, the acetylcholine-activated inward current determined with patch-clamp.


Determinants of protein-energy malnutrition in community-dwelling older adults: a systematic review of observational studies.
Protein-energy malnutrition is associated with numerous poor health outcomes, including high health care costs, mortality rates and poor physical functioning in older adults. This systematic literature review aims to identify and provide an evidence based overview of potential determinants of protein-energy malnutrition in community-dwelling older adults. A systematic search was conducted in PUBMED, EMBASE, CINAHL and COCHRANE from the earliest possible date through January 2013. Observational studies that examined determinants of protein-energy malnutrition were selected and a best evidence synthesis was performed to summarize the results. In total 28 studies were included in this review from which 122 unique potential determinants were derived. Thirty-seven determinants were examined in sufficient number of studies and were included in a best evidence synthesis. The best evidence score comprised design (cross-sectional, longitudinal) and quality of the study (high, moderate) to grade the evidence level. Strong evidence for an association with protein-energy malnutrition was found for poor appetite, and moderate evidence for edentulousness, having no diabetes, hospitalization and poor self-reported health. Strong evidence for no association was found for anxiety, chewing difficulty, few friends, living alone, feeling lonely, death of spouse, high number of diseases, heart failure and coronary failure, stroke (CVA) and the use of anti-inflammatory medications. This review shows that protein-energy malnutrition is a multifactorial problem and that different domains likely play a role in the pathway of developing protein-energy malnutrition. These results provide important knowledge for the development of targeted, multifactorial interventions that aim to prevent the development of protein-energy malnutrition in community-dwelling older adults.


Allergen-specific IL-5 responses in early childhood predict asthma at age eight.
The pattern of development of allergen-specific T cell cytokine responses in early childhood and their relation to later disease is poorly understood. Here we describe longitudinal changes in allergen-stimulated T cell cytokine responses and their relation to asthma and allergic disease during the first 8 years of life. Subjects with a family history of asthma, who were enrolled antenatally in the Childhood Asthma Prevention Study (public trials registration number ACTRN12605000042640), had skin prick tests, clinical evaluation for asthma and eczema, and in vitro assessment of T cell cytokine responses to HDM extract performed at ages 18 months (n = 281), 3 years (n = 349), 5 years (n = 370) and 8 years (n = 275). We measured interleukin (IL-) 13 at 3, 5 and 8 years, and IL-5, IL-10, and interferon-γ (IFN-γ), at 18 months, 3, 5 and 8 years by ELISA. A cohort analysis was undertaken. Independent effects of cytokine responses at each age on the risk of asthma and allergic outcomes at age 8 years were estimated by multivariable logistic regression. HDM-specific IL-5 responses increased with age. HDM-specific IL-13 and IL-10 responses peaked at age 5 years. HDM-specific IL-5 responses at 3 years, 5 years and 8 years were significantly associated with the presence of asthma and atopy at 8 years. IL-13 responses at 3 years, 5 years and 8 years were significantly associated with atopy at 8 years, but this association was not independent of the effect of IL-5. Other HDM-specific cytokine responses were not independently related to asthma or eczema at 8 years. HDM-specific IL-5 responses at age 3 years or later are the best measure of T cell function for predicting asthma at age 8 years.


Insulin secretion-sensitivity index-2 could be a novel marker in the identification of the role of pancreatic iron deposition on beta-cell function in thalassemia major.
The purpose of this study is to evaluate the impact of insulin secretion-sensitivity index-2 (ISSI-2) in the identification of the role of pancreatic iron deposition on beta-cell function in thalassemia major. Tissue iron stores were measured with magnetic resonance imaging (MRI) in the liver (R2), pancreas (R2*), and heart (T2*). ISSI-2 was assessed as a novel oral glucose tolerance test-based measure of beta-cell function. Also, the Stumvoll index showing the insulin sensitivity and Stumvoll index estimating first and second phase insulin secretion were calculated. Fourteen of the 51 Thalassemia Major patients, aged 8-34 (mean 21.1 ± 7.2) years-old, had either an impaired glucose tolerance test (n = 9, 17.6%) or diabetes mellitus (n = 5, 9.8%)-referred to as the glucose dysregulation (GD) group. The median serum ferritin and the mean liver R2 and cardiac T2* values were not significantly different between the GD and normal glucose tolerance (NGT, n = 37) groups whereas pancreas R2* was significantly higher in the GD group compared to the NGT group (p = 0.004). Patients with GD showed significantly lower ISSI-2 index (p < 0.001) as well as the Stumvoll index and Stumvoll first and second phase indices compared to those with NGT (p < 0.001). All patients with GD displayed a pancreas R2* >50 Hz and ISSI-2 <2. In conclusion, Pancreas R2* MRI combined with ISSI-2 index may be valuable parameters to identify patients at the highest risk for developing glucose dysregulation.


Artificial disc replacement and adjacent-segment pathology: 10-year outcomes of a randomized trial.
Artificial disc replacement (ADR) is designed to preserve motion and thus protect against adjacent-segment pathology (ASP) and act as an alternative treatment to fusion surgery. The question remains, how well do ADR devices perform after 10 years of follow-up compared with fusion surgery in terms of patient satisfaction, sustainability, and protection against ASP? This was the 10-year follow-up study of 153 participants who underwent ADR or fusion surgery after anterior decompression due to cervical degenerative radiculopathy (ISRCTN registration no. 44347115). Scores on the Neck Disability Index (NDI), EQ-5D, and visual analog scale for neck and arm pain were obtained from the Swedish Spine Registry and analyzed using ANCOVA. Information about secondary surgical procedures was collected from medical records and presented as Kaplan-Meier curves. MRI and flexion-extension radiography were performed, and ASP was graded according to the Miyazaki classification system. Ten participants were lost to follow-up, which left 143 participants (80 underwent ADR and 65 underwent anterior cervical discectomy and fusion). There were no differences between groups in terms of patient-reported outcome measures (10-year difference in NDI scores 1.7 points, 95% CI -5.1 to 8.5, p = 0.61). Nineteen (24%) participants in the ADR group compared with 9 (14%) in the fusion group underwent secondary surgical procedures. The higher reoperation rate of the ADR group was mainly due to 11 female participants with device loosening. The rates of reoperation due to ASP were similar between groups, which was confirmed with MRI assessment of ASP that also showed no differences between the groups (p = 0.21). This was the first 10-year follow-up study to compare ADR with fusion surgery and to provide MRI information for the assessment of ASP. The authors found no benefit of ADR over fusion surgery after anterior decompression for cervical degenerative radiculopathy.


Single-molecule spectroscopy and imaging of biomolecules in living cells.
The number of reports per year on single-molecule imaging experiments has grown roughly exponentially since the first successful efforts to optically detect a single molecule were completed over two decades ago. Single-molecule spectroscopy has developed into a field that includes a wealth of experiments at room temperature and inside living cells. The fast growth of single-molecule biophysics has resulted from its benefits in probing heterogeneous populations, one molecule at a time, as well as from advances in microscopes and detectors. This Perspective summarizes the field of live-cell imaging of single biomolecules.