Thioctic acid and acetyl-L-carnitine in the treatment of sciatic pain caused by a herniated disc: a randomized, double-blind, comparative study.
Sciatica is a painful condition characterized by radiating leg pain that most commonly originates from a herniated disc in the lumbar or sacral spine. Although sciatic pain is typically self-limiting, pharmacological analgesic therapy forms the mainstay of treatment. Acetyl-L-carnitine (levacecarnine; ALC) is a naturally occurring substance that promotes peripheral nerve regeneration and has been shown to have analgesic effects in patients with peripheral neuropathies of diabetic, HIV-related or chemotherapeutic origin. Thioctic acid, a key compound in oxidative metabolism, has antioxidant properties that may help the recovery of nerve functionality and decrease neuropathic pain. This study aimed to compare, for the first time, the efficacy of oral treatment with ALC or thioctic acid in patients with peripheral neuropathic (sciatic) pain associated with a herniated disc. This was a randomized, double-blind trial conducted in a hospital setting. A total of 64 consecutive patients (mean age 61 years; range 29-85) with acute backache and moderate sciatica were recruited. Patients in group 1 (n = 33) received ALC 1180 mg/day; patients in group 2 (n = 31) received thioctic acid 600 mg/day. The study period was 60 days. The primary efficacy endpoint was change in clinical signs and symptoms of sciatica, as measured on the Neuropathy Impairment Score in the Lower Limbs (NIS-LL) questionnaire, the Neuropathy Symptoms and Change in the Lower Limbs (NSC-LL) questionnaire, and the Total Symptom Score (TSS) questionnaire. The secondary efficacy endpoint was improvement in neurological deficit (as measured by electromyography) compared with baseline. Both treatments produced significant improvements from baseline in neuropathy on electromyography at day 60, and greater mean improvements were observed with thioctic acid (-0.19 +/- 0.29 vs baseline) than with ALC (-0.09 +/- 0.40 vs baseline), although the between-group difference was not statistically significant. Thioctic acid produced significantly greater mean improvements than ALC from baseline for NIS-LL (-2.52 +/- 1.50 vs -1.48 +/- 1.37, respectively), NSC-LL (-2.16 +/- 1.37 vs 1.42 +/- 1.37, respectively) and TSS (-1.90 +/- 1.08 vs 1.18 +/- 1.01, respectively) scores (p < 0.05 for all comparisons). More patients receiving thioctic acid than ALC reported a decreased need for analgesia (71.0% vs 45.5%, respectively; p < 0.05) and neither treatment impacted significantly on sleep quality. Thioctic acid 600 mg/day appears to be at least as effective as ALC in the treatment of sciatic pain caused by a herniated disc and may be associated with an improvement in symptom scores and reduced need for analgesia. However, because of the limited number of patients evaluated and the lack of a placebo control in this trial, further studies are warranted in order to provide more definitive results.


The Diversity of Bacterial Lifestyles Hampers Bacteriophage Tenacity.
Phage therapy is based on a simple concept: the use of a virus (bacteriophage) that is capable of killing specific pathogenic bacteria to treat bacterial infections. Since the pioneering work of Félix d'Herelle, bacteriophages (phages) isolated in vitro have been shown to be of therapeutic value. Over decades of study, a large number of rather complex mechanisms that are used by phages to hijack bacterial resources and to produce their progeny have been deciphered. While these mechanisms have been identified and have been studied under optimal conditions in vitro, much less is known about the requirements for successful viral infections in relevant natural conditions. This is particularly true in the context of phage therapy. Here, we highlight the parameters affecting phage replication in both in vitro and in vivo environments, focusing, in particular, on the mammalian digestive tract. We propose avenues for increasing the knowledge-guided implementation of phages as therapeutic tools.


A novel and innovative hair test to determine glucocorticoid levels in racing camels for use in assessment of doping, health, and disease.
The aim of this project was to develop and validate a new test for the analysis of glucocorticoids in camel hair and to use the new test to analyse hair samples from a variety of camel breeds in sports and racing applications. These findings could be of importance when evaluating racing camels for suspected doping offenses or for injury and disease control. Camel hair samples were collected from 30 non-racing dromedary camels along with 3 racing camels in Al Ain, UAE and were decontaminated, pulverised, sonicated, and extracted prior to analysis. A liquid chromatographic-mass spectrometric method was employed to determine the levels of glucocorticoids in the hair samples. The 4 drugs of interest, namely hydrocortisone, dexamethasone, flumethasone and methylprednisolone, and an internal standard were quantified in camel hair samples. All 4 of the glucocorticoids were detected in camel hair samples with concentrations ranging between 31 and 935 pg/mg for hydrocortisone, 8-59 pg/mg for dexamethasone, 0.7-1034 pg/mg for flumethasone and 5-66 pg/mg for methylprednisolone in non-racing camels. One of the racing camels displayed high concentrations of hydrocortisone (1130 pg/mg), flumethasone (2576 pg/mg), methylprednisone (1156 pg/mg) and dexamethasone (29 pg/mg). The authors believe this is the first report of a test for corticosteroids in camel hair. The new test has been validated according to Food and Drug Administration (FDA) guidelines. This new hair test could be useful for further studies in doping control, toxicological studies, pharmacological studies and other clinical applications in camel health, injury, and disease.


A randomized, double-blind, multicenter trial comparing transdermal scopolamine plus ondansetron to ondansetron alone for the prevention of postoperative nausea and vomiting in the outpatient setting.
Postoperative nausea and vomiting (PONV) are common complications after ambulatory surgery. We sought to determine whether the use of transdermal scopolamine (TDS) in combination with IV ondansetron (OND) is more effective than one alone for reducing PONV in outpatient settings. In a randomized, double blind, multicenter trial, 620 at-risk female patients undergoing outpatient laparoscopic or breast augmentation surgery received either an active TDS patch or a similar appearing sham 2 h before entering the operating room. All patients received IV OND (4 mg) 2-5 min before induction of anesthesia followed by a general anesthetic regimen. Complete antiemetic response, defined as no vomiting/retching or rescue medication use, was measured through 24 h and 48 h after surgery. The proportion of patients with vomiting/retching, nausea, or use of rescue medication, the time from the end of surgery to the first episode of these events and the time to discharge from the hospital/surgery center, as well as the number and severity of vomiting/retching and nausea episodes, and patient satisfaction with antiemetic therapy were also collected. The combination of TDS + OND statistically significantly reduced nausea and vomiting/retching compared with OND alone 24 h after surgery but not at 48 h. The proportion of patients who did not experience vomiting/retching and did not use rescue medication was 48% for TDS + OND and 39% for OND alone (P < 0.02). Total response (no nausea, no vomiting/retching, and no use of rescue medication) was also statistically higher for the TDS + OND group compared with the OND-only group (35% vs 25%, P < 0.01). The time to first nausea, vomiting/retching, or rescue episode was statistically significantly longer for the TDS + OND group compared with the OND-only group (P < 0.05). The cumulative overall incidence of adverse events was lower in the TDS + OND group compared with the OND group (36.7% vs 49%, P < 0.01). TDS + OND reduces PONV compared with OND alone. This is achieved with a reduction in adverse events.


High-throughput decoding of antitrypanosomal drug efficacy and resistance.
The concept of disease-specific chemotherapy was developed a century ago. Dyes and arsenical compounds that displayed selectivity against trypanosomes were central to this work, and the drugs that emerged remain in use for treating human African trypanosomiasis (HAT). The importance of understanding the mechanisms underlying selective drug action and resistance for the development of improved HAT therapies has been recognized, but these mechanisms have remained largely unknown. Here we use all five current HAT drugs for genome-scale RNA interference target sequencing (RIT-seq) screens in Trypanosoma brucei, revealing the transporters, organelles, enzymes and metabolic pathways that function to facilitate antitrypanosomal drug action. RIT-seq profiling identifies both known drug importers and the only known pro-drug activator, and links more than fifty additional genes to drug action. A bloodstream stage-specific invariant surface glycoprotein (ISG75) family mediates suramin uptake, and the AP1 adaptin complex, lysosomal proteases and major lysosomal transmembrane protein, as well as spermidine and N-acetylglucosamine biosynthesis, all contribute to suramin action. Further screens link ubiquinone availability to nitro-drug action, plasma membrane P-type H(+)-ATPases to pentamidine action, and trypanothione and several putative kinases to melarsoprol action. We also demonstrate a major role for aquaglyceroporins in pentamidine and melarsoprol cross-resistance. These advances in our understanding of mechanisms of antitrypanosomal drug efficacy and resistance will aid the rational design of new therapies and help to combat drug resistance, and provide unprecedented molecular insight into the mode of action of antitrypanosomal drugs.


A blended learning teaching strategy strengthens the nursing students' performance and self-reported learning outcome achievement in an anatomy, physiology and biochemistry course - A quasi-experimental study.
In nursing, bioscience is regarded as one of the cornerstones of nursing practice. However, bioscience disciplines as anatomy, physiology and biochemistry are considered challenging for students and the failure rate is high. In this study we explore a blended learning teaching strategy in an anatomy, physiology and biochemistry course for first year Bachelor nursing students. In the blended learning teaching strategy, short narrated online digital resources of bioscientific terms and concepts were integrated into the teaching design along with digital metacognitive evaluations of learning outcomes. Results show that compared to students receiving traditional face-to-face teaching, the students with a blended learning approach performed better on their national exam with a small to medium effect size (Cohen's d=0.23). Student course evaluations supported the blended learning delivery with small to medium effect sizes. The students reported that the digital resources supported their learning outcome achievement, that they better understood the teacher's expectations and that they were more satisfied with their virtual learning environment. This study adds to the growing literature of blended learning effectiveness in higher education, and suggests the use of digital resources as an enrichment of teaching and enhancement of students' study experience.


Response of different genotypes of faba bean plant to drought stress.
Drought stress is one of the major abiotic stresses that are a threat to crop production worldwide. Drought stress impairs the plants growth and yield. Therefore, the aim of the present experiment was to select the tolerant genotype/s on the basis of moprpho-physiological and biochemical characteristics of 10 Vicia faba genotypes (Zafar 1, Zafar 2, Shebam, Makamora, Espan, Giza Blanka, Giza 3, C4, C5 and G853) under drought stress. We studied the effect of different levels of drought stress i.e., (i) normal irrigation (ii) mild stress (iii) moderate stress, and (iv) severe stress on plant height (PH) plant-1, fresh weight (FW) and dry weight (DW) plant-1, area leaf-1, leaf relative water content (RWC), proline (Pro) content, total chlorophyll (Total Chl) content, electrolyte leakage (EL), malondialdehyde (MDA), hydrogen peroxide (H2O2) content, and activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) of genotypes of faba bean. Drought stress reduced all growth parameters and Total Chl content of all genotypes. However, the deteriorating effect of drought stress on the growth performance of genotypes "C5" and "Zafar 1" were relatively low due to its better antioxidant enzymes activities (CAT, POD and SOD), and accumulation of Pro and Total Chl, and leaf RWC. In the study, genotype "C5" and "Zafar 1" were found to be relatively tolerant to drought stress and genotypes "G853" and "C4" were sensitive to drought stress.


THE NEUMANN PROBLEM ON ELLIPSOIDS.
The Neumann problem on an ellipsoid in Rn asks for a function harmonic inside the ellipsoid whose normal derivative is some specified function on the ellipsoid. We solve this problem when the specified function on the ellipsoid is a normalized polynomial (a polynomial divided by the norm of the normal vector arising from the definition of the ellipsoid). Specifically, we give a necessary and sufficient condition for a solution to exist, and we show that if a solution exists then it is a polynomial whose degree is at most the degree of the polynomial giving the specified function. Furthermore, we give an algorithm for computing this solution. We also solve the corresponding generalized Neumann problem and give an algorithm for computing its solution.


Central and Peripheral Mechanism of Acupuncture Analgesia on Visceral Pain: A Systematic Review.
Despite the wide use of acupuncture for the management of visceral pain and the growing interest in the pathophysiology of visceral pain, there is no conclusive elucidation of the mechanisms behind the effects of acupuncture on visceral pain. This systematic review aims to provide an integrative understanding of the treatment mechanism of acupuncture for visceral pain. Electronic and hand searches were conducted to identify studies that involved visceral pain and acupuncture. We retrieved 192 articles, out of which 46 studies were included in our review. The results of our review demonstrated that visceral pain behaviors were significantly alleviated in response to acupuncture treatment in groups treated with this intervention compared to in sham acupuncture or no-treatment groups. Changes in the concentrations of β-endorphin, epinephrine, cortisol, and prostaglandin E2 in plasma, the levels of c-Fos, substance P, corticotropin-releasing hormone, P2X3, acetylcholinesterase (AchE), N-methyl-D-aspartate (NMDA) receptors, and serotonin in the gut/spinal cord, and the neuronal activity of the thalamus were associated with acupuncture treatment in visceral pain. Acupuncture reduced visceral pain behavior and induced significant changes in neuronal activity as well as in the levels of pain/inflammation-related cytokines and neurotransmitters in the brain-gut axis. Further researches on the thalamus and on a standard animal model are warranted to improve our knowledge on the mechanism of acupuncture that facilitates visceral pain modulation.


Altitude, pasture type, and sheep breed affect bone metabolism and serum 25-hydroxyvitamin D in grazing lambs.
This study aimed to investigate the bone development of two mountain sheep breeds during natural summer grazing either in the lowlands or on different characteristic alpine pastures. Pasture types differed in topographic slope, plant species composition, general nutritional feeding value, Ca and P content, and Ca:P ratio of herbage. Twenty-seven Engadine sheep (ES) lambs and 27 Valaisian Black Nose sheep (VS) lambs were divided into four groups of 6 to 7 animals per breed and allocated to three contrasting alpine pasture types and one lowland pasture type. The lambs were slaughtered after 9 wk of experimental grazing. The steep alpine pastures in combination with a high (4.8) to very high (13.6) Ca:P ratio in the forage decreased total bone mineral content as measured in the middle of the left metatarsus of the lambs from both breeds, and cortical bone mineral content and cortical bone mineral density of ES lambs. Breed × pasture type interactions occurred in the development of total and cortical bone mineral content, and in cortical thickness, indicating that bone metabolism of different genotypes obviously profited differently from the varying conditions. An altitude effect occurred for 25-hydroxyvitamin D with notably higher serum concentrations on the three alpine sites, and a breed effect led to higher concentrations for ES than VS. Despite a high variance, there were pasture-type effects on serum markers of bone formation and resorption.


Qualitative analysis of seized cocaine samples using desorption electrospray ionization- mass spectrometry (DESI-MS).
Desorption electrospray ionization - mass spectrometry (DESI-MS) is a useful technique for the qualitative analysis of compounds found in seized drug material. In this study, DESI-MS was utilized in the screening analysis of illicit cocaine samples. The technique was also applied to the geographical origin determination of these samples. The limit of detection was determined to be 24.3 µg (or 3.47 µg/mm(2) ) and the analysis time was less than 1 minute per sample. The intra-day and inter-day precision for the detection of cocaine was 11 % and 42 %, respectively; therefore the quantitative data provided by DESI-MS was limited in its use for accurate determination of cocaine concentration in a sample. Using the quadrupole time-of-flight (QTOF) mass spectrometer, the presence of cocaine and impurities detected were confirmed by accurate tandem MS data. The qualitative chemical profiles obtained using DESI-MS were compared to two popular analysis techniques, GC-MS and LC-MS. The effects of a range of adulterants including caffeine, procaine, levamisole, lignocaine, paracetamol, and atropine on the detectability of cocaine were also investigated. It was found that the addition of these adulterants in a cocaine sample did not prevent the detection of the analyte itself (there was slight enhancement in some samples), which was useful in drug detection. The detection of truxillines in the seized samples by DESI-MS aided in the preliminary determination of geographical origin, i.e., Bolivian, Peruvian or Colombian leaf origin. The application of DESI-MS to the qualitative analysis and screening of seized cocaine samples demonstrates the potential and applicability of the technique to the fast chemical profiling of illicit samples.


Recognition and management of exercise-induced bronchospasm.
Exercise-induced bronchospasm is an obstruction of transient airflow that usually occurs five to 15 minutes after physical exertion. Although this condition is highly preventable, it is still underrecognized and affects aerobic fitness and quality of life. Diagnosis is based on the results of a detailed history, including assessment of asthma triggers, symptoms suggestive of exercise-induced bronchoconstriction, and a normal forced expiratory volume at one second at rest. A trial of therapy with an inhaled beta agonist may be instituted, with the subsequent addition of inhaled anti-inflammatory agents or ipratropium bromide. Nonpharmacologic measures, such as increased physical conditioning, warm-up exercises, and covering the mouth and nose, should be instituted. If symptoms persist, pulmonary function testing is warranted to rule out underlying lung disease.


Linking Joint Impairment and Gait Biomechanics in Patients with Juvenile Idiopathic Arthritis.
Juvenile Idiopathic Arthritis (JIA) is a paediatric musculoskeletal disease of unknown aetiology, leading to walking alterations when the lower-limb joints are involved. Diagnosis of JIA is mostly clinical. Imaging can quantify impairments associated to inflammation and joint damage. However, treatment planning could be better supported using dynamic information, such as joint contact forces (JCFs). To this purpose, we used a musculoskeletal model to predict JCFs and investigate how JCFs varied as a result of joint impairment in eighteen children with JIA. Gait analysis data and magnetic resonance images (MRI) were used to develop patient-specific lower-limb musculoskeletal models, which were evaluated for operator-dependent variability (< 3.6°, 0.05 N kg-1 and 0.5 BW for joint angles, moments, and JCFs, respectively). Gait alterations and JCF patterns showed high between-subjects variability reflecting the pathology heterogeneity in the cohort. Higher joint impairment, assessed with MRI-based evaluation, was weakly associated to overall joint overloading. A stronger correlation was observed between impairment of one limb and overload of the contralateral limb, suggesting risky compensatory strategies being adopted, especially at the knee level. This suggests that knee overloading during gait might be a good predictor of disease progression and gait biomechanics should be used to inform treatment planning.


Improving routine outpatient monitoring for patients with sickle-cell disease on hydroxyurea.
Hydroxyurea is the gold standard treatment for prevention of vaso-occlusive crises in patients with sickle-cell anaemia. It has a narrow therapeutic index and dangerous side effects including cytopenias. There is high variation in dose-response across the population. Therefore, a robust outpatient monitoring programme is crucial to ensure efficacy and safety of treatment. However, there has historically been difficulty engaging the target population in regular laboratory test monitoring programmes. This project aimed to ensure that all patients on hydroxyurea had routine blood tests at least once every 2 months which were reviewed and acted upon within the 3-year project life cycle. A specialist haematology nurse prescriber clinic service was introduced, first informally, and then formally to take blood tests, alter medication dosing, prescribe it and then write a clinic letter. The mean number of tests per patient per year rose from 0.21 at baseline to 9.05 after 2 years of the formal nurse prescriber clinic. This led to an associated increase in dose changes from 0.23 to 1.45 per patient per year. This improved the number of patients on the optimum dose of hydroxyurea. Furthermore, due to increased confidence in the outpatient monitoring, the total number of people being prescribed hydroxyurea increased from 26 to 42. Restriction of prescriptions to only those enrolled in the service has prevented unmonitored patients being at risk of the potential toxicities associated with doses that are too high. The introduction of a formal nurse-led clinic has improved the safety, efficacy and compliance and increased the number of patients on the gold standard preventative treatment for vaso-occlusive crises in sickle-cell anaemia.


Novel immunoassay for quantification of brain natriuretic peptide and its precursor in human blood.
Brain natriuretic peptide (BNP) is an unstable molecule that can rapidly lose immunologic activity in blood. Conventional sandwich BNP immunoassays use 2 antibodies specific to 2 different epitopes. Larger distances between epitopes are associated with a greater probability of proteolysis sites being located between the antibody-binding sites, and thus such assays have an increased susceptibility to underdetect BNP because of the increased likelihood of proteolytic degradation. The purpose of our study was to develop a sandwich immunoassay for the precise quantification of BNP and BNP precursor (proBNP) in human blood that is not susceptible to proteolysis. Mice were immunized with an immune complex consisting of monoclonal antibody (MAb) 24C5 (specific for BNP peptide 11-22) and the entire BNP molecule. The MAb used in our assay (Ab-BNP2) recognizes the immune complex but neither free BNP nor MAb 24C5. We used MAbs 24C5 and Ab-BNP2 to develop a new type of sandwich BNP assay (the "single-epitope sandwich assay"), which requires only a short BNP fragment (fragment 11-22) for immunodetection. This assay recognizes both BNP and proBNP with the same efficiency and sensitivity and demonstrates both considerably less susceptibility to antigen degradation and greater stability of the measured antigen than conventional sandwich BNP immunoassays. We have developed this sensitive single-epitope sandwich assay for detecting BNP, proBNP, and their fragments in human blood. This assay appears promising for use in clinical studies to assist in triage, management, and outcomes assessment in heart failure patients.


Spatiotemporal properties of optic flow and vestibular tuning in the cerebellar nodulus and uvula.
Convergence of visual motion and vestibular information is essential for accurate spatial navigation. Such multisensory integration has been shown in cortex, e.g., the dorsal medial superior temporal (MSTd) and ventral intraparietal (VIP) areas, but not in the parieto-insular vestibular cortex (PIVC). Whether similar convergence occurs subcortically remains unknown. Many Purkinje cells in vermal lobules 10 (nodulus) and 9 (uvula) of the macaque cerebellum are tuned to vestibular translation stimuli, yet little is known about their visual motion responsiveness. Here we show the existence of translational optic flow-tuned Purkinje cells, found exclusively in the anterior part of the nodulus and ventral uvula, near the midline. Vestibular responses of Purkinje cells showed a remarkable similarity to those in MSTd (but not PIVC or VIP) neurons, in terms of both response latency and relative contributions of velocity, acceleration, and position components. In contrast, the spatiotemporal properties of optic flow responses differed from those in MSTd, and matched the vestibular properties of these neurons. Compared with MSTd, optic flow responses of Purkinje cells showed smaller velocity contributions and larger visual motion acceleration responses. The remarkable similarity between the nodulus/uvula and MSTd vestibular translation responsiveness suggests a functional coupling between the two areas for vestibular processing of self-motion information.


Hospitalization for Anxiety and Mood Disorders in HIV-Infected and -Uninfected Gay and Bisexual Men.
Prevalence of anxiety and mood disorders (AMDs) in HIV-infected individuals has varied widely because of the variety of measurements used and differences in risk factor profiles between different populations. We aimed to examine the relationship between HIV status and hospitalization for AMDs in gay and bisexual men (GBM). HIV-infected (n = 557) and HIV-uninfected (n = 1325) GBM recruited in Sydney, Australia were probabilistically linked to their hospital admissions and death notifications (2000-2012). Random-effects Poisson models were used to assess HIV risk factors for hospitalization. Cox regression methods were used to assess risk factors for mortality. We observed 300 hospitalizations for AMDs in 15.3% of HIV-infected and 181 in 5.4% of HIV-uninfected participants. Being infected with HIV was associated with a 2.5-fold increase in risk of hospitalization for AMDs in GBM. Other risk factors in the HIV-infected cohort included previous hospitalization for HIV-related dementia, a more recent HIV diagnosis, and a CD4 T-cell count above 350 cells per cubic millimeter. Being hospitalized for an AMD was associated with a 5.5-fold increased risk of mortality; this association did not differ by HIV status. An association between substance use and mortality was observed in individuals hospitalized for AMDs. There is a need to provide more effective strategies to identify and treat AMDs in HIV-infected GBM. This research highlights the importance of further examination of the effects of substance use, neurocognitive decline, and AMDs on the health of HIV-infected individuals.


Adverse effects of antiretroviral drugs on HIV-1-infected and -uninfected human monocyte-derived macrophages.
Antiretroviral drugs approved for treatment of HIV-1 infection include nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs). Use of these drugs in combinations (highly active antiretroviral therapy) has delayed disease progression. However, long-term therapy is associated with potentially serious adverse effects. NRTIs are thought to contribute to these adverse effects via depletion of mtDNA. Inasmuch as macrophages (major targets for HIV-1) are highly metabolically active with large numbers of mitochondria, we investigated the effects of NRTIs (didanosine, stavudine, lamivudine, and zidovudine) on the viability and function of HIV-1-infected and -uninfected human monocyte-derived macrophages (MDMs). We demonstrate that the combinations didanosine/stavudine and lamivudine/zidovudine decrease mtDNA content in MDMs, with HIV-1-infected MDMs displaying a greater reduction than uninfected cells. This decrease correlated with decreased complement-mediated phagocytosis (C'MP) by MDMs, a process dependent on mitochondrial function. Inasmuch as PIs have previously been reported to interact with cellular proteases and given that cellular proteases are involved in the phagocytic process, we investigated the effects of the PI indinavir on C'MP. We demonstrate that indinavir augments C'MP by uninfected MDMs, but not HIV-1-infected MDMs. This study provides additional understanding on the effects of commonly used antiretroviral drugs on cellular immune function.


Angiotensin-converting enzyme gene insertion/deletion polymorphism and breast cancer risk.
The renin-angiotensin system plays an important role in homeostasis and lately, its main effector, angiotensin II, has been attributed with angiogenic and growth factor actions in the breast tissue. Previous studies have shown that the insertion/deletion (I/D) polymorphism in the angiotensin-converting enzyme (ACE) gene accounts for the variability of ACE plasma concentrations. The use of ACE inhibitors and the ACE I/D polymorphism may be linked to breast cancer risk. In this study, we evaluate the relationship of the ACE I/D polymorphism with breast cancer risk in Caucasian postmenopausal women. The ACE I/D polymorphism was genotyped in 4,117 women participants in the Rotterdam Study. Baseline information was obtained through a questionnaire. We conducted a logistic regression and survival analysis to assess the risk of breast cancer by the ACE genotype. The DD carriers showed a significantly increased risk of developing breast cancer when compared with the II carriers (odds ratio, 1.86; 95% confidence interval, 1.06-3.27; P = 0.03). This association remained after adjusting for other risk factors, including body mass index, age at menarche, age at menopause, hormone replacement therapy, and hypertension. Our survival analysis showed that the cancer-free survival was significantly reduced in DD compared with II carriers (hazard ratio, 1.80; 95% confidence interval, 1.07-3.01; P = 0.03). Our results suggest that the ACE I/D polymorphism plays an important role in breast cancer risk and disease-free survival in Caucasian postmenopausal women.


Highly Sensitive Detection of Organic Molecules on the Basis of a Poly(N-isopropylacrylamide) Microassembly Formed by Plasmonic Optical Trapping.
We demonstrate that a poly(N-isopropylacrylamide) (PNIPAM) microassembly, formed by plasmonic optical trapping, can provide the platform for a highly sensitive detection technique for fluorescent and nonfluorescent organic molecules dissolved in aqueous solution. PNIPAM microassemblies can be easily formed by a combination with a photothermal effect and an enhanced optical force. These physical phenomena were obtained through resonant excitation of localized surface plasmon (LSP). Sparsely distributed fluorescent or nonfluorescent molecules dissolved in solution can be extracted into the PNIPAM assembly, resulting in an increase in fluorescence or Raman signals. In particular, we successfully detected quite small amounts of analytes (rhodamine B) at the 10-9 mol/L level. Using LSP is an alternative approach in analytical chemistry and can be used in addition to surface enhanced Raman scattering and surface enhanced fluorescence.


Preferred provider organizations.
The 1980s has marked the beginning of a new alternative health care delivery system: the preferred provider organization ( PPO ). This system has developed from the health maintenance organization model and is predominant in California and Colorado. A PPO is a group of providers, usually hospitals and doctors, who agree to provide health care to subscribers for a negotiated fee that is usually discounted. Preferred provider organizations are subject to peer review and strict use controls in exchange for a consistent volume of patients and speedy turnaround on claims payments. This article describes the factors leading to the development of PPOs and the implications for occupational therapy.


Communicating clinical research to reduce cancer risk through diet: Walnuts as a case example.
Inflammation is one mechanism through which cancer is initiated and progresses, and is implicated in the etiology of other conditions that affect cancer risk and prognosis, such as type 2 diabetes, cardiovascular disease, and visceral obesity. Emerging human evidence, primarily epidemiological, suggests that walnuts impact risk of these chronic diseases via inflammation. The published literature documents associations between walnut consumption and reduced risk of cancer, and mortality from cancer, diabetes, and cardiovascular disease, particularly within the context of the Mediterranean Diet. While encouraging, follow-up in human intervention trials is needed to better elucidate any potential cancer prevention effect of walnuts, per se. In humans, the far-reaching positive effects of a plant-based diet that includes walnuts may be the most critical message for the public. Indeed, appropriate translation of nutrition research is essential for facilitating healthful consumer dietary behavior. This paper will explore the translation and application of human evidence regarding connections with cancer and biomarkers of inflammation to the development of dietary guidance for the public and individualized dietary advice. Strategies for encouraging dietary patterns that may reduce cancer risk will be explored.


[Differences between Dutch provinces in perinatal mortality and travel time to hospital].
To investigate differences in perinatal mortality between Dutch provinces and to determine the significance of risk factors including travel time from home to the hospital during labour. Cohort study. The study was based on 1,242,725 singleton births in 2000-2006 as recorded in the Netherlands Perinatal Registry. The influence of province on perinatal mortality was estimated, with logistic regression analysis adjusting for risk factors (age, parity, ethnicity, socioeconomic status) and care factors such as start of antenatal care and travel time. The perinatal mortality rate in the Netherlands was 9.9 per 1000 births. The provinces with the highest mortality rates were Friesland (11.3‰), Groningen (11.1‰), Zeeland (10.6‰) and Flevoland (10.4‰). Noord-Brabant (9.2‰) and Limburg (9.2‰) had the lowest mortality rates. These differences were significantly higher for Friesland (odds ratio: 1.16; 95%-CI: 1.05-1.28) and Groningen (odds ratio: 1.13; 95%-CI: 1.02-1.26). Starting late with perinatal care, at 18 weeks of gestation or later was an important risk factor (adjusted odds ratio 1.8; 95%-CI: 1.7-1.8). Low socio-economic status could partly be associated with the higher mortality risk in Groningen. Longer travel time (≥ 20 minutes) was an independent risk factor associated with perinatal mortality. On average 19% of the women travelled ≥ 20 minutes to the hospital. In the provinces Groningen, Friesland, Flevoland and Zeeland these percentages ranged between 32 and 36%. The adjusted odds ratio of travel time was 1.7 (95%-CI 1.6-1.7). The perinatal mortality differs per province. This can be explained by longer travel time to the hospital during labour. Late start of perinatal care and low socio-economic status also affect the mortality rate. These risk factors need to be taken into account during registration, investigation, audit and obstetric policy.


Multidrug-resistant tuberculosis in panama is driven by clonal expansion of a multidrug-resistant Mycobacterium tuberculosis strain related to the KZN extensively drug-resistant M. tuberculosis strain from South Africa.
Multidrug-resistant tuberculosis (MDR-TB) is a significant health problem in Panama. The extent to which such cases are the result of primary or acquired resistance and the strain families involved are unknown. We performed whole-genome sequencing of a collection of 66 clinical MDR isolates, along with 31 drug-susceptible isolates, that were isolated in Panama between 2001 and 2010; 78% of the MDR isolates belong to the Latin American-Mediterranean (LAM) family. Drug resistance mutations correlated well with drug susceptibility profiles. To determine the relationships among these strains and to better understand the acquisition of resistance mutations, a phylogenetic tree was constructed based on a genome-wide single-nucleotide polymorphism analysis. The phylogenetic tree shows that the isolates are highly clustered, with a single strain (LAM9-c1) accounting for nearly one-half of the MDR isolates (29/66 isolates). The LAM9-c1 strain was most prevalent among male patients of working age and was associated with high mortality rates. Members of this cluster all share identical mutations conferring resistance to isoniazid (KatG S315T mutation), rifampin (RpoB S531L mutation), and streptomycin (rrs C517T mutation). This evidence of primary resistance supports a model in which MDR-TB in Panama is driven by clonal expansion and ongoing transmission of several strains in the LAM family, including the highly successful MDR strain LAM9-c1. The phylogenetic analysis also shows that the LAM9-c1 strain is closely related to the KwaZulu-Natal (KZN) extensively drug-resistant TB strain identified in KwaZulu-Natal, South Africa. The LAM9-c1 and KZN strains likely arose from a recent common ancestor that was transmitted between Panama and South Africa and had the capacity to tolerate an accumulation of multiple resistance mutations.


Fitness measures in selection analyses: sensitivity to the overall number of offspring produced in a lifetime.
Age at first (alpha) and last (omega) breeding are important life-history traits; however, the direction and strength of selection detected on traits may vary depending on the fitness measure used. We provide the first estimates of lifetime breeding success (LBS) and lambda(ind) (the population growth rate of an individual) of European badgers Meles meles, by genotyping 915 individuals, sampled over 18 years, for 22 microsatellites. Males are slightly larger than females, and the opportunity for selection was slightly greater for males, as predicted. lambda(ind) and LBS both performed well in predicting the number of grand-offspring, and both detected selection for a late omega, until the age of eight. Differential selection (S'(alpha)) for an early alpha, however, was only detected using LBS, not with lambda(ind). In declining populations (lambda(ind) < 1) selection favours reproduction later in life, whereas early reproduction is selected in increasing populations (lambda(ind) > 1). As 41% of badgers were assigned only one offspring (lambda(ind) < 1), whereas 40% were assigned more than two (lambda(ind) > 1), this cancelled out S'(alpha) measured by lambda(ind).


A regulatory archipelago controls Hox genes transcription in digits.
The evolution of digits was an essential step in the success of tetrapods. Among the key players, Hoxd genes are coordinately regulated in developing digits, where they help organize growth and patterns. We identified the distal regulatory sites associated with these genes by probing the three-dimensional architecture of this regulatory unit in developing limbs. This approach, combined with in vivo deletions of distinct regulatory regions, revealed that the active part of the gene cluster contacts several enhancer-like sequences. These elements are dispersed throughout the nearby gene desert, and each contributes either quantitatively or qualitatively to Hox gene transcription in presumptive digits. We propose that this genetic system, which we call a "regulatory archipelago," provides an inherent flexibility that may partly underlie the diversity in number and morphology of digits across tetrapods, as well as their resilience to drastic variations.


Toll like receptor (TLR)-4 as a regulator of peripheral endogenous opioid-mediated analgesia in inflammation.
Leukocytes containing opioid peptides locally control inflammatory pain. In the early phase of complete Freund's adjuvant (CFA)-induced hind paw inflammation, formyl peptides (derived e.g. from Mycobacterium butyricum) trigger the release of opioid peptides from neutrophils contributing to tonic basal antinociception. In the later phase we hypothesized that toll-like-receptor-(TLR)-4 activation of monocytes/macrophages triggers opioid peptide release and thereby stimulates peripheral opioid-dependent antinociception. In Wistar rats with CFA hind paw inflammation in the later inflammatory phase (48-96 h) systemic leukocyte depletion by cyclophosphamide (CTX) or locally injected naloxone (NLX) further decreased mechanical and thermal nociceptive thresholds. In vitro β-endorphin (β-END) content increased during human monocyte differentiation as well as in anti-inflammatory CD14+CD16- or non-classical M2 macrophages. Monocytes expressing TLR4 dose-dependently released β-END after stimulation with lipopolysaccharide (LPS) dependent on intracellular calcium. Despite TLR4 expression proinflammatory M1 and anti-inflammatory M2 macrophages only secreted opioid peptides in response to ionomycin, a calcium ionophore. Intraplantar injection of LPS as a TLR4 agonist into the inflamed paw elicited an immediate opioid- and dose-dependent antinociception, which was blocked by TAK-242, a small-molecule inhibitor of TLR4, or by peripheral applied NLX. In the later phase LPS lowered mechanical and thermal nociceptive thresholds. Furthermore, local peripheral TLR4 blockade worsened thermal and mechanical nociceptive pain thresholds in CFA inflammation. Endogenous opioids from monocytes/macrophages mediate endogenous antinociception in the late phase of inflammation. Peripheral TLR4 stimulation acts as a transient counter-regulatory mechanism for inflammatory pain in vivo, and increases the release of opioid peptides from monocytes in vitro. TLR4 antagonists as new treatments for sepsis and neuropathic pain might unexpectedly transiently enhance pain by impairing peripheral opioid analgesia.


Theoretical studies on the interaction of partial agonists with the 5-HT2A receptor.
A series of 51 5-HT(2A) partial agonistic arylethylamines (primary or benzylamines) from different structural classes (indoles, methoxybenzenes, quinazolinediones) was investigated by fragment regression analysis (FRA), docking and 3D-QSAR approaches. The data, pEC(50) values and intrinsic activities (E(max)) on rat arteries, show high variability of pEC(50) from 4 to 10 and of E(max) from 15 to 70%. FRA indicates which substructures affect potency or intrinsic activity. The high contribution of halogens in para position of phenethylamines to pEC(50) points to a specific hydrophobic pocket. Other results suggest the significance of hydrogen bonds of the aryl moiety for activation and the contrary effect of benzyl groups on affinity (increasing) and intrinsic activity (decreasing). Results from fragment regression and data on all available mutants were considered to derive a common binding site at the rat 5-HT(2A) receptor. After generation and MD simulations of a receptor model based on the β(2)-adrenoceptor structure, typical derivatives were docked, leading to the suggestion of common interactions, e.g., with serines in TM3 and TM5 and with a cluster of aromatic amino acids in TM5 and TM6. The whole series was aligned by docking and minimization of the complexes. The pEC(50) values correlate well with Sybyl docking energies and hydrophobicity of the aryl moieties. With this alignment, CoMFA and CoMSIA approaches based on a training set of 36 and a test set of 15 compounds were performed. The correlation of pEC(50) with steric, electrostatic, hydrophobic and H-bond acceptor fields resulted in sufficient fit (q (2): 0.75-0.8, r (2): 0.92-0.95) and predictive power (r (pred) (2) : 0.85-0.88). The important interaction regions largely reflect the patterns provided by the putative binding site. In particular, the fit of the aryl moieties and benzyl substituents to two hydrophobic pockets is evident.


Pan-genomic analyses identify key Helicobacter pylori pathogenic loci modified by carcinogenic host microenvironments.
Helicobacter pylori is the strongest risk factor for gastric cancer; however, the majority of infected individuals do not develop disease. Pathological outcomes are mediated by complex interactions among bacterial, host and environmental constituents, and two dietary factors linked with gastric cancer risk are iron deficiency and high salt. We hypothesised that prolonged adaptation of H. pylori to in vivo carcinogenic microenvironments results in genetic modification important for disease. Whole genome sequencing of genetically related H. pylori strains that differ in virulence and targeted H. pylori sequencing following prolonged exposure of bacteria to in vitro carcinogenic conditions were performed. A total of 180 unique single nucleotide polymorphisms (SNPs) were identified among the collective genomes when compared with a reference H. pylori genome. Importantly, common SNPs were identified in isolates harvested from iron-depleted and high salt carcinogenic microenvironments, including an SNP within fur (FurR88H). To investigate the direct role of low iron and/or high salt, H. pylori was continuously cultured in vitro under low iron or high salt conditions to assess fur genetic variation. Exposure to low iron or high salt selected for the FurR88H variant after only 5 days. To extend these results, fur was sequenced in 339 clinical H. pylori strains. Among the isolates examined, 17% (40/232) of strains isolated from patients with premalignant lesions harboured the FurR88H variant, compared with only 6% (6/107) of strains from patients with non-atrophic gastritis alone (p=0.0034). These results indicate that specific genetic variation arises within H. pylori strains during in vivo adaptation to conditions conducive for gastric carcinogenesis.


Infusion of Melatonin Into the Paraventricular Nucleus Ameliorates Myocardial Ischemia-Reperfusion Injury by Regulating Oxidative Stress and Inflammatory Cytokines.
Melatonin, the receptors for which are abundant in the hypothalamic paraventricular nucleus (PVN), can protect the heart from myocardial ischemia-reperfusion (MI/R) injury. The aim of this study was to determine whether the infusion of melatonin into the PVN protects the heart from MI/R injury by suppressing oxidative stress or regulating the balance between proinflammatory cytokines and anti-inflammatory cytokines in MI/R rats. Male Sprague-Dawley rats were treated with a bilateral PVN infusion of melatonin. MI/R operation was performed 1 week after infusion. At the end of the third week after the infusion, all the rats were euthanized. This was followed by immunohistochemistry and immunofluorescence studies of the rats. MI/R rats showed larger infarct size, increased left ventricular (LV) end-diastolic volume, and decreased LV ejection fraction and LV fractional shortening. Moreover, MI/R rats had a higher level of norepinephrine in the plasma, heart, and PVN; higher PVN levels of reactive oxygen species, NOX2, NOX4, IL-1β, and NF-κB activity; and lower PVN levels of copper/zinc superoxide dismutase (Cu/Zn-SOD) and IL-10 compared with the sham group. Melatonin infusion in PVN reduced LV end-diastolic volume, norepinephrine, reactive oxygen species, NOX2, NOX4, IL-1β, and NF-κB activity, and increased LV ejection fraction, LV fractional shortening, Cu/Zn-SOD, and IL-10. Overall, these results suggest that the infusion of melatonin ameliorates sympathetic nerve activity and MI/R injury by attenuating oxidative stress and inflammatory cytokines in the PVN of MI/R rats.


Identification and characterization of petiolule-like pulvinus mutants with abolished nyctinastic leaf movement in the model legume Medicago truncatula.
Leaves of many plant species open during the day and fold at night. Diurnal leaf movement, named nyctinasty, has been of great interest to researchers since Darwin's time. Nyctinastic leaf movement is generated by the pulvinus, which is a specialized motor organ located at the base of leaf and leaflet. The molecular basis and functional reason behind nyctinasty are unknown. In a forward screening of a retrotransposon-tagged mutant population of Medicago truncatula, four petiolule-like pulvinus (plp) mutant lines with defects in leaf movement were identified and characterized. Loss of function of PLP results in the change of pulvini to petiolules. PLP is specifically expressed in the pulvinus, as demonstrated by quantitative reverse-transcription polymerase chain reaction analysis, expression analysis of a PLP promoter-β-glucuronidase construct in transgenic plants and in situ hybridization. Microarray analysis revealed that the expression levels of many genes were altered in the mutant during the day and at night. Crosses between the plp mutant and several leaf pattern mutants showed that the developmental mechanisms of pulvini and leaf patterns are likely independent. Our results demonstrated that PLP plays a crucial role in the determination of pulvinus development. Leaf movement generated by pulvini may have an impact on plant vegetative growth.


Spectrum of acute coronary syndromes: history and clinical presentation in relation to sex and age.
To study sex-based differences in the clinical presentation in relation to age and type of acute coronary syndrome (ACS) in patients under 80 years of age. The study includes 1,744 consecutive patients with the full spectrum of ACS (ST elevation myocardial infarction (MI), non-ST elevation MI, and unstable angina of high- and low-risk types) admitted to the coronary care unit in a university hospital. The women were older than the men and were as likely to present with ST elevation MI. They had lower rates of prior MI and prior coronary artery bypass surgery than men but similar rates of percutaneous coronary interventions. Further, women were less likely to have a short delay before admission to hospital and they were attended to less rapidly in the emergency department. The prevalence of risk factors, prior cardiovascular disease and ongoing treatment with cardiovascular drugs were strongly associated with less severe type of ACS with no significant sex interaction. Presentation with non-ST elevation MI was significantly associated with older age while the opposite was true for unstable low-risk angina. ECG signs of acute ischemia were not associated with age. Significant interactions between age and sex were observed for the prevalence of treatment with diuretics as well as hypotension at presentation, both more prevalent among women than men below 65 years of age. Women are struck by ACS at a higher age than men, are less likely to present early for hospital care, and at younger age women are more likely to present with hypotension. There is a striking difference in risk factors and previous history depending on type of ACS in both sexes.


Combined effects of fibrinogen genetic variability on atherosclerosis in patients with or without stable angina pectoris: focus on the coagulation cascade and endothelial function.
Fibrinogen is a coagulation/inflammatory biomarker strongly associated with atherogenesis. Data have reported that the genetic variability on fibrinogen chains may affect the atherosclerotic process and the risk of coronary artery disease (CAD). We examined the combined effects of the G455A and the G58A fibrinogen genetic polymorphisms on prothrombotic profile, endothelial function and the risk of CAD in a Caucasian population. We recruited 422 patients with angiographically documented CAD and 277 controls matched for age and gender. The two polymorphisms were genotyped by polymerase chain reaction and restriction endonuclease digestion. Fibrinogen and D-Dimers levels, as well as factors' (f) V, X activity were measured by standard coagulometry techniques. Endothelial function was assessed by the flow mediated dilatation (FMD) of the brachial artery. The two polymorphisms had no significant effect on the risk for CAD. Although the 58AA subjects had not significantly different levels of fibrinogen compared with the 58GG+GA in both groups (p=NS), we importantly found that the 455AA homozygosity was associated with increased fibrinogen levels not only in the control group (p=0.035), but also in the CAD group (p<0.001) compared to the G allele carriers. Moreover, both the 58AA (p=0.016) and 455AA homozygotes (p=0.022) presented with higher levels of D-Dimers in the CAD group. Interestingly, the 455AA homozygotes had increased fV activity in the CAD group (p=0.048). However, no significant effects were observed on fX activity and FMD. Both fibrinogen polymorphisms are capable to modify the atherosclerotic process via their effects on the coagulation cascade.


Contribution of selected vitamins and trace elements to immune function.
Adequate intakes of vitamins and trace elements are required for the immune system to function efficiently. Micronutrient deficiency suppresses immune functions by affecting the innate T-cell-mediated immune response and adaptive antibody response, and leads to dysregulation of the balanced host response. This increases the susceptibility to infections, with increased morbidity and mortality. In turn, infections aggravate micronutrient deficiencies by reducing nutrient intake, increasing losses, and interfering with utilization by altering metabolic pathways. Insufficient intake of micronutrients occurs in people with eating disorders, in smokers (both active and passive), in individuals with chronic alcohol abuse, in patients with certain diseases, during pregnancy and lactation, and in the elderly. With aging a variety of changes are observed in the immune system, which translate into less effective innate and adaptive immune responses and increased susceptibility to infections. Antioxidant vitamins and trace elements (vitamins C, E, selenium, copper, and zinc) counteract potential damage caused by reactive oxygen species to cellular tissues and modulate immune cell function through regulation of redox-sensitive transcription factors and affect production of cytokines and prostaglandins. Adequate intake of vitamins B(6), folate, B(12), C, E, and of selenium, zinc, copper, and iron supports a Th1 cytokine-mediated immune response with sufficient production of proinflammatory cytokines, which maintains an effective immune response and avoids a shift to an anti-inflammatory Th2 cell-mediated immune response and an increased risk of extracellular infections. Supplementation with these micronutrients reverses the Th2 cell-mediated immune response to a proinflammatory Th1 cytokine-regulated response with enhanced innate immunity. Vitamins A and D play important roles in both cell-mediated and humoral antibody response and support a Th2-mediated anti-inflammatory cytokine profile. Vitamin A deficiency impairs both innate immunity (mucosal epithelial regeneration) and adaptive immune response to infection resulting in an impaired ability to counteract extracellular pathogens. Vitamin D deficiency is correlated with a higher susceptibility to infections due to impaired localized innate immunity and defects in antigen-specific cellular immune response. Overall, inadequate intake and status of these vitamins and minerals may lead to suppressed immunity, which predisposes to infections and aggravates malnutrition.


Effects of inhibition of nitric oxide synthesis and of hypercapnia on blood pressure and brain blood flow in the turtle.
In the mammalian brain, nitric oxide (NO) is responsible for a vasodilatory tonus as well as the elevation of cerebral blood flow (CBF) induced by hypercapnia. There have been few comparative studies of cerebral vasoregulation in lower vertebrates. Using epi-illumination microscopy in vivo to observe CBF velocity on the brain surface (cerebral cortex), we show that turtles (Trachemys scripta) exposed to hypercapnia (inspired PCO2 = 4.9 kPa) displayed a 62% increase in CBF velocity, while systemic blood pressure remains constant. Exposing turtles to a PCO2 of 14.9 kPa caused an additional increase in CBF velocity, to 104% above control values, as well as a 30% increase in systemic blood pressure. The elevated CBF velocity during hypercapnia could not be blocked by a systemic injection of the NO synthase (NOS) inhibitor NG-nitro-L-arginine (L-NA). However, L-NA injection caused a temporary stop in CBF as well as a persistent increase in systemic blood pressure, suggesting that there is a NO tonus that is attenuated by the NOS inhibitor and that CBF is strongly dependent on this tonus, although compensatory mechanisms exist. Thus, although the cerebrovascular reaction to hypercapnia appeared to be NO-independent, the results suggest that there is a NO-dependent vasodilatory tonus affecting both cerebral and systemic blood circulation in this species.


Limited Use of Adjuvant Therapy in Patients With Resected Gallbladder Cancer Despite a Strong Association With Survival.
There are no randomized data to guide clinicians treating patients with gallbladder cancer (GBC). Several retrospective studies reported the survival benefits of adjuvant radiotherapy (RT) and chemoradiation (CRT). In this paper, we examine whether these publications have impacted the utilization of adjuvant therapies and whether their survival benefits are evident in a contemporary cohort of patients. Using the National Cancer Data Base, we identified 5029 patients diagnosed with T1-3N0-1 GBC and treated with surgical resection from 2005 to 2013. We described trends in receipt of adjuvant treatments for three time periods (2005-2007, 2008-2010, 2011-2013) and calculated three-year overall survival (OS) probabilities for 2989 patients treated in 2005-2010. All statistical tests were two-sided. The percentage of patients who received no adjuvant treatments was unchanged from 2005 to 2013. Adjuvant RT decreased from 4.2% to 1.7% ( P < .001), adjuvant chemotherapy increased from 8.3% to 13.8% ( P < .001), and adjuvant CRT remained stable at 15.9% ( P = .98). Adjuvant treatments were associated with improved three-year OS, with adjusted hazard ratio of 0.47 (95% confidence interval [CI] = 0.39 to 0.58) for CRT, 0.77 (95% CI = 0.61 to 0.97) for chemotherapy, and 0.63 (95% CI = 0.44 to 0.92) for RT. Adjuvant CRT was associated with improved survival in all categories, except T1N0, and in patients with negative and positive margins. Over the past decade there was no increase in the utilization of adjuvant therapies in the United States for patients with resected GBC. Adjuvant therapy is associated with statistically significantly improved three-year OS. This analysis should form the basis for current clinical recommendations and support future prospective trials.


Identification of the GPR55 antagonist binding site using a novel set of high-potency GPR55 selective ligands.
GPR55 is a class A G protein-coupled receptor (GPCR) that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development, and cancer. Initially deorphanized as a cannabinoid receptor, GPR55 has been shown to be activated by non-cannabinoid ligands such as l-α-lysophosphatidylinositol (LPI). While there is a growing body of evidence of physiological and pathophysiological roles for GPR55, the paucity of specific antagonists has limited its study. In collaboration with the Molecular Libraries Probe Production Centers Network initiative, we identified a series of GPR55 antagonists using a β-arrestin, high-throughput, high-content screen of ~300000 compounds. This screen yielded novel, GPR55 antagonist chemotypes with IC50 values in the range of 0.16-2.72 μM [Heynen-Genel, S., et al. (2010) Screening for Selective Ligands for GPR55: Antagonists (ML191, ML192, ML193) (Bookshelf ID NBK66153; PMID entry 22091481)]. Importantly, many of the GPR55 antagonists were completely selective, with no agonism or antagonism against GPR35, CB1, or CB2 up to 20 μM. Using a model of the GPR55 inactive state, we studied the binding of an antagonist series that emerged from this screen. These studies suggest that GPR55 antagonists possess a head region that occupies a horizontal binding pocket extending into the extracellular loop region, a central ligand portion that fits vertically in the receptor binding pocket and terminates with a pendant aromatic or heterocyclic ring that juts out. Both the region that extends extracellularly and the pendant ring are features associated with antagonism. Taken together, our results provide a set of design rules for the development of second-generation GPR55 selective antagonists.


Isolation of urinary exosomes for RNA biomarker discovery using a simple, fast, and highly scalable method.
Urinary exosomes are nanovesicles (40-100 nm) of endocytic origin that are secreted into the urine when a multivesicular body fuses with the membrane of cells from all nephron segments. Interest in urinary exosomes intensified after the discovery that they contain not only protein and mRNA but also microRNA (miRNA) markers of renal dysfunction and structural injury. Currently, the most widely used protocol for the isolation of urinary exosomes is based on ultracentrifugation, a method that is time consuming, requires expensive equipment, and has low scalability, which limits its applicability in the clinical practice. In this chapter, a simple, fast, and highly scalable step-by-step method for isolation of urinary exosomes is described. This method starts with a 10-min centrifugation of 10 ml urine, then the supernatant is saved (SN1), and the pellet is treated with dithiothreitol and heat to release and recover those exosomes entrapped by polymeric Tamm-Horsfall protein. The treated pellet is then resuspended and centrifuged, and the supernatant obtained (SN2) is combined with the first supernatant, SN1. Next, 3.3 ml of ExoQuick-TC, a commercial exosome precipitation reagent, is added to the total supernatant (SN1 + SN2), mixed well, and saved for at least 12 h at 4 °C. Finally, a pellet of exosomes is obtained after a 30-min centrifugation of the supernatant/ExoQuick-TC mix. We previously compared this method with five others used to isolate urinary exosomes and found that this is the simplest, fastest, and most effective alternative to ultracentrifugation-based protocols if the goal of the study is RNA profiling. A method for isolation and quantification of miRNAs and mRNAs from urinary exosomes is also described here. In addition, we provide a step-by-step description of exosomal miRNA profiling using universal reverse transcription and SYBR qPCR.


Is the link between socioeconomic status and resilience mediated by reserve-building activities: mediation analysis of web-based cross-sectional data from chronic medical illness patient panels.
The purpose of this study is to test the hypothesis that the link between socioeconomic status (SES) and resilience is mediated by reserve-building activities. Cross-sectional observational study. Structural equation modelling (SEM) was used to test the mediation hypothesis. Web-based survey. Participants with a chronic medical condition were recruited from Rare Patient Voice. DeltaQuest Reserve-Building Measure; demographic variables to capture SES; Centers for Disease Control Healthy Days Core Module; Self-Administered Comorbidity Questionnaire. Resilience was operationalised using residual modelling. The study sample included 442 patients (mean age 49, 85% female). SES was modelled as a bifactor model composed of general SES and specific factors for personal finance and parent's education. A series of simple mediation models predicting resilience led to the selection of three reserve-building activities for subsequent SEM-based mediation models: Active in the World, Outdoor and Exercise. The full SEM model supported the hypothesis that the relationships from both general SES and personal finance to resilience were mediated by engaging in the three reserve-building activities. In addition, the number of comorbidities partially mediated the relationship between personal finance and reserve-building. Those with more comorbidities generally had lower levels of resilience. This study provides suggestive evidence that reserve-building activities may be one pathway by which SES is associated with resilience: people of higher SES are more likely to engage in reserve-building activities that are intellectually stimulating, involve Outdoor pursuits and include physical Exercise. These reserve-building activities are not costly to pursue. These findings may empower patients to introduce more such reserve-building activities into their lives.


Chain of care development in Sweden: results of a national study.
Chains of Care are today an important counterbalance to the ever-increasing fragmentation of Swedish health care, and the ongoing development work has high priority. Improved quality of care is the most important reason for developing Chains of Care. Despite support in the form of goals and activity plans, seven out of ten county councils are uncertain whether they have been quite successful in the development work. Strong departmentalisation of responsibilities between different medical professions and departments, types of responsibilities and power still remaining in the vertical organisation structure, together with limited participation from the local authorities, are some of the most commonly mentioned reasons for the lack of success. Even though there is hesitation regarding the development work up to today, all county councils will continue developing Chains of Care. The main reason is, as was the case with Chain of Care development up to today, to improve quality of care. Although one of the main purposes is to make health care more patient-focused, patients in general seem to have limited impact on the development work. Therefore, the challenge is to design Chains of Care, which regards patients as partners instead of objects.


A Single phenylalanine residue in the main intracellular loop of α1 γ-aminobutyric acid type A and glycine receptors influences their sensitivity to propofol.
The intravenous anesthetic propofol acts as a positive allosteric modulator of glycine (GlyRs) and γ-aminobutyric acid type A (GABAARs) receptors. Although the role of transmembrane residues is recognized, little is known about the involvement of other regions in the modulatory effects of propofol. Therefore, the influence of the large intracellular loop in propofol sensitivity of both receptors was explored. The large intracellular loop of α1 GlyRs and α1β2 GABAARs was screened using alanine replacement. Sensitivity to propofol was studied using patch-clamp recording in HEK293 cells transiently transfected with wild type or mutant receptors. Alanine mutation of a conserved phenylalanine residue within the α1 large intracellular loop significantly reduced propofol enhancement in both GlyRs (360 ± 30 vs. 75 ± 10%, mean ± SEM) and GABAARs (361 ± 49% vs. 80 ± 23%). Remarkably, propofol-hyposensitive mutant receptors retained their sensitivity to other allosteric modulators such as alcohols, etomidate, trichloroethanol, and isoflurane. At the single-channel level, the ability of propofol to increase open probability was significantly reduced in both α1 GlyR (189 ± 36 vs. 22 ± 13%) and α1β2 GABAAR (279 ± 29 vs. 29 ± 11%) mutant receptors. In this study, it is demonstrated that the large intracellular loop of both GlyR and GABAAR has a conserved single phenylalanine residue (F380 and F385, respectively) that influences its sensitivity to propofol. Results suggest a new role of the large intracellular loop in the allosteric modulation of two members of the Cys-loop superfamily. Thus, these data provide new insights into the molecular framework behind the modulation of inhibitory ion channels by propofol.


The Impact of Pulmonary Hypertension in Preterm Infants with Severe Bronchopulmonary Dysplasia through 1 Year.
To assess the effect of pulmonary hypertension on neonatal intensive care unit mortality and hospital readmission through 1 year of corrected age in a large multicenter cohort of infants with severe bronchopulmonary dysplasia. This was a multicenter, retrospective cohort study of 1677 infants born <32 weeks of gestation with severe bronchopulmonary dysplasia enrolled in the Children's Hospital Neonatal Consortium with records linked to the Pediatric Health Information System. Pulmonary hypertension occurred in 370 out of 1677 (22%) infants. During the neonatal admission, pulmonary hypertension was associated with mortality (OR 3.15, 95% CI 2.10-4.73, P < .001), ventilator support at 36 weeks of postmenstrual age (60% vs 40%, P < .001), duration of ventilation (72 IQR 30-124 vs 41 IQR 17-74 days, P < .001), and higher respiratory severity score (3.6 IQR 0.4-7.0 vs 0.8 IQR 0.3-3.3, P < .001). At discharge, pulmonary hypertension was associated with tracheostomy (27% vs 9%, P < .001), supplemental oxygen use (84% vs 61%, P < .001), and tube feeds (80% vs 46%, P < .001). Through 1 year of corrected age, pulmonary hypertension was associated with increased frequency of readmission (incidence rate ratio [IRR] = 1.38, 95% CI 1.18-1.63, P < .001). Infants with severe bronchopulmonary dysplasia-associated pulmonary hypertension have increased morbidity and mortality through 1 year of corrected age. This highlights the need for improved diagnostic practices and prospective studies evaluating treatments for this high-risk population.


Characterization of the SKN7 ortholog of Aspergillus fumigatus.
Reactive oxidant intermediates play a major role in the killing of Aspergillus fumigatus by phagocytes. In yeasts, SKN7 is a transcription factor contributing to the oxidative stress response. We investigated here the role of afSkn7p in the adaptation of A. fumigatus against oxidative stress. To analyze functionally the afSKN7 in A. fumigatus, we modified a quick PCR fusion methodology for targeted deletion in A. fumigatus. The afskn7Delta mutant was morphologically similar to the wild-type strain, but showed a growth inhibition phenotype associated with hydrogen peroxide and tert-butyl hydroperoxide. However, no significant virulence differences were observed between wild type, mutant and reconstituted strains in a murine model of pulmonary aspergillosis. This result indicated that an increased sensitivity of A. fumigatus to peroxides in vitro is not correlated with a modification of fungal virulence.


Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line.
Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we report a simple, adherent cell culture protocol for differentiation of hPSCs to RGCs using a CRISPR-engineered RGC fluorescent reporter stem cell line. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. Additionally, we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. Lastly, using this protocol we identified forskolin as a potent promoter of RGC differentiation.


Relationship between asymmetric dimethylarginine and endothelial dysfunction in patients with rheumatoid arthritis.
In rheumatoid arthritis (RA), endothelial dysfunction caused by the inflammatory process increases the risk of cardiovascular disease. Asymmetric Dimethylarginine (ADMA) leads to vascular dysfunction, whereas atherosclerosis and increased ADMA is associated with cardiovascular disease risk factors. Flow-mediated Dilation (FMD) is a radiological method to demonstrate endothelial dysfunction. In the present study, we assessed the availability of ADMA as a marker for endothelial dysfunction in RA patients. ADMA can be used as a simple and cheaper method for the determination of endothelial dysfunction. Forty patients (1 male, 39 female) diagnosed with RA according to the classification criteria and 29 healthy volunteers (2 males, 27 females) were included in this study. ADMA was studied by enzyme-linked immunosorbent assay (ELISA). Chi-square, Fisher's exact test, Mann-Whitney U test, and Spearman's correlation tests were used for analytical analysis, and p<0.05 was considered as the level of statistical significance. In our study, ADMA levels were significantly higher in RA patients. The ADMA level was inversely correlated with FMD. Although high levels of both C-reactive protein and ADMA were detected in patients with high disease activity, there was no statistically significant difference between these parameters (p=0.18). There were statistically significant negative correlations between FMD and age and disease duration (p=0.01, p=0.01). However, there were no statistically significant correlations with erythrocyte sedimentation rate, rheumatoid factor, and disease activity score (p=0.68). In RA patients, there was a statistically significant positive correlation between disease duration and ADMA, whereas a negative correlation was found between FMD and ADMA (p<0.05). Our results support the hypothesis that ADMA may be used in the assessment of endothelial dysfunction in patients with RA. It will be cost-effective when commonly used. ADMA may be used in the assessment of endothelial dysfunction in patients with RA.


Implementing the Affordable Care Act: State Action to Establish SHOP Marketplaces.
The Affordable Care Act seeks to help small employers offer coverage by reforming the small-group market and establishing Small Business Health Options Program (SHOP) marketplaces. Seventeen states and the District of Columbia chose to operate their own SHOP marketplaces in 2014, with the federal government operating the SHOP marketplace in 33 states. This brief examines state decisions to enhance the value of SHOP marketplaces for small employers and finds that most have set predictable participation and eligibility requirements and will offer a competitive choice of insurers and plans. States also are seeking to facilitate small employers' shopping experience through online tools and access to personalized assistance. While not all SHOP marketplaces are yet functioning as intended, their establishment offers an opportunity to identify successful strategies for improving the affordability and accessibility of coverage for small employers.


When and how to image a suspected broken rib.
Radiographic confirmation of rib fracture is often clinically irrelevant in patients who appear to have no complications or associated injuries. However, it is essential in a number of cases, especially when the clinical presentation and history raise suspicion of complications such as organ damage, or if the patient has other risk factors or conditions for which the precise information would help in management decisions. The authors discuss when to order imaging and which imaging test to order.


Functionalized poly(γ-Glutamic Acid) fibrous scaffolds for tissue engineering.
Poly(γ-glutamic acid) (γ-PGA) is a biocompatible, enzymatically-degradable, natural polymer with a higher resistance to hydrolysis than polyesters commonly used for tissue engineering scaffolds such as poly(L-lactide) (PLLA). Notably, γ-PGA's free carboxyl side groups allow for simple chemical functionalization, making it a versatile candidate for producing scaffolds. Here, a series of water-resistant fibrous scaffolds were engineered from ethyl (Et), propyl (Pr) and benzyl (Bn) esterifications of γ-PGA. All scaffolds were non-cytotoxic and γ-PGA-Bn showed an increase in cell adhesion of hMSCs compared to γ-PGA-Et and γ-PGA-Pr. Moreover, cells on γ-PGA-Bn showed three-fold higher viability at day 14 and significantly higher adhesion when compared with PLLA scaffolds, despite having a similar hydrophobicity. Cell attachment decreased by 40% when the polymer was only partially modified with benzyl groups (γ-PGA-Bn-77%), but was restored when integrin-binding RGD peptide was conjugated to the remaining free carboxylic groups, indicating the peptide was accessible and able to bind integrins. The mechanism behind the cell-material interactions on γ-PGA-Bn scaffolds was further investigated through protein adsorption and fibronectin conformation experiments. These results, in addition to the cell-adhesion studies, suggest an inherent effect of the benzyl modification in the mechanism of cell attachment to γ-PGA-Bn scaffolds. Finally, γ-PGA-Bn scaffolds cultured in osteogenic media were also efficient in supporting hMSCs differentiation towards an osteogenic lineage as determined by alkaline phosphatase and Runx2 gene expression. Taken together these data suggest that esterified γ-PGA polymer scaffolds are new and versatile candidates for tissue engineering applications and that, intriguingly, aromatic functionality plays a key role in the cell-scaffold interaction.


Adipose-derived mesenchymal stem cells from the sand rat: transforming growth factor beta and 3D co-culture with human disc cells stimulate proteoglycan and collagen type I rich extracellular matrix.
Adult mesenchymal stem cell therapy has a potential application in the biological treatment of disc degeneration. Our objectives were: to direct adipose-derived mesenchymal stem cells (AD-MSC) from the sand rat to produce a proteoglycan and collagen type I extracellular matrix (ECM) rich in known ECM components of the annulus fibrosis of disc; and to stimulate proteoglycan production by co-culture of human annulus cells with AD-MSC. AD-MSC were isolated and characterised by adherence to plastic, appropriate expression of cluster of differentiation (CD) markers, and differentiation to osteoblasts and chondrocytes in vitro. AD-MSC were grown in three-dimensional (3D) culture and treated with or without transforming growth factor beta (TGFbeta) to direct them to produce annulus-like ECM as determined by proteoglycan content and collagen expression. AD-MSC were co-cultured with human annulus cells and grown in 3D culture. AD-MSC produced a proteoglycan and collagen type I rich ECM after treatment with TGFbeta in 3D culture as confirmed by a 48% increase in proteoglycan content assayed by 1,9-dimethylmethylene blue (DMB), and by immunohistochemical identification of ECM components. Co-culture of human annulus and sand rat AD-MSC in 3D culture resulted in a 20% increase in proteoglycan production compared with the predicted value of the sum of the individual cultures. Results support the hypothesis that AD-MSC have potential in cell-based therapy for disc degeneration.


Relationship between neutrophil-lymphocyte ratio and isolated low high-density lipoprotein cholesterol.
Several studies showed that high-density lipoprotein cholesterol (HDL-C) has anti-inflammatory effect. The neutrophil-lymphocyte ratio (NLR) is a new biomarker of systemic inflammation. The aim of this study was to assess the NLR in healthy participants with low HDL-C. We studied 69 patients with low HDL-C (≤ 35 mg/dL) and 59 control participants (HDL-C >35 mg/dL) with similar cardiovascular risk factors. Hematological indices were measured in all patients with low HDL-C and control participants. The HDL-C was significantly lower among the patients with low HDL-C than that of the control group (31.7 ± 2.7 vs 50.7 ± 5.0 mg/dL, respectively; P < .001). The NLR was significantly higher among the patients with low HDL-C than that of the control group (2.6 ± 0.8 vs 1.6 ± 0.4, respectively; P < .001). The NLR was negatively correlated with HDL-C (P < .001, r = -.53). The NLR is significantly elevated in patients with low HDL-C when compared with control participants.


The 'super-resolution' revolution.
We are currently in the midst of an exciting revolution in microscopy. In many ways, this has been happening for several decades, but it is the rate of development of new methods that has increased recently. The last few years have seen an impressive proliferation of new instruments for imaging at higher resolution, imaging single molecules and faster and more sensitive multidimensional live cell imaging. These include light sheet microscopy, stimulated emission depletion, structured illumination and live cell imaging on the OMX (optical microscopy experimental) platform. However, new probes and image analysis methods have also been crucial for the development of these revolutionary methods.


Trigeminal nerve-mediated reflex arterial blood pressure decrease and vasodilatation in lower lip of the rabbit.
We measured the effects of electrical stimulation of the central cut end of the lingual nerve on lower lip blood flow (LBF) and on arterial blood pressure in urethane-anesthetized, artificially ventilated, cervically vagosympathectomized rabbits. Different effects were observed depending on the stimulus frequency. Increasing the stimulus frequency above 5 Hz produced progressively larger ipsilateral LBF increases until the optimal frequency was reached at 20 Hz. In contrast, stimulation at above 0.5 Hz evoked progressively larger decreases in both contralateral LBF and arterial blood pressure until the optimal frequency was reached at around 10 and 2 Hz, respectively. Thus, the optimal stimulus frequencies for the ipsilateral LBF increase and the arterial blood pressure decrease were widely different. The lingual nerve-evoked change (i.e., fall) in arterial blood pressure showed a significant correlation with the contralateral LBF decrease, but not with the ipsilateral LBF increase. Prior administration of hexamethonium at 10 mg/kg markedly reduced both the ipsilateral LBF increase and arterial blood pressure decrease, although it was more effective against the former than against the latter. Pretreatment with scopolamine (muscarinic-receptor antagonist, 0.1 mg/kg), phentolamine (alpha-adrenoceptor antagonist, 0.1 mg/kg), or propranolol (beta-adrenoceptor antagonist, 0.1 mg/kg) failed to affect either response. However, 1.0 mg/kg phentolamine significantly reduced both responses (P<0.05). These results indicate that, in the rabbit, the LN-evoked reflex increase in ipsilateral LBF is (a) largely independent of any concomitant arterial blood pressure change and (b) probably due to active vasodilatation mediated via parasympathetic mechanisms. In contrast, the evoked decrease in contralateral LBF was proportional to the decrease in arterial blood pressure, suggesting that the former was secondary to the latter.


Introduction.
During the last few years the physics of ultracold atomic gases has experienced an extraordinary development, mostly due to the experimental realization of Bose-Einstein condensation (BEC). This development has being certainly interdisciplinary, bridging the gaps among very different areas, including atomic and molecular physics, quantum optics, condensed matter and solid-state physics, nonlinear physics, quantum information theory, etc.


Electrophysiological ON and OFF Responses in Autosomal Dominant Optic Atrophy.
To assess the effect of autosomal dominant optic atrophy (ADOA) on ON and OFF retinal ganglion cell (RGC) function by evaluating the ON and OFF components of the photopic negative response (PhNR). Twelve participants from six families with OPA1 ADOA and 16 age-matched controls were recruited. Electrophysiological assessment involved pattern ERGs (PERGs), focal (20°) and full-field long-duration (250 ms) flash ERGs using a red light-emitting diode flash on a rod-saturating blue background, and full-field brief (300 μs) xenon flash ERGs using a red filter over a continuous rod saturating blue background. Amplitudes and implicit times of the ERG components were analyzed and the diagnostic potential of each electrophysiological technique was determined by generating receiver operating characteristic (ROC) curves. Mean amplitudes of the N95 and all PhNRs, except the full-field PhNRON, were significantly reduced in participants with ADOA (P < 0.01). Subtraction of the group-averaged focal ERG of ADOA participants from that of controls showed an equal loss in the focal PhNRON and PhNROFF components, whereas in the full-field ERG the loss in the PhNROFF was greater than that in the PhNRON component. The areas under the ROC curve (AUC) for the focal PhNRON (0.92), focal PhNROFF (0.95), and full-field PhNROFF (0.83), were not significantly different from that of the PERG N95 (0.99). In patients with ADOA, the PhNRON and PhNROFF components are nearly symmetrically reduced in the long-duration ERG, suggesting that ON- and OFF-RGC pathways may be equally affected.


Inhibitory effects of alpha-cyclodextrin and its derivative against sucrose-induced hyperglycemia in an in vivo evaluation system.
Cyclodextrins (CyDs) are cyclic oligosaccharides consisting of six to eight glucose residues. Administration of α-CyD (six glucose residues) inhibits sucrose-induced hyperglycemia in humans. Here we show that oral administration of α-CyD and dimethyl α-CyD suppresses sucrose-induced hyperglycemia in an in vivo evaluation system using silkworms. On the other hand, β-CyD (seven glucose residues), γ-CyD (eight glucose residues), and their derivatives did not show the suppressive effect. These findings suggest that dimethyl α-CyD is a new inhibitor against sucrose-induced hyperglycemia and the silkworm system is useful for evaluation of suppressive activities of α-CyD derivatives against postprandial hyperglycemia.


Enantioselective Synthesis of D-α-Amino Amides from Aliphatic Aldehydes.
Peptides consisting of D-amino amides are highly represented among both biologically active natural products and non-natural small molecules used in therapeutic development. Chemical synthesis of D-amino amides most often involves approaches based on enzymatic resolution or fractional recrystallization of their diastereomeric amine salts, techniques that produce an equal amount of the L-amino acid. Enantioselective synthesis, however, promises selective and general access to a specific α-amino amide, and may enable efficient peptide synthesis regardless of the availability of the corresponding α-amino acid. This report describes the use of a cinchona alkaloid-catalyzed aza-Henry reaction using bromonitromethane, and the integration of its product with Umpolung Amide Synthesis. The result is a straightforward 3-step protocol beginning from aliphatic aldehydes that provides homologated peptides bearing an aliphatic side chain at the resulting D-α-amino amide.


Temperament, parenting, and depressive symptoms in a population sample of preadolescents.
Depressive symptoms can be triggered by negative social experiences and individuals' processing of these experiences. This study focuses on the interaction between temperament, perceived parenting, and gender in relation to depressive problems in a Dutch population sample of preadolescents. The sample consisted of 2230 ten-to-twelve-year-olds from the North of The Netherlands. Perceived parenting (overprotection, rejection, emotional warmth) was assessed by the EMBU (a Swedish acronym for My Memories of Upbringing) for Children, temperament (fearfulness and frustration) by the parent version of the Early Adolescent Temperament Questionnaire-Revised, and depressive problems by the Child Behavior Checklist (parent report) and the Youth Self-Report (child report). All parenting and temperament factors were significantly associated with depressive problems. Frustration increased the depressogenic effect of parental overprotection and lack of emotional warmth. Fearfulness increased the effect of rejection in girls, but not in boys. Furthermore, the association between frustration and depression was stronger in boys. These findings support the hypothesis that the effect of specific parenting behaviors depends on the temperament and gender of the child.


Raman spectroscopy to differentiate between fresh tissue samples of glioma and normal brain: a comparison with 5-ALA-induced fluorescence-guided surgery.
Raman spectroscopy is a biophotonic tool that can be used to differentiate between different tissue types. It is nondestructive and no sample preparation is required. The aim of this study was to evaluate the ability of Raman spectroscopy to differentiate between glioma and normal brain when using fresh biopsy samples and, in the case of glioblastomas, to compare the performance of Raman spectroscopy to predict the presence or absence of tumor with that of 5-aminolevulinic acid (5-ALA)-induced fluorescence. A principal component analysis (PCA)-fed linear discriminant analysis (LDA) machine learning predictive model was built using Raman spectra, acquired ex vivo, from fresh tissue samples of 62 patients with glioma and 11 glioma-free brain samples from individuals undergoing temporal lobectomy for epilepsy. This model was then used to classify Raman spectra from fresh biopsies from resection cavities after functional guided, supramaximal glioma resection. In cases of glioblastoma, 5-ALA-induced fluorescence at the resection cavity biopsy site was recorded, and this was compared with the Raman spectral model prediction for the presence of tumor. The PCA-LDA predictive model demonstrated 0.96 sensitivity, 0.99 specificity, and 0.99 accuracy for differentiating tumor from normal brain. Twenty-three resection cavity biopsies were taken from 8 patients after supramaximal resection (6 glioblastomas, 2 oligodendrogliomas). Raman spectroscopy showed 1.00 sensitivity, 1.00 specificity, and 1.00 accuracy for predicting tumor versus normal brain in these samples. In the glioblastoma cases, where 5-ALA-induced fluorescence was used, the performance of Raman spectroscopy was significantly better than the predictive value of 5-ALA-induced fluorescence, which showed 0.07 sensitivity, 1.00 specificity, and 0.24 accuracy (p = 0.0009). Raman spectroscopy can accurately classify fresh tissue samples into tumor versus normal brain and is superior to 5-ALA-induced fluorescence. Raman spectroscopy could become an important intraoperative tool used in conjunction with 5-ALA-induced fluorescence to guide extent of resection in glioma surgery.


Pyrrolidine Dithiocarbamate Attenuates Cardiocyte Apoptosis and Ameliorates Heart Failure Following Coronary Microembolization in Rats
Nuclear factor-kB is highly activated in cardiovascular disorders. However, few articles have targeted at the role of nuclear factor-kB inhibitor in heart failure. To evaluate the effects of nuclear factor-kB inhibitor pyrrolidine dithiocarbamate on cardiocyte apoptosis and cardiac function in a rat heart failure model. Animal experiment. A stable and reproducible rat heart failure model (n=64) was prepared by injecting homologous microthrombotic particles into the left ventricle of Sprague–Dawley rats while obstructing the ascending aorta to produce coronary microembolization. Rats with heart failure were randomized into untreated (HFu) and pyrrolidine dithiocarbamate-treated (HFp) groups; the latter received an intraperitoneal injection of pyrrolidine dithiocarbamate (100 mg/kg/day) 1 h prior to surgery as well as on postoperative days 1-7. The sham group comprised 32 Sprague–Dawley rats. Eight rats from each group were sacrificed on days 1, 3, 7, and 14 postoperatively. Masson's trichrome staining was used to determine the micro-fibrotic area to indicate the severity of myocardial loss. Terminal transferase uridine triphosphate nick end labeling staining was used to detect apoptotic cardiomyocytes. Echocardiography and hemodynamics were performed to evaluate left ventricular function. Rats with heart failure exhibited pathological changes evidenced by patchy myocardial fibrosis, remarkably elevated severity of myocardial loss, and persistently reduced left ventricular function. At the end of the study, compared with the HFu group, myocardial infarct size was reduced by 28% (p=0.001), cardiocyte apoptosis was suppressed (7.17%±1.47% vs 2.83%±0.75%, p<0.001), cardiac function parameters such as left ventricular ejection fraction (80%±4% vs 61%±6%), left ventricular + dP/dt max (4828±289 vs 2918±76 mmHg.s−1), left ventricular - dP/dt max (4398±269 vs 2481±365 mmHg.s−1), and left ventricular systolic pressure (126±13 vs 100±10 mmHg) were significantly increased, and left ventricular end-diastolic pressure was reduced (18±2 vs 13±1 mmHg) (p<0.001, for all) in the HFu group. Our rat model can adequately mimic heart failure via coronary vessel embolization. Moreover, pyrrolidine dithiocarbamate treatment can reduce cardiocyte apoptosis and improve cardiac function, which may be beneficial for patients with heart failure secondary to myocardial infarction.


Identification of fibroblast growth factor receptor 3 (FGFR3) as a protein receptor for botulinum neurotoxin serotype A (BoNT/A).
Botulinum neurotoxin serotype A (BoNT/A) causes transient muscle paralysis by entering motor nerve terminals (MNTs) where it cleaves the SNARE protein Synaptosomal-associated protein 25 (SNAP25206) to yield SNAP25197. Cleavage of SNAP25 results in blockage of synaptic vesicle fusion and inhibition of the release of acetylcholine. The specific uptake of BoNT/A into pre-synaptic nerve terminals is a tightly controlled multistep process, involving a combination of high and low affinity receptors. Interestingly, the C-terminal binding domain region of BoNT/A, HC/A, is homologous to fibroblast growth factors (FGFs), making it a possible ligand for Fibroblast Growth Factor Receptors (FGFRs). Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3) as a high affinity receptor for BoNT/A in neuronal cells. HC/A binds with high affinity to the two extra-cellular loops of FGFR3 and acts similar to an agonist ligand for FGFR3, resulting in phosphorylation of the receptor. Native ligands for FGFR3; FGF1, FGF2, and FGF9 compete for binding to FGFR3 and block BoNT/A cellular uptake. These findings show that FGFR3 plays a pivotal role in the specific uptake of BoNT/A across the cell membrane being part of a larger receptor complex involving ganglioside- and protein-protein interactions.


Metastatic Disease in Polyploid Uveal Melanoma Patients Is Associated With BAP1 Mutations.
Most of the uvea melanoma (UM) display a near-diploid (normal, -2N) karyotype with only a few chromosomal changes. In contrast to these simple aberrations 18% of the UM samples show a polyploid character (>2N) and this was associated with an unfavorable prognosis. This study attempts to gain insight in the prognostic value of polyploidy in UM. In 202 patients the ploidy status of the UM was determined using cytogenetic analysis, fluorescence-in-situ-hybridization (FISH), multiplex ligation dependent probe amplification (MLPA), and/or single nucleotide polymorphism (SNP) array analysis. Immunohistochemistry was used to determine the BAP1 expression and mutation analyses of BAP1 (coding regions) and the mutation hotspots for the SF3B1, EIF1AX, GNAQ, and GNA11 genes was carried out using Sanger sequencing or whole-exome sequencing. Twenty-three patients had a polyploid UM karyotype (11.4%). Patients with a polyploid tumor had larger tumors (15.61 vs. 13.13 mm, P = 0.004), and more often loss of heterozygosity of chromosome 3 (P = 0.003). No difference in occurrence of mutations between polyploid and diploid tumors was observed for BAP1, SF3B1, EIF1AX, GNAQ, and GNA11. Polyploidy did not affect survival (P = 0.143). BAP1 deficiency was the only significant independent prognostic predictor for patients with polyploid tumors, with a 16-fold increased hazard ratio (HR 15.90, P = 0.009). The prevalence of mutations in the UM related genes is not different in polyploid UM compared with diploid UM. Moreover, similar to patients with diploid UM, BAP1 mutation is the most significant prognostic predictor of metastasis in patients with polyploid UM.


Prevalence and correlates of Kaposi's sarcoma-associated herpesvirus infection in a sample of men who have sex with men in Eastern China.
This study aimed to determine the seroprevalence of Kaposi's sarcoma-associated herpesvirus(KSHV) and related factors in men who have sex with men (MSM) in China. A total of 208 subjects were recruited from eastern China from 2008 to 2009. Plasma samples were collected and screened for KSHV, HIV and syphilis. Overall, KSHV seroprevalence was 32.7% in the subjects. About 92.7% of subjects reported having ever had anal sex with a man, of whom 67.8% had receptive anal sex and 77.9% had insertive anal sex. About 93.1% of the study participants reported having had oral sex with a man, of whom 76.0% had receptive oral sex and 56.7% had insertive oral sex. KSHV seropositivity was independently associated with receptive anal sex[odds ratio (OR) 2.68, 95% confidence interval (CI) 1.15–6.23], syphilis (OR 3.25, 95% CI1.56–6.76) and HSV-2 infection (OR 2.40, 95% CI 1.09–5.26). KSHV infection is highly prevalent and is probably sexually transmitted among MSM in eastern China.