Genetic structure and systematic relationships within the Ophrys fuciflora aggregate (Orchidaceae: Orchidinae): high diversity in Kent and a wind-induced discontinuity bisecting the Adriatic.
A recent phylogenetic study based on multiple datasets is used as the framework for a more detailed examination of one of the ten molecularly circumscribed groups identified, the Ophrys fuciflora aggregate. The group is highly morphologically variable, prone to phenotypic convergence, shows low levels of sequence divergence and contains an unusually large proportion of threatened taxa, including the rarest Ophrys species in the UK. The aims of this study were to (a) circumscribe minimum resolvable genetically distinct entities within the O. fuciflora aggregate, and (b) assess the likelihood of gene flow between genetically and geographically distinct entities at the species and population levels. Fifty-five accessions sampled in Europe and Asia Minor from the O. fuciflora aggregate were studied using the AFLP genetic fingerprinting technique to evaluate levels of infraspecific and interspecific genetic variation and to assess genetic relationships between UK populations of O. fuciflora s.s. in Kent and in their continental European and Mediterranean counterparts. The two genetically and geographically distinct groups recovered, one located in England and central Europe and one in south-eastern Europe, are incongruent with current species delimitation within the aggregate as a whole and also within O. fuciflora s.s. Genetic diversity is higher in Kent than in the rest of western and central Europe. Gene flow is more likely to occur between populations in closer geographical proximity than those that are morphologically more similar. Little if any gene flow occurs between populations located in the south-eastern Mediterranean and those dispersed throughout the remainder of the distribution, revealing a genetic discontinuity that runs north-south through the Adriatic. This discontinuity is also evident in other clades of Ophrys and is tentatively attributed to the long-term influence of prevailing winds on the long-distance distribution of pollinia and especially seeds. A cline of gene flow connects populations from Kent and central and southern Europe; these individuals should therefore be considered part of an extensive meta-population. Gene flow is also evident among populations from Kent, which appear to constitute a single metapopulation. They show some evidence of hybridization, and possibly also introgression, with O. apifera.


U box proteins as a new family of ubiquitin-protein ligases.
The U box is a domain of approximately 70 amino acids that is present in proteins from yeast to humans. The prototype U box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor that cooperates with a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-protein ligase (E3) to catalyze ubiquitin chain formation on artificial substrates. E3 enzymes are thought to determine the substrate specificity of ubiquitination and have been classified into two families, the HECT and RING finger families. Six mammalian U box proteins have now been shown to mediate polyubiquitination in the presence of E1 and E2 and in the absence of E3. These U box proteins exhibited different specificities for E2 enzymes in this reaction. Deletion of the U box or mutation of conserved amino acids within it abolished ubiquitination activity. Some U box proteins catalyzed polyubiquitination by targeting lysine residues of ubiquitin other than lysine 48, which is utilized by HECT and RING finger E3 enzymes for polyubiquitination that serves as a signal for proteolysis by the 26 S proteasome. These data suggest that U box proteins constitute a third family of E3 enzymes and that E4 activity may reflect a specialized type of E3 activity.


The effect on survival of continuing chemotherapy to near death.
Overuse of anti-cancer therapy is an important quality-of-care issue. An aggressive approach to treatment can have negative effects on quality of life and cost, but its effect on survival is not well-defined. Using the Surveillance, Epidemiology, and End Results-Medicare database, we identified 7,879 Medicare-enrolled patients aged 65 or older who died after having survived at least 3 months after diagnosis of advanced non-small cell lung cancer (NSCLC) between 1991 and 1999. We used Cox proportional hazards regression analysis, propensity scores, and instrumental variable analysis (IVA) to compare survival among patients who never received chemotherapy (n = 4,345), those who received standard chemotherapy but not within two weeks prior to death (n = 3,235), and those who were still receiving chemotherapy within 14 days of death (n = 299). Geographic variation in the application of chemotherapy was used as the instrument for IVA. Receipt of chemotherapy was associated with a 2-month improvement in overall survival. However, based on three different statistical approaches, no additional survival benefit was evident from continuing chemotherapy within 14 days of death. Moreover, patients receiving chemotherapy near the end of life were much less likely to enter hospice (81% versus 51% with no chemotherapy and 52% with standard chemotherapy, P < 0.001), or were more likely to be admitted within only 3 days of death. Continuing chemotherapy for advanced NSCLC until very near death is associated with a decreased likelihood of receiving hospice care but not prolonged survival. Oncologists should strive to discontinue chemotherapy as death approaches and encourage patients to enroll in hospice for better end-of-life palliative care.


The Effects of 24 weeks of Resistance Training with Simultaneous Elastic and Free Weight Loading on Muscular Performance of Novice Lifters.
The purpose of this investigation was to assess the effectiveness of variable resistance as provided through elastic plus free weight techniques in college aged males and females. Twenty novice lifters were randomly assigned to a traditional free weight only (6 males and 5 females) or elastic band plus free weight group (5 males and 5 females) and 9 more normally active controls (5 males and 4 females), were recruited to maintain normal activity for the duration of the study. No differences existed between control, free weight and elastic band at baseline for age, body height, body mass, body mass index, and body fat percentage. One-repetition maximums were performed for squat and bench press while both strength and power were assessed using isokinetic dynamometry. Elastic groups and free-weight groups completed 24 weeks of whole body, periodized, high intensity resistance (65-95% of one-repetition maximum) training three times/week. Training programs were identical except that the elastic group trained the barbell squat, bench press and stiff-legged deadlift with 20-35% of their total prescribed training loads coming from band resistance (assessed at the top of the range of motion) with the remainder from free weight resistance. A mixed-model analysis revealed that peak torque, average power and one-repetition maximums for squat were significantly greater after training for the elastic group compared to the control (p<0.05). In addition, the free weight group also showed significantly greater improvements over the control in peak torque and one-repetition maximums for squat and bench press. No significant differences were observed between the elastic band and free weight groups. Combined variable elastic band plus free weight exercises are effective at increasing strength and power similar to free-weights alone in novice college aged males and females. However, due to complexity in set-up and load assignment elastic adoption by novice lifters in an unsupervised situation is not advised.


Prevalence, treatment patterns, and stay characteristics associated with hospitalizations for major depressive disorder.
Hospitalizations for major depressive disorder (MDD) are a significant burden on patients, their families, and to healthcare systems. This study characterized the prevalence of MDD hospitalizations in the US and described clinical characteristics, treatment patterns, length of stay, costs, and MDD-related hospitalization readmissions. A retrospective analysis of the Premier Perspective® Hospital Database was conducted using records of hospital admissions for MDD from January 1, 2014 to December 31, 2015. To supplement this analysis, healthcare claims data from Truven MarketScan® Research Database were also evaluated between January 1, 2013 and December 31, 2014. Among adult hospital stays in the Premier network, 1.3% included a primary diagnosis of MDD. The mean length of MDD-related stays was 6 days, with a mean total hospital charge per stay of $6713. Of those with hospital stays, 5.2% of patients had at least 1 readmission for MDD within 30 days of discharge. In the MarketScan database, 4% of adults with MDD had a MDD-related hospital stay, with a mean length of stay of 6 days and total reimbursed amount per stay of $8441. Of those with hospital stays, 5.4% had at least 1 readmission for MDD within 30 days. Results may not be generalizable to hospitals outside of those represented by these databases. Adult MDD hospitalizations are costly and associated with high rates of readmission. There is a need for new treatments that may help reduce hospitalizations and costs related to hospitalizations in patients with MDD.


Magnetic Levitation of MC3T3 Osteoblast Cells as a Ground-Based Simulation of Microgravity.
Diamagnetic samples placed in a strong magnetic field and a magnetic field gradient experience a magnetic force. Stable magnetic levitation occurs when the magnetic force exactly counter balances the gravitational force. Under this condition, a diamagnetic sample is in a simulated microgravity environment. The purpose of this study is to explore if MC3T3-E1 osteoblastic cells can be grown in magnetically simulated hypo-g and hyper-g environments and determine if gene expression is differentially expressed under these conditions. The murine calvarial osteoblastic cell line, MC3T3-E1, grown on Cytodex-3 beads, were subjected to a net gravitational force of 0, 1 and 2 g in a 17 T superconducting magnet for 2 days. Microarray analysis of these cells indicated that gravitational stress leads to up and down regulation of hundreds of genes. The methodology of sustaining long-term magnetic levitation of biological systems are discussed.


Temporary internal distraction as an aid to correction of severe scoliosis. Surgical technique.
Halo traction is a well-recognized adjunct for correcting severe, complex, rigid scoliotic curves, but it is associated with complications and is contraindicated in the presence of fixed cervical instability, kyphosis, or stenosis. In addition, halo traction often requires prolonged hospital stays and is not welcomed by all families. These limitations led to consideration of temporary internal distraction as an alternative. We retrospectively reviewed the records of children in whom severe scoliosis had been treated with temporary internal distraction. Our goals were to (1) assess whether the use of temporary internal distraction can aid in the correction of severe scoliosis and (2) identify complications associated with temporary internal distraction and compare them with those associated with halo traction. The mean preoperative curve was 104 degrees. All patients underwent initial posterior release of the rigid portion of the spine (with six also having anterior release) and placement of spinal instrumentation under distraction during spinal cord monitoring. Of the ten patients, four had one distraction procedure (i.e., the initial surgery [or first distraction]) followed by definitive fusion and the remaining six had two distraction procedures (i.e., the initial surgery [or first distraction] followed by the second distraction) followed by definitive fusion. After distraction, all patients underwent posterior spinal fusion with definitive dual-rod fixation. The amount of correction was determined by measuring the curve on plain radiographs made preoperatively, after each internal distraction procedure, after definitive fusion, and at the time of final follow-up. Curve correction after use of internal distraction, and before definitive fusion, averaged 53% (from 104 degrees to 49 degrees) (range, 39% [from 70 degrees to 43 degrees] to 79% [from 70 degrees to 15 degrees]). This method facilitated safe, gradual deformity correction in all ten patients. The mean time between the initial procedure and the definitive fusion was 2.4 weeks. The mean final curve correction was 80% (from 104 degrees to 20 degrees) (range, 73% [from 131 degrees to 35 degrees] to 91% [from 110 degrees to 10 degrees]). No neurologic deficits or infections resulted. Temporary internal distraction is a viable alternative approach to maximizing curve correction in patients undergoing spinal fusion for severe scoliosis.


Managing usual and unexpected pain with physical disability: a qualitative analysis.
With physical disabilities, persons often experience secondary pain that adds to restrictions in activity and participation. We investigated pain-management strategies used by those with physical disabilities. Qualitative phenomenological inquiry was used in multiple interviews with (N= 28) adults with physical disabilities (9 with amputation, 7 with cerebral palsy, and 12 with spinal cord injury) and subsequent thematic analysis. Among those with physical disabilities, a distinction is made between usual and unexpected pains. Usual pain is experienced consistently or as a consequence of not getting adequate rest, exercise, or stress-free time. Managing usual pain involves uses of prevention (e.g., exercise/fitness) and/or pragmatic actions through scheduling and pacing daily activities and taking interim retreats. Unexpected pain is experienced periodically and requires an immediate response and change of activity. To manage, persons describe making efforts to create a mind and body disassociation, activating safety nets to support function and alleviate pain, and making decisions to persevere with activity and participation. Advising those with physical disabilities to do proactive planning for both usual and unexpected pain may help them to use varied pain-management strategies to enhance function and minimize negative impacts on participation.


Comparative analysis of cell killing and autosomal mutation in mouse kidney epithelium exposed to 1 GeV protons in vitro or in vivo.
Human exposure to high-energy protons occurs in space flight scenarios or, where necessary, during radiotherapy for cancer or benign conditions. However, few studies have assessed the mutagenic effectiveness of high-energy protons, which may contribute to cancer risk. Mutations cause cancer and most cancer-associated mutations occur at autosomal loci. This study addresses the cytotoxic and mutagenic effects of 1 GeV protons in mouse kidney epithelium. Mutant fractions were measured for an endogenous autosomal locus (Aprt) that detects all types of mutagenic events. Results for kidneys irradiated in vivo are compared with the results for kidney cells from the same strain exposed in vitro. The results demonstrate dose-dependent cell killing in vitro and for cells explanted 3-4 months postirradiation in vivo. Incubation in vivo for longer periods (8-9 months) further attenuates proton-induced cell killing. Protons are mutagenic to cells in vitro and for in vivo irradiated kidneys. The dose-response for Aprt mutation is curvilinear after in vitro or in vivo exposure, bending upward at the higher doses. While the absolute mutant fractions are higher in vivo, the fold-increase over background is similar for both in vitro and in situ exposures. Results are also presented for a limited study on the effect of dose fractionation on the induction of Aprt mutations in kidney epithelial cells. Dose-fractionation reduces the fraction of proton-induced Aprt mutants in vitro and in vivo and also results in less cell killing. Taken together, the mutation burden in the epithelium is slightly reduced by dose-fractionation. Autosomal mutations accumulated during clinical exposure to high-energy protons may contribute to the risk of treatment-associated neoplasms, thereby highlighting the need for rigorous treatment planning to reduce the dose to normal tissues. For low dose exposures that occur during most space flight scenarios, the mutagenic effects of protons appear to be modest.


Arthroscopic hip surgery compared with physiotherapy and activity modification for the treatment of symptomatic femoroacetabular impingement: multicentre randomised controlled trial.
To compare arthroscopic hip surgery with physiotherapy and activity modification for improving patient reported outcome measures in patients with symptomatic femoroacetabular impingement (FAI). Two group parallel, assessor blinded, pragmatic randomised controlled trial. Secondary and tertiary care centres across seven NHS England sites. 222 participants aged 18 to 60 years with symptomatic FAI confirmed clinically and with imaging (radiography or magnetic resonance imaging) were randomised (1:1) to receive arthroscopic hip surgery (n=112) or a programme of physiotherapy and activity modification (n=110). Exclusion criteria included previous surgery, completion of a physiotherapy programme targeting FAI within the preceding 12 months, established osteoarthritis (Kellgren-Lawrence grade ≥2), and hip dysplasia (centre-edge angle <20 degrees). Participants in the physiotherapy group received a goal based programme tailored to individual patient needs, with emphasis on improving core stability and movement control. A maximum of eight physiotherapy sessions were delivered over five months. Participants in the arthroscopic surgery group received surgery to excise the bone that impinged during hip movements, followed by routine postoperative care. The primary outcome measure was the hip outcome score activities of daily living subscale (HOS ADL) at eight months post-randomisation, with a minimum clinically important difference between groups of 9 points. Secondary outcome measures included additional patient reported outcome measures and clinical assessment. At eight months post-randomisation, data were available for 100 patients in the arthroscopic hip surgery group (89%) and 88 patients in the physiotherapy programme group (80%). Mean HOS ADL was 78.4 (95% confidence interval 74.4 to 82.3) for patients randomised to arthroscopic hip surgery and 69.2 (65.2 to 73.3) for patients randomised to the physiotherapy programme. After adjusting for baseline HOS ADL, age, sex, and study site, the mean HOS ADL was 10.0 points higher (6.4 to 13.6) in the arthroscopic hip surgery group compared with the physiotherapy programme group (P<0.001)). No serious adverse events were reported in either group. Patients with symptomatic FAI referred to secondary or tertiary care achieve superior outcomes with arthroscopic hip surgery than with physiotherapy and activity modification. ClinicalTrials.gov NCT01893034.


Days alive and out of hospital and the patient journey in patients with heart failure: Insights from the candesartan in heart failure: assessment of reduction in mortality and morbidity (CHARM) program.
Conventional composite outcomes in heart failure (HF) trials, for example, time to cardiovascular death or first HF hospitalization, have recognized limitations. We propose an alternative outcome, days alive and out of hospital (DAOH), which incorporates mortality and all hospitalizations into a single measure. A refinement, the patient journey, also uses functional status (New York Heart Association [NYHA] class) measured during follow-up. The CHARM program is used to illustrate the methodology. CHARM randomized 7,599 patients with symptomatic HF to placebo or candesartan, with median follow-up of 38 months. We related DAOH and percent DAOH (ie, percentage of time spent alive and out of hospital) to treatment using linear regression adjusting for follow-up time. Mean increase in DAOH for patients on candesartan versus placebo was 24.1 days (95% CI 9.8-38.3 days, P < .001). The corresponding mean increase in percent DAOH was 2.0% (95% CI 0.8%-3.1%, P < .001). These findings were dominated by reduced mortality (23 days) but enhanced by reduced time in hospital (1 day). Percent time spent in hospital because of HF was reduced by 0.10% (95% CI 0.04%-0.14%, P < .001). The patient journey analysis showed that patients in the candesartan group spent more follow-up time in NYHA classes I and II and less in NYHA class IV. Days alive and out of hospital, especially percent DAOH, provide a valuable tool for summarizing the overall absolute treatment effect on mortality and morbidity. In future HF trials, percent DAOH can provide a useful alternative perspective on the effects of treatment.


[A case of primary small cell neuroendocrine carcinoma of the liver].
Small cell neuroendocrine carcinoma is a type of undifferentiated, malignant neuroendocrine tumor. Most of neuroendocrine tumors exhibit well-differentiated features and are classified as carcinoid tumors. However, carcinomas of the liver with anaplastic characters, which are classified as small-cell carcinomas are extremely rare and only few cases have been reported in the literature. We report an unusual case of primary small cell neuroendocrine carcinoma of the liver in a 67-year-old man. The patient was found to have a palpable mass on right upper quadrant of abdomen on physical examination. The diagnosis was made by immunohistochemical stains of biopsied specimen from the liver. Other possible primary site was excluded by radiologic and endoscopic evaluations. The tumor was composed of small monotonous and hyperchromatic poorly differentiated cells with higher nuclear to cytoplasmic ratio, and were positive for neuroendocrine tissue markers such as synaptophysin, c-kit, and CD56.


Development of Comparative Toxicity Potentials of 14 cationic metals in freshwater.
Site-dependent and site-generic Comparative Toxicity Potentials (CTPs) (also known as Characterization Factors (CFs)) were calculated for 14 cationic metals (Al(III), Ba, Be, Cd, Co, Cr(III), Cs, Cu(II), Fe(II), Fe(III), Mn(II), Ni, Pb, Sr and Zn), to be applied in Life Cycle Impact Assessment. CTPs were calculated for 7 EU-archetypes, taking bioavailability and speciation pattern into account. The resulting site-dependent CTPs showed up to 2.4-6.5 orders of magnitude variation across archetypes for those metals that form stable hydroxyl compounds in slightly alkaline waters (Al(III), Be, Cr(III), Cu(II) and Fe(III)), emphasizing the importance of using site-dependent CTPs for these metals where possible. For the other metals, CTPs stayed within around 0.9 orders of magnitude, making spatial differentiation less important. In acidic waters (pH<6.4), Al(III) and Cu(II) had the highest CTPs, while Cd ranked highest in other waters. Based on the site-dependent CTPs, site-generic CTPs were developed applying different averaging principle. Emission weighted average of 7 EU-archetype CTPs was recommended as site-generic CTP for use in LCA studies, where receiving location is unclear. Compared to previous studies by Gandhi et al. (2010, 2011a), new site-dependent CTPs were similar or slightly higher for Cd, Co, Ni, Pb and Zn, but 1-2 orders of magnitude higher for Cu. Compared to the default site-generic CTPs in the frequently used characterization models USES-LCA and USEtox, new site-generic CTPs were mostly higher or similar, within up to ∼2 orders of magnitude difference.


Subcellular stretch-induced cytoskeletal response of single fibroblasts within 3D designer scaffolds.
In vivo, cells are exposed to mechanical forces in many different ways. These forces can strongly influence cell functions or may even lead to diseases. Through their sensing machinery, cells are able to perceive the physical information of the extracellular matrix and translate it into biochemical signals resulting in cellular responses. Here, by virtue of two-component polymer scaffolds made via direct laser writing, we precisely control the cell matrix adhesions regarding their spatial arrangement and size. This leads to highly controlled and uniform cell morphologies, thereby allowing for averaging over the results obtained from several different individual cells, enabling quantitative analysis. We transiently deform these elastic structures by a micromanipulator, which exerts controlled stretching forces on primary fibroblasts grown in these scaffolds on a subcellular level. We find stretch-induced remodeling of both actin cytoskeleton and cell matrix adhesions. The responses to static and periodic stretching are significantly different. The amount of paxillin and phosphorylated focal adhesion kinase increases in cell matrix adhesions at the manipulated pillar after static stretching whereas it decreases after periodic stretching.


Predictors of failure in infants with viral bronchiolitis treated with high-flow, high-humidity nasal cannula therapy*.
Viral bronchiolitis is an acute infection and inflammatory disease of the respiratory tract, with infants typically presenting with the most severe symptoms. Medical management of bronchiolitis is mostly supportive. Several preliminary studies suggest potential benefit from the use of high-flow nasal cannula systems. Although high-flow nasal cannula is a well-established modality in the newborn intensive care unit, its use in the pediatric intensive care unit for acute respiratory failure is far less established. The objective of this study was to identify any laboratory and clinical variables that may predict high-flow nasal cannula failure in management of bronchiolitis in the pediatric intensive care unit. The study design was a retrospective chart review of all patients admitted to the pediatric intensive care unit from 2006 to 2010 with a diagnosis of viral bronchiolitis. Inclusion criteria included the initiation of high flow nasal cannula therapy at the time of admission and age ≤ 12 months. Exclusion criteria were intubation prior to admission, age >12 months, and the presence of a tracheostomy. A total of 113 patients with viral bronchiolitis met the inclusion criteria. Academic free standing Children's Hospital in the Midwest. Retrospective chart review. The data were analyzed by comparing those patients who responded to high-flow nasal cannula (n = 92) with those who were nonresponders to high-flow nasal cannula and required intubation (n = 21). No differences were noted between the groups for age, sex, or ethnicity. Mean weight and weight-for-corrected-age percentiles were significantly lower for patients who failed high-flow nasal cannula (p = .016 and .031, respectively), but weight-for-corrected-age percentile was not significant in logistic regression controlling for other variables. Respiratory rate prior to the initiation of high-flow nasal cannula also correlated strongly with respiratory deterioration (p < .001). The PCO2 was significantly higher for both before (p < .001) and after (p < .001) initiation of therapy in the nonresponder group. Pediatric Risk of Mortality III scores for the patients who failed high-flow nasal cannula were significantly higher (p < .001) than those of patients who tolerated this therapy. History of prematurity and the patient's age did not increase a patient's risk of failure. Nonresponders to high-flow nasal cannula therapy were on the onset, more hypercarbic, were less tachypnic prior to the start of high-flow nasal cannula, and had no change in their respiratory rate after the initiation of high-flow nasal cannula therapy. Nonresponders had higher pediatric risk of mortality scores in the first 24 hrs.


B-type natriuretic peptide and high sensitive C-reactive protein predict 2-year all cause mortality in chest pain patients: a prospective observational study from Salta, Argentina.
Several mechanisms are involved in the pathophysiology of the Acute Coronary Syndrome (ACS). We have addressed whether B-type natriuretic peptide (BNP) and high-sensitive C-reactive protein (hsCRP) in admission samples may improve risk stratification in chest pain patients with suspected ACS. We included 982 patients consecutively admitted with chest pain and suspected ACS at nine hospitals in Salta, Northern Argentina. Total and cardiac mortality were recorded during a 2-year follow up period. Patients were divided into quartiles according to BNP and hsCRP levels, respectively, and inter quartile differences in mortality were statistically evaluated applying univariate and multivariate analyses. 119 patients died, and the BNP and hsCRP levels were significantly higher among these patients than in survivors. In a multivariable Cox regression model for total death and cardiac death in all patients, the hazard ratio (HR) in the highest quartile (Q4) as compared to the lowest quartile (Q1) of BNP was 2.32 (95% confidence interval (CI), 1.24-4.35), p = 0.009 and 3.34 (95% CI, 1.26-8.85), p = 0.015, respectively. In the TnT positive patients (TnT > 0.01 ng/mL), the HR for total death and cardiac death in Q4 as compared to Q1 was 2.12 (95% CI, 1.07-4.18), p = 0.031 and 3.42 (95% CI, 1.13-10.32), p = 0.029, respectively.The HR for total death for hsCRP in Q4 as compared to Q1 was 1.97 (95% CI, 1.17-3.32), p = 0.011, but this biomarker did not predict cardiac death (p = 0.21). No prognostic impact of these two biomarkers was found in the TnT negative patients. BNP and hsCRP may act as clinically useful biomarkers when obtained at admission in a population with suspected ACS.


Short-term estrogen treatment in ovariectomized rats augments hippocampal acetylcholine release during place learning.
Estrogen modulates learning and memory in ovariectomized and naturally cycling female rats, especially in tasks using spatial learning and navigation. Estrogen also modulates cholinergic function in various forebrain structures. Past studies have shown positive correlations between hippocampal ACh output and performance on hippocampus-dependent tasks. The present study examined whether estradiol replacement would potentiate hippocampal ACh release during place learning. In vivo microdialysis and HPLC were used to measure extracellular ACh levels in the hippocampus of ovariectomized female rats that had received s.c. injections of 17beta-estradiol (10 microg) or sesame oil (vehicle treatment) 48 and 24h prior to training on a place task. Estrogen did not alter baseline levels of extracellular ACh in the hippocampus. During training, hippocampal ACh increased in ovariectomized rats regardless of estrogen status. However, while estradiol did not enhance learning in this experiment, estradiol significantly potentiated the increase in hippocampal ACh release seen during place training. This represents the first demonstration of on-line assessment of ACh output in hippocampus during learning in female rats and suggests that estrogen-dependent modulation of ACh release during training might control activation of different neural systems used during learning.


Long-term add-on pregabalin treatment in patients with partial-onset epilepsy: pooled analysis of open-label clinical trials.
To evaluate the safety, tolerability, and efficacy of long-term pregabalin as add-on therapy for patients with poorly controlled partial seizures. Analysis of data from six long-term clinical trials involving 2,061 patients receiving open-label pregabalin 75-600 mg/day adjunctive therapy for partial onset epilepsy refractory to multiple antiepileptic drugs. Total pregabalin exposure was 3,877 person-years. The mean duration of pregabalin treatment was 534 days (range 0.3-8 years) and 59% completed 1 year. One-third of patients discontinued for lack of efficacy. The most common dose was >or=300 mg/day; over half took >or=450 mg/day. There was a mean reduction in the 28-day seizure rate of 25-40%, and more than 40% of all patients had a >or=50% reduction in seizures from baseline during the last 3 months of treatment. Twelve percent of all patients had a 6-month period continuously free of seizures. In the last year, 6% were seizure-free for the entire year. Pregabalin was generally well-tolerated and the safety profile favorable in patients treated for up to several years, with an adverse event (AE) profile similar to short-term placebo-controlled trials. Common AEs included CNS symptoms (dizziness, somnolence, headache, and asthenia), accidental injury, and weight gain. CNS AEs tended to be mild and transient. Rates of sudden unexpected death in epilepsy (SUDEP), mortality, cancer, and status epilepticus were within the expected range for this population. Adjunctive pregabalin was effective, generally well tolerated, and safe in the long-term treatment of partial seizures, and provided clinically meaningful seizure reduction and freedom without evidence of tolerance over 2 years of follow-up.


Preliminary infrared radiometry of the night side of mercury from mariner 10.
The infrared radiometer on Mariner 10 measured the thermal emission from the planet with a spatial resolution element as small as 40 kilometers in a broad wavelength band centered at 45 micrometers. The minimum brightness temperature (near local midnight) in these near-equatorial scans was 100 degrees K. Along the track observed, the temperature declined steadily from local sunset to near midnight, behaving as would be expected for a homogeneous, porous material with a thermal inertia of 0.0017 cal cm(-2) sec(-(1/2)) degrees K(-1), a value only slightly larger than that of the moon. From near midnight to dawn, however, the temperature fluctuated over a range of about 10 degrees K, implying the presence of regions having thermal inertia as high as 0.003 cal cm(-2) sec-(1/2) degrees K(-1).


The protein-folding problem, 50 years on.
The protein-folding problem was first posed about one half-century ago. The term refers to three broad questions: (i) What is the physical code by which an amino acid sequence dictates a protein's native structure? (ii) How can proteins fold so fast? (iii) Can we devise a computer algorithm to predict protein structures from their sequences? We review progress on these problems. In a few cases, computer simulations of the physical forces in chemically detailed models have now achieved the accurate folding of small proteins. We have learned that proteins fold rapidly because random thermal motions cause conformational changes leading energetically downhill toward the native structure, a principle that is captured in funnel-shaped energy landscapes. And thanks in part to the large Protein Data Bank of known structures, predicting protein structures is now far more successful than was thought possible in the early days. What began as three questions of basic science one half-century ago has now grown into the full-fledged research field of protein physical science.


[Renal protection with amifostine during intraoperative peritoneal chemohyperthermia (IPCH) with cisplatin (CDDP) for peritoneal carcinosis. Phase 1 study].
An innovative approach to peritoneal neoplasm therapy is based on the surgical exeresis of visceral neoplasms, peritonectomy and lastly perfusion of the abdominal cavity with cytostatic drugs in hypothermia (1PCH). Cisplatin (CDDP) is one of the most frequently used drugs for peritoneal perfusion owing to its excellent synergy with hypothermia, reduced ability to penetrate the peritoneal- plasma barrier and its demonstrated efficacy against the majority of neoplasms causing peritoneal carcinosis. A major restriction is that CDDP causes renal toxicity. In order to minimise the risk of renal damage, the authors studied the use of amifostin to protect the renal system. A phase-1 study was performed to find the dose of amifostin that guarantees effective renal protection without causing hypotension. A total of 67 cytoreductions were performed at our centre associated with abdominal cavity perfusion using cytostatic compounds in hyperthermia (1PCH) with CDDP for peritoneal carcinosis. Among the first 9 patients undergoing IPCH without nephroprotection, Grade 2 (WHO) renal toxicity was observed in 4 cases (44%) and Grade IV-WHO toxicity in one patient (11%) leading to hemodialysis and death. Arnifastin was then administered to 18 patients. The administration protocol was as follows: cytoreduction, im. administration of amifostin 910 mg/m2 in 15 minutes, execution of IPCH. All patients treated using this dose presented hypotension with systolic arterial pressure <70 mmHg and amifostin administration was consequently suspended. Amifostin was then administered to a further 18 patients divided into groups of three. The dose used for the first triplet was 400 mg/m2; we then increased the dose by 50 mg/m2 in each subsequent triplet. The maximum dose tolerated was 50 mg/m2 less than the initial dose that caused systolic pressure to fall below 70 mmHg. Patients treated with doses = or <500 mg/m2 did not present hypotension and it was therefore possible to administer the entire dose. Patients treated with 600 mg/m2 of amifostin all presented hypotension <70 mg Hg, leading to the suspension of the drug. A new triplet of patients was treated at a dose of 550 mg/m2 and none showed hypotension. Taking 550 mg/m2 as the maximum tolerable dose, a further 22 patients then received amifostin infusion with 550 mg/m2 prior to IPCH. Creatinemia was assayed daily for two weeks and creatinine clearance was measured twice a week to evaluate the efficacy of nephroprotection. None of the patients treated with amifostin during the study died from causes correlated to renal failure: 1 patient died from TEP and 1 from septic shock. No patient treated with a dose of 550 mg/m2 developed arterial hypotension. None of the 18 patients in the dose-finding study presented postoperative creatinemia >1.6 (WHO grade 1 toxicity). In the group of 22 patients treated later, 2 cases (9%) presented creatinemia >1.6 (1.8 and 2.1) for a few days; both had undergone severe debulking and one of the patients subsequently underwent resection and anastomosis of the left renal artery invaded by the neoplasm. Some patients undergoing cytoreduction+IPCH without the use of amifostin developed severe renal toxicity; acute renal failure occurred in 1 patient requiring hemodialysis and was the main cause of death. None of the 28 patients treated with amifostin 550 mg/m2 developed hypotension or renal insufficiency; only 2 cases showed a slight transient increase in renal function markers. Amifostin appears to be an effective drug for protecting renal emunctory from the toxic effects of CDDP used in cytoreduction+IPCH in patients with peritoneal carcinosis. The dose of 550 mg/m2 used in this study does not cause hypotension and is recommended for this type of clinical use.


Clinical and molecular evaluation of 16 patients with Rett syndrome.
Zengin-Akkuş P, Taşkıran EZ, Kabaçam S, Şimşek-Kiper PÖ, Haliloğlu G, Boduroğlu K, Utine GE. Clinical and molecular evaluation of 16 patients with Rett syndrome. Turk J Pediatr 2018; 60: 1-9. Rett syndrome is a neurodevelopmental disorder caused by mutations in MECP2. The disease is characterized by early neurological regression following a normal initial development. The diagnosis is a clinical one, based on major and minor diagnostic criteria. This study, in a group of patients from a single tertiary center, aimed to evaluate the efficiency of clinical diagnosis and to see if there was a diagnostic delay. A second aim was to investigate genotype-phenotype correlations, based on Pineda scores. In this study, sixteen patients with a median age of 6.5 years (2.5-22 years) were included, following molecular confirmation of clinical diagnosis. The median age at the onset of symptoms and the median age at clinical diagnosis was 1.5 years and 2.5 years, respectively, the difference being statistically significant. Considering the Rett syndrome diagnostic criteria, initially regulated in 2002 and revised in 2010, seven and two patients in our group, respectively, did not meet the main criteria. Pineda scores among mutation groups were statistically not different. To conclude, the present study revealed presence of a diagnostic delay. The challenge may be that the patients do not exhibit full-blown clinical picture initially. No genotype-phenotype correlations were detected in clinical severity, as measured by Pineda scores. Moreover, the diagnostic criteria revised in 2010 are more comprehensive as compared to the 2002 criteria; however, further revision may increase diagnostic sensitivity.


The role of splicing factor mutations in the pathogenesis of the myelodysplastic syndromes.
Accurate pre-mRNA splicing by the spliceosome is a fundamental cellular mechanism required to remove introns that are present in most protein-coding transcripts. The recent discovery of a variety of somatic spliceosomal mutations in the myelodysplastic syndromes (MDS), a heterogeneous group of myeloid malignancies, has revealed a new leukemogenic pathway involving spliceosomal dysfunction. Spliceosome mutations are found in over half of all MDS patients and are likely founder mutations. The spliceosome mutations are highly specific to MDS and closely related conditions and, to some extent, appear to define distinct clinical phenotypes in MDS. The high frequency of mutations in different components of the RNA splicing machinery in MDS suggests that abnormal RNA splicing is the common consequence of these mutations. The identification of the downstream targets of the spliceosome mutations is an active area of research. Emerging data from the study of the MDS transcriptome suggests that spliceosomal mutations have effects on specific genes, including some previously shown to play a role in MDS pathogenesis. The effects of the spliceosomal mutations on RNA splicing and cell growth have been evaluated only in a limited context to date, however, and the determination of the impact of these mutations in primary human hematopoietic cells is essential in order to elucidate fully the molecular mechanism by which they contribute to MDS pathogenesis.


[Emergency contraception in Latin America and the Caribbean].
Introducing the post-coital birth control method in the family-planning services of Latin American countries has not been an easy task. Catholic and other conservative groups with great influence in the political arena have time and again stopped it from being adopted as an alternative method and have even succeeded in having it removed from official directives after formal acceptance by health authorities. The main objections are triggered by the erroneous supposition that "emergency contraception" pills are abortifacients. However, a large dose of cultural discrimination against women seems also to be involved. It has been extremely difficult to register dedicated products and make them available in drug-stores and even more difficult to distribute them without charge at public health centers. They are hard to find, expensive, and unavailable to adolescents at risk for unwanted pregnancies and to most low-income women, especially in rural areas. Dissemination of appropriate information has been scarce and slow and there are still great numbers of people that do not understand how or why the method works. Brazil has been the only exception, as its open society has readily accepted this method of contraception. The Latin American Consortium on Emergency Contraception founded in the year 2000 and its regional conference two years later had an important impact on the situation, as they encouraged the coordination of efforts by governmental and nongovernmental entities with those of women's groups to fight for sexual and reproductive rights. A number of studies have shown that the more people learn about emergency contraception, the more they find it acceptable and necessary, and radio spots and other media techniques have begun to educate the public about this matter. In spite of the many difficulties encountered, in the last few years several countries have made strides to include this method in their public health guidelines. However, because of the powerful forces against it, accessibility and distribution of the emergency pills are not always implemented as planned and there are still many areas that require work. Details are given on the situation in Argentina, Bolivia, Chile, Colombia, Ecuador, Honduras, Mexico, Paraguay, and Peru.


Evaluation of local media surveillance for improved disease recognition and monitoring in global hotspot regions.
Digital disease detection tools are technologically sophisticated, but dependent on digital information, which for many areas suffering from high disease burdens is simply not an option. In areas where news is often reported in local media with no digital counterpart, integration of local news information with digital surveillance systems, such as HealthMap (Boston Children's Hospital), is critical. Little research has been published in regards to the specific contribution of local health-related articles to digital surveillance systems. In response, the USAID PREDICT project implemented a local media surveillance (LMS) pilot study in partner countries to monitor disease events reported in print media. This research assessed the potential of LMS to enhance digital surveillance reach in five low- and middle-income countries. Over 16 weeks, select surveillance system attributes of LMS, such as simplicity, flexibility, acceptability, timeliness, and stability were evaluated to identify strengths and weaknesses in the surveillance method. Findings revealed that LMS filled gaps in digital surveillance network coverage by contributing valuable localized information on disease events to the global HealthMap database. A total of 87 health events were reported through the LMS pilot in the 16-week monitoring period, including 71 unique reports not found by the HealthMap digital detection tool. Furthermore, HealthMap identified an additional 236 health events outside of LMS. It was also observed that belief in the importance of the project and proper source selection from the participants was crucial to the success of this method. The timely identification of disease outbreaks near points of emergence and the recognition of risk factors associated with disease occurrence continue to be important components of any comprehensive surveillance system for monitoring disease activity across populations. The LMS method, with its minimal resource commitment, could be one tool used to address the information gaps seen in global 'hot spot' regions.


Linear wave dynamics explains observations attributed to dark solitons in a polariton quantum fluid.
We investigate the propagation and scattering of polaritons in a planar GaAs microcavity in the linear regime under resonant excitation. The propagation of the coherent polariton wave across an extended defect creates phase and intensity patterns with identical qualitative features previously attributed to dark and half-dark solitons of polaritons. We demonstrate that these features are observed for negligible nonlinearity (i.e., polariton-polariton interaction) and are, therefore, not sufficient to identify dark and half-dark solitons. A linear model based on the Maxwell equations is shown to reproduce the experimental observations.


Conjugated polymer nanoparticles for effective siRNA delivery to tobacco BY-2 protoplasts.
Post transcriptional gene silencing (PTGS) is a mechanism harnessed by plant biologists to knock down gene expression. siRNAs contribute to PTGS that are synthesized from mRNAs or viral RNAs and function to guide cellular endoribonucleases to target mRNAs for degradation. Plant biologists have employed electroporation to deliver artificial siRNAs to plant protoplasts to study gene expression mechanisms at the single cell level. One drawback of electroporation is the extensive loss of viable protoplasts that occurs as a result of the transfection technology. We employed fluorescent conjugated polymer nanoparticles (CPNs) to deliver siRNAs and knockdown a target gene in plant protoplasts. CPNs are non toxic to protoplasts, having little impact on viability over a 72 h period. Microscopy and flow cytometry reveal that CPNs can penetrate protoplasts within 2 h of delivery. Cellular uptake of CPNs/siRNA complexes were easily monitored using epifluorescence microscopy. We also demonstrate that CPNs can deliver siRNAs targeting specific genes in the cellulose biosynthesis pathway (NtCesA-1a and NtCesA-1b). While prior work showed that NtCesA-1 is a factor involved in cell wall synthesis in whole plants, we demonstrate that the same gene plays an essential role in cell wall regeneration in isolated protoplasts. Cell wall biosynthesis is central to cell elongation, plant growth and development. The experiments presented here shows that NtCesA is also a factor in cell viability. We show that CPNs are valuable vehicles for delivering siRNAs to plant protoplasts to study vital cellular pathways at the single cell level.


Recombination between small X chromosome duplications and the X chromosome in Caenorhabditis elegans.
Twelve new X chromosome duplications were identified and characterized. Eight are translocated to autosomal sites near four different telomeres, and four are free. Ten include unc-1(+), which in wild type is near the left end of the X chromosome, and two of these, mnDp72(X;IV) and mnDp73(X;f), extend rightward past dpy-3. Both mnDp72 and mnDp73 recombined with the one X chromosome in males in the unc-1-dpy-3 interval at a frequency 15- to 30-fold higher than was observed for X-X recombination in hermaphrodites in the same interval. Recombinant duplications and recombinant X chromosomes were both recovered. Recombination with the X chromosome in the unc-1-dpy-3 interval was also detected for five other unc-1(+) duplications, even though their right breakpoints lie within the interval. In hermaphrodites, mnDp72 and mnDp73 promoted meiotic X nondisjunction and recombined with an X chromosome in the unc-1-dpy-3 interval at frequencies comparable to that found for X-X recombination; mnDp72(X;IV) also promoted trisomy for chromosome IV. A mutation in him-8 IV was identified that severely reduced recombination between the two X chromosomes in hermaphrodites and between mnDp73 and the X chromosome in males. Recombination between the X chromosome and duplications of either the right end of the X or a region near but not including the left end was rare. We suggest that the X chromosome has one or more elements near its left end that promote meiotic chromosome pairing.


Physiological barriers to delivery of monoclonal antibodies and other macromolecules in tumors.
The efficacy in cancer treatment of monoclonal antibodies or other macromolecules bound to radionuclides, chemotherapeutic agents, toxins, enzymes, growth factors, or effector antibodies has been limited by their inability to reach their target in vivo in adequate quantities. Heterogeneity of tumor-associated antigen expression alone has failed to explain the nonuniform uptake of antibodies. As a result, only in recent years have the peculiarities of tumor physiology been recognized as determinants of antibody distribution. Three physiological barriers responsible for the poor localization of macromolecules in tumors have been identified: (a) heterogeneous blood supply; (b) elevated interstitial pressure; and (c) large transport distances in the interstitium. The first barrier limits the delivery of blood-borne molecules to well-perfused regions of a tumor; the second barrier reduces extravasation of fluid and macromolecules in the high interstitial pressure regions and also leads to an experimentally verifiable, radially outward convection in the tumor periphery which opposes the inward diffusion; and the third barrier increases the time required for slowly moving macromolecules to reach distal regions of a tumor. Binding of antibody to an antigen further lowers the effective diffusion rate of the antibody by reducing the amount of mobile antibody. Due to micro- and macroscopic heterogeneities in tumors, the relative magnitude of each of these barriers would vary from one location to another and from one day to the next in the same tumor and from one tumor to another. If the genetically engineered macromolecules, e.g., lymphokines, and other new modalities, e.g., killer lymphocytes, as well as low molecular weight cytotoxic agents, are to fulfill their clinical promise, methods must be developed to overcome these physiological barriers. Some of these methods are discussed, and situations wherein these barriers may not be a problem are pointed out.


Certain and progressive methylation of histone H4 at lysine 20 during the cell cycle.
Methylation of histone H4 at lysine 20 (K20) has been implicated in transcriptional activation, gene silencing, heterochromatin formation, mitosis, and DNA repair. However, little is known about how this modification is regulated or how it contributes to these diverse processes. Metabolic labeling and top-down mass spectrometry reveal that newly synthesized H4 is progressively methylated at K20 during the G(2), M, and G(1) phases of the cell cycle in a process that is largely inescapable and irreversible. Approximately 98% of new H4 becomes dimethylated within two to three cell cycles, and K20 methylation turnover in vivo is undetectable. New H4 is methylated regardless of prior acetylation, and acetylation occurs predominantly on K20-dimethylated H4, refuting the hypothesis that K20 methylation antagonizes H4 acetylation and represses transcription epigenetically. Despite suggestions that it is required for normal mitosis and cell cycle progression, K20 methylation proceeds normally during colchicine treatment. Moreover, delays in PR-Set7 synthesis and K20 methylation which accompany altered cell cycle progression during sodium butyrate treatment appear to be secondary to histone hyperacetylation or other effects of butyrate since depletion of PR-Set7 did not affect cell cycle progression. Together, our data provide an unbiased perspective of the regulation and function of K20 methylation.


Prediction of future Alzheimer's disease dementia using plasma phospho-tau combined with other accessible measures.
A combination of plasma phospho-tau (P-tau) and other accessible biomarkers might provide accurate prediction about the risk of developing Alzheimer's disease (AD) dementia. We examined this in participants with subjective cognitive decline and mild cognitive impairment from the BioFINDER (n = 340) and Alzheimer's Disease Neuroimaging Initiative (ADNI) (n = 543) studies. Plasma P-tau, plasma Aβ42/Aβ40, plasma neurofilament light, APOE genotype, brief cognitive tests and an AD-specific magnetic resonance imaging measure were examined using progression to AD as outcome. Within 4 years, plasma P-tau217 predicted AD accurately (area under the curve (AUC) = 0.83) in BioFINDER. Combining plasma P-tau217, memory, executive function and APOE produced higher accuracy (AUC = 0.91, P < 0.001). In ADNI, this model had similar AUC (0.90) using plasma P-tau181 instead of P-tau217. The model was implemented online for prediction of the individual probability of progressing to AD. Within 2 and 6 years, similar models had AUCs of 0.90-0.91 in both cohorts. Using cerebrospinal fluid P-tau, Aβ42/Aβ40 and neurofilament light instead of plasma biomarkers did not improve the accuracy significantly. The clinical predictions by memory clinic physicians had significantly lower accuracy (4-year AUC = 0.71). In summary, plasma P-tau, in combination with brief cognitive tests and APOE genotyping, might greatly improve the diagnostic prediction of AD and facilitate recruitment for AD trials.


Passive mitigation strategies for mode instabilities in high-power fiber laser systems.
Mode instabilities have quickly become the most limiting effect when it comes to scaling the output average power of fiber laser systems. In consequence, there is an urgent need for effective strategies to mitigate it and, thus, to increase the power threshold at which it appears. Passive mitigation strategies can be classified into intrinsic, which are related to the fiber design, and extrinsic, which require a modification of the setup. In order to evaluate the impact of mitigation strategies, a means to calculate its power threshold and predict its behavior is required. In this paper we present a simple semi-analytic formula that is able to predict the changes of the mode instability threshold by analyzing the strength of the thermally-induced waveguide perturbations. Furthermore, we propose two passive mitigation strategies, one intrinsic and one extrinsic, that should lead to a significant increase of the power threshold of mode instabilities.


Alterations of elastic architecture in human aortic dissecting aneurysm.
Elastin of the ascending aortic media of 10 cases with type A dissecting aneurysm, 14 hypertensive cases, and 30 control cases were prepared by the treatment of aortas with hot formic acid, and three-dimensional architecture was observed by scanning electron microscopy. In the control cases, elastin showed framework-like continuous structure consisting of elastic laminae, and interlaminar fibers that interconnected the laminae. In 6 of 10 cases of dissecting aneurysm, the interlaminar fibers were apparently irregular in arrangement and shape, and decreased in number, especially in the outer media. This architectural alteration resulted in a rarefaction of interconnection between the elastic laminae in the media, and possibly resulted in the local weakness against the dissecting force of the laminae. This medial weakness may be related to the mechanism of initiation and progression of dissecting aneurysm. The cystic medial necrosis (CMN) was found in 3 cases, but only 1 of them was accompanied by a mild decrease of the interlaminar fibers in the area outside of CMN, suggesting that the initiation of CMN did not directly relate to the decrease of the interlaminar fibers. The aortic media of hypertensives generally showed an increase of interlaminar fibers, but their focal decrease was encountered in the outer media of 3 cases. These findings suggest that the decrease of the interlaminar fibers of medial elastin seen in dissecting aneurysms were related to hypertension.


Backbone assignment of the three dimers of HU from Escherichia coli at 293 K: EcHUα2, EcHUβ2 and EcHUαβ.
HU is one of the major nucleoid-associated proteins involved in bacterial chromosome structure and in all DNA-dependent cellular activities. Similarly to eukaryotic histones, this small dimeric basic protein wraps DNA in a non-sequence specific manner, promoting DNA super-structures. In most bacteria, HU is a homodimeric protein encoded by a single gene. However, in enterobacteria such as Escherichia coli, the presence of two genes coding for two peptidic chains, HUα and HUβ, lead to the coexistence of three forms: two homodimers EcHUα2 and EcHUβ2, as well as a heterodimer EcHUαβ. Genetic and biochemical investigation suggest that each EcHU dimer plays a specific physiological role in bacteria. Their relative abundance depends on the environmental conditions and is driven by an essential, yet unknown, fast outstanding chain-exchange mechanism at physiological temperature. Our goal is to understand this fundamental mechanism from a structural and kinetics standpoint using NMR. For this purpose, the first steps are the assignment of each dimer in their native and intermediate states. Here, we report the backbone assignment of each HU dimers from E. coli at 293 K in their native state.


Drug Dosing and Pharmacokinetics in Children With Obesity: A Systematic Review.
Obesity affects nearly one-sixth of US children and results in alterations to body composition and physiology that can affect drug disposition, possibly leading to therapeutic failure or toxic side effects. The depth of available literature regarding obesity's effect on drug safety, pharmacokinetics, and dosing in obese children is unknown. To perform a systematic literature review describing the current evidence of the effect of obesity on drug disposition in children. We searched the MEDLINE, Cochrane, and EMBASE databases (January 1, 1970-December 31, 2012) and included studies if they contained data on drug clearance, volume of distribution, or drug concentration in obese children (aged ≤18 years). We compared exposure and weight-normalized volume of distribution and clearance between obese and nonobese children. We explored the association between drug physicochemical properties and clearance and volume of distribution. Twenty studies met the inclusion criteria and contained pharmacokinetic data for 21 drugs. The median number of obese children studied per drug was 10 (range, 1-112) and ages ranged from newborn to 29 years (1 study described pharmacokinetics in children and adults together). Dosing schema varied and were either a fixed dose (6 [29%]) or based on body weight (10 [48%]) and body surface area (4 [19%]). Clinically significant pharmacokinetic alterations were observed in obese children for 65% (11 of 17) of the studied drugs. Pharmacokinetic alterations resulted in substantial differences in exposure between obese and nonobese children for 38% (5 of 13) of the drugs. We found no association between drug lipophilicity or Biopharmaceutical Drug Disposition Classification System class and changes in volume of distribution or clearance due to obesity. Consensus is lacking on the most appropriate weight-based dosing strategy for obese children. Prospective pharmacokinetic trials in obese children are needed to ensure therapeutic efficacy and enhance drug safety.


Estrogen receptor silencing induces epithelial to mesenchymal transition in human breast cancer cells.
We propose the hypothesis that loss of estrogen receptor function which leads to endocrine resistance in breast cancer, also results in trans-differentiation from an epithelial to a mesenchymal phenotype that is responsible for increased aggressiveness and metastatic propensity. siRNA mediated silencing of the estrogen receptor in MCF7 breast cancer cells resulted in estrogen/tamoxifen resistant cells (pII) with altered morphology, increased motility with rearrangement and switch from a keratin/actin to a vimentin based cytoskeleton, and ability to invade simulated components of the extracellular matrix. Phenotypic profiling using an Affymetrix Human Genome U133 plus 2.0 GeneChip indicated geometric fold changes ≥ 3 in approximately 2500 identifiable unique sequences, with about 1270 of these being up-regulated in pII cells. Changes were associated with genes whose products are involved in cell motility, loss of cellular adhesion and interaction with the extracellular matrix. Selective analysis of the data also showed a shift from luminal to basal cell markers and increased expression of a wide spectrum of genes normally associated with mesenchymal characteristics, with consequent loss of epithelial specific markers. Over-expression of several peptide growth factors and their receptors are indicative of an increased contribution to the higher proliferative rates of pII cells as well as aiding their potential for metastatic activity. Signalling molecules that have been identified as key transcriptional drivers of epithelial to mesenchymal transition were also found to be elevated in pII cells. These data support our hypothesis that induced loss of estrogen receptor in previously estrogen/antiestrogen sensitive cells is a trigger for the concomitant loss of endocrine dependence and onset of a series of possibly parallel events that changes the cell from an epithelial to a mesenchymal type. Inhibition of this transition through targeting of specific mediators may offer a useful supplementary strategy to circumvent the effects of loss of endocrine sensitivity.


Putative cancer stem cells in malignant pleural mesothelioma show resistance to cisplatin and pemetrexed.
Malignant pleural mesothelioma (MPM) is a lethal cancer of the mesothelium with high chemotherapeutic resistance via unknown mechanisms. A prevailing hypothesis states that cancer stem cells (CSCs) persist in tumors causing relapse after chemotherapy, thus, rendering these cells as critical targets responsible for tumor resistance and recurrence. We selected candidate CSC markers based on expansion under hypoxic conditions, a hallmark for the selection of chemoresistant cells; and investigated the expression of CSC markers: CD133, Bmi-1, uPAR and ABCG2 in three MPM cell lines and normal mesothelial cells by quantitative RT-PCR. Furthermore, we evaluated the chemotherapeutic resistance associated with each CSC marker by determining the change in CSC marker-mRNA levels as an index of drug-resistance following treatment with either cisplatin or pemetrexed. We demonstrate the expression of CSC markers: CD133, Bmi-1, uPAR and ABCG2 in both normal and MPM cell lines. Bmi-1+, uPAR+ and ABCG2+ cells show a distinct role in conferring chemoresistance to cisplatin and pemetrexed in the malignant setting. By contrast, these markers have no apparent participation in chemoresistance to drug treatments in normal mesothelial cells. Intriguingly, CD133 revealed chemoresistant properties in both normal mesothelial and malignant pleural mesothelioma cells. This study provides evidence of putative CSCs conferring drug-resistance to cisplatin and pemetrexed in MPM cell lines. Specific targeting of these drug-resistant cells, while considering the functional heterogeneity of the MPM subtypes, may contribute to more focused and effective chemotherapeutic regimens for malignant pleural mesothelioma.


Towards "evidence-making intervention" approaches in the social science of implementation science: The making of methadone in East Africa.
In this commentary, we take the recent introduction of methadone treatment in response to emerging problems of HIV linked to heroin addiction in Kenya as a case for reflecting on the social science of implementation science. We offer a framework of 'evidence-making intervention' which we hold as distinct from mainstream 'evidence-based intervention' approaches. Whilst accepting that interventions are shaped in their contexts, evidence-based intervention approaches tend to imagine a stable intervention object with universal effect potential. By contrast, an evidence-making intervention approach investigates how an intervention, and the knowledge which constitutes it, is made locally, through its processes of implementation. Drawing on qualitative research generated in Kenya prior to (2012-2013) and during (2014-2015) the implementation of methadone treatment, we explore the making of 'methadone promise' as a case of evidence-making intervention. We show how enactments of methadone promise make multiple methadones, through which a binary is negotiated between the narratives of methadone as hope for addiction recovery and methadone as hope for HIV prevention. Addiction recovery narratives predominate, despite methadone's incorporation into policy via its globally supported HIV prevention evidence-base. Key practices in the making of methadone promise in Kenya include its medicalization, and renaming, as 'medically assisted treatment' - or simply 'MAT' - which distance it from prior constitutions elsewhere as a drug of substitution, and the visualisation of its effects wherein unhealthy people can be seen and shown to have become well. We also show how actors seek to protect the story of methadone promise from counter narratives, including through mass media projects. We conclude that there is no single biomedical object of methadone intervening on a single biological body across contexts, and no single universe of evidence. By giving weight to local rather than outside expert knowledge, and by tracing how the meaning of intervention is made locally through its implementation, we can make visible the multiple enactments of an intervention and how these shape local ecologies of care, including in ways beyond those foreseen by an intervention's evidencing elsewhere.


A 52-Year-Old Man With Vertigo: A Common Symptom or an Urgent Syndrome?
Basilar occlusion syndrome is an uncommon cause of posterior circulation cerebrovascular syndromes. Approximately one-fifth of the ischemic strokes occur in the posterior cerebrovascular system and it is associated with an increased morbidity and mortality. The symptoms and signs are non-specific, resulting in delays in diagnosis, and a high index of suspicion is required that will lead to the correct diagnosis. Herein, we present a case of a 52-year-old patient who presented to the emergency department with acute onset of dizziness and vertigo due to partial thrombosis of the basilar artery.


Do changes in the pace of events affect one-off judgments of duration?
Five experiments examined whether changes in the pace of external events influence people's judgments of duration. In Experiments 1a-1c, participants heard pieces of music whose tempo accelerated, decelerated, or remained constant. In Experiment 2, participants completed a visuo-motor task in which the rate of stimulus presentation accelerated, decelerated, or remained constant. In Experiment 3, participants completed a reading task in which facts appeared on-screen at accelerating, decelerating, or constant rates. In all experiments, the physical duration of the to-be-judged interval was the same across conditions. We found no significant effects of temporal structure on duration judgments in any of the experiments, either when participants knew that a time estimate would be required (prospective judgments) or when they did not (retrospective judgments). These results provide a starting point for the investigation of how temporal structure affects one-off judgments of duration like those typically made in natural settings.


DNA barcodes for species identification in the hyperdiverse ant genus Pheidole (Formicidae: Myrmicinae).
DNA sequencing is increasingly being used to assist in species identification in order to overcome taxonomic impediment. However, few studies attempt to compare the results of these molecular studies with a more traditional species delineation approach based on morphological characters. Mitochondrial DNA Cytochrome oxidase subunit 1 (CO1) gene was sequenced, measuring 636 base pairs, from 47 ants of the genus Pheidole (Formicidae: Myrmicinae) collected in the Brazilian Atlantic Forest to test whether the morphology-based assignment of individuals into species is supported by DNA-based species delimitation. Twenty morphospecies were identified, whereas the barcoding analysis identified 19 Molecular Operational Taxonomic Units (MOTUs). Fifteen out of the 19 DNA-based clusters allocated, using sequence divergence thresholds of 2% and 3%, matched with morphospecies. Both thresholds yielded the same number of MOTUs. Only one MOTU was successfully identified to species level using the CO1 sequences of Pheidole species already in the Genbank. The average pairwise sequence divergence for all 47 sequences was 19%, ranging between 0-25%. In some cases, however, morphology and molecular based methods differed in their assignment of individuals to morphospecies or MOTUs. The occurrence of distinct mitochondrial lineages within morphological species highlights groups for further detailed genetic and morphological studies, and therefore a pluralistic approach using several methods to understand the taxonomy of difficult lineages is advocated.


Effects of smell loss on daily life and adopted coping strategies in patients with nasal polyposis with asthma.
Results from prior studies of quality of life (QoL) in heterogeneous patient groups (regarding disorder type and etiology) with olfactory disorders may be useful also for understanding QoL in homogeneous patient groups. Diagnosis and treatment of smell loss should be given high priority in polyposis with asthma, and coping strategies can be suggested to these patients. To investigate the effects of smell loss on daily life and coping strategies in patients with smell loss without dysosmia and with nasal polyposis with asthma as the only primary etiology, and to compare these results with those from a prior study of a patient group with heterogeneous olfactory disorders and etiology. Fifty patients with smell loss and with nasal polyposis and asthma responded to questions about consequences of smell loss, QoL, psychological well-being and distress, and coping strategies. Negative consequences of smell loss, associated risks, and diminished food enjoyment were commonly reported, and various aspects of QoL were rated as being deteriorated. Psychological well-being was found to be poorer than normal, and use of both problem- and emotion-focused strategies was common. The results from this homogeneous patient group are very similar to those previously obtained from a heterogeneous group.


Comparison of nonballistic active knee extension in neural slump position and static stretch techniques on hamstring flexibility.
Nonballistic, active range of motion exercises have been advocated as more effective than static stretching for increasing range of motion, yet no published data exist to support this claim. This study compared the effect of nonballistic, repetitive active knee extension movements performed in a neural slump sitting position with static stretching technique on hamstring flexibility. Forty healthy, adult volunteer subjects with limited right hamstring flexibility (i.e., minimum of 15 degrees loss of active knee extension measured with femur held at 90 degrees of hip flexion) were randomly assigned to one of three groups. Group 1 (static stretch) performed a 30-second stretch twice daily. Group 2 (active stretch) performed 30 repetitions of active knee extension while sitting in a neural slump position twice daily. Group 3 served as a control. Hamstring flexibility was determined by an active knee extension test before and after 6 weeks of stretching. Goniometric measurement of knee joint flexion angle was obtained from videotape recording of the active knee extension test. A 3 (group) x 2 (test) repeated measures analysis of variance and subsequent Tukey post hoc testing revealed no significant difference in knee joint range of motion gains between the static (mean = 8.9 degrees) and active stretch (mean = 10.2 degrees). Both stretch groups' knee joint range of motion improved significantly (p < .05) more than the control group. We conclude that 6 weeks of nonballistic, repetitive active knee extensions (30 repetitions, twice daily) performed in a neural slump sitting position improves hamstring flexibility in uninjured subjects, but is no different compared with static stretching (30 seconds, twice daily).


Development of environmental tools for anopheline larval control.
Malaria mosquitoes spend a considerable part of their life in the aquatic stage, rendering them vulnerable to interventions directed to aquatic habitats. Recent successes of mosquito larval control have been reported using environmental and biological tools. Here, we report the effects of shading by plants and biological control agents on the development and survival of anopheline and culicine mosquito larvae in man-made natural habitats in western Kenya. Trials consisted of environmental manipulation using locally available plants, the introduction of predatory fish and/or the use of Bacillus thuringiensis var. israelensis (Bti) in various combinations. Man-made habitats provided with shade from different crop species produced significantly fewer larvae than those without shade especially for the malaria vector Anopheles gambiae. Larval control of the African malaria mosquito An. gambiae and other mosquito species was effective in habitats where both predatory fish and Bti were applied, than where the two biological control agents were administered independently. We conclude that integration of environmental management techniques using shade-providing plants and predatory fish and/or Bti are effective and sustainable tools for the control of malaria and other mosquito-borne disease vectors.


Nicotinic acid receptor abnormalities in human skin cancer: implications for a role in epidermal differentiation.
Chronic UV skin exposure leads to epidermal differentiation defects in humans that can be largely restored by pharmacological doses of nicotinic acid. Nicotinic acid has been identified as a ligand for the human G-protein-coupled receptors GPR109A and GPR109B that signal through G(i)-mediated inhibition of adenylyl cyclase. We have examined the expression, cellular distribution, and functionality of GPR109A/B in human skin and skin derived epidermal cells. Nicotinic acid increases epidermal differentiation in photodamaged human skin as judged by the terminal differentiation markers caspase 14 and filaggrin. Both GPR109A and GPR109B genes are transcribed in human skin and in epidermal keratinocytes, but expression in dermal fibroblasts is below limits of detection. Receptor transcripts are greatly over-expressed in squamous cell cancers. Receptor protein in normal skin is prominent from the basal through granular layers of the epidermis, with cellular localization more dispersive in the basal layer but predominantly localized at the plasma membrane in more differentiated epidermal layers. In normal human primary and immortalized keratinocytes, nicotinic acid receptors show plasma membrane localization and functional G(i)-mediated signaling. In contrast, in a squamous cell carcinoma derived cell line, receptor protein shows a more diffuse cellular localization and the receptors are nearly non-functional. The results of these studies justify future genetic and pharmacological intervention studies to define possible specific role(s) of nicotinic acid receptors in human skin homeostasis.


A normal colposcopy examination fails to provide psychological reassurance for women who have had low-grade abnormal cervical cytology.
Worldwide, each year, large numbers of women are referred for colposcopy following low-grade abnormal cervical cytology. Many have no visible abnormality on examination. The risk of cervical intra-epithelial neoplasia grade 2/3 (CIN2/3) in these women is low. It is unknown whether, for women, a normal colposcopy resolves the anxiety which often follows the receipt of an abnormal cytology result. We investigated the prevalence of adverse psychological outcomes over 30 months following a normal colposcopy. This cohort study was nested within the UK TOMBOLA randomized controlled trial. Women aged 20-59 years, with recent low-grade cytology, who had a satisfactory colposcopy examination and normal transformation zone, completed the Hospital Anxiety and Depression Scale (HADS) and Process Outcome Specific Measure (POSM) at recruitment and during follow-up (12, 18, 24 and 30 months post-recruitment). Outcomes included percentages reporting significant anxiety (HADS anxiety subscale score ≥11), significant depression (HADS depression subscale score ≥8) or worries about the result of the next cytology test, cervical cancer, having sex, future fertility and general health at each time point (point prevalence) and during follow-up (cumulative prevalence). The study included 727 women. All psychological measures (except depression) had high prevalence at recruitment, falling substantially by 12 months. During follow-up, the cumulative prevalence of significant anxiety was 27% and significant depression was 21%. The most frequently reported worry was that the next cytology test would be abnormal (cumulative prevalence of 71%; point prevalence of ≥50% at 12 and 18 months). The cumulative prevalence values of worries about cervical cancer, having sex and future fertility were 33%, 20% and 16%, respectively. For some women who have low-grade cytology, a normal colposcopy does not appear to provide psychological reassurance.


Bortezomib augments lymphocyte stimulatory cytokine signaling in the tumor microenvironment to sustain CD8+T cell antitumor function.
Tumor-induced immune tolerance poses a major challenge for therapeutic interventions aimed to manage cancer. We explored approaches to overcome T-cell suppression in murine breast and kidney adenocarcinomas, and lung fibrosarcoma expressing immunogenic antigens. We observed that treatment with a reversible proteasome inhibitor bortezomib (1 mg/kg body weight) in tumor-bearing mice significantly enhanced the expression of lymphocyte-stimulatory cytokines IL-2, IL-12, and IL-15. Notably, bortezomib administration reduced pulmonary nodules of mammary adenocarcinoma 4T1.2 expressing hemagglutinin (HA) model antigen (4T1HA) in mice. Neutralization of IL-12 and IL-15 cytokines with a regimen of blocking antibodies pre- and post-adoptive transfer of low-avidity HA518-526-specific CD8+T-cells following intravenous injection of 4T1HA cells increased the number of pulmonary tumor nodules. This neutralization effect was counteracted by the tumor metastasis-suppressing action of bortezomib treatments. In bortezomib-treated 4T1HA tumor-bearing mice, CD4+T-cells showed increased IL-2 production, CD11c+ dendritic cells showed increased IL-12 and IL-15 production, and HA-specific activated CD8+T-cells showed enhanced expression of IFNγ, granzyme-B and transcription factor eomesodermin. We also noted a trend of increased expression of IL-2, IL-12 and IL-15 receptors as well as increased phosphorylation of STAT5 in tumor-infiltrating CD8+T-cells following bortezomib treatment. Furthermore, bortezomib-treated CD8+T-cells showed increased phosphorylation of mitogen-activated protein kinase p38, and Akt, which was abrogated by phosphatidylinositide 3-kinase (PI3K) inhibitor. These data support the therapeutic potential of bortezomib in conjunction with other immunotherapies to augment the strength of convergent signals from CD8+T-cell signaling molecules including TCR, cytokine receptors and downstream PI3K/Akt/STAT5 pathways to sustain CD8+T-cell effector function in the tumor microenvironment.


ADP-ribosyltransferase activity of pertussis toxin and immunomodulation by Bordetella pertussis.
Pertussis toxin is produced by the causative agent of whooping cough, Bordetella pertussis, and is an adenosine diphosphate (ADP)-ribosyltransferase capable of covalently modifying and thereby inactivating many eukaryotic G proteins involved in cellular metabolism. The toxin is a principal determinant of virulence in whooping cough and is a primary candidate for an acellular pertussis vaccine, yet it is unclear whether the ADP-ribosyltransferase activity is required for both pathogenic and immunoprotective activities. A B. pertussis strain that produced an assembled pertussis holotoxin with only 1 percent of the ADP-ribosyltransferase activity of the native toxin was constructed and was found to be deficient in pathogenic activities associated with B. pertussis including induction of leukocytosis, potentiation of anaphylaxis, and stimulation of histamine sensitivity. Moreover, this mutant strain failed to function as an adjuvant and was less effective in protecting mice from intracerebral challenge infection. These data suggest that the ADP-ribosyltransferase activity is necessary for both pathogenicity and optimum immunoprotection. These findings bear directly on the design of a nontoxic pertussis vaccine.


SNP identification in unamplified human genomic DNA with gold nanoparticle probes.
Single nucleotide polymorphisms (SNPs) comprise the most abundant source of genetic variation in the human genome. SNPs may be linked to genetic predispositions, frank disorders or adverse drug responses, or they may serve as genetic markers in linkage disequilibrium analysis. Thus far, established SNP detection techniques have utilized enzymes to meet the sensitivity and specificity requirements needed to overcome the high complexity of the human genome. Herein, we present for the first time a microarray-based method that allows multiplex SNP genotyping in total human genomic DNA without the need for target amplification or complexity reduction. This direct SNP genotyping methodology requires no enzymes and relies on the high sensitivity of the gold nanoparticle probes. Specificity is derived from two sequential oligonucleotide hybridizations to the target by allele-specific surface-immobilized capture probes and gene-specific oligonucleotide-functionalized gold nanoparticle probes. Reproducible multiplex SNP detection is demonstrated with unamplified human genomic DNA samples representing all possible genotypes for three genes involved in thrombotic disorders. The assay format is simple, rapid and robust pointing to its suitability for multiplex SNP profiling at the 'point of care'.


False-positive results with third-generation monoclonal hepatitis B surface antigen enzyme immunoassay.
Nonspecific hepatitis B surface antigen reactions with a third-generation enzyme immunoassay (Auszyme Monoclonal; Abbott Laboratories, North Chicago, Ill.) were investigated with 9,577 serum specimens in a clinical laboratory setting. Of the 196 serum specimens found reactive in Auszyme screen by the overnight procedure, 103 turned out to be true-positives, 71 were nonrepeatably reactive, and 22 were repeatably reactive but actually falsely positive (false-positive rate, 22 of 196, or 11.2%). Verification of the 196 screen reactives by the Auszyme 3-h incubation assay detected all but 4 true-positives, with a false-negative rate of 3.9% (4 of 103), and was negative for the rest. These observations reinforce the need for retesting all reactive specimens and confirming repeatedly reactive samples when the Auszyme Monoclonal test is used to detect hepatitis B surface antigen.


Pharmacogenetics of Cannabinoids.
Although the application of medical marijuana and cannabinoid drugs is controversial, it is a part of modern-day medicine. The list of diseases in which cannabinoids are promoted as a treatment is constantly expanding. Cases of significant improvement in patients with a very poor prognosis of glioma or epilepsy have already been described. However, the occurrence of side effects is still difficult to estimate, and the current knowledge of the therapeutic effects of cannabinoids is still insufficient. In our opinion, the answers to many questions and concerns regarding the medical use of cannabis can be provided by pharmacogenetics. Knowledge based on proteins and molecules involved in the transport, action, and metabolism of cannabinoids in the human organism leads us to predict candidate genes which variations are responsible for the presence of the therapeutic and side effects of medical marijuana and cannabinoid-based drugs. We can divide them into: receptor genes-CNR1, CNR2, TRPV1, and GPR55, transporters-ABCB1, ABCG2, SLC6A, biotransformation, biosynthesis, and bioactivation proteins encoded by CYP3A4, CYP2C19, CYP2C9, CYP2A6, CYP1A1, COMT, FAAH, COX2, ABHD6, ABHD12 genes, and also MAPK14. This review organizes the current knowledge in the context of cannabinoids pharmacogenetics according to individualized medicine and cannabinoid drugs therapy.


Cellulose degradation and ethanol production by thermophilic bacteria using mineral growth medium.
Growth of thermophilic cellulase-utilizing bacteria in a vitamin-free growth medium is reported for both a previously described strain, Clostridium thermoclelum 31549, and now isolates HJA1 and HJA2. Formation of fermentation products with and without vitamins was similar for strains HJA1 and HJA2 as well as for the enrichment cultures from which these stains were derived. Strain HJA2 was maintained in continuous culture on a vitamin-free mineral medium with Avicel as the carbon source for over a week. At a 38 h residence time, Avicel conversion was higher (81%) at pH 6.42 than at pH 6.97 (73%) or at 6.01 (58%). Ethanol and acetate were produced in significant amounts by strain JHA2 at all pH values tested (6.97, 6.42, 6.01). Lactic acid was the primary fermentation product at pH 6.97, but was not a significant product at both the lower values. Efforts to grow thermophilic, cellulose-utilizing bacteria at pH < 6.0 were unsuccessful for described strains, new isolates, and enrichment cultures.


Agreement between spirometers: a challenge in the follow-up of patients and populations?
Long-term cohort studies and lung function laboratories are confronted with the need for replacement of spirometers. Lack of agreement between spirometers might affect the longitudinal comparison of data, notably when replacing conventional by portable spirometers. To compare the handheld EasyOne (EO) with the conventional SensorMedics (SM) spirometer, and to analyze the interdevice reproducibility of EO spirometers. In total, 82 volunteers completed spirometry sessions with 1 SM and 2 of 3 EO spirometers following a Latin square design. Analyses of differences in forced vital capacity (FVC), forced expiratory flow in 1 s (FEV1), FEV1/FVC and mean forced expiratory flow calculated between 25 and 75% of the FVC between spirometers used a mixed effect model with a random intercept for each subject and the effect of the device as fixed effect adjusted for sex, age, height and order of spirometer tested. Bland-Altman plots show the 95% limits of agreement. Comparisons between EO and SM showed relatively small mean differences of <3%, but systematically lower values for FVC and FEV1 in all EO devices. The 95% agreement exceeded the limits for FEV1 by 50 ml in 2 EO spirometers. The EO interdevice comparisons showed mean differences and limits of agreement within established thresholds, thus indicating fair accuracy when comparing devices. Repeats with the same spirometer did not result in statistically significant differences. This study suggests fair agreement between the handheld and the conventional spirometer. Differences slightly exceeding limits for FEV1 in 2 EO devices might be considered mostly irrelevant for clinical practice. However, the systematically lower FVC and FEV1 observed with EO may be significant for epidemiological studies, thus justifying inspection before replacing devices.


Transcriptomics in Toxicogenomics, Part I: Experimental Design, Technologies, Publicly Available Data, and Regulatory Aspects.
The starting point of successful hazard assessment is the generation of unbiased and trustworthy data. Conventional toxicity testing deals with extensive observations of phenotypic endpoints in vivo and complementing in vitro models. The increasing development of novel materials and chemical compounds dictates the need for a better understanding of the molecular changes occurring in exposed biological systems. Transcriptomics enables the exploration of organisms' responses to environmental, chemical, and physical agents by observing the molecular alterations in more detail. Toxicogenomics integrates classical toxicology with omics assays, thus allowing the characterization of the mechanism of action (MOA) of chemical compounds, novel small molecules, and engineered nanomaterials (ENMs). Lack of standardization in data generation and analysis currently hampers the full exploitation of toxicogenomics-based evidence in risk assessment. To fill this gap, TGx methods need to take into account appropriate experimental design and possible pitfalls in the transcriptomic analyses as well as data generation and sharing that adhere to the FAIR (Findable, Accessible, Interoperable, and Reusable) principles. In this review, we summarize the recent advancements in the design and analysis of DNA microarray, RNA sequencing (RNA-Seq), and single-cell RNA-Seq (scRNA-Seq) data. We provide guidelines on exposure time, dose and complex endpoint selection, sample quality considerations and sample randomization. Furthermore, we summarize publicly available data resources and highlight applications of TGx data to understand and predict chemical toxicity potential. Additionally, we discuss the efforts to implement TGx into regulatory decision making to promote alternative methods for risk assessment and to support the 3R (reduction, refinement, and replacement) concept. This review is the first part of a three-article series on Transcriptomics in Toxicogenomics. These initial considerations on Experimental Design, Technologies, Publicly Available Data, Regulatory Aspects, are the starting point for further rigorous and reliable data preprocessing and modeling, described in the second and third part of the review series.


Functional expression of the thiolase gene thl from Clostridium beijerinckii P260 in Lactococcus lactis and Lactobacillus buchneri.
The first step of the butanol pathway involves an acetyl-CoA acetyltransferase (ACoAAT), which controls the key branching point from acetyl-CoA to butanol. ACoAAT, also known as thiolase (EC 126.96.36.199), is encoded by the thl gene and catalyzes ligation of two acetyl-CoA into acetoacetyl-CoA. Bioinformatics analyses suggest there are no thl in the genomes of lactic acid bacteria (LAB), in this study we aimed to introduce the thl gene into selected LAB strains and analyze the fermentation products. The thl gene from Clostridium beijerinckii P260 was amplified by genomic PCR using gene-specific primers designed from the published genome sequences of C. beijerinckii NCIMB 8025. The 1.2 kb thl gene was cloned into the pETBlue vector and overexpressed in Escherichia coli Tuner (DE3) pLacI cells. Functional enzyme activity was detected spectrophotometrically by measuring the decrease in absorbance at 303 nm, which reflects the change in acetoacetyl-CoA concentrations. The thl gene was subsequently introduced into Lactococcus lactis and Lactobacillus buchneri strains, and GC analysis indicated about 28 mg/L and 66 mg/L of butanol was produced in the recombinant strains, respectively. This study reports the first step toward developing a butanolgenic LAB through the introduction of the butanol pathway into butanol-tolerant strains of LAB.


Peripheral nerves in the rat exhibit localized heterogeneity of tensile properties during limb movement.
Peripheral nerves in the limbs stretch to accommodate changes in length during normal movement. The aim of this study was to determine how stretch is distributed along the nerve relative to local variations in mechanical properties. Deformation (strain) in joint and non-joint regions of rat median and sciatic nerves was measured in situ during limb movement using optical image analysis. In each nerve the strain was significantly greater in the joint rather than the non-joint regions (2-fold in the median nerve, 5- to 10-fold in the sciatic). In addition, this difference in strain was conserved in the median nerve ex vivo, demonstrating an in-built longitudinal heterogeneity of mechanical properties. Tensile testing of isolated samples of joint and non-joint regions of both nerves showed that joint regions were less stiff (more compliant) than their non-joint counterparts with joint: non-joint stiffness ratios of 0.5 +/- 0.07 in the median nerve, and 0.8 +/- 0.02 in the sciatic. However, no structural differences identified at the light microscope level in fascicular/non-fascicular tissue architecture between these two nerve regions could explain the observed tensile heterogeneity. This identification of localized functional heterogeneity in tensile properties is particularly important in understanding normal dynamic nerve physiology, provides clues to why peripheral nerve repair outcomes are variable, and suggests potential novel therapeutic targets.


Lixisenatide for the treatment of type 2 diabetes.
Lixisenatide (trade name Lyxumia®), a short-acting glucagon-like peptide 1 receptor (GLP-1R) agonist, was approved for the treatment of type 2 diabetes by the European Medicines Agency in early 2013. In preclinical investigations, acceptable toxicity and carcinogenicity profiles were demonstrated, as well as pancreatic beta cell-preserving actions and favorable effects on glycemic control. Following subcutaneous administration in humans, lixisenatide displays linear pharmacokinetics and an absorption-dependent elimination half-life of 2-3 hours. In clinical trials of up to 1 year duration in patients with type 2 diabetes, treatment with lixisenatide alone and in combination with insulin and various oral antidiabetics conferred significant reductions in HbA1c, fasting and postprandial plasma glucose. In direct comparison with the other GLP-1R agonists on the market (exenatide and liraglutide), lixisenatide appears to be less efficient, or at best non-inferior in terms of reducing HbA1c, fasting plasma glucose and body weight. Nevertheless, lixisenatide confers fewer adverse events than the other currently marketed GLP-1R agonists, while exhibiting a clinically valuable effect on postprandial hyperglycemia.


Sponge biomass and bioerosion rates increase under ocean warming and acidification.
The combination of ocean warming and acidification as a result of increasing atmospheric carbon dioxide (CO2 ) is considered to be a significant threat to calcifying organisms and their activities on coral reefs. How these global changes impact the important roles of decalcifying organisms (bioeroders) in the regulation of carbonate budgets, however, is less understood. To address this important question, the effects of a range of past, present and future CO2 emission scenarios (temperature + acidification) on the excavating sponge Cliona orientalis Thiele, 1900 were explored over 12 weeks in early summer on the southern Great Barrier Reef. C. orientalis is a widely distributed bioeroder on many reefs, and hosts symbiotic dinoflagellates of the genus Symbiodinium. Our results showed that biomass production and bioerosion rates of C. orientalis were similar under a pre-industrial scenario and a present day (control) scenario. Symbiodinium population density in the sponge tissue was the highest under the pre-industrial scenario, and decreased towards the two future scenarios with sponge replicates under the 'business-as-usual' CO2 emission scenario exhibiting strong bleaching. Despite these changes, biomass production and the ability of the sponge to erode coral carbonate materials both increased under the future scenarios. Our study suggests that C. orientalis will likely grow faster and have higher bioerosion rates in a high CO2 future than at present, even with significant bleaching. Assuming that our findings hold for excavating sponges in general, increased sponge biomass coupled with accelerated bioerosion may push coral reefs towards net erosion and negative carbonate budgets in the future.