The evolution of genomic and epigenomic features in two Pleurotus fungi.
Pleurotus tuoliensis (Bailinggu, designated Pt) and P. eryngii var. eryngii (Xingbaogu, designated Pe) are highly valued edible mushrooms. We report de novo assemblies of high-quality genomes for both mushrooms based on PacBio RS II sequencing and annotation of all identified genes. A comparative genomics analysis between Pt and Pe with P. ostreatus as an outgroup taxon revealed extensive genomic divergence between the two mushroom genomes primarily due to the rapid gain of taxon-specific genes and disruption of synteny in either taxon. The re-appraised phylogenetic relationship between Pt and Pe at the genome-wide level validates earlier proposals to designate Pt as an independent species. Variation of the identified wood-decay-related gene content can largely explain the variable adaptation and host specificity of the two mushrooms. On the basis of the two assembled genome sequences, methylomes and the regulatory roles of DNA methylation in gene expression were characterized and compared. The genome, methylome and transcriptome data of these two important mushrooms will provide valuable information for advancing our understanding of the evolution of Pleurotus and related genera and for facilitating genome- and epigenome-based strategies for mushroom breeding.


Lipid composition dictates serum stability of reconstituted high-density lipoproteins: implications for in vivo applications.
Nanolipoprotein particles (NLPs) are reconstituted high-density lipoproteins, consisting of a phospholipid bilayer stabilized by an apolipoprotein scaffold protein. This class of nanoparticle has been a vital tool in the study of membrane proteins, and in recent years has been increasingly used for in vivo applications. Previous work demonstrated that the composition of the lipid bilayer component affects the stability of these particles in serum solutions. In the current study, NLPs assembled with phosphatidylcholine lipids featuring different acyl chain structures were systematically tested to understand the effect that lipid composition has on NLP stability in both neat serum and cell culture media supplemented with 10% serum by volume. The time at which 50% of the particles dissociate, as well as the fraction of the initial population that remains resistant to dissociation, were correlated to key parameters obtained from all-atom simulations of the corresponding lipid bilayers. A significant correlation was observed between the compressibility modulus of the lipid bilayer and particle stability in these complex biological milieu. These results can be used as a reference to tune the stability of these versatile biological nanoparticles for in vitro and in vivo applications.


Populus tremula (European aspen) shows no evidence of sexual dimorphism.
Evolutionary theory suggests that males and females may evolve sexually dimorphic phenotypic and biochemical traits concordant with each sex having different optimal strategies of resource investment to maximise reproductive success and fitness. Such sexual dimorphism would result in sex biased gene expression patterns in non-floral organs for autosomal genes associated with the control and development of such phenotypic traits. We examined morphological, biochemical and herbivory traits to test for sexually dimorphic resource allocation strategies within collections of sexually mature and immature Populus tremula (European aspen) trees. In addition we profiled gene expression in mature leaves of sexually mature wild trees using whole-genome oligonucleotide microarrays and RNA-Sequencing. We found no evidence of sexual dimorphism or differential resource investment strategies between males and females in either sexually immature or mature trees. Similarly, single-gene differential expression and machine learning approaches revealed no evidence of large-scale sex biased gene expression. However, two significantly differentially expressed genes were identified from the RNA-Seq data, one of which is a robust diagnostic marker of sex in P. tremula.


Patterns of treated non-melanoma skin cancer in Queensland--the region with the highest incidence rates in the world.
The incidence of non-melanoma skin cancer, comprising basal cell carcinoma and squamous cell carcinoma, was studied in Queensland during 1984. The world-standardised annual incidence rates (per 100,000 population) for the number of persons with non-melanoma skin cancer were estimated to be 1372 for men and 702 for women, the highest recorded incidence rates in the world. Rates in men were nearly double the rates in women and age-specific incidence rates increased curvilinearly with age. There were, on average, 1.4 skin cancers per person with non-melanoma skin cancer and the ratio of basal cell carcinomas to squamous cell carcinomas was approximately three to one. The age-standardised annual incidence rate (per 100,000 population) of basal cell carcinoma for residents of the Gold Coast was 1.83 times the Brisbane rate for men and 1.57 times that for women, indicating significant differences between the two regions. For squamous cell carcinoma the regional differences were not statistically significant. The average potential number of non-melanoma skin cancers (per person) treated during the lifetime of a cohort of 100,000 was estimated to be 0.014 for men and 0.009 for women by age 40. By age 65, these numbers increased to 0.22 for men and 0.11 for women. At age 90, these average numbers were 1.09 and 0.42, respectively. Although the incidence of non-melanoma skin cancer is much higher in the older age groups, it should be kept in mind that it also affects the younger population; 1028 Queenslanders under 40 required treatment for 2300 non-melanoma skin cancers in 1984. This study which provides baseline information about the occurrence of non-melanoma skin cancer in Queensland emphasises the importance of developing safe sun-exposure habits, detecting non-melanoma skin cancer early and protecting and restoring the atmosphere.


On nitrogen fixation and "residual nitrogen content" in cellulosic pulps.
Cellulosic material is capable of permanently retaining nitrogen compounds (mostly having amino functions), which is reflected in a residual nitrogen content (in the low per mille range to the low percent range) of some pulps and certain lab samples. Merely adsorptively bound compounds can be removed by mild acidic washing, but part of the nitrogen seems to be resistant and very tightly bound, and thus not accessible for removal by washing. Tertiary and aromatic amines are not retained in this way, but only primary and secondary amines. There is only a weak correlation between the "firmly bound nitrogen" and the carbonyl content in cellulosics (because of oxidative damage), so that possible aminal, Schiff base and enamine structures can hardly be relevant as major nitrogen sources. However, there is a very good linear correlation between the ISO brightness (chromophore content) in aged pulps and the residual nitrogen content. In particular the concentration of the cellulosic key chromophore 2,5-dihydroxy-[1,4]-benzoquinone (DHBQ) determines the permanent N-binding capacity of the pulp. DHBQ reacts very readily with primary and secondary amines under ambient conditions to 2,5-diamino-substituted [1,4]-benzoquinones, which have very low solubility (because of zwitterionic resonance contributions) and thus remain on/in the pulp. Examples of nitrogen fixation in pulps are the binding of piperidine (a common amine catalyst in derivatization reactions), amine degradation products of the cellulose solvent NMMO, dimethylamine in materials processed from the cellulose solvent DMAc/LiCl, imidazole (a degradation product of 1-alkyl-3-methylimidazolium ionic liquids), and of amino groups in proteins after enzymatic treatment. The nature of the respective DHBQ-amine addition compound has been verified by complete structure determination.


A regression-based differential expression detection algorithm for microarray studies with ultra-low sample size.
Global gene expression analysis using microarrays and, more recently, RNA-seq, has allowed investigators to understand biological processes at a system level. However, the identification of differentially expressed genes in experiments with small sample size, high dimensionality, and high variance remains challenging, limiting the usability of these tens of thousands of publicly available, and possibly many more unpublished, gene expression datasets. We propose a novel variable selection algorithm for ultra-low-n microarray studies using generalized linear model-based variable selection with a penalized binomial regression algorithm called penalized Euclidean distance (PED). Our method uses PED to build a classifier on the experimental data to rank genes by importance. In place of cross-validation, which is required by most similar methods but not reliable for experiments with small sample size, we use a simulation-based approach to additively build a list of differentially expressed genes from the rank-ordered list. Our simulation-based approach maintains a low false discovery rate while maximizing the number of differentially expressed genes identified, a feature critical for downstream pathway analysis. We apply our method to microarray data from an experiment perturbing the Notch signaling pathway in Xenopus laevis embryos. This dataset was chosen because it showed very little differential expression according to limma, a powerful and widely-used method for microarray analysis. Our method was able to detect a significant number of differentially expressed genes in this dataset and suggest future directions for investigation. Our method is easily adaptable for analysis of data from RNA-seq and other global expression experiments with low sample size and high dimensionality.


The Components of Bone and What They Can Teach Us about Regeneration.
The problem of bone regeneration has engaged both physicians and scientists since the beginning of medicine. Not only can bone heal itself following most injuries, but when it does, the regenerated tissue is often indistinguishable from healthy bone. Problems arise, however, when bone does not heal properly, or when new tissue is needed, such as when two vertebrae are required to fuse to stabilize adjacent spine segments. Despite centuries of research, such procedures still require improved therapeutic methods to be devised. Autologous bone harvesting and grafting is currently still the accepted benchmark, despite drawbacks for clinicians and patients that include limited amounts, donor site morbidity, and variable quality. The necessity for an alternative to this "gold standard" has given rise to a bone-graft and substitute industry, with its central conundrum: what is the best way to regenerate bone? In this review, we dissect bone anatomy to summarize our current understanding of its constituents. We then look at how various components have been employed to improve bone regeneration. Evolving strategies for bone regeneration are then considered.


Modeling deterministic effects in hematopoietic system caused by chronic exposure to ionizing radiation in large human cohorts.
A new model of the hematopoietic system for humans chronically exposed to ionizing radiation allows for quantitative description of the initial hematopoiesis inhibition and subsequent increase in the risks of late stochastic effects such as leukemia. This model describes the dynamics of the hematopoietic stem cell compartment as well as the dynamics of each of the three blood cell types (leukocytes, erythrocytes, and platelets). The model parameters are estimated from the results of other experiments. They include the steady-state numbers of hematopoietic stem cells and peripheral blood cell lines for an unexposed organism, amplification parameters for each blood cell line, parameters describing the proliferation and apoptosis, parameters of feedback functions regulating the steady-state numbers, and characteristics of radiosensitivity in respect to cell death and non-lethal cell damages. The dynamic model of hematopoiesis is applied to the data on a subcohort of the Techa River residents with hematological measurements (e.g., blood counts) performed in 1950-1956 (which totals to about 3,500 exposed individuals). Among well-described effects observed in these data are the slope values of the dose-effect curves describing the hematopoietic inhibition and the dose rate patterns of the fractions of cytopenic states (e.g., leukopenia, thrombocytopenia). The model has been further generalized by inclusion of the component describing the risk of late stochastic effects. The risks of the development of late effects (such as leukemia) in population groups with specific patterns of early reactions in hematopoiesis (such as leukopenia induced by ionizing radiation) are investigated using simulation studies and compared to data.


A control/experimental trial of an operant treatment for early stuttering.
A preliminary study (Onslow, Costa, & Rue, 1990) suggested that a parent-conducted program of verbal response-contingent stimulation would be an effective treatment for stuttering children younger than 5 years. The present study was designed to expand those preliminary findings by using a larger group of children and by comparing them to a control group of children. Twelve children in the experimental group achieved median percent syllables stuttered (%SS) scores below 1.0 for a 12-month posttreatment period. The children's treatments were completed in a median of 10.5 1-hour clinic sessions and a median of 84.5 days from the start of treatment. The majority of parents of the control children withdrew from the study and elected to have treatment begin for their child. These results suggest that the program may be a cost-effective method for managing a clinical caseload of stuttering children younger than 5 years. It is suggested that controlled clinical trials cannot be used validly or ethically to determine the number of cases of early stuttering who recover without formal intervention.


Role of the central cholinergic system in the therapeutics of schizophrenia.
The therapeutic agents currently used to treat schizophrenia effectively improve psychotic symptoms; however, they are limited by adverse effects and poor efficacy when negative symptoms of the illness and cognitive dysfunction are considered. While optimal pharmacotherapy would directly target the neuropathology of schizophrenia neither the underlying neurobiological substrates of the behavioral symptoms nor the cognitive deficits have been clearly established. Abnormalities in the neurotransmitters dopamine, serotonin, glutamate, and GABA are commonly implicated in schizophrenia; however, it is not uncommon for alterations in the brain cholinergic system (e.g., choline acetyltransferase, nicotinic and muscarinic acetylcholine receptors) to also be reported. Further, there is now considerable evidence in the animal literature to suggest that both first and second generation antipsychotics (when administered chronically) can alter the levels of several cholinergic markers in the brain as well as impair memory-related task performance. Given the well-established importance of central cholinergic neurons to information processing and cognition, it is important that cholinergic function in schizophrenia be further elucidated and that the mechanisms of the chronic effects of antipsychotic drugs on this important neurotransmitter system be identified. A better understanding of these mechanisms would be expected to facilitate optimal treatment strategies for schizophrenia as well as the identification of novel therapeutic targets. In this review, the following topics are discussed: 1) the central cholinergic system in schizophrenia 2) effects of antipsychotic drugs on central cholinergic neurons 3) important neurotrophins in schizophrenia, especially those that support central cholinergic neurons; 4) novel strategies to optimize the therapeutics of schizophrenia via the use of cholinergic compounds as primary (i.e., antipsychotic) treatments as well as adjunctive, pro-cognitive agents.


Decoherence, control and attosecond probing of XUV-induced charge migration in biomolecules. A theoretical outlook.
The sudden ionization of a molecule by an attosecond pulse is followed by charge redistribution on a time scale from a few femtoseconds down to hundreds of attoseconds. This ultrafast redistribution is the result of the coherent superposition of electronic continua associated with the ionization thresholds that are reached by the broadband attosecond pulse. Thus, a correct theoretical description of the time evolution of the ensuing wave packet requires the knowledge of the actual ionization amplitudes associated with all open ionization channels, a real challenge for large and medium-size molecules. Recently, the first calculation of this kind has come to light, allowing for interpretation of ultrafast electron dynamics observed in attosecond pump-probe experiments performed on the amino acid phenylalanine [Calegari et al., Science 2014, 346, 336]. However, as in most previous theoretical works, the interpretation was based on various simplifying assumptions, namely, the ionized electron was not included in the description of the cation dynamics, the nuclei were fixed at their initial position during the hole migration process, and the effect of the IR probe pulse was ignored. Here we go a step further and discuss the consequences of including these effects in the photoionization of the glycine molecule. We show that (i) the ionized electron does not affect hole dynamics beyond the first femtosecond, and (ii) nuclear dynamics has only a significant effect after approximately 8 fs, but does not destroy the coherent motion of the electronic wave packet during at least few additional tens of fs. As a first step towards understanding the role of the probe pulse, we have considered an XUV probe pulse, instead of a strong IR one, and show that such an XUV probe does not introduce significant distortions in the pump-induced dynamics, suggesting that pump-probe strategies are suitable for imaging and manipulating charge migration in complex molecules. Furthermore, we show that hole dynamics can be changed by shaping the attosecond pump pulse, thus opening the door to the control of charge dynamics in biomolecules.


Inhibition of thyroid hormone sulfotransferase activity by brominated flame retardants and halogenated phenolics.
Many halogenated organic contaminants (HOCs) are considered endocrine disruptors and affect the hypothalamic-pituitary-thyroid axis, often by interfering with circulating levels of thyroid hormones (THs). We investigated one potential mechanism for TH disruption, inhibition of sulfotransferase activity. One of the primary roles of TH sulfation is to support the regulation of biologically active T3 through the formation of inactive THs. We investigated TH sulfotransferase inhibition by 14 hydroxylated polybrominated diphenyl ethers (OH BDEs), BDE 47, triclosan, and fluorinated, chlorinated, brominated, and iodinated analogues of 2,4,6-trihalogenated phenol and bisphenol A (BPA). A new mass spectrometry-based method was also developed to measure the formation rates of 3,3'-T2 sulfate (3,3'-T2S). Using pooled human liver cytosol, we investigated the influence of these HOCs on the sulfation of 3,3'-T2, a major substrate for TH sulfation. For the formation of 3,3'-T2S, the Michaelis constant (Km) was 1070 ± 120 nM and the Vmax was 153 ± 6.6 pmol min(-1) (mg of protein)(-1). All chemicals investigated inhibited sulfotransferase activity with the exception of BDE 47. The 2,4,6-trihalogenated phenols were the most potent inhibitors followed by the OH BDEs and then halogenated BPAs. The IC50 values for the OH BDEs were primarily in the low nanomolar range, which may be environmentally relevant. In silico molecular modeling techniques were also used to simulate the binding of OH BDE to SULT1A1. This study suggests that some HOCs, including antimicrobial chemicals and metabolites of flame retardants, may interfere with TH regulation through inhibition of sulfotransferase activity.


The sero-epidemiology of Coxiella burnetii (Q fever) across livestock species and herding contexts in Laikipia County, Kenya.
Coxiella burnetii, the causative agent of Query fever (Q fever), is among the most highly infectious zoonotic pathogens transmitted among livestock, with chronic effects challenging to veterinary and medical detection and care systems. Transmission among domestic livestock species can vary regionally due to herd management practices that determine which livestock species are raised, whether or not livestock are in contact with wildlife, and the susceptibility of these livestock to infection. To explore how different livestock management practices are associated with the risk of infection in multispecies environments, we carried out a comparative study of three types of herd management systems in the central Kenyan county of Laikipia: agro-commercial, mixed conservancy/commercial, and smallholder ranches. We tested C. burnetii antibody seroprevalence in four common livestock species. Across all management types, the highest seroprevalence was in camels (20%), followed by goats (18%), sheep (13%), and cattle (6%). We observed a lower odds of testing seropositive for young compared to adult animals (adjusted OR = 0.44 [95% CI 0.24, 0.76]), and for males compared to females (adjusted OR = 0.52 [95% CI 0.33, 0.80]). Animals from mixed conservancy/commercial and smallholder operations had a higher odds of testing seropositive compared to animals from agro-commercial ranches (adjusted OR = 5.17 [95% CI 2.71, 10.44] and adjusted OR = 2.21 [95% CI 1.17, 4.43] respectively). These data suggest that herd management practices might affect the transmission dynamics of C. burnetiiin arid African ecosystems like those seen in Kenya where several transmission modes are possible, risk of drought has promoted new livestock species such as camels, and multiple wildlife species may co-occur with livestock on the landscape. Further longitudinal studies are needed to disentangle the mechanisms underlying these patterns, and further explore transmission patterns between wildlife, domestic animal, and human populations.


Significant association between clinical characteristics and changes in peripheral immuno-phenotype in large vessel vasculitis.
Large vessel vasculitis (LVV) is a type of vasculitis characterized by granulomatous inflammation of medium- and large-sized arteries. Clinical assessment of acute phase reactants has been conventionally used to diagnose and monitor diseases; however, accurate assessment of vascular disease activity status can be difficult. In this study, we investigated comprehensive immuno-phenotyping to explore useful biomarkers associated with clinical characteristics. Consecutive patients with newly diagnosed LVV who visited our institution between May 2016 and May 2019 were enrolled. The number of circulating T cells, B cells, natural killer cells, dendritic cells, monocytes, and granulocytes was examined and chronologically followed. Baseline and time-course changes in immuno-phenotyping associated with disease activity were assessed. Comprehensive immuno-phenotyping data from 90 samples from each of 20 patients with LVV were compared with those from healthy controls (HCs). The number of helper T (Th), follicular helper T (Tfh), CD8+ T, CD14++ CD16+ monocytes, and neutrophils were higher in patients with giant cell arteritis (GCA) and/or Takayasu arteritis (TAK) than in HCs. Among them, the number of CD8+ T and CD8+ Tem were higher in patients with TAK than in GCA. Notably, memory CD4+ and CD8+ T cells in patients with TAK remained high even in the remission phase. Further analysis revealed that the number of Th1, Th17, and Tfh cells was associated with disease relapse in GCA and TAK and that the number of CD8+ T cells was associated with relapse in TAK. Th1, Th17, and Tfh cells decreased after treatment with biologic agents, while CD8+ T cells did not. Our results from peripheral immuno-phenotyping analysis indicate that the numbers of Th and Tfh cells changed along with the disease condition in both GCA and TAK, while that of CD8+ T cells did not, especially in TAK. Treatment with biologic agents decreased the proportion of Th and Tfh cells, but not CD8+ T cells, in the patients. Chronological immuno-phenotyping data explained the difference in therapeutic response, such as reactivities against biologics, between GCA and TAK.


Plant immunity directly or indirectly restricts the injection of type III effectors by the Pseudomonas syringae type III secretion system.
Plants perceive microorganisms by recognizing microbial molecules known as pathogen-associated molecular patterns (PAMPs) inducing PAMP-triggered immunity (PTI) or by recognizing pathogen effectors inducing effector-triggered immunity (ETI). The hypersensitive response (HR), a programmed cell death response associated with ETI, is known to be inhibited by PTI. Here, we show that PTI-induced HR inhibition is due to direct or indirect restriction of the type III protein secretion system's (T3SS) ability to inject type III effectors (T3Es). We found that the Pseudomonas syringae T3SS was restricted in its ability to inject a T3E-adenylate cyclase (CyaA) injection reporter into PTI-induced tobacco (Nicotiana tabacum) cells. We confirmed this restriction with a direct injection assay that monitored the in planta processing of the AvrRpt2 T3E. Virulent P. syringae strains were able to overcome a PAMP pretreatment in tobacco or Arabidopsis (Arabidopsis thaliana) and continue to inject a T3E-CyaA reporter into host cells. In contrast, ETI-inducing P. syringae strains were unable to overcome PTI-induced injection restriction. A P. syringae pv tomato DC3000 mutant lacking about one-third of its T3E inventory was less capable of injecting into PTI-induced Arabidopsis plant cells, grew poorly in planta, and did not cause disease symptoms. PTI-induced transgenic Arabidopsis expressing the T3E HopAO1 or HopF2 allowed higher amounts of the T3E-CyaA reporter to be injected into plant cells compared to wild-type plants. Our results show that PTI-induced HR inhibition is due to direct or indirect restriction of T3E injection and that T3Es can relieve this restriction by suppressing PTI.


EULAR recommendations for the management of systemic lupus erythematosus with neuropsychiatric manifestations: report of a task force of the EULAR standing committee for clinical affairs.
To develop recommendations for the diagnosis, prevention and treatment of neuropsychiatric systemic lupus erythematosus (NPSLE) manifestations. The authors compiled questions on prevalence and risk factors, diagnosis and monitoring, therapy and prognosis of NPSLE. A systematic literature search was performed and evidence was categorised based on sample size and study design. Systemic lupus erythematosus (SLE) patients are at increased risk of several neuropsychiatric manifestations. Common (cumulative incidence > 5%) manifestations include cerebrovascular disease (CVD) and seizures; relatively uncommon (1-5%) are severe cognitive dysfunction, major depression, acute confusional state (ACS), peripheral nervous disorders psychosis. Strong risk factors (at least fivefold increased risk) are previous or concurrent severe NPSLE (for cognitive dysfunction, seizures) and antiphospholipid antibodies (for CVD, seizures, chorea). The diagnostic work-up of suspected NPSLE is comparable to that in patients without SLE who present with the same manifestations, and aims to exclude causes unrelated to SLE. Investigations include cerebrospinal fluid analysis (to exclude central nervous system infection), EEG (to diagnose seizure disorder), neuropsychological tests (to assess cognitive dysfunction), nerve conduction studies (for peripheral neuropathy) and MRI (T1/T2, fluid-attenuating inversion recovery, diffusion-weighted imaging, enhanced T1 sequence). Glucocorticoids and immunosuppressive therapy are indicated when NPSLE is thought to reflect an inflammatory process (optic neuritis, transverse myelitis, peripheral neuropathy, refractory seizures, psychosis, ACS) and in the presence of generalised lupus activity. Antiplatelet/anticoagulation therapy is indicated when manifestations are related to antiphospholipid antibodies, particularly thrombotic CVD. Neuropsychiatric manifestations in SLE patients should be first evaluated and treated as in patients without SLE, and secondarily attributed to SLE and treated accordingly.


The effects of aquatic therapy on mobility of individuals with neurological diseases: a systematic review.
To summarize evidence on the effects of aquatic therapy on mobility in individuals with neurological diseases. MEDLINE, EMBASE, PsycInfo, CENTRAL, CINAHL, SPORTDiscus, PEDro, PsycBITE and OT Seeker were searched from inception to 15 September 2014. Hand-searching of reference lists was performed in the selected studies. The search included randomized controlled trials and quasi-experimental studies that investigated the use of aquatic therapy and its effect on mobility of adults with neurological diseases. One reviewer screened titles and abstracts of retrieved studies from the search strategy. Two reviewers independently examined the full texts and conducted the study selection, data extraction and quality assessment. A narrative synthesis of data was applied to summarize information from included studies. The Downs and Black Scale was used to assess methodological quality. A total of 116 articles were obtained for full text eligibility. Twenty studies met the specified inclusion criteria: four Randomized Controlled Trials (RCTs), four non-randomized studies and 12 before-and-after tests. Two RCTs (30 patients with stroke in the aquatic therapy groups), three non-randomized studies and three before-and-after studies showed "fair" evidence that aquatic therapy increases dynamic balance in participants with some neurological disorders. One RCT (seven patients with stroke in the aquatic therapy group) and two before-and-after tests (20 patients with multiple sclerosis) demonstrated "fair" evidence on improvement of gait speed after aquatic therapy. Our synthesis showed "fair" evidence supporting the use of aquatic therapy to improve dynamic balance and gait speed in adults with certain neurological conditions.


Essential role of protein phosphatase 2A in metaphase II arrest and activation of mouse eggs shown by okadaic acid, dominant negative protein phosphatase 2A, and FTY720.
Vertebrate eggs arrest at second meiotic metaphase. The fertilizing sperm causes meiotic exit through Ca(2+)-mediated activation of the anaphase-promoting complex/cyclosome (APC/C). Although the loss in activity of the M-phase kinase CDK1 is known to be an essential downstream event of this process, the contribution of phosphatases to arrest and meiotic resumption is less apparent, especially in mammals. Therefore, we explored the role of protein phosphatase 2A (PP2A) in mouse eggs using pharmacological inhibition and activation as well as a functionally dominant-negative catalytic PP2A subunit (dn-PP2Ac-L199P) coupled with live cell imaging. We observed that PP2A inhibition using okadaic acid induced events normally observed at fertilization: degradation of the APC/C substrates cyclin B1 and securin resulting from loss of the APC/C inhibitor Emi2. Although sister chromatids separated, chromatin remained condensed, and polar body extrusion was blocked as a result of a rapid spindle disruption, which could be ameliorated by non-degradable cyclin B1, suggesting that spindle integrity was affected by CDK1 loss. Similar cell cycle effects to okadaic acid were also observed using dominant-negative PP2Ac. Preincubation of eggs with the PP2A activator FTY720 could block many of the actions of okadaic acid, including Emi2, cyclin B1, and securin degradation and sister chromatid separation. Therefore, in conclusion, we used okadaic acid, dn-PP2Ac-L199P, and FTY720 on mouse eggs to demonstrate that PP2A is needed to for both continued metaphase arrest and successful exit from meiosis.


Effect of a novel saponin adjuvant derived from Quillaja saponaria on the immune response to recombinant hepatitis B surface antigen.
Adjuvant activity of saponins extracted from the South American tree Quillaja saponaria has been demonstrated with many antigens. Recently, four saponin fractions (designated as QS-7, QS-17, QS-18, and QS-21) with adjuvant activity were purified by reverse phase chromatography. In particular, efficacy of the less toxic QS-21 fraction has been demonstrated with several recombinant viral antigens including HIV gp120. Here, we report a novel saponin fraction (designated as QS-L1) derived from Quillaja saponaria. Unlike previously identified saponins, QS-L1 had a different chemical structure and showed adjuvant activity only when administered in the presence of alum-precipitated antigen. Interestingly, the QS-L1 greatly increased not only a humoral immune response but also cellular immune response to recombinant hepatitis B virus surface antigen (HBsAg). Furthermore, QS-L1 showed lower toxicity in vivo and in vitro than the previously identified saponin fraction, QS-21. Finally, we examined the chemical structure of the QS-L1 using mass spectroscopic analysis, carbohydrate composition analysis and NMR spectroscopic analysis. Thus, our results indicated that this novel QS-L1 saponin fraction had several desirable properties required for an effective adjuvant.


The results of interventional catheterization in infants weighing under 2,000 g.
The aim of this study was to evaluate the early and mid-term results of interventional cardiac catheterization and procedure-related complications in infants weighing <2,000 g. Between May 1998 and April 2017, 22 patients (14 males, 8 females; mean age 14±8.4 days; range, 1 to 30 days) weighing <2,000 g who underwent a total of 23 interventional cardiac catheterization were retrospectively analyzed. Procedures were balloon coarctation angioplasty in 14, balloon atrial septostomy in five, balloon aortic valvuloplasty in one, balloon pulmonary valvuloplasty in one, patent ductus arteriosus closure in one, and stent placement in the ductus in one patient. Another patient underwent balloon coarctation angioplasty and balloon aortic valvuloplasty in the same session. The overall success rate of the interventional procedures was 95.6%. The mean follow-up was 3.2±1.6 years (range, 1 to 5.5) for 18 patients with available records. The rate of serious complications was 18%. The most frequent complications in the early period were low hemoglobin levels requiring erythrocyte suspension transfusion (54.5%) and vascular injury (54.5%). Two patients required reintervention, one patient required surgery after the second intervention, and three patients required only surgery. Six patients underwent palliative interventional procedures, and interventional procedures led to definitive treatment in five patients. The mortality and morbidity rate of surgery is high in premature under 2,000 g infants and interventional heart catheterization can be life-saving in this patient group, although it is associated with significant complications in low birth weight newborns.


Carbon dioxide-catalyzed peroxynitrite reactivity - The resilience of the radical mechanism after two decades of research.
Peroxynitrite, ONOO-, formed in tissues that are simultaneously generating NO• and O2•-, is widely regarded as a major contributor to oxidative stress. Many of the reactions involved are catalyzed by CO2 via formation of an unstable adduct, ONOOC(O)O-, that undergoes O-O bond homolysis to produce NO2• and CO3•- radicals, whose yields are equal at about 0.33 with respect to the ONOO- reactant. Since its inception two decades ago, this radical-based mechanism has been frequently but unsuccessfully challenged. The most recent among these [Serrano-Luginbuehl et al. Chem. Res. Toxicol.31:721-730; 2018] claims that ONOOC(O)O- is stable, predicts a yield of NO2•/CO3•- of less than 0.01 under physiological conditions and, contrary to widely accepted viewpoints, suggests that radical generation is inconsequential to peroxynitrite-induced oxidative damage. Here we review the experimental and theoretical evidence that support the radical model and show this recently proposed alternative mechanism to be incorrect.


The role of protein structural analysis in the next generation sequencing era.
Proteins are macromolecules that serve a cell's myriad processes and functions in all living organisms via dynamic interactions with other proteins, small molecules and cellular components. Genetic variations in the protein-encoding regions of the human genome account for >85% of all known Mendelian diseases, and play an influential role in shaping complex polygenic diseases. Proteins also serve as the predominant target class for the design of small molecule drugs to modulate their activity. Knowledge of the shape and form of proteins, by means of their three-dimensional structures, is therefore instrumental to understanding their roles in disease and their potentials for drug development. In this chapter we outline, with the wide readership of non-structural biologists in mind, the various experimental and computational methods available for protein structure determination. We summarize how the wealth of structure information, contributed to a large extent by the technological advances in structure determination to date, serves as a useful tool to decipher the molecular basis of genetic variations for disease characterization and diagnosis, particularly in the emerging era of genomic medicine, and becomes an integral component in the modern day approach towards rational drug development.


Effect of co-exposure to nickel and particulate matter on insulin resistance and mitochondrial dysfunction in a mouse model.
It has been well recognized that toxicity of fine ambient air particulate matter (PM(2.5)) may depend on its chemical constituents, including components such as soluble metals that may theoretically exert distinctive effects. We have recently demonstrated an important effect of PM(2.5) on metabolic function. Since transition metals, such as nickel (Ni), represent an important component of exposure in certain environments, and may significantly influence the toxicity of inhalational exposure, we investigated the effects of Ni as a variable component of ambient PM(2.5) exposure. Male ApoE knockout mice were exposed to filtered air (FA), fine-sized nickel sulfate particles alone (Ni) at 0.44 μg/m(3), concentrated ambient air PM(2.5) (CAPs) at a mean of 70 μg/m(3), or CAPs+Ni in Tuxedo, NY, 6 hours/day, 5 days/week, for 3 months. Exposure to Ni, irrespective of co-exposure to CAPs, resulted in body weight gain, while exposure to CAPs+Ni significantly enhanced fasting glucose and worsened insulin resistance measures (HOMA-IR), when compared with exposure to CAPs alone. CAPs+Ni exposure induced a significant decrease in phosphorylation of AMP-activated protein kinase (AMPK) α. Exposure to Ni or CAPs+Ni significantly induced microcirculatory dysfunction and increased monocytic cell infiltration into lung and adipose, and decreased uncoupling protein 1 expression at gene and protein levels and several brown adipocyte-specific genes in adipose tissue. Ni exposure has effects on metabolic and inflammatory parameters that are comparable to that of CAPs. Additionally, Ni synergistically exacerbates CAPs-induced adverse effects on some of, but not all of, these parameters, that may be mediated via the AMPK signaling pathway. These findings have important implications for inhaled transition metal toxicity that may exert synergistic effects with other PM(2.5) components.


MRI findings in the young infant with brainstem disconnection and extracerebral features. Report of one case and review of the literature.
We present the young infant with the extremely rare brain abnormality-brainstem disconnection. Additionally, several extracerebral abnormalities were diagnosed: bilateral anotia, micrognatia, hypertelorism, scoliosis, ribs and vertebral anomalies. MR brain examination precisely demonstrated absence of the pons, with disruption between midbrain and hypoplastic medulla oblongata. The thin strands connecting the medulla with the midbrain and medulla with both cerebellar hemispheres were revealed. The large hamartoma of the tuber cinereum was found. In this study we review case reports published previously.


Multilocus sequence typing of Bartonella henselae in the United Kingdom indicates that only a few, uncommon sequence types are associated with zoonotic disease.
Bartonella henselae is one of the most common zoonotic agents acquired from companion animals (cats) in industrialized countries. Nonetheless, although the prevalence of infections in cats is high, the number of human cases reported is relatively low. One hypothesis for this discrepancy is that B. henselae strains vary in their zoonotic potential. To test this hypothesis, we employed structured sampling to explore the population structure of B. henselae in the United Kingdom and to determine the distribution of strains associated with zoonotic disease within this structure. A total of 118 B. henselae strains were delineated into 12 sequence types (STs) using multilocus sequence typing. We observed that most (85%) of the zoonosis-associated strains belonged to only three genotypes, i.e., ST2, ST5, and ST8. Conversely, most (74%) of the feline isolates belonged to ST4, ST6, and ST7. The difference in host association of ST2, ST5, and ST8 (zoonosis associated) and ST6 (feline) was statistically significant (P < 0.05), indicating that a few, uncommon STs were responsible for the majority of symptomatic human infections.


Great apes distinguish true from false beliefs in an interactive helping task.
Understanding the behavior of others in a wide variety of circumstances requires an understanding of their psychological states. Humans' nearest primate relatives, the great apes, understand many psychological states of others, for example, perceptions, goals, and desires. However, so far there is little evidence that they possess the key marker of advanced human social cognition: an understanding of false beliefs. Here we demonstrate that in a nonverbal (implicit) false-belief test which is passed by human 1-year-old infants, great apes as a group, including chimpanzees (Pan troglodytes), bonobos (Pan paniscus), and orangutans (Pongo abelii), distinguish between true and false beliefs in their helping behavior. Great apes thus may possess at least some basic understanding that an agent's actions are based on her beliefs about reality. Hence, such understanding might not be the exclusive province of the human species.


Association of ambulatory blood pressure and dietary caffeine in adolescents.
Although relatively little is known about the responsible factors, there is an increased prevalence of essential hypertension in youth. Our previous research using casual blood pressure (BP) suggests a role for caffeine intake. The objective of this study was to assess the association between caffeine intake and ambulatory BP patterns among adolescents and to replicate our previous findings that compared caffeine intake to BP values obtained at a single time point. Eighty-two African-American and non-Hispanic white adolescents (15 to 19 years old) with normal systolic BP selected foods and beverages for a 4-day sodium-controlled diet. Subjects were stratified into three groups based on the amount of caffeine in these foods. Ambulatory BP measures (24-h) were recorded during 1 day of the 4-day diet. The effects of ethnicity, caffeine, and the interaction of ethnicity and caffeine on BP were assessed for daytime and nighttime hours controlling for gender and body mass index. The level of dietary caffeine was positively associated with daytime systolic BP (F(2,76) = 3.1, P = .05, partial R(2) = 0.07) and daytime diastolic BP (F = 3.53(2,76), P = .03, partial R(2) = 0.07). Caffeine's effect on systolic BP was most pronounced for African-American subjects. These results replicated our earlier findings. There was no association between caffeine intake and nighttime BP. This investigation replicates and extends our previous findings that caffeine consumption impacts the BP of adolescents, during the daytime when sympathetic nervous system responses dominate BP control. Controlled studies that examine the pressor effects of caffeine intake at levels typical of the dietary patterns of today's adolescents are needed.


Clinical spectrum of clonal proliferations of T-large granular lymphocytes: a T-cell clonopathy of undetermined significance?
We identified 68 patients with clonal T-large granular lymphocyte (T-LGL) proliferations who were seen at the Mayo Clinic between 1984 and 1992. Nineteen (28%) were asymptomatic at diagnosis, while the rest experienced fatigue (60%), B-symptoms (12%), and recurrent infections (15%). Associated comorbid conditions included rheumatoid arthritis (RA) in 26%. Severe anemia (hemoglobin [Hb] < 8g/dL) and neutopenia (absolute neutrophil count [ANC] < 500/microL) were seen in 19% and 40% of patients, respectively. Immunophenotypic studies showed CD3+, CD8+ phenotype in the majority (72%). Twenty-one patients (31%) have required no therapy, and remain relatively stable with a median follow-up period of 50 months. Treatment was required at either diagnosis (36 patients) or at subsequent follow-up (11 patients). Initial response rates were similar in patients treated with cyclophosphamide (CTX) with or without prednisone (69%), or prednisone alone (73%). Overall, 61 patients (90%) are alive with a median follow-up of 44 months. Actuarial median survival of this entire cohort is 161 months. The presence of anemia or symptoms does not appear to correlate with the tumor burden. In patients requiring therapy, a lower ANC and the presence of B-symptoms/infection were independently associated with a significantly lower probability of achieving a molecular or hematologic complete remission (H-CR). Intermittent immunosuppressive therapy is effective in achieving durable responses in a number of patients. T-LGL proliferations are associated with a favorable prognosis and response to therapy. However, significant heterogeneity exists in clinical presentation and associated comorbid conditions. These disorders should be included in the differential diagnosis of patients with unexplained cytopenias, particularly in the setting of RA and other autoimmune disorders. Analogous to the situation with monoclonal gammopathies, a term such as T-cell clonopathy of undetermined significance (TCUS) may be more appropriate to describe these patients.


Influenza vaccination in children being treated with chemotherapy for cancer.
Influenza infection is a potential cause of severe morbidity in children with cancer, therefore vaccination against influenza is recommended. However, there are conflicting data concerning the immune response to influenza vaccination in children with cancer and the value of vaccination remains unclear. 1. To assess the efficacy of influenza vaccination in stimulating immunological response in children with cancer during chemotherapy, compared to control groups. 2. To assess the efficacy of influenza vaccination in preventing confirmed influenza and influenza-like illness and/or stimulating immunological response in children with cancer treated with chemotherapy, compared to placebo, no intervention or different dosage schedules. 3. To determine the adverse effects associated with influenza vaccination in children with cancer. We searched CENTRAL, MEDLINE (1966 to 2007) and EMBASE (1980 to 2007) up to February 2007. We also searched reference lists of relevant articles and conference proceedings of ICAAC, IDSA, MASCC and SIOP. We considered randomised controlled trials (RCTs) and controlled clinical trials (CCTs) in which the serologic response to influenza vaccination of children with cancer was compared to other control groups. We also considered RCTs and CCTs comparing the effects of influenza vaccination on clinical response and/or immunological response in children with cancer, with placebo, no intervention or different dosage schedules. Two independent authors assessed the methodological quality of included studies and extracted data. We included 1 RCT and 8 CCTs ( total number of participants=708). None of the included studies reported on clinical outcome. All included studies reported on influenza immunity and adverse reactions to vaccination. In five studies, immune responses to influenza vaccine were compared in 272 children on chemotherapy with 166 children not on chemotherapy. In three studies, responses to influenza vaccine were assessed in 204 children on chemotherapy compared with responses in 112 healthy children. The measures used to assess immune responses were: a four-fold rise in antibody titre after vaccination, development of haemagglutination inhibition (HI) titre > 32, and pre- and post-vaccination geometric mean titres (GMT). Immune responses in children receiving chemotherapy were consistently weaker (four-fold rise of 25% to 52%) than in those children who had completed chemotherapy (50% to 86%) and in healthy children (71% to 89%). Concerning adverse effects, 359 paediatric oncology patients received influenza vaccine and the side effects described were mild local reactions and low grade fever. No life-threatening or persistent adverse effects were reported. Paediatric oncology patients receiving chemotherapy are able to generate an immune response to the influenza vaccine, but it remains unclear whether this immune response protects them from influenza infection or its complications. We are awaiting results from well-designed RCTs addressing the clinical benefit of influenza vaccination in these patients.


Pyrimidine salvage pathway in Mycobacterium tuberculosis.
The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, infects one-third of the world population. TB remains the leading cause of mortality due to a single bacterial pathogen. The worldwide increase in incidence of M. tuberculosis has been attributed to the high proliferation rates of multi and extensively drug-resistant strains, and to co-infection with the human immunodeficiency virus. There is thus a continuous requirement for studies on mycobacterial metabolism to identify promising targets for the development of new agents to combat TB. Singular characteristics of this pathogen, such as functional and structural features of enzymes involved in fundamental metabolic pathways, can be evaluated to identify possible targets for drug development. Enzymes involved in the pyrimidine salvage pathway might be attractive targets for rational drug design against TB, since this pathway is vital for all bacterial cells, and is composed of enzymes considerably different from those present in humans. Moreover, the enzymes of the pyrimidine salvage pathway might have an important role in the mycobacterial latent state, since M. tuberculosis has to recycle bases and/or nucleosides to survive in the hostile environment imposed by the host. The present review describes the enzymes of M. tuberculosis pyrimidine salvage pathway as attractive targets for the development of new antimycobacterial agents. Enzyme functional and structural data have been included to provide a broader knowledge on which to base the search for compounds with selective biological activity.


Esophagectomy with Esophagocoloplasty for Malignancies: Indications, Technique (with Video), and Results. Systematic Review of the Literature.
Esophagocoloplasty represents a useful technique to restore the intestinal continuity after esophagogastrectomy. This technique has been used mainly after esophagogastric caustic injuries. The aim of this review is to assess the role of esophagogastrectomy with esophagocoloplasty for esophageal or gastric neoplasms. A systematic literature search was performed using Embase, Medline, Cochrane, and PubMed databases to identify all studies published in the previous 25 years (1991-2016) reporting cases of esophagocoloplasty after esophagogastrectomy for malignancies. The systematic review was conducted according to the PRISMA guidelines. The systematic review of the literature shows a morbidity rate of 57% and a mortality rate of 15% in the 93 reported cases of esophagocoloplasty performed for malignant diseases. However, R0 rate ranged from 76.1 to 85%, and 5-year survival was obtained in 11.9-32.8% of patients in the different series. In highly selected cases of primary or relapsing gastric or esophageal neoplasms, esophagogastrectomy with esophagocoloplasty is a viable and useful option, which may guarantee complete tumor resection and long-term survival.


Occupation: the keystone of a curriculum for a self-defined profession.
The idea of occupation is proposed as the basis for constructing a curricular renaissance for occupational therapy in preparation for a new millennium. Implementing an occupation-centered curriculum could create a more integrated profession in which practice, ideas, scholarship, and education nurture and support one another, increasing the autonomy of both the occupational therapy profession and recipients of its services. A practice-oriented rationale for curricular design includes explication of the idea of occupation, a view of the person as an occupational being embedded in that concept, and the thought process of occupational therapy. Recommendations are provided to create an integrated curriculum that will contribute to future-oriented practice and a self-defined profession.


Autoantibodies against heat shock proteins as biomarkers for the diagnosis and prognosis of cancer.
Thanks to their specificity and stability in the sera, autoantibodies (AAbs) against tumor-associated antigens (TAAs) are very attractive biomarkers for the development of less invasive serological tests for the diagnosis and prognosis of cancer. Heat shock proteins (HSP) belong to TAAs and they are over-expressed in various human cancers. Elevated HSP can stimulate the immune system to produce anti-HSP antibodies. So far, AAbs against HSP have been identified in the circulation of various cancer patients. Here we will review current literature on the use of anti-HSP antibodies for cancer diagnosis and prognosis. The challenges as well as future directions of AAbs identification in oncology are also discussed.


Fabrication of Gelatin-ZnO Nanofibers for Antibacterial Applications.
In this study, GNF@ZnO composites (gelatin nanofibers (GNF) with zinc oxide (ZnO) nanoparticles (NPs)) as a novel antibacterial agent were obtained using a wet chemistry approach. The physicochemical characterization of ZnO nanoparticles (NPs) and GNF@ZnO composites, as well as the evaluation of their antibacterial activity toward Gram-positive (Staphyloccocus aureus and Bacillus pumilus) and Gram-negative (Escherichia coli and Pseudomonas fluorescens) bacteria were performed. ZnO NPs were synthesized using a facile sol-gel approach. Gelatin nanofibers (GNF) were obtained by an electrospinning technique. GNF@ZnO composites were obtained by adding previously produced GNF into a Zn2+ methanol solution during ZnO NPs synthesis. Crystal structure, phase, and elemental compositions, morphology, as well as photoluminescent properties of pristine ZnO NPs, pristine GNF, and GNF@ZnO composites were characterized using powder X-ray diffraction (XRD), FTIR analysis, transmission and scanning electron microscopies (TEM/SEM), and photoluminescence spectroscopy. SEM, EDX, as well as FTIR analyses, confirmed the adsorption of ZnO NPs on the GNF surface. The pristine ZnO NPs were highly crystalline and monodispersed with a size of approximately 7 nm and had a high surface area (83 m2/g). The thickness of the pristine gelatin nanofiber was around 1 µm. The antibacterial properties of GNF@ZnO composites were investigated by a disk diffusion assay on agar plates. Results show that both pristine ZnO NPs and their GNF-based composites have the strongest antibacterial properties against Pseudomonas fluorescence and Staphylococcus aureus, with the zone of inhibition above 10 mm. Right behind them is Escherichia coli with slightly less inhibition of bacterial growth. These properties of GNF@ZnO composites suggest their suitability for a range of antimicrobial uses, such as in the food industry or in biomedical applications.


Keratinocyte-derived cytokine TSLP promotes growth and metastasis of melanoma by regulating the tumor-associated immune microenvironment.
Malignant melanoma is a major public health issue displaying frequent resistance to targeted therapy and immunotherapy. A major challenge lies in better understanding how melanoma cells evade immune elimination and how tumor growth and metastasis is facilitated by the tumor microenvironment. Here, we show that expression of the cytokine thymic stromal lymphopoietin (TSLP) by epidermal keratinocytes is induced by cutaneous melanoma in both mice and humans. Using genetically engineered models of melanoma and tumor cell grafting combined with TSLP-KO or overexpression, we defined a crosstalk between melanoma cells, keratinocytes, and immune cells in establishing a tumor-promoting microenvironment. Keratinocyte-derived TSLP is induced by signals derived from melanoma cells and subsequently acts via immune cells to promote melanoma progression and metastasis. Furthermore, we show that TSLP signals through TSLP receptor-expressing (TSLPR-expressing) DCs to play an unrecognized role in promoting GATA3+ Tregs expressing a gene signature including ST2, CCR8, ICOS, PD-1, CTLA-4, and OX40 and exhibiting a potent suppressive activity on CD8+ T cell proliferation and IFN-γ production. An analogous population of GATA3-expressing Tregs was also identified in human melanoma tumors. Our study provides insights into the role of TSLP in programming a protumoral immune microenvironment in cutaneous melanoma.


Phospholipid Levels at Seroconversion Are Associated With Resolution of Persistent Islet Autoimmunity: The Diabetes Autoimmunity Study in the Young.
Reversion of islet autoimmunity (IA) may point to mechanisms that prevent IA progression. We followed 199 individuals who developed IA during the Diabetes Autoimmunity Study in the Young. Untargeted metabolomics was performed in serum samples following IA. Cox proportional hazards models were used to test whether the metabolites (2,487) predicted IA reversion: two or more consecutive visits negative for all autoantibodies. We conducted a principal components analysis (PCA) of the top metabolites; |hazard ratio (HR) >1.25| and nominal P < 0.01. Phosphatidylcholine (16:0_18:1(9Z)) was the strongest individual metabolite (HR per 1 SD 2.16, false discovery rate (FDR)-adjusted P = 0.0037). Enrichment analysis identified four clusters (FDR P < 0.10) characterized by an overabundance of sphingomyelin (d40:0), phosphatidylcholine (16:0_18:1(9Z)), phosphatidylcholine (30:0), and l-decanoylcarnitine. Overall, 63 metabolites met the criteria for inclusion in the PCA. PC1 (HR 1.4, P < 0.0001), PC2 (HR 0.85, P = 0.0185), and PC4 (HR 1.28, P = 0.0103) were associated with IA reversion. Given the potential influence of diet on the metabolome, we investigated whether nutrients were correlated with PCs. We identified 20 nutrients that were correlated with the PCs (P < 0.05). Total sugar intake was the top nutrient. Overall, we identified an association between phosphatidylcholine, sphingomyelin, and carnitine levels and reversion of IA.


Chronic cold exposure increases liver oxidative capacity in the marsupial Monodelphis domestica.
Marsupials lack brown adipose tissue, and therefore rely exclusively on other tissues for thermogenesis. To determine the magnitude of phenotypic plasticity of the liver in response to changing metabolic demand, gray short-tailed opossums (M. domestica) were exposed to thermoneutral (28 degrees C) or cold (9-12 degrees C) conditions continuously for 6 weeks. Half of each group was also endurance trained with a treadmill program during their respective temperature exposure. Mass specific summit metabolism (VO(2)summit) increased 11% following cold acclimation, though there was no significant main effect by training on VO(2)summit. To estimate the contribution of the liver to whole animal oxidative activity, we determined liver mass, mitochondrial volume density, and total mitochondrial volume. Relative liver mass was 48% greater in cold-acclimated animals, whereas training had no effect on liver mass. The stereological analysis of hepatocyte ultrastructure suggests the percentage of intracellular volumes remained unchanged in response to either aerobic challenge. Thus, following cold-acclimation, there is a 20% increase in the total mitochondrial volume of the liver. This increase could account for nearly half (44%) of the observed increase in whole animal VO(2)summit following cold exposure.


The development of a mouse model to explore resistance and susceptibility to early Ascaris suum infection.
Ascaris suum and Ascaris lumbricoides exhibit an over-dispersed frequency distribution in their host populations in both the adult and larval stages. The impact of host factors on this observed distribution is still poorly understood and difficult to investigate in the natural host populations. The use of a mouse model has been supported by the observations that the larval migratory pattern, in this host, mimics the pattern observed in the pig. We explored the extrinsic factors that might affect the quantitative recovery of larvae during this migration in order to standardize a model system facilitating accurate future assessment of host genetic variation on this phase of the infection. In Exp. 1 larvae accumulated in the livers of both C57BL/6j and BALB/c mice up to and including days 4-5 p.i. and then declined in both strains until day 9. Loss of larvae from the livers corresponded to arrival in the lungs and maximum accumulation on day 7 p.i. but recovery was considerably higher in C57BL/6j mice. It was concluded that day 7 recoveries gave the best indication of relative resistance/susceptibility to this parasite. In Exp. 2 A/J, BALB/c, CBA/Ca, C57BL/6j, C3H/HeN, DBA/2, NIH, SJL, and SWR mice were compared. C57BL/6j mice were identified as the most susceptible strain and CBA/Ca mice as having the most contrasting phenotype, but with a similar kinetic pattern of migration. Finally, in Exp. 3, a strong positive correlation between the size of the inoculum and the mean worm recovery from the lungs was found in CBA/Ca and C57BL/6j mice, but the difference between these strains was highly consistent, 66.6-80%, regardless of the initial dose. These results demonstrate that, using our protocols for infection and recovery, between-experiment variation in A. suum worm burdens is minimal, and that C57BL/6j mice are highly susceptible to infection compared to other strains. The mechanistic basis of this susceptibility in relation to the resistance of other strains is unknown, but the possibilities are reviewed.


Multilayer three-dimensional filter paper constructs for the culture and analysis of aortic valvular interstitial cells.
Culturing aortic valvular interstitial cells in an environment that models the aortic valve is an essential step towards understanding the progression of calcific aortic valve disease. Here the adaption of a three-dimensional (3-D) stacked paper-based culture system is presented for analyzing valve cells in a thick collagen gel matrix. Filter paper layers, modeled after a 96-well plate design, were printed with a wax well-plate template and then seeded with valve cell and collagen mixtures that quickly gelled into 3-D cultures. Stacking these layers permitted extensive customization of culture thickness and cell density profiles to model the full thickness of native valve tissue. Aortic valvular interstitial cells seeded into the paper-based constructs consistently demonstrated high survival up to 14 days of culture with significant increases in cell number through the first 3 days of culture. After 4 days following seeding, valve cells in single layer cultures showed reduced smooth muscle α-actin expression with a stabilized cell density, suggesting a transition from an activated phenotype to a more quiescent state. Valve cells in multilayer cultures demonstrated the ability to migrate from layer to layer and had the highest smooth muscle α-actin expression in areas with predicted low oxygen tensions. These results establish the filter-paper-based method as a viable culture system for analyzing valve cells in an in vitro 3-D model of the aortic valve.


Identification and function of long non-coding RNA.
Long non-coding (lnc) RNAs are defined as non-protein coding RNAs distinct from housekeeping RNAs such as tRNAs, rRNAs, and snRNAs, and independent from small RNAs with specific molecular processing machinery such as micro- or piwi-RNAs. Recent studies of lncRNAs across different species have revealed a diverse population of RNA molecules of differing size and function. RNA sequencing studies suggest transcription throughout the genome, so there is a need to understand how sequence relates to functional and structural relationships amongst RNA molecules. Our synthesis of recent studies suggests that neither size, presence of a poly-A tail, splicing, direction of transcription, nor strand specificity are of importance to lncRNA function. Rather, relative genomic position in relation to a target is fundamentally important. In this review, we describe issues of key importance in functional assessment of lncRNA and how this might apply to lncRNAs important in neurodevelopment.


National variation in pulmonary metastasectomy for colorectal cancer.
Evidence on patterns of use of pulmonary metastasectomy in colorectal cancer patients is limited. This population-based study aims to investigate the use of pulmonary metastasectomy in the colorectal cancer population across the English National Health Service (NHS) and quantify the extent of any variations in practice and outcome. All adults who underwent a major resection for colorectal cancer in an NHS hospital between 2005 and 2013 were identified in the COloRECTal cancer data Repository (CORECT-R). All inpatient episodes corresponding to pulmonary metastasectomy, occurring within 3 years of the initial colorectal resection, were identified. Multi-level logistic regression was used to determine patient and organizational factors associated with the use of pulmonary metastasectomy for colorectal cancer, and Kaplan-Meier and Cox models were used to assess survival following pulmonary metastasectomy. In all, 173 354 individuals had a major colorectal resection over the study period, with 3434 (2.0%) undergoing pulmonary resection within 3 years. The frequency of pulmonary metastasectomy increased from 1.2% of patients undergoing major colorectal resection in 2005 to 2.3% in 2013. Significant variation was observed across hospital providers in the risk-adjusted rates of pulmonary metastasectomy (0.0%-6.8% of patients). Overall 5-year survival following pulmonary resection was 50.8%, with 30-day and 90-day mortality of 0.6% and 1.2% respectively. This study shows significant variation in the rates of pulmonary metastasectomy for colorectal cancer across the English NHS.


Do clinical outcomes suffer during transition to an ultrasound-first paradigm for the evaluation of acute appendicitis in children?
The objective of our study was to examine whether the rate of complicated appendicitis and the hospital length of stay (LOS) increased during the transition to an ultrasound-first paradigm for the imaging evaluation of acute appendicitis. All pediatric patients with surgically proven appendicitis from 2005 to 2011 were identified by searching the hospital billing database for the discharge diagnosis codes for simple appendicitis (ICD-9 [International Classification of Diseases, 9th revision] code 540.9) and complicated appendicitis (ICD-9 codes 540.0 and 540.1). Annual trends of the proportions of these patients who underwent ultrasound and CT were determined and plotted for the study period. Correlation of complicated appendicitis and median hospital LOS with calendar year was assessed using the Spearman (ρ) rank correlation test. RESULTS. Eight hundred four patients met the inclusion criteria. The percentage of patients who underwent CT only showed a moderate downward association with year (ρ = -0.32, p < 0.01), and the percentage of patients who underwent ultrasound first showed a moderate upward trend (ρ = 0.44, p < 0.01). The percentage of patients with ultrasound as the only study performed before appendectomy increased moderately over the 7-year study period (ρ = 0.33, p < 0.01). The percentage of patients with complicated appendicitis and the median hospital LOS did not increase significantly over the study duration (ρ = -0.01, p = 0.74 and ρ = -0.04, p = 0.25, respectively). The transition to an ultrasound-first pathway for the imaging workup of acute appendicitis in children occurred without evidence of a corresponding increase in the proportion of patients with complicated appendicitis or in the median hospital LOS.


Unexpected contribution of cytochrome P450 enzymes CYP11B2 and CYP21, as well as CYP3A4 in xenobiotic androgen elimination - insights from metandienone metabolism.
The metabolism of a variety of anabolic steroids frequently misused for doping purposes has been investigated in the last years. This research mainly focused on main and long-term metabolites suitable for detection, but detailed clearance mechanisms have rarely been elucidated. Recent studies on metandienone focused on the identification of 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one (20βOH-NorMD) as long-term metabolite, however, the metabolic pathway of its generation remained unclear. Metandienone and its Wagner-Meerwein rearrangement product 17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one (NorMD) were hydroxylated by different human cytochrome P450 enzymes (CYPs). Some of their hydroxylation products were chemically synthesized and characterized by mass spectrometry to allow for their trace detection in urine samples. Following oral administration of metandienone or NorMD in one human volunteer each the post administration urines were checked for the presence of those hydroxylated metabolites using GC-MS/MS analysis. The human mitochondrial steroid hydroxylating enzymes CYP11B1 and CYP11B2 were capable to metabolize metandienone leading to the formation of 11β-hydroxymetandienone and 18-hydroxymetandienone. Following Wagner-Meerwein rearrangement, the resulting products could be assigned to 20βOH-NorMD and 11βOH-NorMD. The contribution of CYP11B1 and CYP11B2 in human metabolism of metandienone was confirmed by analysis of post-administration samples of metandienone and NorMD. Combined with the results from a previous study, enzymatic pathways were identified that involve CYP21 and CYP3A4 in the hydroxylation of NorMD, while CYP21, CYP3A4 and CYP11B2 take part in 20βOH-NorMD generation from MD. The current study represents a valuable contribution to the elucidation of clearance mechanisms of anabolic steroids and also indicates that mainly non-liver CYPs seem to be involved in these processes.


Characterization of a hotspot for mimicry: assembly of a butterfly wing transcriptome to genomic sequence at the HmYb/Sb locus.
The mimetic wing patterns of Heliconius butterflies are an excellent example of both adaptive radiation and convergent evolution. Alleles at the HmYb and HmSb loci control the presence/absence of hindwing bar and hindwing margin phenotypes respectively between divergent races of Heliconius melpomene, and also between sister species. Here, we used fine-scale linkage mapping to identify and sequence a BAC tilepath across the HmYb/Sb loci. We also generated transcriptome sequence data for two wing pattern forms of H. melpomene that differed in HmYb/Sb alleles using 454 sequencing technology. Custom scripts were used to process the sequence traces and generate transcriptome assemblies. Genomic sequence for the HmYb/Sb candidate region was annotated both using the MAKER pipeline and manually using transcriptome sequence reads. In total, 28 genes were identified in the HmYb/Sb candidate region, six of which have alternative splice forms. None of these are orthologues of genes previously identified as being expressed in butterfly wing pattern development, implying previously undescribed molecular mechanisms of pattern determination on Heliconius wings. The use of next-generation sequencing has therefore facilitated DNA annotation of a poorly characterized genome, and generated hypotheses regarding the identity of wing pattern at the HmYb/Sb loci.


Comparison of proximal femur and vertebral body strength improvements in the FREEDOM trial using an alternative finite element methodology.
Denosumab reduced the incidence of new fractures in postmenopausal women with osteoporosis by 68% at the spine and 40% at the hip over 36 months compared with placebo in the FREEDOM study. This efficacy was supported by improvements from baseline in vertebral (18.2%) strength in axial compression and femoral (8.6%) strength in sideways fall configuration at 36 months, estimated in Newtons by an established voxel-based finite element (FE) methodology. Since FE analyses rely on the choice of meshes, material properties, and boundary conditions, the aim of this study was to independently confirm and compare the effects of denosumab on vertebral and femoral strength during the FREEDOM trial using an alternative smooth FE methodology. Unlike the previous FE study, effects on femoral strength in physiological stance configuration were also examined. QCT data for the proximal femur and two lumbar vertebrae were analyzed by smooth FE methodology at baseline, 12, 24, and 36 months for 51 treated (denosumab) and 47 control (placebo) subjects. QCT images were segmented and converted into smooth FE models to compute bone strength. L1 and L2 vertebral bodies were virtually loaded in axial compression and the proximal femora in both fall and stance configurations. Denosumab increased vertebral body strength by 10.8%, 14.0%, and 17.4% from baseline at 12, 24, and 36 months, respectively (p<0.0001). Denosumab also increased femoral strength in the fall configuration by 4.3%, 5.1%, and 7.2% from baseline at 12, 24, and 36 months, respectively (p<0.0001). Similar improvements were observed in the stance configuration with increases of 4.2%, 5.2%, and 5.2% from baseline (p≤0.0007). Differences between the increasing strengths with denosumab and the decreasing strengths with placebo were significant starting at 12 months (vertebral and femoral fall) or 24 months (femoral stance). Using an alternative smooth FE methodology, we confirmed the significant improvements in vertebral body and proximal femur strength previously observed with denosumab. Estimated increases in strength with denosumab and decreases with placebo were highly consistent between both FE techniques.


Fully Implanted Brain-Computer Interface in a Locked-In Patient with ALS.
Options for people with severe paralysis who have lost the ability to communicate orally are limited. We describe a method for communication in a patient with late-stage amyotrophic lateral sclerosis (ALS), involving a fully implanted brain-computer interface that consists of subdural electrodes placed over the motor cortex and a transmitter placed subcutaneously in the left side of the thorax. By attempting to move the hand on the side opposite the implanted electrodes, the patient accurately and independently controlled a computer typing program 28 weeks after electrode placement, at the equivalent of two letters per minute. The brain-computer interface offered autonomous communication that supplemented and at times supplanted the patient's eye-tracking device. (Funded by the Government of the Netherlands and the European Union; ClinicalTrials.gov number, NCT02224469 .).


Unexpected rates of chromosomal instabilities and alterations of hormone levels in Namibian uranium miners.
A common problem in determining the health consequences of radiation exposure is factoring out other carcinogenic influences. The conditions in Namibia provide a test case for distinguishing the effects of long-term low-dose exposure to uranium from the other environmental factors because of good air quality and the lack of other industries with negative health effects. Present records indicate a much higher prevalence of cancer among male workers in the open-pit uranium mine in Namibia compared with the general population. The objective of the present study was to determine whether long-term exposure to low doses of uranium increases the risk of a biological radiation damage which would lead to malignant diseases and to derive a dose-response model for these miners. To investigate this risk, we measured uranium excretion in urine, neutrophil counts and the serum level of FSH, LH and testosterone and analyzed chromosome aberrations in whole blood cells using fluorescence in situ hybridization. A representative cohort of 75 non-smoking, HIV-negative miners was compared to a control group of 31 individuals with no occupational history in mining. A sixfold increase in uranium excretion among the miners compared to the controls was recorded (P < 0.001). Furthermore, we determined a significant reduction in testosterone levels (P < 0.008) and neutrophil count (P < 0.004) in miners compared to the unexposed controls. A threefold increase in chromosome aberrations in the miners compared to the nonexposed controls was recorded (P < 0.0001). Most remarkably, cells with multiple aberrations such as "rogue" cells were observed for the first time in miners; these cells had previously been found only after short-term high-dose radiation exposure, e.g. from the Hiroshima atomic bomb or the Chernobyl accident. We conclude that the miners exposed to uranium are at an increased risk to acquire various degrees of genetic damage, and that the damage may be associated with an increased risk for malignant transformation. As expected, the chronic radiation injury of the hematopoietic system resulted in low neutrophil counts. Also, low hormone levels probably reflect damage to the gonadal endocrine system.


Organelle-specific control of intracellular transport: distinctly targeted isoforms of the regulator Klar.
Microtubule-based transport in cells is powered by a small set of distinct motors, yet timing and destination of transport can be controlled in a cargo-specific manner. The mechanistic basis for this specificity is not understood. To address this question, we analyzed the Drosophila Klarsicht (Klar) protein that regulates distinct microtubule-based transport processes. We find that localization of Klar to its cargoes is crucial for Klar function. Using mutations, we identify functionally important regions of Klar that confer distinct cargo specificity. In ovaries, Klar is present on the nuclear envelope, a localization that requires the C-terminal KASH domain. In early embryos, Klar is attached to lipid droplets, a localization mediated by a novel C-terminal domain encoded by an alternatively spliced exon. In cultured cells, these two domains are sufficient for targeting to the correct intracellular location. Our analysis disentangles Klar's modular organization: we propose that a core region integral to motor regulation is attached to variable domains so that the cell can target regulators with overlapping, yet distinct functions to specific cargoes. Such isoform variation may be a general strategy for adapting a common regulatory mechanism to specifically control motion and positioning of multiple organelles.


Biliverdin Reductase-A Deficiency Brighten and Sensitize Biliverdin-binding Chromoproteins.
Tissue absorbance, light scattering, and autofluorescence are significantly lower in the near-infrared (NIR) range than in the visible range. Because of these advantages, NIR fluorescent proteins (FPs) are in high demand for in vivo imaging. Nevertheless, application of NIR FPs such as iRFP is still limited due to their dimness in mammalian cells. In contrast to GFP and its variants, iRFP requires biliverdin (BV) as a chromophore. The dimness of iRFP is at least partly due to rapid reduction of BV by biliverdin reductase-A (BLVRA). Here, we established biliverdin reductase-a knockout (Blvra-/-) mice to increase the intracellular BV concentration and, thereby, to enhance iRFP fluorescence intensity. As anticipated, iRFP fluorescence intensity was significantly increased in all examined tissues of Blvra-/- mice. Similarly, the genetically encoded calcium indicator NIR-GECO1, which is engineered based on another NIR FP, mIFP, exhibited a marked increase in fluorescence intensity in mouse embryonic fibroblasts derived from Blvra-/- mice. We expanded this approach to an NIR light-sensing optogenetic tool, the BphP1-PpsR2 system, which also requires BV as a chromophore. Again, deletion of the Blvra gene markedly enhanced the light response in HeLa cells. These results indicate that the Blvra-/- mouse is a versatile tool for the in vivo application of NIR FPs and NIR light-sensing optogenetic tools.Key words: in vivo imaging, near-infrared fluorescent protein, biliverdin, biliverdin reductase, optogenetic tool.


Feeding preterm infants during red blood cell transfusion is associated with a decline in postprandial mesenteric oxygenation.
To evaluate the mesenteric tissue oxygenation response in preterm infants fed and not fed during red blood cell (RBC) transfusions. Prospective, observational comparison of mesenteric oxygenation using near-infrared spectroscopy in preterm infants (<33 weeks' at birth) who were fed or not fed during RBC transfusion. Tissue oxygenation means were examined up to 48 hours after each transfusion event. Mean mesenteric regional oxygen saturation (rSO2) slopes during RBC transfusion of fed (n = 9) vs not fed (n = 8) infants ranged from -0.23 to +0.23 (mean 0.04) with no differences between groups (P = .480). However, following transfusions, postprandial mesenteric oxygenation means significantly declined in infants fed during transfusion compared with infants not fed during transfusion (P < .001). Infants fed during RBC transfusion had a mean 2.16 point decrease in rSO2 mesenteric oxygenation with each sequential feeding post-transfusion, whereas infants not fed during RBC transfusion increased their rSO2 postprandial mesenteric oxygenation by a mean of 2.09 points. Mesenteric tissue oxygenation during RBC transfusion is not influenced by feeding status. However, infants fed during RBC transfusion had, for the next 15 hours, decreasing postprandial mesenteric tissue oxygenation patterns compared with infants not fed during RBC transfusion. Feeding during RBC transfusions may increase the risk for mesenteric ischemia and the development of transfusion-related necrotizing enterocolitis in preterm infants.


A simple perturbative analysis for fast design of an electrically pumped micro-disk laser.
A perturbative analysis is proposed to estimate optical losses for electrically pumped micro-disk lasers. The optical field interaction with the electrical contacts and the optimization of their implementation is investigated. Our model shows a good agreement with 3D Finite Difference Time Domain (FDTD) computation and can be used for designing contacts for thin micro-disks, with a considerably reduced calculation time. We also demonstrate that losses induced by the contacts can be exploited to select the optical mode of a micro-laser.


A study protocol for the N-ICE trial: A randomised double-blind placebo-controlled study of the safety and efficacy of N-acetyl-cysteine (NAC) as a pharmacotherapy for methamphetamine ("ice") dependence.
There are currently no approved pharmacotherapies for managing methamphetamine dependence. N-acetylcysteine (NAC) has been found to reduce the craving for methamphetamine and other drugs, but its effect on methamphetamine use and other clinically related endpoints are uncertain. The N-ICE trial is evaluating the safety and efficacy of NAC as a take-home pharmacotherapy for methamphetamine dependence. This is a two-arm parallel double-blind placebo-controlled three-site randomised trial (ratio 1:1) using permuted block randomisation, with variable block sizes. It is stratified by site, sex and whether the methamphetamine is injected or not. Participants (N = 180; 60 per site) need to be dependent on methamphetamine, interested in reducing their methamphetamine use and not currently receiving treatment for substance use disorders. The trial is being conducted in outpatient settings in Melbourne, Geelong and Wollongong, Australia. Participants will receive either 2400 mg oral NAC or a matched placebo, delivered as a take-home medication for 12 weeks. Two 600 mg capsules are self-administered in the morning and two more in the evening. Adherence is being monitored using eCAP™ medication bottle lids, which record the date and time of each occasion the bottle is opened. The primary outcome is methamphetamine use during the 12-week trial medication period, measured as (a) days of use, assessed using the timeline followback, and (b) methamphetamine-positive saliva tests, taken weekly. Secondary measures include weekly assessment of methamphetamine craving, severity of methamphetamine dependence, methamphetamine withdrawal symptoms and psychiatric symptoms (depression, suicidality, psychotic symptoms and hostility). Adverse events are monitored at each weekly assessment. Tolerability is assessed using the Treatment Satisfaction Questionnaire for Medication. The N-ICE trial is the first clinical trial to assess whether NAC can reduce methamphetamine use. This trial will improve our understanding of the potential utility of NAC in managing methamphetamine dependence and clinically related outcomes. If found to be effective, take-home NAC could be a potentially scalable and affordable pharmacotherapy option for treating methamphetamine dependence. Australian and New Zealand Clinical Trials Registry, ACTRN12618000366257 . Registered on 29 May 2018.


Etiologies of NICU deaths.
Infant mortality is an indicator of overall societal health, and a significant proportion of infant deaths occur in NICUs. The objectives were to identify causes of death and to define potentially preventable factors associated with death as areas for quality improvement efforts in the NICU. In a prospectively defined study, the principal investigator in 46 level III NICUs agreed to review health care records of infants who died. For each infant, the principal investigator reviewed the medical record to identify the primary cause of death and to look for preventable factors associated with the infant's death. Infants born at ≥22 weeks estimated gestational age who were born alive were included. Stillborn infants were excluded. Data were collected on 641 infants who died. At lower gestational ages, mortality was most commonly due to extreme prematurity and the complications of premature birth (respiratory distress progressing to respiratory failure, intraventricular hemorrhage, necrotizing enterocolitis, and sepsis). With increasing gestational age, the etiology of mortality shifted to hypoxic-ischemic encephalopathy and genetic or structural anomalies. Reviewers of clinical care identified 197 (31%) infants with potentially modifiable factors that may have contributed to their deaths. The factors associated with death in infants admitted for intensive care are multifactorial and diverse, and they change with gestational age. In 31% of the deaths, potentially modifiable factors were identified, and these factors suggest important targets for reducing infant mortality.


Electrochemical performance of hierarchical porous carbon materials obtained from the infiltration of lignin into zeolite templates.
Hierarchical porous carbon materials prepared by the direct carbonization of lignin/zeolite mixtures and the subsequent basic etching of the inorganic template have been electrochemically characterized in acidic media. These lignin-based templated carbons have interesting surface chemistry features, such as a variety of surface oxygen groups and also pyridone and pyridinic groups, which results in a high capacitance enhancement compared to petroleum-pitch-based carbons obtained by the same procedure. Furthermore, they are easily electro-oxidized in a sulfuric acid electrolyte under positive polarization to produce a large amount of surface oxygen groups that boosts the pseudocapacitance. The lignin-based templated carbons showed a specific capacitance as high as 250 F g(-1) at 50 mA g(-1) , with a capacitance retention of 50 % and volumetric capacitance of 75 F cm(-3) at current densities higher than 20 A g(-1) thanks to their suitable porous texture. These results indicate the potential use of inexpensive biomass byproducts, such as lignin, as carbon precursors in the production of hierarchical carbon materials for electrodes in electrochemical capacitors.


Prevalence, segregation, and phenotype of the mitochondrial DNA 3243A>G mutation in children.
We studied the prevalence, segregation, and phenotype of the mitochondrial DNA 3243A>G mutation in children in a defined population in Northern Ostrobothnia, Finland. Children with diagnoses commonly associated with mitochondrial diseases were ascertained. Blood DNA from 522 selected children was analyzed for 3243A>G. Children with the mutation were clinically examined. Information on health history before the age of 18 years was collected from previously identified adult patients with 3243A>G. Mutation segregation analysis in buccal epithelial cells was performed in mothers with 3243A>G and their children whose samples were analyzed anonymously. Eighteen children were found to harbor 3243A>G in a population of 97,609. A minimum estimate for the prevalence of 3243A>G was 18.4 in 100,000 (95% confidence interval, 10.9-29.1/100,000). Information on health in childhood was obtained from 37 adult patients with 3243A>G. The first clinical manifestations appearing in childhood were sensorineural hearing impairment, short stature or delayed maturation, migraine, learning difficulties, and exercise intolerance. Mutation analysis from 13 mothers with 3243A>G and their 41 children gave a segregation rate of 0.80. The mothers with heteroplasmy greater than 50% tended to have offspring with lower or equal heteroplasmy, whereas the opposite was true for mothers with heteroplasmy less than or equal to 50% (p = 0.0016). The prevalence of 3243A>G is relatively high in the pediatric population, but the morbidity in children is relatively low. The random genetic drift model may be inappropriate for the transmission of the 3243A>G mutation.


Ultrafast quantitative MS-based method for ceritinib analysis in human plasma samples from clinical trial.
An ultrafast, sensitive, selective and robust LDTD-APCI-MS/MS method was developed for the quantification of ceritinib in human plasma. Samples were protein precipitated using acetonitrile containing [(13)C6]-ceritinib as internal standard. The assay was validated over a concentration range from 5.00 to 1000 ng/ml. Intra- and inter-day precision and accuracy met acceptance from EMA and US FDA guidelines. The normalized recovery was 69%, whereas no carryover and matrix effects were observed. The method was applied to clinical samples and resultant data were consistent with the LC-ESI-MS/MS reference method. The new assay is suitable for ceritinib quantification in clinical trials, whereas the analysis time is significantly reduced to 10 s.


Placing ocular mutants into a functional context: a chronobiological approach.
The behavior of mammals is characterized by a 24-h cycle of rest and activity which is a fundamental adaption to the solar cycle of light and darkness. The pacemaker of this circadian clock is localized in the ventral part of the hypothalamus, the so-called suprachiasmatic nuclei (SCN), and is entrained by light signals mediated by the eye. The eye is directly connected via the retinohypothalamic tract (RHT) to the SCN. Light that reaches the retina elicits glutamate release at the synaptic terminals of the RHT and influences the neurons in the SCN in a manner that alters the behavioral state of the animal. A light pulse that reaches the retina at the beginning of the night elicits a delay of the clock phase, whereas a light pulse that reaches the retina at the end of the dark period leads to an advance of the clock phase. This advance or delay can be quantified by measuring the change in onset of wheel-running activity. Such measurements have, and continue to provide, a remarkably powerful assay of how light is detected and transduced to regulate circadian rhythms. The methods used for such measurements in mice are described in the following article.


Mapping allergenic pollen vegetation in UK to study environmental exposure and human health.
Allergenic pollen is produced by the flowers of a number of trees, grasses and weeds found throughout the UK. Exposure to such pollen grains can exacerbate pollen-related asthma and allergenic conditions such as allergic rhinitis (hay fever). Maps showing the location of these allergenic taxa have many applications: they can be used to provide advice on risk assessments; combined with health data to inform research on health impacts such as respiratory hospital admissions; combined with weather data to improve pollen forecasting systems; or as inputs to pollen emission models. In this study we present 1km resolution maps of 12 taxa of trees, grass and weeds found in the UK. We have selected the main species recorded by the UK pollen network. The taxa mapped in this study were: Alnus (alder), Fraxinus (ash), Betula (birch), Corylus (hazel), Quercus (oak), Pinus (pine) and Salix (willow), Poaceae (grass), Artemisia (mugwort), Plantago (plantain), Rumex (dock, sorrels) and Urtica (nettle). We also focus on one high population centre and present maps showing local level detail around the city of London. Our results show the different geographical distributions of the 12 taxa of trees, weeds and grass, which can be used to study plants in the UK associated with allergy and allergic asthma. These maps have been produced in order to study environmental exposure and human health, although there are many possible applications. This novel method not only provides maps of many different plant types, but also at high resolution across regions of the UK, and we uniquely present 12 key plant taxa using a consistent methodology. To consider the impact on human health due to exposure of the pollen grains, it is important to consider the timing of pollen release, and its dispersal, as well as the effect on air quality, which is also discussed here.


Fasting-induced FGF21 signaling activates hepatic autophagy and lipid degradation via JMJD3 histone demethylase.
Autophagy is essential for cellular survival and energy homeostasis under nutrient deprivation. Despite the emerging importance of nuclear events in autophagy regulation, epigenetic control of autophagy gene transcription remains unclear. Here, we report fasting-induced Fibroblast Growth Factor-21 (FGF21) signaling activates hepatic autophagy and lipid degradation via Jumonji-D3 (JMJD3/KDM6B) histone demethylase. Upon FGF21 signaling, JMJD3 epigenetically upregulates global autophagy-network genes, including Tfeb, Atg7, Atgl, and Fgf21, through demethylation of histone H3K27-me3, resulting in autophagy-mediated lipid degradation. Mechanistically, phosphorylation of JMJD3 at Thr-1044 by FGF21 signal-activated PKA increases its nuclear localization and interaction with the nuclear receptor PPARα to transcriptionally activate autophagy. Administration of FGF21 in obese mice improves defective autophagy and hepatosteatosis in a JMJD3-dependent manner. Remarkably, in non-alcoholic fatty liver disease patients, hepatic expression of JMJD3, ATG7, LC3, and ULK1 is substantially decreased. These findings demonstrate that FGF21-JMJD3 signaling epigenetically links nutrient deprivation with hepatic autophagy and lipid degradation in mammals.


The Health Extension Program and Its Association with Change in Utilization of Selected Maternal Health Services in Tigray Region, Ethiopia: A Segmented Linear Regression Analysis.
In 2003, the Ethiopian Ministry of Health established the Health Extension Program (HEP), with the goal of improving access to health care and health promotion activities in rural areas of the country. This paper aims to assess the association of the HEP with improved utilization of maternal health services in Northern Ethiopia using institution-based retrospective data. Average quarterly total attendances for antenatal care (ANC), delivery care (DC) and post-natal care (PNC) at health posts and health care centres were studied from 2002 to 2012. Regression analysis was applied to two models to assess whether trends were statistically significant. One model was used to estimate the level and trend changes associated with the immediate period of intervention, while changes related to the post-intervention period were estimated by the other. The total number of consultations for ANC, DC and PNC increased constantly, particularly after the late-intervention period. Increases were higher for ANC and PNC at health post level and for DC at health centres. A positive statistically significant upward trend was found for DC and PNC in all facilities (p<0.01). The positive trend was also present in ANC at health centres (p = 0.04), but not at health posts. Our findings revealed an increase in the use of antenatal, delivery and post-natal care after the introduction of the HEP. We are aware that other factors, that we could not control for, might be explaining that increase. The figures for DC and PNC are however low and more needs to be done in order to increase the access to the health care system as well as the demand for these services by the population. Strengthening of the health information system in the region needs also to be prioritized.