Imipramine exploits histone deacetylase 11 to increase the IL-12/IL-10 ratio in macrophages infected with antimony-resistant Leishmania donovani and clears organ parasites in experimental infection.
The efflux of antimony through multidrug resistance protein (MDR)-1 is the key factor in the failure of metalloid treatment in kala-azar patients infected with antimony-resistant Leishmania donovani (Sb(R)LD). Previously we showed that MDR-1 upregulation in Sb(R)LD infection is IL-10-dependent. Imipramine, a drug in use for the treatment of depression and nocturnal enuresis in children, inhibits IL-10 production from Sb(R)LD-infected macrophages (Sb(R)LD-Mϕs) and favors accumulation of surrogates of antimonials. It inhibits IL-10-driven nuclear translocation of c-Fos/c-Jun, critical for enhanced MDR-1 expression. The drug upregulates histone deacetylase 11, which inhibits acetylation of IL-10 promoter, leading to a decrease in IL-10 production from Sb(R)LD-Mϕs. It abrogates Sb(R)LD-mediated p50/c-Rel binding to IL-10 promoter and preferentially recruits p65/RelB to IL-12 p35 and p40 promoters, causing a decrease in IL-10 and overproduction of IL-12 in Sb(R)LD-Mϕs. Histone deacetylase 11 per se does not influence IL-12 promoter activity. Instead, a imipramine-mediated decreased IL-10 level allows optimal IL-12 production in Sb(R)LD-Mϕs. Furthermore, exogenous rIL-12 inhibits intracellular Sb(R)LD replication, which can be mimicked by the presence of Ab to IL-10. This observation indicated that reciprocity exists between IL-10 and IL-12 and that imipramine tips the balance toward an increased IL-12/IL-10 ratio in Sb(R)LD-Mϕs. Oral treatment of infected BALB/c mice with imipramine in combination with sodium stibogluconate cleared organ Sb(R)LD parasites and caused an expansion of the antileishmanial T cell repertoire where sodium stibogluconate alone had no effect. Our study deciphers a detailed molecular mechanism of imipramine-mediated regulation of IL-10/IL-12 reciprocity and its impact on Sb(R)LD clearance from infected hosts.


American Joint Committee on Cancer acceptance criteria for inclusion of risk models for individualized prognosis in the practice of precision medicine.
The American Joint Committee on Cancer (AJCC) has increasingly recognized the need for more personalized probabilistic predictions than those delivered by ordinal staging systems, particularly through the use of accurate risk models or calculators. However, judging the quality and acceptability of a risk model is complex. The AJCC Precision Medicine Core conducted a 2-day meeting to discuss characteristics necessary for a quality risk model in cancer patients. More specifically, the committee established inclusion and exclusion criteria necessary for a risk model to potentially be endorsed by the AJCC. This committee reviewed and discussed relevant literature before creating a checklist unique to this need of AJCC risk model endorsement. The committee identified 13 inclusion and 3 exclusion criteria for AJCC risk model endorsement in cancer. The emphasis centered on performance metrics, implementation clarity, and clinical relevance. The facilitation of personalized probabilistic predictions for cancer patients holds tremendous promise, and these criteria will hopefully greatly accelerate this process. Moreover, these criteria might be useful for a general audience when trying to judge the potential applicability of a published risk model in any clinical domain. CA Cancer J Clin 2016;66:370-374. © 2016 American Cancer Society.


A Paal-Knorr agent for chemoproteomic profiling of targets of isoketals in cells.
Natural systems produce various γ-dicarbonyl-bearing compounds that can covalently modify lysine in protein targets via the classic Paal-Knorr reaction. Among them is a unique class of lipid-derived electrophiles - isoketals that exhibit high chemical reactivity and critical biological functions. However, their target selectivity and profiles in complex proteomes remain unknown. Here we report a Paal-Knorr agent, 4-oxonon-8-ynal (herein termed ONAyne), for surveying the reactivity and selectivity of the γ-dicarbonyl warhead in biological systems. Using an unbiased open-search strategy, we demonstrated the lysine specificity of ONAyne on a proteome-wide scale and characterized six probe-derived modifications, including the initial pyrrole adduct and its oxidative products (i.e., lactam and hydroxylactam adducts), an enlactam adduct from dehydration of hydroxylactam, and two chemotypes formed in the presence of endogenous formaldehyde (i.e., fulvene and aldehyde adducts). Furthermore, combined with quantitative chemoproteomics in a competitive format, ONAyne permitted global, in situ, and site-specific profiling of targeted lysine residues of two specific isomers of isoketals, levuglandin (LG) D2 and E2. The functional analyses reveal that LG-derived adduction drives inhibition of malate dehydrogenase MDH2 and exhibits a crosstalk with two epigenetic marks on histone H2B in macrophages. Our approach should be broadly useful for target profiling of bioactive γ-dicarbonyls in diverse biological contexts.


Physiological aspects of surfboard riding performance.
Surfboard riding (surfing) has experienced a 'boom' in participants and media attention over the last decade at both the recreational and the competitive level. However, despite its increasing global audience, little is known about physiological and other factors related to surfing performance. Time-motion analyses have demonstrated that surfing is an intermittent sport, with arm paddling and remaining stationary representing approximately 50% and 40% of the total time, respectively. Wave riding only accounts for 4-5% of the total time when surfing. It has been suggested that these percentages are influenced mainly by environmental factors. Competitive surfers display specific size attributes. Particularly, a mesomorphic somatotype and lower height and body mass compared with other matched-level aquatic athletes. Data available suggest that surfers possess a high level of aerobic fitness. Upper-body ergometry reveals that peak oxygen uptake (VO2peak) values obtained in surfers are consistently higher than values reported for untrained subjects and comparable with those reported for other upper-body endurance-based athletes. Heart rate (HR) measurements during surfing practice have shown an average intensity between 75% and 85% of the mean HR values measured during a laboratory incremental arm paddling VO2peak test. Moreover, HR values, together with time-motion analysis, suggest that bouts of high-intensity exercise demanding both aerobic and anaerobic metabolism are intercalated with periods of moderate- and low-intensity activity soliciting aerobic metabolism. Minor injuries such as lacerations are the most common injuries in surfing. Overuse injuries in the shoulder, lower back and neck area are becoming more common and have been suggested to be associated with the repetitive arm stroke action during board paddling. Further research is needed in all areas of surfing performance in order to gain an understanding of the sport and eventually to bring surfing to the next level of performance.


μRALP and Beyond: Micro-Technologies and Systems for Robot-Assisted Endoscopic Laser Microsurgery.
Laser microsurgery is the current gold standard surgical technique for the treatment of selected diseases in delicate organs such as the larynx. However, the operations require large surgical expertise and dexterity, and face significant limitations imposed by available technology, such as the requirement for direct line of sight to the surgical field, restricted access, and direct manual control of the surgical instruments. To change this status quo, the European project μRALP pioneered research towards a complete redesign of current laser microsurgery systems, focusing on the development of robotic micro-technologies to enable endoscopic operations. This has fostered awareness and interest in this field, which presents a unique set of needs, requirements and constraints, leading to research and technological developments beyond μRALP and its research consortium. This paper reviews the achievements and key contributions of such research, providing an overview of the current state of the art in robot-assisted endoscopic laser microsurgery. The primary target application considered is phonomicrosurgery, which is a representative use case involving highly challenging microsurgical techniques for the treatment of glottic diseases. The paper starts by presenting the motivations and rationale for endoscopic laser microsurgery, which leads to the introduction of robotics as an enabling technology for improved surgical field accessibility, visualization and management. Then, research goals, achievements, and current state of different technologies that can build-up to an effective robotic system for endoscopic laser microsurgery are presented. This includes research in micro-robotic laser steering, flexible robotic endoscopes, augmented imaging, assistive surgeon-robot interfaces, and cognitive surgical systems. Innovations in each of these areas are shown to provide sizable progress towards more precise, safer and higher quality endoscopic laser microsurgeries. Yet, major impact is really expected from the full integration of such individual contributions into a complete clinical surgical robotic system, as illustrated in the end of this paper with a description of preliminary cadaver trials conducted with the integrated μRALP system. Overall, the contribution of this paper lays in outlining the current state of the art and open challenges in the area of robot-assisted endoscopic laser microsurgery, which has important clinical applications even beyond laryngology.


Beta-arrestin binding to the beta2-adrenergic receptor requires both receptor phosphorylation and receptor activation.
Homologous desensitization of beta2-adrenergic receptors has been shown to be mediated by phosphorylation of the agonist-stimulated receptor by G-protein-coupled receptor kinase 2 (GRK2) followed by binding of beta-arrestins to the phosphorylated receptor. Binding of beta-arrestin to the receptor is a prerequisite for subsequent receptor desensitization, internalization via clathrin-coated pits, and the initiation of alternative signaling pathways. In this study we have investigated the interactions between receptors and beta-arrestin2 in living cells using fluorescence resonance energy transfer. We show that (a) the initial kinetics of beta-arrestin2 binding to the receptor is limited by the kinetics of GRK2-mediated receptor phosphorylation; (b) repeated stimulation leads to the accumulation of GRK2-phosphorylated receptor, which can bind beta-arrestin2 very rapidly; and (c) the interaction of beta-arrestin2 with the receptor depends on the activation of the receptor by agonist because agonist withdrawal leads to swift dissociation of the receptor-beta-arrestin2 complex. This fast agonist-controlled association and dissociation of beta-arrestins from prephosphorylated receptors should permit rapid control of receptor sensitivity in repeatedly stimulated cells such as neurons.


Fluctuation domains in adaptive evolution.
We derive an expression for the variation between parallel trajectories in phenotypic evolution, extending the well known result that predicts the mean evolutionary path in adaptive dynamics or quantitative genetics. We show how this expression gives rise to the notion of fluctuation domains-parts of the fitness landscape where the rate of evolution is very predictable (due to fluctuation dissipation) and parts where it is highly variable (due to fluctuation enhancement). These fluctuation domains are determined by the curvature of the fitness landscape. Regions of the fitness landscape with positive curvature, such as adaptive valleys or branching points, experience enhancement. Regions with negative curvature, such as adaptive peaks, experience dissipation. We explore these dynamics in the ecological scenarios of implicit and explicit competition for a limiting resource.


Comparative cost-effectiveness analysis of voriconazole and fluconazole for prevention of invasive fungal infection in patients receiving allogeneic hematopoietic cell transplants.
The cost-effectiveness of voriconazole versus fluconazole prophylaxis against fungal infections in hematopoietic cell transplant (HCT) recipients is investigated. A decision-analytic model was developed to estimate the drug costs associated with planned or supplemental prophylaxis and empirical therapy and the costs of treating suspected or documented invasive fungal infections (IFIs) in HCT recipients. Published clinical trial data on 599 patients who received 100-180 days of prophylactic therapy with voriconazole or fluconazole were used to model specified IFI-prevention and mortality outcomes; 6-month, 12-month, and lifetime incremental cost-effectiveness ratios (ICERs) were estimated, with a bootstrap analysis performed to reffect the uncertainty of the clinical trial data. Estimated mean total prophylaxis and IFI-related costs associated with voriconazole versus fluconazole prophylaxis over 12 months were higher in the entire study population and among patients receiving HCT for diagnoses other than acute myeloid leukemia (AML) but were not significantly different for patients with AML. The cost per IFI avoided ($66,919) and the cost per life-year gained ($5,453) were lower among patients with AML who received voriconazole relative to the full study population. ICERs were more favorable for voriconazole over a 6-month time frame and when modeling was conducted using generic price data. Assuming a threshold value of $50,000 for one year of life gained, the calculated probability of voriconazole being cost-effective was 33% for the full study population and 85% for the AML subgroup. The decision model indicated that voriconazole prophylaxis was cost-effective for patients undergoing allogeneic HCT for AML.


From maid to mother: transforming facilities, staff training, and caregiver dignity in an institutional facility for young children in Nepal.
This article provides a case study of a project to improve the health, safety, and development of children birth to 6 years old in a large orphanage in Nepal. Two interventions were conducted: improvement of physical infrastructure and training, mentoring, and support for caregiving staff. As a result of these interventions, positive outcomes in terms of children's health and development have been observed, including reduction of communicable diseases and increased social interactions with caregivers. As part of the new training initiative, the caregivers began to meet regularly to share their ideas and experiences, and came to realize their vital role in the holistic development of the children in their care. One important change was a greater sense of dignity for the caregivers. The caregivers were formerly called Maids (Aaya), but asked to be called Mothers (Aama). The project also faced challenges, including communication barriers related to organizational structure.


Function of the MYND Domain and C-Terminal Region in Regulating the Subcellular Localization and Catalytic Activity of the SMYD Family Lysine Methyltransferase Set5.
SMYD lysine methyltransferases target histones and nonhistone proteins for methylation and are critical regulators of muscle development and implicated in neoplastic transformation. They are characterized by a split catalytic SET domain and an intervening MYND zinc finger domain, as well as an extended C-terminal domain. Saccharomyces cerevisiae contains two SMYD proteins, Set5 and Set6, which share structural elements with the mammalian SMYD enzymes. Set5 is a histone H4 lysine 5, 8, and 12 methyltransferase, implicated in the regulation of stress responses and genome stability. While the SMYD proteins have diverse roles in cells, there are many gaps in our understanding of how these enzymes are regulated. Here, we performed mutational analysis of Set5, combined with phosphoproteomics, to identify regulatory mechanisms for its enzymatic activity and subcellular localization. Our results indicate that the MYND domain promotes Set5 chromatin association in cells and is required for its role in repressing subtelomeric genes. Phosphoproteomics revealed extensive phosphorylation of Set5, and phosphomimetic mutations enhance Set5 catalytic activity but diminish its ability to interact with chromatin in cells. These studies uncover multiple regions within Set5 that regulate its localization and activity and highlight potential avenues for understanding mechanisms controlling the diverse roles of SMYD enzymes.


Protein structure determination by electron diffraction using a single three-dimensional nanocrystal.
Three-dimensional nanometre-sized crystals of macromolecules currently resist structure elucidation by single-crystal X-ray crystallography. Here, a single nanocrystal with a diffracting volume of only 0.14 µm3, i.e. no more than 6 × 105 unit cells, provided sufficient information to determine the structure of a rare dimeric polymorph of hen egg-white lysozyme by electron crystallography. This is at least an order of magnitude smaller than was previously possible. The molecular-replacement solution, based on a monomeric polyalanine model, provided sufficient phasing power to show side-chain density, and automated model building was used to reconstruct the side chains. Diffraction data were acquired using the rotation method with parallel beam diffraction on a Titan Krios transmission electron microscope equipped with a novel in-house-designed 1024 × 1024 pixel Timepix hybrid pixel detector for low-dose diffraction data collection. Favourable detector characteristics include the ability to accurately discriminate single high-energy electrons from X-rays and count them, fast readout to finely sample reciprocal space and a high dynamic range. This work, together with other recent milestones, suggests that electron crystallography can provide an attractive alternative in determining biological structures.


Effects of hypercalcemia on serum concentrations of magnesium, potassium, and phosphate and urinary excretion of electrolytes in horses.
To determine effects of experimentally induced hypercalcemia on serum concentrations and urinary excretion of electrolytes, especially ionized magnesium (iMg), in healthy horses. 21 clinically normal mares. Horses were assigned to 5 experimental protocols (1, hypercalcemia induced with calcium gluconate; 2, hypercalcemia induced with calcium chloride; 3, infusion with dextrose solution; 4, infusion with sodium gluconate; and 5, infusion with saline [0.9% NaCl] solution). Hypercalcemia was induced for 2 hours. Dextrose, sodium gluconate, and saline solution were infused for 2 hours. Blood samples were collected to measure serum concentrations of electrolytes, creatinine, parathyroid hormone, and insulin. Urine samples were collected to determine the fractional excretion of ionized calcium (iCa), iMg, sodium, phosphate, potassium, and chloride. Hypercalcemia induced by administration of calcium gluconate or calcium chloride decreased serum iMg, potassium, and parathyroid hormone concentrations; increased phosphate concentration; and had no effect on sodium, chloride, and insulin concentrations. Hypercalcemia increased urinary excretion of iCa, iMg, sodium, phosphate, potassium, and chloride; increased urine output; and decreased urine osmolality and specific gravity. Dextrose administration increased serum insulin; decreased iMg, potassium, and phosphate concentrations; and decreased urinary excretion of iMg. Sodium gluconate increased the excretion of iCa, sodium, and potassium. Hypercalcemia resulted in hypomagnesemia, hypokalemia, and hyperphosphatemia; increased urinary excretion of calcium, magnesium, potassium, sodium, phosphate, and chloride; and induced diuresis. This study has clinical implications because hypercalcemia and excessive administration of calcium have the potential to increase urinary excretion of electrolytes, especially iMg, and induce volume depletion.


Association of lipoprotein-associated phospholipase A2 with coronary calcification among American and Japanese men.
We have previously reported that the prevalence of coronary artery calcification (CAC) was substantially lower among Japanese than American men despite a less favorable profile of many traditional risk factors in Japanese men. To determine whether lipoprotein-associated phospholipase A2 (Lp-PLA2) levels are related to the difference in the prevalence of CAC between the two populations. A total of 200 men aged 40-49 years were examined: 100 residents in Allegheny County, Pennsylvania, United States, and 100 residents in Kusatsu City, Shiga, Japan. Coronary calcium score (CCS) was evaluated by electron-beam tomography, Lp-PLA2 levels, nuclear magnetic resonance (NMR) lipoprotein subclasses, and other factors were assessed in 2001-2002. Lp-PLA2 levels were higher among American than Japanese men (Mean +/- standard deviation 301.7 +/- 82.6 versus 275.9 +/- 104.7 ng/mL, respectively, p=0.06). Among all Japanese men and those with low density lipoprotein (LDL) cholesterol > or =130 mg/dL, there was an inverse association of the prevalence of CCS>0 with the tertile groups of Lp-PLA2 levels (p=0.08 and p=0.03, respectively). American men did not have any association between CCS>0 with the tertile groups of Lp-PLA2 (p=0.62). Although Lp-PLA2 among both populations correlated positively with LDL and total cholesterol, American and Japanese men had different correlations with NMR lipoprotein subclasses. Reported high odds ratio for CCS>0 among American compared to Japanese men was not reduced after adjusting for Lp-PLA2 levels. Lp-PLA2 may have different mechanisms of action among American and Japanese men. Lp-PLA2 levels can not explain the observed CAC differences between the two populations.


Effect of vitamin D supplementation on pain and physical function in patients with knee osteoarthritis (OA): an OA Trial Bank protocol for a systematic review and individual patient data (IPD) meta-analysis.
Observational data suggest that vitamin D deficiency is associated with the onset and progression of knee osteoarthritis (OA). However, randomised controlled trials (RCTs) to date investigating the efficacy of vitamin D supplementation in knee OA have reported conflicting results. Further research is needed to clarify the effects of vitamin D on patient-reported outcomes and determine whether there are patient subgroups who may benefit from the supplementation. The aim of this individual patient data (IPD) meta-analysis is to identify patient-level predictors of treatment response to vitamin D supplementation on pain and physical function. A systematic literature search will be conducted for RCTs of vitamin D supplementation on knee OA. Authors of original RCTs will be contacted to obtain the IPD. The primary outcomes will include long-term (≥12 months) pain and physical function. Secondary outcomes will include medium-term (≥6 months and <12 months) and short-term (<6 months) pain and physical function, as well as patient global assessment, quality of life and adverse events. Potential treatment effect modifiers to be examined in the subgroup analyses include age, gender, body mass index, baseline knee pain severity and physical function, baseline vitamin D level, radiographic stage, presence of bone marrow lesions on MRI, presence of clinical signs of local inflammation and concomitant depressive symptoms. Both one-step and two-step modelling methods will be used to determine the possible modifiable effect of each subgroup of interest. Research ethical or governance approval is exempt for this study as no new data are being collected. This study will be the first IPD meta-analysis to clarify the effect of vitamin D supplementation on clinical symptoms in different subgroups of patients with knee OA. The findings will be disseminated through peer-review publications and conference presentations. CRD42018107740.


Clinical experience in adults and children treated with intravenous peramivir for 2009 influenza A (H1N1) under an Emergency IND program in the United States.
Peramivir, an investigational intravenous neuraminidase inhibitor in Phase 3 trials for hospitalized patients, was made available during the 2009 H1N1 influenza pandemic under the Emergency Investigational New Drug (eIND) regulations. We describe the clinical characteristics and outcomes of all patients for whom peramivir was requested under the eIND. After obtaining eIND approval from the Food and Drug Administration and local institutional review board approval, clinicians caring for hospitalized patients with influenza administered intravenous peramivir and collected information on demographic characteristics, clinical characteristics, and outcomes. From April through October 2009, peramivir was requested for 42 patients and administered to 20 adults and 11 children. At hospitalization, all patients had rapidly progressing, radiographically confirmed viral pneumonia with respiratory failure, and all but 1 patient required mechanical ventilation. In most patients, including 1 person with documented oseltamivir-resistant infection, the illness had progressed despite oseltamivir treatment. Peramivir was administered for 1-14 days (median duration, 10 days). The 14-day, 28-day, and 56-day survival rates were 76.7%, 66.7%, and 59.0%, respectively. Peramivir was generally well tolerated. Intravenous peramivir was well tolerated and was associated with recovery in most patients hospitalized with severe 2009 H1N1 influenza viral pneumonia and treated under an eIND.


X-ray crystallographic and site-directed mutagenesis analysis of the mechanism of Schiff-base formation in phosphonoacetaldehyde hydrolase catalysis.
Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolytic P-C bond cleavage of phosphonoacetaldehyde (Pald) to form orthophosphate and acetaldehyde. The reaction proceeds via a Schiff-base intermediate formed between Lys-53 and the Pald carbonyl. The x-ray crystal structures of the wild-type phosphonatase complexed with Mg(II) alone or with Mg(II) plus vinylsulfonate (a phosphonoethylenamine analog) were determined to 2.8 and 2.4 A, respectively. These structures were used to determine the identity and positions of active site residues surrounding the Lys-53 ammonium group and the Pald carbonyl. These include Cys-22, His-56, Tyr-128, and Met-49. Site-directed mutagenesis was then employed to determine whether or not these groups participate in catalysis. Based on rate contributions, Tyr-128 and Cys-22 were eliminated as potential catalytic groups. The Lys-53 epsilon-amino group, positioned for reaction with the Pald carbonyl, forms a hydrogen bond with water 120. Water 120 is also within hydrogen bond distance of an imidazole nitrogen of His-56 and the sulfur atom of Met-49. Kinetic constants for mutants indicated that His-56 (1000-fold reduction in k(cat)/K(m) upon Ala substitution) and Met-49 (17,000-fold reduction in k(cat)/K(m) upon Leu substitution) function in catalysis of Schiff-base formation. Based on these results, it is proposed that a network of hydrogen bonds among Lys-53, water 120, His-56, and Met-49 facilitate proton transfer from Lys-53 to the carbinolamine intermediate. Comparison of the vinylsulfonate complex versus unliganded structures indicated that association of the cap and core domains is essential for the positioning of the Lys-53 for attack at the Pald carbonyl and that substrate binding at the core domain stabilizes cap domain binding.


A global search reveals epistatic interaction between QTL for early growth in the chicken.
We have identified quantitative trait loci (QTL) explaining a large proportion of the variation in body weights at different ages and growth between chronological ages in an F(2) intercross between red junglefowl and White Leghorn chickens. QTL were mapped using forward selection for loci with significant marginal genetic effects and with a simultaneous search for epistatic QTL pairs. We found 22 significant loci contributing to these traits, nine of these were only found by the simultaneous two-dimensional search, which demonstrates the power of this approach for detecting loci affecting complex traits. We have also estimated the relative contribution of additive, dominance, and epistasis effects to growth and the contribution of epistasis was more pronounced prior to 46 days of age, whereas additive genetic effects explained the major portion of the genetic variance later in life. Several of the detected loci affected either early or late growth but not both. Very few loci affected the entire growth process, which points out that early and late growth, at least to some extent, have different genetic regulation.


Functional and structural studies of the vaccinia virus virulence factor N1 reveal a Bcl-2-like anti-apoptotic protein.
Vaccinia virus (VACV) encodes many immunomodulatory proteins, including inhibitors of apoptosis and modulators of innate immune signalling. VACV protein N1 is an intracellular homodimer that contributes to virus virulence and was reported to inhibit nuclear factor (NF)-kappaB signalling. However, analysis of NF-kappaB signalling in cells infected with recombinant viruses with or without the N1L gene showed no difference in NF-kappaB-dependent gene expression. Given that N1 promotes virus virulence, other possible functions of N1 were investigated and this revealed that N1 is an inhibitor of apoptosis in cells transfected with the N1L gene and in the context of VACV infection. In support of this finding virally expressed N1 co-precipitated with endogenous pro-apoptotic Bcl-2 proteins Bid, Bad and Bax as well as with Bad and Bax expressed by transfection. In addition, the crystal structure of N1 was solved to 2.9 A resolution (0.29 nm). Remarkably, although N1 shows no sequence similarity to cellular proteins, its three-dimensional structure closely resembles Bcl-x(L) and other members of the Bcl-2 protein family. The structure also reveals that N1 has a constitutively open surface groove similar to the grooves of other anti-apoptotic Bcl-2 proteins, which bind the BH3 motifs of pro-apoptotic Bcl-2 family members. Molecular modelling of BH3 peptides into the N1 surface groove, together with analysis of their physico-chemical properties, suggests a mechanism for the specificity of peptide recognition. This study illustrates the importance of the evolutionary conservation of structure, rather than sequence, in protein function and reveals a novel anti-apoptotic protein from orthopoxviruses.


Towards Computational Models and Applications of Insect Visual Systems for Motion Perception: A Review.
Motion perception is a critical capability determining a variety of aspects of insects' life, including avoiding predators, foraging, and so forth. A good number of motion detectors have been identified in the insects' visual pathways. Computational modeling of these motion detectors has not only been providing effective solutions to artificial intelligence, but also benefiting the understanding of complicated biological visual systems. These biological mechanisms through millions of years of evolutionary development will have formed solid modules for constructing dynamic vision systems for future intelligent machines. This article reviews the computational motion perception models originating from biological research on insects' visual systems in the literature. These motion perception models or neural networks consist of the looming-sensitive neuronal models of lobula giant movement detectors (LGMDs) in locusts, the translation-sensitive neural systems of direction-selective neurons (DSNs) in fruit flies, bees, and locusts, and the small-target motion detectors (STMDs) in dragonflies and hoverflies. We also review the applications of these models to robots and vehicles. Through these modeling studies, we summarize the methodologies that generate different direction and size selectivity in motion perception. Finally, we discuss multiple systems integration and hardware realization of these bio-inspired motion perception models.


Antigene-block strategy: effective regulation of gene expression by 2',4'-BNA-modified TFOs with an additional stem-loop structure.
Antigene strategy is promising technology to regulate gene expression. We have previously reported that 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification of triplex-forming oligonucleotides (TFOs) significantly enhanced the binding affinity towards the target dsDNA. In spite of its usefulness, the TFO-binding site may not completely overlay the protein-binding site because of the limitation of TFOs targeting sequences. To overcome this problem, we developed an antigene-based new methodology called "antigene-block" strategy. In this methodology, the TFOs bearing a bulky hairpin tail are used for efficient inhibition of protein-DNA interaction. The antigene-block TFOs having 2',4'-BNA modifications formed stable triplexes with the homopurine-homopyrimidine sequence which partially overlap the transcription factor NF-kappaB binding site. In addition, the antigene-block TFOs significantly reduced the expression level of the target-gene in living cells, while conventional 2',4'-BNA-modified or unmodified TFOs showed no effect on the target-gene expression.


One year remission of type 1 diabetes mellitus in a patient treated with sitagliptin.
Type 1 diabetes mellitus (T1DM) is a chronic disease characterized by the autoimmune destruction of pancreatic β-cells. This paper describes the case of a 19-year-old male patient who presented with glutamic acid decarboxylase (GAD) antibody positive and diabetic ketoacidosis, which mandated intensive insulin treatment. Once the ketoacidosis was controlled, an oral dose of 100 mg of sitagliptin was administered once a day. Ketoacidosis was managed by insulin and insulin daily requirement began to dwindle after one month, until its complete withdrawal at 8 weeks, when partial remission was reached. The patient has now remained on sitagliptin treatment alone for a year, without requiring insulin. The benefit observed with this medication is possibly associated with its immunological effects. Inhibition of dipeptidyl peptidase 4 in animal models deregulates the Th1 immune response, increases secretion of Th2 cytokines, activates CD4+CD25+FoxP3+ regulatory T-cells, and prevents IL17 production. The use of insulin-dose-adjusted HbA1c constitutes the best way to define partial remission in T1DM patients.The use of sitagliptin in T1DM patients could help to decrease daily requirement of insulin by delaying β-cell loss and improving endogenous insulin production.The determination of antibodies against insulin, islet cells, and GAD permits differentiation of T1DM patients from those with atypical or ketosis-prone diabetes.


Simultaneous fine-needle aspiration and core-needle biopsy of thyroid nodules.
To evaluate the efficacy of simultaneous fine-needle aspiration (FNA) and core-needle (CN) biopsies of thyroid nodules, the clinical course and operative findings in 100 patients having both tests were reviewed. Each patient had a diagnosis made with this approach. Both specimens were adequate for diagnosis in 95 patients. In the remaining 5 patients, a diagnosis was provided by FNA in four and CN biopsy in one. FNA and CN biopsies gave the same diagnosis of either a benign nodule in 53 patients or neoplasia in 30 patients. When both FNA and CN biopsies showed a benign nodule, a nonoperative approach was taken in 43 of the 53 patients. Forty-two patients were operated on either because of biopsy findings (32) or clinical indications (10). The surgical specimens were used to determine the false positive and false negative rates, the sensitivity, specificity, and accuracy for the diagnosis of neoplasia. The false (+) and false (-) rates in diagnosing neoplasia were 33 per cent and 7 per cent for FNA, 20 per cent and 4 per cent for CN biopsy, and 20 per cent and 0 per cent for both. The sensitivity, specificity, and accuracy for FNA were 93 per cent, 67 per cent, and 83 per cent, for CN biopsy 96 per cent, 80 per cent, and 90 per cent, and 100 per cent, 80 per cent, and 93 per cent for both FNA and CN. All neoplasms were detected, and no thyroid carcinomas were missed by this combination. There was only one complication: bleeding after a CN biopsy. Combined FNA and CN biopsies allow most patients with thyroid nodules to avoid an unnecessary operation and accurately diagnose those with thyroid carcinoma. These two procedures are safe and complementary.


Searching for regulatory elements in human noncoding sequences.
Important progress has been made in the past two years in the identification of Pol II promoters. For most other regulatory elements, however, current biological knowledge is still insufficient to allow the development of prediction tools. The phylogenetic-footprinting strategy, which is based on the comparative analysis of homologous sequences, is a very efficient approach to identify new unknown regulatory elements. The recent organization of large-scale sequencing projects for some model vertebrate organisms will be extremely valuable for the prediction of regulatory elements in the human genome.


Toward a Consensus in the Repertoire of Hemocytes Identified in Drosophila.
The catalog of the Drosophila immune cells was until recently limited to three major cell types, based on morphology, function and few molecular markers. Three recent single cell studies highlight the presence of several subgroups, revealing a large diversity in the molecular signature of the larval immune cells. Since these studies rely on somewhat different experimental and analytical approaches, we here compare the datasets and identify eight common, robust subgroups associated to distinct functions such as proliferation, immune response, phagocytosis or secretion. Similar comparative analyses with datasets from different stages and tissues disclose the presence of larval immune cells resembling embryonic hemocyte progenitors and the expression of specific properties in larval immune cells associated with peripheral tissues.


Effect of First Ray Insufficiency and Metatarsal Index on Metatarsalgia in Hallux Valgus.
Two concepts have been proposed to explain the etiology of metatarsalgia in hallux valgus patients: First, as the magnitude of hallux valgus increases, there is a mechanical overload of the lesser metatarsals. Second, increased relative lesser metatarsal length is a factor in the development of metatarsalgia. However, there is no current evidence that these structural factors lead to primary metatarsalgia. The purpose of the study was to evaluate the factors associated with metatarsalgia in hallux valgus patients. A cross-sectional study of 121 consecutive adult patients with non-arthritic hallux valgus was carried out. Binary logistic regression was performed to identify the effect of the clinical and demographic factors on the occurrence of metatarsalgia. One hundred twenty-one patients (184 feet) with hallux valgus were analyzed. The median weight was 65 kg (interquartile range 58-72). Metatarsalgia was present in 84 (45.6%) feet. The binary logistic regression showed that lesser toe deformity (OR 2.6, 95% CI 0.2-0.5), gastrocnemius shortening (OR 5.8, 95% CI 2.8-12.3), metatarsal index (OR 0.3, 95% CI 0.2-0.5), and weight (OR 2.5, 95% CI 1.2-5.3) were significantly associated. Metatarsalgia occurs in almost half of hallux valgus patients. It has a multifactorial etiology. Our findings contradict the common theory that both the magnitude of hallux valgus deformity and an increased length of the lesser metatarsals, by themselves, lead to primary metatarsalgia. Metatarsalgia was associated with Achilles shortening, excessive weight, and associated lesser toe deformity. These factors should be addressed in order to treat this disorder adequately. Level III, comparative series.


Production and characterization of high-affinity human monoclonal antibodies to human immunodeficiency virus type 1 envelope glycoproteins in a mouse model expressing human immunoglobulins.
Human (Hu) monoclonal antibodies (MAbs) against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) are useful tools in the structural and functional analysis of Env, are under development both as potential prophylaxis and as therapy for established HIV-1 infection, and have crucial roles in guiding the design of preventative vaccines. Despite representing more than 50% of infections globally, no MAbs have been generated in any species against C clade HIV-1 Env. To generate HuMAbs to a novel Chinese C clade Env vaccine candidate (primary isolate strain HIV-1(97CN54)), we used BAB5 mice that express a human immunoglobulin (Ig) M antibody repertoire in place of endogenous murine immunoglobulins. When immunized with HIV-1(97CN54) Env, these mice developed antigen-specific IgM antibodies. Hybridoma fusions using splenocytes from these mice enabled the isolation of two Env-specific IgM HuMAbs: N3C5 and N03B11. N3C5 bound to HIV-1 Env from clades A and C, whereas N03B11 bound two geographically distant clade C isolates but not Env from other clades. These HuMAbs bind conformational epitopes within the immunodominant region of the gp41 ectodomain. N3C5 weakly neutralized the autologous isolate in the absence of complement and weakly enhanced infection in the presence of complement. N03B11 has no effect on infectivity in either the presence or the absence of complement. These novel HuMAbs are useful reagents for the study of HIV-1 Env relevant to the global pandemic, and mice producing human immunoglobulin present a tool for the production of such antibodies.


Genetic variability and natural selection at the ligand domain of the Duffy binding protein in Brazilian Plasmodium vivax populations.
Plasmodium vivax malaria is a major public health challenge in Latin America, Asia and Oceania, with 130-435 million clinical cases per year worldwide. Invasion of host blood cells by P. vivax mainly depends on a type I membrane protein called Duffy binding protein (PvDBP). The erythrocyte-binding motif of PvDBP is a 170 amino-acid stretch located in its cysteine-rich region II (PvDBPII), which is the most variable segment of the protein. To test whether diversifying natural selection has shaped the nucleotide diversity of PvDBPII in Brazilian populations, this region was sequenced in 122 isolates from six different geographic areas. A Bayesian method was applied to test for the action of natural selection under a population genetic model that incorporates recombination. The analysis was integrated with a structural model of PvDBPII, and T- and B-cell epitopes were localized on the 3-D structure. The results suggest that: (i) recombination plays an important role in determining the haplotype structure of PvDBPII, and (ii) PvDBPII appears to contain neutrally evolving codons as well as codons evolving under natural selection. Diversifying selection preferentially acts on sites identified as epitopes, particularly on amino acid residues 417, 419, and 424, which show strong linkage disequilibrium. This study shows that some polymorphisms of PvDBPII are present near the erythrocyte-binding domain and might serve to elude antibodies that inhibit cell invasion. Therefore, these polymorphisms should be taken into account when designing vaccines aimed at eliciting antibodies to inhibit erythrocyte invasion.


Confirmed exposure to tick-borne encephalitis virus and probable human cases of tick-borne encephalitis in Central/Northern Anatolia, Turkey.
Tick-borne encephalitis virus (TBEV) is the aetiological agent of tick-borne encephalitis (TBE), a potentially fatal central nervous system infection of humans. TBE is endemic in many areas of Europe and Asia; however, very scarce data on TBEV activity are available from Turkey. We aimed to identify TBEV exposure in healthy blood donors and the impact of TBEV in central nervous system infections in Central/Northern Anatolia. Two-thousand four hundred and fifty four sera, collected from blood donors at Ankara, Konya, Eskişehir and Zonguldak branches of the Turkish Red Crescent Middle Anatolia Regional Blood Center, were analysed for TBEV serosurveillance. Paired serum and cerebrospinal fluid samples from 108 patients with the diagnosis of aseptic meningitis/encephalitis of unknown aetiology were also evaluated to identify TBE and neuroborreliosis cases. Commercial enzyme-linked immunosorbent assays and indirect immunofluorescence tests were employed for antibody detection. Forty-seven donor samples (1.9%) were reactive for TBEV IgG. In 25 persons with IgG reactivity (53.1%), risk factors for tick-borne infections were revealed. One sample from Zonguldak province (1/198; 0.5%) in the Black Sea region of Turkey was confirmed to possess neutralizing antibodies via plaque reduction neutralization test. TBEV IgM was detected in 9.2% (8/108) of the patients. IgM was accompanied by IgG reactivity in two persons where, in one, recent history of a tick bite was also identified. Intrathecal antibody production for TBEV could not be demonstrated. No evidence for Borrelia infections could be found. Confirmed exposure to TBEV and/or an antigenically similar tick-borne flavivirus is documented for the first time in blood donors in Zonguldak in Northern Anatolia. Probable cases of TBE have also been identified from Central Anatolia. The epidemiology of TBEV activity in Turkey needs to be assessed and benefits of vaccination for general population, risk groups or travellers must be considered.


Pitfalls in MR morphology of the sterno-costo-clavicular region using whole-body MRI.
To analyse the imaging findings at the sterno-costo-clavicular (SCC) joint region using whole-body (WB) magnetic resonance imaging (MRI) in healthy individuals to minimize misinterpretation as changes due to spondyloarthritis (SpA). As part of a cross-sectional study of 122 SpA patients, 75 healthy individuals (42/33 males/females; median age 30.3 years; range 17.7-63.8 years) were scanned using sagittal and coronal WB short tau inversion recovery (STIR) and T1-weighted MRI sequences. The SCC region was analysed independently by seven readers for bone marrow oedema (BMO), erosions, subchondral fat signal intensity (FSI), and joint fluid accumulation. SCC changes simulating inflammation were reported by four or more of the seven readers in 15 (20%) healthy individuals (12 male/three female; median age 32.1 years; range 20.2-48 years). Thirteen individuals (17%) had changes at the manubriosternal joint (MSJ); five had BMO, one BMO + erosion, four erosion, two erosion + FSI, and one FSI only. Changes at the sternoclavicular joint occurred in three individuals (4%) encompassing erosion, erosion + FSI + BMO, and joint fluid accumulation, respectively. One patient had both MSJ and sternoclavicular joint changes. Findings mimicking inflammatory changes occurred in healthy individuals, particularly in the MSJ. Awareness of this is important in recognition of SCC inflammation in SpA.


Hybridization-promoted and cytidine-selective activation for cross-linking with the use of 2-amino-6-vinylpurine derivatives.
Recently, we have proposed a new concept for cross-linking agents with inducible reactivity, in which the highly reactive cross-linking agent, the 2-amino-6-vinylpurine nucleoside analogue (1), can be regenerated in situ from its stable precursors, the phenylsulfide (4) and the phenylsulfoxide (3) derivatives, by a hybridization-promoted activation process with selectivity to cytidine. The phenylsulfide precursor (4) exhibited cross-linking ability despite its high stability toward strong nucleophiles such as amines and thiols. In this study, we investigated the substituent effects of the phenylsulfide group on the cross-linking reaction, and determined the 2-carboxy substituent of the phenylsulfide derivative (11k) as an efficient cross-linking agent with inducible reactivity. Detailed investigations have shown that the phenylsulfoxide (3) and phenylsulfide (4) precursors produce the 2-amino-6-vinylpurine nucleoside (1) as the common reactive species. It has been concluded that the nature of the inducible reactivity of the precursors (3 and 4) is acceleration of their elimination to the 2-amino-6-vinylpurine nucleoside (1) through the selective process in the duplex with the ODN having cytidine at the target site.


Ethical dilemmas in online research and treatment of sexually abused adolescents.
In a recent uncontrolled trial of a new therapist-assisted Web-based treatment of adolescent victims of sexual abuse, the treatment effects were found to be promising. However, the study suffered a large pretreatment withdrawal rate that appeared to emanate from reluctance among the participants to disclose their identity and obtain their parents' consent. Our objectives were to confirm the effects of the online treatment in a controlled trial and to evaluate measures to reduce pretreatment withdrawal in vulnerable populations including young victims of sexual abuse. The study was designed as a within-subject baseline-controlled trial. Effects of an 8-week attention-placebo intervention were contrasted with the effects of an 8-week treatment episode. Several measures were taken to reduce pretreatment dropout. Pretreatment withdrawal was reduced but remained high (82/106, 77%). On the other hand, treatment dropout was low (4 out of 24 participants), and improvement during treatment showed significantly higher effects than during the attention placebo control period (net effect sizes between 0.5 and 1.6). In treatment of vulnerable young populations, caregivers and researchers will have to come to terms with high pretreatment withdrawal rates. Possible measures may reduce pretreatment withdrawal to some degree. Providing full anonymity is not a viable option since it is incompatible with the professional responsibility of the caregiver and restricts research possibilities.


Association of PET-based estradiol-challenge test for breast cancer progesterone receptors with response to endocrine therapy.
Estrogen receptor (ER) testing of breast cancer imperfectly predicts response to endocrine therapy (ET). We hypothesize that a brief estradiol challenge will increase tumor progesterone receptor (PgR) levels only in tumors with functional ER. In this prospective, phase 2, single-center, single-arm trial (NCT02455453), we report the association of response to ET with change in tumor uptake of the progestin analog, 21-[18F]fluorofuranylnorprogesterone (FFNP), before and after a one-day estradiol challenge. In 43 postmenopausal women with advanced ER+ breast cancer, we show a post-challenge increase in tumor FFNP uptake only in 28 subjects with clinical benefit from ET (responders), but not in 15 without clinical benefit (nonresponders) (p < 0.0001), indicating 100% sensitivity and specificity. We further show significantly longer survival (p < 0.0001) in the responding subjects. Our results demonstrate that change in tumor FFNP uptake after estradiol challenge is highly predictive of response to ET in women with ER+ breast cancer.


Improved Patch-Based Automated Liver Lesion Classification by Separate Analysis of the Interior and Boundary Regions.
The bag-of-visual-words (BoVW) method with construction of a single dictionary of visual words has been used previously for a variety of classification tasks in medical imaging, including the diagnosis of liver lesions. In this paper, we describe a novel method for automated diagnosis of liver lesions in portal-phase computed tomography (CT) images that improves over single-dictionary BoVW methods by using an image patch representation of the interior and boundary regions of the lesions. Our approach captures characteristics of the lesion margin and of the lesion interior by creating two separate dictionaries for the margin and the interior regions of lesions ("dual dictionaries" of visual words). Based on these dictionaries, visual word histograms are generated for each region of interest within the lesion and its margin. For validation of our approach, we used two datasets from two different institutions, containing CT images of 194 liver lesions (61 cysts, 80 metastasis, and 53 hemangiomas). The final diagnosis of each lesion was established by radiologists. The classification accuracy for the images from the two institutions was 99% and 88%, respectively, and 93% for a combined dataset. Our new BoVW approach that uses dual dictionaries shows promising results. We believe the benefits of our approach may generalize to other application domains within radiology.


Gene transfer from bacteria and archaea facilitated evolution of an extremophilic eukaryote.
Some microbial eukaryotes, such as the extremophilic red alga Galdieria sulphuraria, live in hot, toxic metal-rich, acidic environments. To elucidate the underlying molecular mechanisms of adaptation, we sequenced the 13.7-megabase genome of G. sulphuraria. This alga shows an enormous metabolic flexibility, growing either photoautotrophically or heterotrophically on more than 50 carbon sources. Environmental adaptation seems to have been facilitated by horizontal gene transfer from various bacteria and archaea, often followed by gene family expansion. At least 5% of protein-coding genes of G. sulphuraria were probably acquired horizontally. These proteins are involved in ecologically important processes ranging from heavy-metal detoxification to glycerol uptake and metabolism. Thus, our findings show that a pan-domain gene pool has facilitated environmental adaptation in this unicellular eukaryote.


Anti-depressant effect of hesperidin in diabetic rats.
This study aimed to investigate the anti-depressant effect of hesperidin (Hsp) in streptozotocin (STZ)-induced diabetic rats. Additionally, the effect of Hsp on hyperglycaemia, oxidative stress, inflammation, brain-derived neurotrophic factor (BDNF), and brain monoamines in diabetic rats was also assessed. The Wistar rats in the experimental groups were rendered hyperglycaemic with a single dose of STZ (52.5 mg·(kg body mass)(-1), by intraperitoneal injection). The normal group received the vehicle only. Hyperglycaemic rats were treated with Hsp (25.0, 50.0, or 100.0 mg·(kg body mass)(-1)·day(-1), per oral) and fluoxetine (Flu) (5.0 mg·(kg body mass)(-1)·day(-1), per oral) 48 h after the STZ injection, for 21 consecutive days. The normal and STZ control groups received the vehicle (distilled water). Behavioral and biochemical parameters were then assessed. When Hsp was administered to the STZ-treated rats, this reversed the STZ-induced increase in immobility duration in the forced swimming test (FST) and attenuated hyperglycaemia, decreased malondialdehyde (MDA), increased reduced glutathione (GSH) decreased interleukin-6 (IL-6), and increased BDNF levels in the brain. Treatment with Hsp attenuated STZ-induced neurochemical alterations, as indicated by increased levels of monoamines in the brain, namely, norepinephrine (NE), dopamine (DA), and serotonin (5-hydroxytryptamine; 5-HT). All of these effects of Hsp were similar to those observed with the established anti-depressant Flu. This study shows that Hsp exerted anti-depressant effect in diabetic rats, which may have been partly mediated by its amelioration of hyperglycaemia as well as its anti-oxidant and anti-inflammatory activities, the enhancement of neurogenesis, and changes in the levels of monoamines in the brain.


Indel variant analysis of short-read sequencing data with Scalpel.
As the second most common type of variation in the human genome, insertions and deletions (indels) have been linked to many diseases, but the discovery of indels of more than a few bases in size from short-read sequencing data remains challenging. Scalpel (http://scalpel.sourceforge.net) is an open-source software for reliable indel detection based on the microassembly technique. It has been successfully used to discover mutations in novel candidate genes for autism, and it is extensively used in other large-scale studies of human diseases. This protocol gives an overview of the algorithm and describes how to use Scalpel to perform highly accurate indel calling from whole-genome and whole-exome sequencing data. We provide detailed instructions for an exemplary family-based de novo study, but we also characterize the other two supported modes of operation: single-sample and somatic analysis. Indel normalization, visualization and annotation of the mutations are also illustrated. Using a standard server, indel discovery and characterization in the exonic regions of the example sequencing data can be completed in ∼5 h after read mapping.


Why do bacteria regulate public goods by quorum sensing?-How the shapes of cost and benefit functions determine the form of optimal regulation.
Many bacteria secrete compounds which act as public goods. Such compounds are often under quorum sensing (QS) regulation, yet it is not understood exactly when bacteria may gain from having a public good under QS regulation. Here, we show that the optimal public good production rate per cell as a function of population size (the optimal production curve, OPC) depends crucially on the cost and benefit functions of the public good and that the OPC will fall into one of two categories: Either it is continuous or it jumps from zero discontinuously at a critical population size. If, e.g., the public good has accelerating returns and linear cost, then the OPC is discontinuous and the best strategy thus to ramp up production sharply at a precise population size. By using the example of public goods with accelerating and diminishing returns (and linear cost) we are able to determine how the two different categories of OPSs can best be matched by production regulated through a QS signal feeding back on its own production. We find that the optimal QS parameters are different for the two categories and specifically that public goods which provide accelerating returns, call for stronger positive signal feedback.


Combinatorial transcriptional control in blood stem/progenitor cells: genome-wide analysis of ten major transcriptional regulators.
Combinatorial transcription factor (TF) interactions control cellular phenotypes and, therefore, underpin stem cell formation, maintenance, and differentiation. Here, we report the genome-wide binding patterns and combinatorial interactions for ten key regulators of blood stem/progenitor cells (SCL/TAL1, LYL1, LMO2, GATA2, RUNX1, MEIS1, PU.1, ERG, FLI-1, and GFI1B), thus providing the most comprehensive TF data set for any adult stem/progenitor cell type to date. Genome-wide computational analysis of complex binding patterns, followed by functional validation, revealed the following: first, a previously unrecognized combinatorial interaction between a heptad of TFs (SCL, LYL1, LMO2, GATA2, RUNX1, ERG, and FLI-1). Second, we implicate direct protein-protein interactions between four key regulators (RUNX1, GATA2, SCL, and ERG) in stabilizing complex binding to DNA. Third, Runx1(+/-)::Gata2(+/-) compound heterozygous mice are not viable with severe hematopoietic defects at midgestation. Taken together, this study demonstrates the power of genome-wide analysis in generating novel functional insights into the transcriptional control of stem and progenitor cells.


Aggregatibacter actinomycetemcomitans targets NLRP3 and NLRP6 inflammasome expression in human mononuclear leukocytes.
Periodontitis is an inflammatory condition that destroys the tooth-supporting tissues, as a result of local bacterial infection. Aggregatibacter actinomycetemcomitans is a Gram-negative facultative anaerobic species, highly associated with aggressive periodontitis. Periodontal inflammation is dominated by cytokines of the Interleukin (IL)-1 family. Prior to their secretion by mononuclear cells, IL-1 cytokines are processed by intracellular protein complexes, known as "inflammasomes", which can sense the bacterial challenge. The aim of this study was to investigate which inflammasomes are regulated in mononuclear cells in response to A. actinomycetemcomitans. The D7SS strain and its derivative leukotoxin and cytolethal distending toxin knock-out mutant strains were used to infect human mononuclear cells at a 1:10 cell: bacteria ratio, for 3 h. The expression of various inflammasome components in the cells was investigated by TaqMan quantitative real-time polymerase chain reaction (qPCR). The expressions of NOD-like receptor protein (NLRP)1, NLRP2 and Absent In Melanoma (AIM)2 inflammasome sensors, as well as their effector Caspase-1 were not affected. However, NLRP3 was up-regulated, while NLRP6 was down-regulated. This effect was not dependent on the leukotoxin or the cytolethal distending toxin, as demonstrated by the use of specific gene knock-out mutant strains. IL-1β and IL-18 expressions were also up-regulated by the bacterial challenge. In conclusion, A. actinomycetemcomitans enhances NLRP3 and reduces NLRP6 inflammasome expression, irrespective of its major virulence factors, confirming the high pathogenic profile of this species, and providing further insights to the mechanisms of periodontal inflammation.


Female-biased sex allocation and lack of inbreeding avoidance in Cubitermes termites.
Sexually reproducing organisms face a strong selective pressure to find a mate and ensure reproduction. An important criterion during mate-selection is to avoid closely related individuals and subsequent potential fitness costs of resulting inbred offspring. Inbreeding avoidance can be active through kin recognition during mate choice, or passive through differential male and female-biased sex ratios, which effectively prevents sib-mating. In addition, sex allocation, or the resources allotted to male and female offspring, can impact mating and reproductive success. Here, we investigate mate choice, sex ratios, and sex allocation in dispersing reproductives (alates) from colonies of the termite Cubitermes tenuiceps. Termites have a short time to select a mate for life, which should intensify any fitness consequences of inbreeding. However, alates did not actively avoid inbreeding through mate choice via kin recognition based on genetic or environmental cues. Furthermore, the majority of colonies exhibited a female-biased sex ratio, and none exhibited a male-bias, indicating that differential bias does not reduce inbreeding. Sex allocation was generally female-biased, as females also were heavier, but the potential fitness effect of this costly strategy remains unclear. The bacterium Wolbachia, known in other insects to parasitically distort sex allocation toward females, was present within all alates. While Wolbachia is commonly associated with termites, parasitism has yet to be demonstrated, warranting further study of the nature of the symbiosis. Both the apparent lack of inbreeding avoidance and potential maladaptive sex allocation implies possible negative effects on mating and fitness.


Scanning and transmission electron microscopic study of the tracheal air sac system in a grasshopper Chrotogonus senegalensis (Kraus)--Orthoptera: Acrididae: Pyrgomorphinae.
The morphology of the trachea-air sac system in a species of grasshopper Chrotogonus senegalensis has been studied by using scanning and transmission electron microscopes. Capacious air sacs were formed as dilatations along the primary tracheal trunks. Narrower secondary trachea arose either directly from the primary trachea that bypassed the air sacs or from the air sacs themselves. At or close to the organ or tissue supplied with air, the secondary trachea gave rise to the notably smaller tertiary trachea that penetrated the tissue, giving rise terminally to the extremely small tracheoles that indent some cells. The trachea and the air sacs were basically made up of an inner cuticular lining, helical taenidial rings, and an overlying epithelial cell cover. The air sacs may be important in efficient ventilation of the respiratory system. The supply of air directly to the tissue cells was viewed as an exemplary efficient design when compared to that prevailing in the nontracheate air-breathing animals, where the vascular system is interposed between the respiratory organ and the target tissue cells. A similarity in the general morphological design of the insect and avian respiratory systems has been observed, mainly in respect to the presence of the air sacs and that of the respiratory shunts. This, together with the reported functional features like the unidirectional mode of ventilation, has been interpreted as a classic case of structural and functional convergent evolution leading to the evolution of similar and comparably efficient respiratory systems capable of providing the large amount of oxygen demanded by flight.


Interfacial Characteristics and Cytocompatibility of Hydraulic Sealer Cements.
The stability and long-term success of root canal obturation depends on the choice of sealer because the sealer bonds to the dentin and stabilizes the solid cone. Furthermore, the sealer needs to be nontoxic because sealer toxicity will certainly lead to treatment failure. The aim of this study was to assess the sealer-dentin interface of 3 hydraulic root canal sealers and to evaluate their cytocompatibility compared with AH Plus (Dentsply DeTrey GmbH, Konstanz, Germany). Four dental root canal sealers were assessed. AH Plus, MTA Fillapex (Angelus, Londrina, Brazil), BioRoot RCS (Septodont, Saint-Maur-des-Fossés, France), and Endoseal (Maruchi, Wonju-si, Gangwon-do, South Korea) were characterized using scanning electron microscopy and energy-dispersive spectroscopy. The sealer-tooth interface was assessed by confocal microscopy and scanning electron microscopy, and biocompatibility was measured by assessing the cell metabolic function using direct contact assays and alkaline phosphatase activity. The tricalcium silicate-based sealers presented a different microstructure and elemental composition despite their similar chemistry and classification. BioRoot RCS was free of aluminum, and all sealers presented different radiopacifying elements. The sealer penetration in the dentinal tubules and interfacial characteristics were different. The migration of silicon was evident from sealer to tooth for all sealers containing tricalcium silicate. MTA Fillapex and BioRoot RCS exhibited the best cytocompatibility in both the direct contact test and alkaline phosphatase activity. The use of hydraulic calcium silicate-based sealers has introduced a different material type to endodontics. These materials are different than other sealers mostly because of their hydraulic nature and their interaction with the environment. Although the sealers tested had a similar chemistry, their cytocompatibility and bonding mechanisms were diverse.


Sumoylation of Oct4 enhances its stability, DNA binding, and transactivation.
Transcription factor Oct4 is a master regulator affecting the fate of pluripotent stem cells and germ cell precursors. Oct4 expression is tightly regulated, and small changes in expression level can have dramatic effects on differentiation or oncogenesis. Post-translational modifications including phosphorylation and ubiquitination have been reported to regulate Oct4 transcriptional activity. Here we show that Oct4 is a target for small ubiquitin-related modifier (SUMO)-1 modification in vivo and in vitro. Sumoylation of Oct4 occurs at a single lysine, Lys(118), located at the end of the amino-terminal transactivation domain and next to the Pit1-Oct-Unc86 (POU) DNA binding domain. SUMO-1 and Oct4 colocalize at several promoter sequences in vivo, and a fraction of Oct4 molecules colocalized with SUMO-1 in nuclear aggregates. Sumoylation of Oct4 led to significantly increased Oct4 stability and increased DNA binding. In addition, SUMO-1 cotransfection led to augmented Oct4 transactivation potential that was reduced when the Oct4 sumoylation target site was mutated. Therefore, sumoylation of Oct4 results in increased stability, DNA binding, and transactivation and provides an important mechanism to regulate Oct4 activity.


Brain structural connectivity network alterations in insomnia disorder reveal a central role of the right angular gyrus.
Insomnia Disorder (ID) is a prevalent and persistent condition, yet its neural substrate is not well understood. The cognitive, emotional, and behavioral characteristics of ID suggest that vulnerability involves distributed brain networks rather than a single brain area or connection. The present study utilized probabilistic diffusion tractography to compare the whole-brain structural connectivity networks of people with ID and those of matched controls without sleep complaints. Diffusion-weighted images and T1-weighed images were acquired in 51 people diagnosed with ID (21-69 years of age, 37 female) and 48 matched controls without sleep complaints (22-70 years of age, 31 female). Probabilistic tractography was performed to construct the whole-brain structural connectivity network of each participant. Case-control differences in connectivity strength and network efficiency were evaluated with permutation tests. People with ID showed structural hyperconnectivity within a subnetwork that spread over frontal, parietal, temporal, and subcortical regions and was anchored at the right angular gyrus. The result was robust across different edge-weighting strategies. Moreover, converging support was given by the finding of heightened right angular gyrus nodal efficiency (harmonic centrality) across varying graph density in people with ID. Follow-up correlation analyses revealed that subnetwork connectivity was associated with self-reported reactive hyperarousal. The findings demonstrate that the right angular gyrus is a hub of enhanced structural connectivity in ID. Hyperconnectivity within the identified subnetwork may contribute to increased reactivity to stimuli and may signify vulnerability to ID.


Intermittent foot claudication with active dorsiflexion: the seminal case of dorsalis pedis artery entrapment.
Atypical claudication is a relatively uncommon problem within the general population. However, suspicion for the diagnosis is raised when young and athletic patients present with symptoms of claudication during exercise. The most common causes of atypical claudication are anatomical variants, including popliteal artery entrapment syndrome and tarsal tunnel syndrome. These variants result in impaired arterial flow and nerve compression, respectively. In this report, we present a seminal case of dorsalis pedis artery entrapment by the extensor hallucis brevis tendon during active dorsiflexion of the foot. The patient was a 42-year-old male without significant past medical history, who presented with claudication in both feet upon active dorsiflexion. He underwent dynamic arterial duplex studies that first revealed normal flow in the neutral position and then revealed complete cessation of flow in both duplex and Doppler modes on dorsiflexion of the foot. He also underwent dynamic magnetic resonance angiography of bilateral lower extremities that revealed an incomplete pedal arch with early termination of the posterior tibial artery on static images and termination of the dorsalis pedis artery at notching on the dorsum of the foot during dorsiflexion. The patient was taken to the operating room for bilateral dorsalis pedis artery exploration. During exploration, the patient was found to have entrapment of the dorsalis pedis artery by the extensor hallucis brevis (EHB) tendon. This was documented by both direct visualization and intraoperative cessation of Doppler signal on dorsiflexion. Since the EHB tendon provides only secondary function to the extensor hallucis longus (EHL) tendon, the EHB was transected near its insertion and transposed directly to the EHL tendon. This allowed for normal extensor function of the great toe and restored triphasic Doppler signals during dorsiflexion. Dorsalis pedis arterial entrapment is a novel cause of atypical claudication. It is extremely uncommon as patients must have both abnormal anatomy and an incomplete pedal arch to display symptoms. Similar to other entrapment syndromes, if identified before permanent arterial scarring, the treatment does not require a bypass procedure. Removal of the tendon along with transposition will allow cessation of symptoms without impaired dorsiflexion of the great toe.


A novel mechanism of dasatinib-induced apoptosis in chronic myeloid leukemia; ceramide synthase and ceramide clearance genes.
Sphingolipids are bioeffector molecules that control various aspects of cell growth, proliferation, apoptosis, and drug resistance. Ceramides, the central molecule of sphingolipid metabolism, are inducer of apoptosis and inhibitors of proliferation. Sphingosine-1-phosphate (S1P) and glucosyleceramide, converted from ceramides by sphingosine kinase-1 (SK-1) and glucosyleceramide synthase (GCS) enzymes, respectively, inhibit apoptosis and develop resistance to chemotherapeutic drugs. In this study, we examined the therapeutic potentials of bioactive sphingolipids in chronic myeloid leukemia (CML) alone and in combination with dasatinib in addition to investigate the roles of ceramide-metabolizing genes in dasatinib-induced apoptosis. Cytotoxic effects of dasatinib, C8:ceramide, PDMP, and SK-1 inhibitor were determined by XTT cell proliferation assay. Changes in caspase-3 enzyme activity and mitochondrial membrane potential (MMP) were measured using caspase-3 colorimetric assay and JC-1 MMP detection kit. Expression levels of ceramide-metabolizing genes were examined by qRT-PCR. Application of ceramide analogs and inhibitors of ceramide clearance genes decreased cell proliferation and induced apoptosis. Targeting bioactive sphingolipids towards generation/accumulation of ceramides increased apoptotic effects of dasatinib, synergistically. It was shown for the first time that dasatinib induces apoptosis through downregulating expression levels of antiapoptotic SK-1 but not GCS, and upregulating expression levels of ceramide synthase (CerS) genes, especially CerS1, in K562 cells. On the other hand, dasatinib downregulates expression levels of both GCS and SK-1 and upregulate apoptotic CerS2, -5 and -6 genes in Meg-01 cells. Increasing endogenous ceramide levels and decreasing prosurvival lipids, S1P, and GC, can open the way of more effective treatment of CML.


Evolution of poor reporting and inadequate methods over time in 20 920 randomised controlled trials included in Cochrane reviews: research on research study.
Objective To examine how poor reporting and inadequate methods for key methodological features in randomised controlled trials (RCTs) have changed over the past three decades.Design Mapping of trials included in Cochrane reviews.Data sources Data from RCTs included in all Cochrane reviews published between March 2011 and September 2014 reporting an evaluation of the Cochrane risk of bias items: sequence generation, allocation concealment, blinding, and incomplete outcome data.Data extraction For each RCT, we extracted consensus on risk of bias made by the review authors and identified the primary reference to extract publication year and journal. We matched journal names with Journal Citation Reports to get 2014 impact factors.Main outcomes measures We considered the proportions of trials rated by review authors at unclear and high risk of bias as surrogates for poor reporting and inadequate methods, respectively.Results We analysed 20 920 RCTs (from 2001 reviews) published in 3136 journals. The proportion of trials with unclear risk of bias was 48.7% for sequence generation and 57.5% for allocation concealment; the proportion of those with high risk of bias was 4.0% and 7.2%, respectively. For blinding and incomplete outcome data, 30.6% and 24.7% of trials were at unclear risk and 33.1% and 17.1% were at high risk, respectively. Higher journal impact factor was associated with a lower proportion of trials at unclear or high risk of bias. The proportion of trials at unclear risk of bias decreased over time, especially for sequence generation, which fell from 69.1% in 1986-1990 to 31.2% in 2011-14 and for allocation concealment (70.1% to 44.6%). After excluding trials at unclear risk of bias, use of inadequate methods also decreased over time: from 14.8% to 4.6% for sequence generation and from 32.7% to 11.6% for allocation concealment.Conclusions Poor reporting and inadequate methods have decreased over time, especially for sequence generation and allocation concealment. But more could be done, especially in lower impact factor journals.


Solvent Networks Tune Thermodynamics of Oligosaccharide Complex Formation in an Extended Protein Binding Site.
The principles of protein-glycan binding are still not well understood on a molecular level. Attempts to link affinity and specificity of glycan recognition to structure suffer from the general lack of model systems for experimental studies and the difficulty to describe the influence of solvent. We have experimentally and computationally addressed energetic contributions of solvent in protein-glycan complex formation in the tailspike protein (TSP) of E. coli bacteriophage HK620. HK620TSP is a 230 kDa native trimer of right-handed, parallel beta-helices that provide extended, rigid binding sites for bacterial cell surface O-antigen polysaccharides. A set of high-affinity mutants bound hexa- or pentasaccharide O-antigen fragments with very similar affinities even though hexasaccharides introduce an additional glucose branch into an occluded protein surface cavity. Remarkably different thermodynamic binding signatures were found for different mutants; however, crystal structure analyses indicated that no major oligosaccharide or protein topology changes had occurred upon complex formation. This pointed to a solvent effect. Molecular dynamics simulations using a mobility-based approach revealed an extended network of solvent positions distributed over the entire oligosaccharide binding site. However, free energy calculations showed that a small water network inside the glucose-binding cavity had the most notable influence on the thermodynamic signature. The energy needed to displace water from the glucose binding pocket depended on the amino acid at the entrance, in agreement with the different amounts of enthalpy-entropy compensation found for introducing glucose into the pocket in the different mutants. Studies with small molecule drugs have shown before that a few active water molecules can control protein complex formation. HK620TSP oligosaccharide binding shows that similar fundamental principles also apply for glycans, where a small number of water molecules can dominate the thermodynamic signature in an extended binding site.


Drugs Repurposed as Antiferroptosis Agents Suppress Organ Damage, Including AKI, by Functioning as Lipid Peroxyl Radical Scavengers.
Ferroptosis, nonapoptotic cell death mediated by free radical reactions and driven by the oxidative degradation of lipids, is a therapeutic target because of its role in organ damage, including AKI. Ferroptosis-causing radicals that are targeted by ferroptosis suppressors have not been unequivocally identified. Because certain cytochrome P450 substrate drugs can prevent lipid peroxidation via obscure mechanisms, we evaluated their antiferroptotic potential and used them to identify ferroptosis-causing radicals. Using a cell-based assay, we screened cytochrome P450 substrate compounds to identify drugs with antiferroptotic activity and investigated the underlying mechanism. To evaluate radical-scavenging activity, we used electron paramagnetic resonance-spin trapping methods and a fluorescence probe for lipid radicals, NBD-Pen, that we had developed. We then assessed the therapeutic potency of these drugs in mouse models of cisplatin-induced AKI and LPS/galactosamine-induced liver injury. We identified various US Food and Drug Administration-approved drugs and hormones that have antiferroptotic properties, including rifampicin, promethazine, omeprazole, indole-3-carbinol, carvedilol, propranolol, estradiol, and thyroid hormones. The antiferroptotic drug effects were closely associated with the scavenging of lipid peroxyl radicals but not significantly related to interactions with other radicals. The elevated lipid peroxyl radical levels were associated with ferroptosis onset, and known ferroptosis suppressors, such as ferrostatin-1, also functioned as lipid peroxyl radical scavengers. The drugs exerted antiferroptotic activities in various cell types, including tubules, podocytes, and renal fibroblasts. Moreover, in mice, the drugs ameliorated AKI and liver injury, with suppression of tissue lipid peroxidation and decreased cell death. Although elevated lipid peroxyl radical levels can trigger ferroptosis onset, some drugs that scavenge lipid peroxyl radicals can help control ferroptosis-related disorders, including AKI.


Outcomes from the first multidrug-resistant tuberculosis programme in Kenya.
In March 2006, the first multidrug-resistant tuberculosis (MDR-TB) treatment programme was implemented in Kenya. To describe patients' treatment outcomes and adverse events. A retrospective case note review of patients started on MDR-TB treatment at two Médecins Sans Frontières-supported sites and the national referral hospital of Kenya was undertaken. Sites operated an ambulatory model of care. Patients were treated for a minimum of 24 months with at least 4-5 drugs for the intensive phase of treatment, including an injectable agent. Of 169 patients, 25.6% were human immunodeficiency virus (HIV) positive and 89.3% were culture-positive at baseline. Adverse events occurred in 67.4% of patients: 45.9% had nausea/vomiting, 43.9% electrolyte disturbance, 41.8% dyspepsia and 31.6% hypothyroidism. The median time to culture conversion was 2 months. Treatment outcomes were as follows: 76.6% success, 14.5% deaths, 8.3% lost to follow-up and 0.7% treatment failure. HIV-positive individuals (adjusted odds ratio [aOR] 3.51, 95% confidence interval [CI] 1.12-11.03) and women (aOR 2.73, 95%CI 1.01-7.39) had a higher risk of unfavourable outcomes, while the risk was lower in those with culture conversion at 6 months (aOR 0.11, 95%CI 0.04-0.32). In Kenya, where an ambulatory model of care is used for MDR-TB treatment, treatment success was high, despite high rates of HIV. Almost half of the patients experienced electrolyte disturbance and one third had hypothyroidism; this supports the view that systematic regular biochemical monitoring is needed in Kenya.


Evaluation of the effect of appropriate antimicrobial therapy on mortality associated with Acinetobacter nosocomialis bacteraemia.
Appropriate antimicrobial therapy is effective for severe infections caused by Acinetobacter baumannii, but efficacy for other Acinetobacter species remains to be established. The current study was designed to determine whether appropriate antimicrobial therapy reduces the mortality of patients with Acinetobacter nosocomialis bacteraemia. A 9-year retrospective study of 266 patients with monomicrobial A. nosocomialis bacteraemia was conducted at a large teaching hospital in Taiwan. Multivariable analysis was performed to evaluate the impact on 14-day mortality according to clinical characteristics, severity of disease and use of appropriate antimicrobial therapy. The influence of APACHE II score on the impact of appropriate antimicrobial therapy was analysed by including an interaction term. The overall 14-day mortality was 9.4%. Multivariable analysis revealed that APACHE II score was the only factor significantly associated with mortality (odds ratio, 1.18; 95% confidence interval, 1.11-1.25; p <0.001). Appropriate antimicrobial therapy was not associated with reduced mortality regardless of disease severity. In the subgroup analyses in patients with different clinical conditions, APACHE II score was consistently an independent factor for 14-day mortality, and appropriate antimicrobial therapy did not affect the mortality in any group. In conclusion, severity of disease, based on the APACHE II score, was the independent risk factor for 14-day mortality for patients with monomicrobial A. nosocomialis bacteraemia, even in different clinical conditions. In contrast, appropriate antimicrobial therapy did not reduce the 14-day mortality. The result highlighted a different effect of appropriate antimicrobial therapy on infections caused by two phenotypically undifferentiated Acinetobacter.


Perceived discrimination, group identification, and life satisfaction among multiracial people: a test of the rejection-identification model.
Like other racial minority groups, multiracial people face discrimination as a function of their racial identity, and this discrimination represents a threat to psychological well-being. Following the Rejection-Identification Model (RIM; Branscombe, Schmitt, & Harvey, 1999), we argue that perceived discrimination will encourage multiracial people to identify more strongly with other multiracials, and that multiracial identification, in turn, fosters psychological well-being. Thus, multiracial identification is conceptualized as a coping response that reduces the overall costs of discrimination on well-being. This study is the first to test the RIM in a sample of multiracial people. Multiracial participants' perceptions of discrimination were negatively related to life satisfaction. Consistent with the RIM, perceived discrimination was positively related to three aspects of multiracial group identification: stereotyping the self as similar to other multiracial people, perceiving people within the multiracial category as more homogenous, and expressing solidarity with the multiracial category. Self-stereotyping was the only aspect of group identification that mediated a positive relationship between perceived discrimination and life satisfaction, suggesting that multiracial identification's protective properties rest in the fact that it provides an collective identity where one "fits."


A copper-mediated oxidative N-cyanation reaction.
Copper-promoted N-cyanation of aliphatic sec-amine by CuCN is achieved via oxidative coupling. This procedure employs O2 as a clean oxidant. Notably, sulfoximines and 1,1,3,3-tetramethylguanidine also worked well in this procedure. Thus, it represents a key progress in the C-N bond formation reaction as well as in the cyanation reaction.


Efficacy of Autologous Platelet-Rich Plasma Combined With Ablative Fractional Carbon Dioxide Laser for Acne Scars: A Systematic Review and Meta-Analysis.
Acne scars are common and challenging clinical complications of acne vulgaris. Ablative fractional carbon dioxide (CO2) laser is a well-established treatment for acne scars; however, some postlaser adverse effects have been noted. Autologous platelet-rich plasma (PRP) can improve tissue regeneration. Several studies have investigated the efficacy of combination therapy of CO2 laser and PRP for acne scars. The authors sought to conduct a meta-analysis of the efficacy of PRP combined with ablative fractional CO2 laser for treating acne scars by examining clinical trial results. A systematic review was performed by searching PubMed, Embase, Cochrane Library, and Web of Science, and a meta-analysis was conducted to assess the clinical outcomes after combination therapy of PRP and ablative fractional CO2 laser compared with laser alone. We identified 4 eligible studies for the meta-analysis, including 3 randomized controlled trials. Our results demonstrated that clinical improvement after combination therapy was significantly higher than that after laser alone (odds ratio = 2.992, P = 0.001). Regarding major side effects, patients who underwent combination therapy experienced significantly shorter duration of crust compared with CO2 laser alone (standard mean difference = -1.140, P < 0.001); relatively shorter durations of erythema and edema were also noted after combination therapy. Furthermore, patient satisfaction rates were significantly higher after combination therapy than after laser alone (odds ratio = 3.169, P = 0.002). The combination of autologous PRP and ablative fractional CO2 laser has synergistic positive effects on the clinical outcomes for acne scars and can accelerate the recovery of laser-damaged skin.


Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes.
Traditionally, the biological fluorination of complex biological systems like proteins is achieved through substitution of canonical amino acids or addition of fluorinated amino acids in the context of the standard genetic code. Ribosomal translation of monofluorinated amino acids into proteins often yields structures with minimal local changes in the interior but, on the same time, results in large global effects on characteristic features of the biopolymers (such as dramatically changed activity profile or folding stability). This is due to the novel and unique local interactions delivered by fluorine atoms such as (i) increase in the covalent radii (ii) changed polarities; (iii) changed hydrogen bond acceptor ability; (iv) altered water solubility as well as water ↔ organic solvent energy transfer. On the other hand, the biological incorporation of tri- or global fluorinated amino acids (such as trifluoroleucine, triflurovaline, and their hexafluoro counterparts, fluoromethionine and trifluoronorleucine etc.) represents still a challenge, as the natural structural scaffolds are optimized for hydrocarbon during evolution but not for fluorocarbon cores. Future work will be focused on the re-design of existing or de novo design of novel protein scaffolds capable of accommodating such building blocks into functional biologically active proteins and proteomes in the context of the viable cells.


Molecular analysis of paramutant plants of Antirrhinum majus and the involvement of transposable elements.
Paramutation is observed when the Antirrhinum majus lines 44 and 53 are crossed. These two lines both have insertions at the nivea locus, which encodes chalcone synthase (chs). The allele niv-53 carries the transposable element Tam1 in the promoter region of the chs gene; niv-44 carries the element Tam2 within the gene. The Tam1 element has previously been extensively characterised. Here the Tam2 element is further characterised, and the arrangement of the nivea locus in paramutant plants is analysed. The complete sequence of Tam2, and that of a partial cDNA complementary to it, have been determined. The cDNA is probably transcribed from a different copy of Tam2 from that present at the nivea locus, and does not encode a functional protein. Genomic Southerns of F1 plants from the 53/44 cross show that no major rearrangements are consistently associated with paramutation at the nivea locus of A. majus. The isolation from a paramutant plant arising from a 53/44 cross of an allele (niv-4432) resulting from the excision of Tam2 is reported. The excision of Tam2 resulted in a 32 bp deletion of chs gene sequences. Plants homozygous for the new niv-4432 allele have white flowers and are still paramutagenic, demonstrating that Tam2 need not be present at the nivea locus for paramutation to occur. Different interactions between Tam1 and Tam2 are discussed, and a possible model for paramutation is presented.


Usefulness and Relevance of an eHealth Tool in Supporting the Self-Management of Chronic Obstructive Pulmonary Disease: Explorative Qualitative Study of a Cocreative Process.
New strategies are urgently needed to support self-management for people with chronic obstructive pulmonary disease (COPD) in primary care. The use of electronic health (eHealth) solutions is promising. However, there is a lack of knowledge about how such eHealth tools should be designed in order to be perceived as relevant and useful and meet the needs and expectations of the health professionals as well as people with COPD and their relatives. The objective of this study was to explore the aspects of an eHealth tool design and content that make it relevant and useful for supporting COPD-related self-management strategies from the perspective of health care professionals, people with COPD and their relatives, and external researchers. Data were collected during the development of an eHealth tool. A cocreation process was carried out with participants from two primary care units in northern Sweden and external researchers. Individual interviews were performed with health care professionals (n=13) as well as people with COPD (n=6) and their relatives (n=2), and focus group discussions (n=9) were held with all groups of participants. Data were analyzed using qualitative content analysis. The overarching theme, reinforcing existing support structures, reflects participant views that the eHealth tool needs to be directly applicable and create a sense of commitment in users. Moreover, participants felt that the tool needs to fit with existing routines and contexts and preferably should not challenge existing hierarchies between health care professionals and people with COPD. Important content for health care professionals and people with COPD included knowledge about self-management strategies. Videos were regarded as the most effective method for communicating such knowledge. The cocreation in the development process enables participant perspectives and priorities to be built into the eHealth tool. This is assumed to contribute to a tool that is useful and relevant and, therefore, adopted into clinical practice and everyday life. Findings from this study can inform the development of eHealth tools for people with COPD in other contexts, as well as the development of eHealth tools for self-management support of other chronic diseases.


Coverage and timeliness of vaccination and the validity of routine estimates: Insights from a vaccine registry in Kenya.
The benefits of childhood vaccines are critically dependent on vaccination coverage. We used a vaccine registry (as gold standard) in Kenya to quantify errors in routine coverage methods (surveys and administrative reports), to estimate the magnitude of survivor bias, contrast coverage with timeliness and use both measures to estimate population immunity. Vaccination records of children in the Kilifi Health and Demographic Surveillance System (KHDSS), Kenya were combined with births, deaths, migration and residence data from 2010 to 17. Using inverse survival curves, we estimated up-to-date and age-appropriate vaccination coverage, calculated mean vaccination coverage in infancy as the area under the inverse survival curves, and estimated the proportion of fully immunised children (FIC). Results were compared with published coverage estimates. Risk factors for vaccination were assessed using Cox regression models. We analysed data for 49,090 infants and 48,025 children aged 12-23 months in 6 birth cohorts and 6 cross-sectional surveys respectively, and found 2nd year of life surveys overestimated coverage by 2% compared to birth cohorts. Compared to mean coverage in infants, static coverage at 12 months was exaggerated by 7-8% for third doses of oral polio, pentavalent (Penta3) and pneumococcal conjugate vaccines, and by 24% for the measles vaccine. Surveys and administrative coverage also underestimated the proportion of the fully immunised child by 10-14%. For BCG, Penta3 and measles, timeliness was 23-44% higher in children born in a health facility but 20-37% lower in those who first attended during vaccine stock outs. Standard coverage surveys in 12-23 month old children overestimate protection by ignoring timeliness, and survivor and recall biases. Where delayed vaccination is common, up-to-date coverage will give biased estimates of population immunity. Surveys and administrative methods also underestimate FIC prevalence. Better measurement of coverage and more sophisticated analyses are required to control vaccine preventable diseases.


Development of an innovative technology to segment luminal borders of intravascular ultrasound image sequences in a fully automated manner.
Although intravascular ultrasound (IVUS) is the commonest intravascular imaging modality, it still is inefficient for clinical use as it requires laborious manual analysis. This study demonstrates the feasibility of a near real-time fully automated technology for accurate identification, detection, and quantification of luminal borders in intravascular images. This technology uses a combination of the novel approaches of a self-tuning engine, dynamic and static masking systems, radar-wise scan, and contour correction cycle method. The performance of the computer algorithm developed based on this technology was tested on a sequence of IVUS and True Vessel Characterization (TVC) images obtained from the left anterior descending (LAD) artery of 6 patients with coronary artery disease. The accuracy of the algorithm was evaluated by comparing luminal borders traced manually with those detected automatically. The processing time of the developed algorithm was also tested on a Dell laptop with an Intel Core i7-8750H Processor (4.1 GHz with 6 cores, 9 MB Cache). Linear regression and Bland-Altman analyses indicated high correlation between manual and automatic tracings (Y = 0.80 × X+1.70, R2 = 0.88 & 0.67 ± 1.31 (bias±SD)). Whereas analysis of 2000 IVUS images using one CPU core with a 30% load took 23.12 min, the same analysis using six CPU cores with 90% load took 1.0 min. The performance, accuracy, and speed of the presented state-of-the-art technology demonstrates its capacity for use in clinical settings.