Reactive γ-ketoaldehydes promote protein misfolding and preamyloid oligomer formation in rapidly-activated atrial cells.
Rapid activation causes remodeling of atrial myocytes resembling that which occurs in experimental and human atrial fibrillation (AF). Using this cellular model, we previously observed transcriptional upregulation of proteins implicated in protein misfolding and amyloidosis. For organ-specific amyloidoses such as Alzheimer's disease, preamyloid oligomers (PAOs) are now recognized to be the primary cytotoxic species. In the setting of oxidative stress, highly-reactive lipid-derived mediators known as γ-ketoaldehydes (γ-KAs) have been identified that rapidly adduct proteins and cause PAO formation for amyloid β1-42 implicated in Alzheimer's. We hypothesized that rapid activation of atrial cells triggers oxidative stress with lipid peroxidation and formation of γ-KAs, which then rapidly crosslink proteins to generate PAOs. To investigate this hypothesis, rapidly-paced and control, spontaneously-beating atrial HL-1 cells were probed with a conformation-specific antibody recognizing PAOs. Rapid stimulation of atrial cells caused the generation of cytosolic PAOs along with a myocyte stress response (e.g., transcriptional upregulation of Nppa and Hspa1a), both of which were absent in control, unpaced cells. Rapid activation also caused the formation of superoxide and γ-KA adducts in atriomyocytes, while direct exposure of cells to γ-KAs resulted in PAO production. Increased cytosolic atrial natriuretic peptide (ANP), and the generation of ANP oligomers with exposure to γ-KAs and rapid atrial HL-1 cell stimulation, strongly suggest a role for ANP in PAO formation. Salicylamine (SA) is a small molecule scavenger of γ-KAs that can protect proteins from modification by these reactive compounds. PAO formation and transcriptional remodeling were inhibited when cells were stimulated in the presence of SA, but not with the antioxidant curcumin, which is incapable of scavenging γ-KAs. These results demonstrate that γ-KAs promote protein misfolding and PAO formation as a component of the atrial cell stress response to rapid activation, and they provide a potential mechanistic link between oxidative stress and atrial cell injury.


Silver acetate for smoking cessation.
Silver acetate produces an unpleasant taste when combined with cigarettes, thereby producing an aversive stimulus. It has been marketed in various forms with the aim of extinguishing the urge to smoke, by pairing the urge with an unpleasant stimulus. The aim of this review was to determine the effectiveness of silver acetate products (gum, lozenge, spray) in promoting smoking cessation. We searched the Cochrane Tobacco Addiction Group specialised trials register. Most recent search was in July 2012. We included randomised trials of silver acetate for smoking cessation with reports of smoking status at least six months after the beginning of treatment. We extracted data in duplicate on the type of subjects, the dose and form of silver acetate, the outcome measures, method of randomisation, and completeness of follow-up.The main outcome measure was biochemically validated abstinence from smoking after at least six months follow-up in patients smoking at baseline. Subjects lost to follow-up were counted as continuing smokers. Where appropriate, we performed meta-analysis using a fixed effects model. Two studies provided long-term follow-up data on patients randomised to silver acetate or placebo. In one of these studies, there was a third arm, randomised to 2mg nicotine gum. The pooled risk ratio for quitting for silver acetate vs placebo was 1.04 (95% confidence interval 0.69 to 1.57). Existing trials show little evidence for a specific effect of silver acetate in promoting smoking cessation. The confidence intervals for the ratio are quite wide. However, the upper limit of the confidence intervals for a positive effect equates to an absolute increase in the smoking cessation rate of about 4%. Any effect of this agent is therefore likely to be smaller than nicotine replacement therapy. The lack of effect of silver acetate may reflect poor compliance with a treatment whose rationale is to create an unpleasant stimulus.


Interaction of the CNC-bZIP factor TCF11/LCR-F1/Nrf1 with MafG: binding-site selection and regulation of transcription.
We have previously shown that the widely expressed human transcription factor TCF11/LCR-F1/Nrf1 interacts with small Maf proteins and binds to a subclass of AP1-sites. Such sites are required for beta-globin 5' DNase I hypersensitive site 2 enhancer activity, erythroid porphobilinogen deaminase inducibility, hemin responsiveness by heme-oxygenase 1 and expression of the gene NAD(P)H:quinone oxidoreductase1. Here we report the optimal DNA-binding sequences for TCF11/LCR-F1/Nrf1 alone and as a heterodimer with MafG, identified by using binding-site selection. The heterodimer recognises a 5'-TGCTgaGTCAT-3' binding-site that is identical to the established NF-E2-site, the antioxidant response element and the heme-responsive element while the binding specificity of the homomer is less stringent. To investigate the activity of TCF11 through this selected site, both alone and in the presence of MafG, we have used a transient transfection assay. TCF11 alone activates transcription while MafG alone acts as a repressor. When co-expressed, MafG interferes with TCF11 transactivation in a dose dependent manner. This indicates that MafG protein, which heterodimerises efficiently with TCF11 in vitro (the heterodimer having a higher affinity for DNA than TCF11 alone), does not co-operate with TCF11 in transactivating transcription. We propose that since both these factors are widely expressed, they may act together to contribute to the negative regulation of this specific target site. Efficient positive regulation by TCF11 may require alternative partners with perhaps more restricted expression patterns.


Precollegiate Knee Surgery Predicts Subsequent Injury Requiring Surgery in NCAA Athletes.
The effect of precollegiate orthopaedic surgery on injury risk in the elite collegiate athlete is unknown. To (1) assess the relationship between precollegiate surgery and subsequent injury requiring surgery in National Collegiate Athletic Association (NCAA) Division I athletes at a single institution and (2) compare the risk of subsequent surgery in the ipsilateral versus contralateral extremity in those with a history of precollegiate surgery. Cohort study; Level of evidence, 3. A retrospective chart review was performed of all athletes who began participation from 2003 to 2009 until completion of eligibility. Athletes who received orthopaedic surgery in college were identified through the Sports Injury Monitoring System and were cross-referenced with medical records. The risk of orthopaedic surgery was evaluated using multivariate Cox and Poisson regression models, with sex and sport as additional covariates. Risk of subsequent surgery in the ipsilateral versus contralateral extremity was compared using Kaplan-Meier survival estimates and Cox proportional hazards regression. Hazard ratios (HRs) and rate ratios (RRs) with corresponding 95% confidence intervals were used to compare groups. In total, 1141 athletes were identified for analysis. Of these, 186 athletes (16.3%) had a history of precollegiate orthopaedic surgery. There were 261 documented intracollegiate orthopaedic surgeries in 181 athletes (15.9%). Precollegiate knee surgery was an independent predictor of orthopaedic surgery (HR, 1.85; 95% CI, 1.16-2.83) in college. When examining only surgeries resulting from acute or primary injuries, precollegiate knee surgery was an independent predictor of primary knee injury requiring surgery in college (HR, 4.45; 95% CI, 2.51-7.59). Athletes with a history of precollegiate surgery were more susceptible to subsequent surgery in their ipsilateral extremity compared with their other extremities (HR, 1.89; 95% CI, 1.03-3.53). In contrast, there was no additional risk of receiving subsequent surgery in the contralateral extremity (P = .54). Precollegiate knee surgery in the Division I athlete is associated with subsequent injury requiring surgery in college. Athletes with a history of precollegiate surgery are at higher risk of subsequent surgery in their ipsilateral extremity compared with other extremities.


Photophysical properties of 2-Phenylindole in poly (vinyl alcohol) film at room temperature. Enhanced phosphorescence anisotropy with direct triplet state excitation.
We report the spectral properties of 2-Phenylindole (2PI) embedded in rigid poly (vinyl alcohol) (PVA) film. The 2PI in PVA film shows relatively strong and structured fluorescence with a maximum at 370 nm and surprisingly strong room temperature phosphorescence with an emission maximum of about 500 nm. The dye is highly immobilized in the polymer matrix, thus presenting high fluorescence anisotropy in an isotropic film of about 0.3 at room temperature. The 2-Phenylindole phosphorescence excited in the usual way through the electronic singlet state excitation (S0 → S1 absorption) results in a very low, near zero anisotropy. We now report that we can directly excite the dye to the triplet state T1 and observe high phosphorescence anisotropy similar to the fluorescence anisotropy. The extinction coefficient for S0 → T1 absorption in the PVA matrix is unusually high- only about 3 orders of magnitude lower than S0 → S1 absorption. We consider this direct excitation to indole's triplet state a very significant finding that may lead to many practical applications. The unusually long-wavelength of excitation around 400 nm, much above typical UV absorption, results in a high phosphorescence anisotropy. This provides a new way to study rotational motion of larger biological objects in the microsecond time scale not accessible through typical fluorescence studies.


RETRACTED: Secondary siRNAs result from unprimed RNA synthesis and form a distinct class.
In Caenorhabditis elegans, an effective RNA interference (RNAi) response requires the production of secondary short interfering RNAs (siRNAs) by RNA-directed RNA polymerases (RdRPs). We cloned secondary siRNAs from transgenic C. elegans lines expressing a single 22-nucleotide primary siRNA. Several secondary siRNAs start a few nucleotides downstream of the primary siRNA, indicating that non-RISC (RNA-induced silencing complex)-cleaved mRNAs are substrates for secondary siRNA production. In lines expressing primary siRNAs with single-nucleotide mismatches, secondary siRNAs do not carry the mismatch but contain the nucleotide complementary to the mRNA. We infer that RdRPs perform unprimed RNA synthesis. Secondary siRNAs are only of antisense polarity, carry 5' di- or triphosphates, and are only in the minority associated with RDE-1, the RNAi-specific Argonaute protein. Therefore, secondary siRNAs represent a distinct class of small RNAs. Their biogenesis depends on RdRPs, and we propose that each secondary siRNA is an individual RdRP product.


Phagocyte function and cytokine production in community acquired pneumonia.
It is possible that many deaths from pneumonia may involve the generation of inflammatory mediators and tissue damage by activated phagocytes. To test this hypothesis phagocyte function, plasma levels of interleukin 6 (IL-6), tumour necrosis factor alpha (TNF alpha), and soluble interleukin 2 receptor (IL-2R), disease severity, and outcome have been examined in 46 patients with community acquired pneumonia. Polymorphonuclear leucocyte (PMNL) and monocyte function were measured daily by chemiluminescence in these patients during the first week of admission, and cytokine levels were subsequently determined by ELISA. A series of 61 healthy individuals were used as a control group for the chemiluminescence results. There was evidence of phagocyte, particularly PMNL, activation on admission in 76% of the patients. Most patients (86%) also had raised IL-2R levels on admission. IL-6 and unbound TNF alpha were present in 23% and 41% of patients at varying times during the course of the disease. There was little correlation between measurements of cytokine or phagocyte levels and outcome or indicators of disease severity, although this may be because of the small number of patients included in this preliminary study. These results are consistent with the hypothesis that activated phagocyte function and raised levels of circulating cytokines may contribute to the pathogenesis of community acquired pneumonia. There are striking similarities in this respect between pneumonia, adult respiratory distress syndrome, and sepsis.


Influence of oily vehicles on fetal testis and lipid profile of rats exposed to di-butyl phthalate.
It has been hypothesized that oils containing high levels of omega-3 polyunsaturated fatty acids, such as canola and fish oil, could counteract some of the adverse effects induced by phthalates. In the present study, the influence of different oily vehicles on di-butyl phthalate (DBP)-induced testicular toxicity and lipid profile was investigated. Pregnant Wistar rats were treated by oral gavage from gestation days 13 to 20 with DBP (500 mg/kg/day) diluted in three different vehicles: corn, canola or fish oil. Male fetuses were analyzed on gestation day 20. DBP exposure lowered intratesticular testosterone levels and anogenital distance, regardless of the vehicle used. The percentage of seminiferous cords containing multinucleated gonocytes and cord diameter was increased in DBP-exposed groups, compared with vehicle controls, with no difference between the three DBP-exposed groups. Clustering of Leydig cells was seen in all DBP groups. Lipid profile indicated that administration of canola and fish oil can increase the content of omega-3 fatty acids in rat testis. However, content of omega-3 was diminished in DBP-treated groups. Overall, our results indicate that different oily vehicles did not alter fetal rat testicular toxicity induced by a high DBP dose.


Patent foramen ovale, ischemic stroke and migraine: systematic review and stratified meta-analysis of association studies.
Observational data have reported associations between patent foramen ovale (PFO), cryptogenic stroke and migraine. However, randomized trials of PFO closure do not demonstrate a clear benefit either because the underlying association is weaker than previously suggested or because the trials were underpowered. In order to resolve the apparent discrepancy between observational data and randomized trials, we investigated associations between (1) migraine and ischemic stroke, (2) PFO and ischemic stroke, and (3) PFO and migraine. Eligibility criteria were consistent; including all studies with specifically defined exposures and outcomes unrestricted by language. We focused on studies at lowest risk of bias by stratifying analyses based on methodological design and quantified associations using fixed-effects meta-analysis models. We included 37 studies of 7,686 identified. Compared to reports in the literature as a whole, studies with population-based comparators showed weaker associations between migraine with aura and cryptogenic ischemic stroke in younger women (OR 1.4; 95% CI 0.9-2.0; 1 study), PFO and ischemic stroke (HR 1.6; 95 CI 1.0-2.5; 2 studies; OR 1.3; 95% CI 0.9-1.9; 3 studies), or PFO and migraine (OR 1.0; 95% CI 0.6-1.6; 1 study). It was not possible to look for interactions or effect modifiers. These results are limited by sources of bias within individual studies. The overall pairwise associations between PFO, cryptogenic ischemic stroke and migraine do not strongly suggest a causal role for PFO. Ongoing randomized trials of PFO closure may need larger numbers of participants to detect an overall beneficial effect.


Genomic characterisation of Leptospira inadai serogroup Lyme isolated from captured rat in Brazil and comparative analysis with human reference strain.
Leptospira inadai is classified as a species of the Leptospira intermediate group that has been poorly studied due to its apparent insignificance to human and animal health. Nevertheless, over the last two decades the species has been described in human cases in India and in carrier animals in Ecuador. Here, we present the first identification and genomic characterisation of L. inadai serogroup Lyme isolated from captured rodent in Brazil. Even though the M34/99 strain was not pathogenic for hamsters, it was able to establish renal colonisation. The M34/99 strain presented high similarity with L. inadai serogroup Lyme human reference indicating that animal strain could also infect humans, although it does not represent high risk of severe disease. An extrachromosomal sequence was also identified in M34/99 strain and presented high identity with previously described L. inadai phage LinZ_10, suggesting that phage-like extrachromosomal sequence may be another feature of this understudied species.


Adaptive and non-adaptive models of depression: A comparison using register data on antidepressant medication during divorce.
Divorce is associated with an increased probability of a depressive episode, but the causation of events remains unclear. Adaptive models of depression propose that depression is a social strategy in part, whereas non-adaptive models tend to propose a diathesis-stress mechanism. We compare an adaptive evolutionary model of depression to three alternative non-adaptive models with respect to their ability to explain the temporal pattern of depression around the time of divorce. Register-based data (304,112 individuals drawn from a random sample of 11% of Finnish people) on antidepressant purchases is used as a proxy for depression. This proxy affords an unprecedented temporal resolution (a 3-monthly prevalence estimates over 10 years) without any bias from non-compliance, and it can be linked with underlying episodes via a statistical model. The evolutionary-adaptation model (all time periods with risk of divorce are depressogenic) was the best quantitative description of the data. The non-adaptive stress-relief model (period before divorce is depressogenic and period afterwards is not) provided the second best quantitative description of the data. The peak-stress model (periods before and after divorce can be depressogenic) fit the data less well, and the stress-induction model (period following divorce is depressogenic and the preceding period is not) did not fit the data at all. The evolutionary model was the most detailed mechanistic description of the divorce-depression link among the models, and the best fit in terms of predicted curvature; thus, it offers most rigorous hypotheses for further study. The stress-relief model also fit very well and was the best model in a sensitivity analysis, encouraging development of more mechanistic models for that hypothesis.


Lp82 is the dominant form of calpain in young mouse lens.
The purpose of this study was to characterize Lp82 calpain in normal mouse. Lp82 is a lens-specific, calcium-activated isozyme from the calpain super family of cysteine proteases (EC 34.22.17). RT-PCR and molecular cloning were performed on total RNA from 12 day-old mice. Lp82 and m-calpain protein levels and proteolytic activities in lenses were measured by casein zymography, immunoblotting, and ELISA after partial purification by DEAE-HPLC. The 2334-bp cDNA encoding for mouse Lp82 contained a single large open reading frame encoding a protein of 709 amino acid residues with a calculated molecular weight of 82.2 kDa and a predicted pI of 5.8. The amino acid sequence of mouse lens Lp82 was 99% homologous to rat lens Lp82. As in rat, mouse lens Lp82 showed a unique N -terminus and deletion of the IS1 and IS2 regions. In contrast to rat, Lp82 was the dominant calpain in young mouse lens. Lp82 was lens-specific, and the lens nucleus contained the highest specific activity of Lp82 and very little m-calpain. Endogenous Lp82 in lens soluble proteins was activated by addition of calcium and caused limited proteolysis of crystallins even in the presence of large amounts of recombinant domain I from the natural calpain inhibitor calpastatin. Loss of Lp82 protein accompanied aging of mouse lens. Lp82 may be responsible for a major portion of crystallin proteolysis occurring during normal lens development and maturation, or during cataract formation in young mice.


Clinical anatomy of the maxillary nerve block in pediatric patients.
Anatomical landmarks in children are mostly extrapolated from studies in adults. Despite this, complex regional anesthetic procedures are frequently performed on pediatric patients. Sophisticated imaging techniques are available but the exact position, course and/or relationships of the structures are best understood with appropriate anatomical dissections. Maxillary nerve blocks are being used for peri-operative analgesia after cleft palate repair in infants. However, the best approach for blocking the maxillary nerve in pediatric patients has yet to be established. To determine the best approach for blocking the maxillary nerve within the pterygopalatine fossa. In an attempt to define an optimal approach for maxillary nerve block in this age group three approaches were simulated and compared on 10 dried pediatric skulls as well as 30 dissected pediatric cadavers. The needle course, including depth and angles, to block the maxillary nerve, as it exits the skull at the foramen rotundum within the pterygopalatine fossa, was measured and compared. Two groups were studied: Group 1 consisted of skulls and cadavers of neonates (0-28 days after birth) and Group 2 consisted of skulls and cadavers from 28 days to 1 year after birth. No statistically significant difference (P > 0.05) was found between the left and right side of each skull or cadaver. Only technique B, the suprazygomatic approach from the frontozygomatic angle towards the pterygopalatine fossa, exhibited no statistical significance (P > 0.05) when other measurements made on the skulls and cadavers were compared. Technique A, a suprazygomatic approach from the midpoint on the lateral border of the orbit, as well as technique C, an infrazygomatic approach with an entry at a point on a vertical line extending along the lateral orbit wall, showed statistical significant differences when measurements of the skulls and cadavers were compared. On the basis of these findings technique B produces the most consistent data for age groups 1 and 2 and supports the clinical findings recently reported.


Regional Amyloid-β Load and White Matter Abnormalities Contribute to Hypometabolism in Alzheimer's Dementia.
We investigated the association between amyloid-β deposition and white matter (WM) integrity as a determinant of brain glucose hypometabolism across the Alzheimer's disease (AD) spectrum. We assessed ninety-six subjects (27 cognitively normal, 49 mild cognitive impairment, and 20 AD dementia) who underwent [18F]FDG and [18F]Florbetapir positron emission tomography (PET) as well as magnetic resonance imaging (MRI) with diffusion tensor imaging. Among the regions with reduced fractional anisotropy (FA) in the AD group, we selected a voxel of interest in the angular bundle bilaterally for subsequent analyses. Using voxel-based interaction models at voxel level, we tested whether the regional hypometabolism is associated with FA in the angular bundle and regional amyloid-β deposition. In the AD patients, [18F]FDG hypometabolism in the striatum, mesiobasal temporal, orbitofrontal, precuneus, and cingulate cortices were associated with the interaction between high levels of [18F]Florbetapir standard uptake value ratios (SUVR) in these regions and low FA in the angular bundle. We found that the interaction between, rather than the independent effects of, high levels of amyloid-β deposition and WM integrity disruption determined limbic hypometabolism in patients with AD. This finding highlights a more integrative model for AD, where the interaction between partially independent processes determines the glucose hypometabolism.


The experiences of sexually assaulted people attending Saint Mary's Sexual Assault Referral Centre for a forensic medical examination.
This study explores the experiences of people who have attended Saint Mary's Sexual Assault Referral Centre (SARC) for a forensic medical examination (FME). Within the United Kingdom, SARCs support complainants following a sexual assault, delivering specialised care and gathering medico-legal evidence for court proceedings. To date, there has been limited research evaluating SARCs responses towards complainants. 863 Feedback and Evaluation forms, from a three-year period, completed by clients who had accessed Saint Mary's SARC's forensic medical examination service were evaluated. Descriptive statistical analysis found a large majority of clients were 'very satisfied' with the crisis worker, forensic physician and police. Content analysis of the free text responses found more nuanced experiences, which impacted clients overall experience within the SARC. These comments were split into two themes, 'Compliments' and 'Suggestions for improvement'. There were 404 comments focusing on compliments of the service and the staff. Compliments included messages of thanks, with praise for professionalism of the staff and importance of the service. Clients noted in particular that the FME service was delivered in challenging circumstances i.e. the immediate aftermath of a sexual assault. There were 34 comments which made suggestions for improvements. These suggestions focused on pragmatic and logistic issues.


Rapid detection of fluoroquinolone-resistant and heteroresistant Mycobacterium tuberculosis by use of sloppy molecular beacons and dual melting-temperature codes in a real-time PCR assay.
Fluoroquinolones (FQ) are important second-line drugs to treat tuberculosis; however, FQ resistance is an emerging problem. Resistance has been mainly attributed to mutations in a 21-bp region of the Mycobacterium tuberculosis gyrA gene, often called the quinolone resistance-determining region (QRDR). We have developed a simple, rapid, and specific assay to detect FQ resistance-determining QRDR mutations. The assay amplifies the M. tuberculosis gyrA QRDR in an asymmetrical PCR followed by probing with two sloppy molecular beacons (SMBs) spanning the entire QRDR. Mutations are detected by melting temperature (T(m)) shifts that occur when the SMBs bind to mismatched sequences. By testing DNA targets corresponding to all known QRDR mutations, we found that one or both of the SMBs produced a T(m) shift of at least 3.6°C for each mutation, making mutation detection very robust. The assay was also able to identify mixtures of wild-type and mutant DNA, with QRDR mutants identified in samples containing as little as 5 to 10% mutant DNA. The assay was blindly validated for its ability to identify the QRDR mutations on DNA extracted from clinical M. tuberculosis strains. Fifty QRDR wild-type samples, 34 QRDR mutant samples, and 8 heteroresistant samples containing mixtures of wild-type and mutant DNA were analyzed. The results showed 100% concordance to conventional DNA sequencing, including a complete identification of all of the mixtures. This SMB T(m) shift assay will be a valuable molecular tool to rapidly detect FQ resistance and to detect the emergence of FQ heteroresistance in clinical samples from tuberculosis patients.


Dysregulation in IGF-1R, FGFR4 and βKlotho signaling in patients with medullary thyroid cancer.
Medullary thyroid cancer (MTC) is a relatively rare thyroid neoplasm derived from neuroendocrine C cells which secrete calcitonin. αKlotho (αKL) and βKlotho (βKL) are transmembrane proteins which modulate different signaling systems, such as endocrine FGFs and IGF1 pathways. Dysregulation of the FGF19/FGFR4/βKL and IGF-1/IGF-1R/αKL signaling axes has been implicated in the pathogenesis of several cancers. However, their role in the pathogenesis of MTC has not been determined. The aim of this study was to assess αKL, βKL, FGF19, IGF-1, FGFR4, and IGF-1R concentrations in a group of 11 patients with medullary thyroid cancer (MTC). The control group consisted of 20 healthy volunteers. Serum concentrations of these factors were measured using specific ELISA methods. Significantly lower concentrations of βKL and higher concentrations of FGFR4 and IGF-1R were found in patients with MTC as compared to controls. Our results indicate that a disrupted signaling pathway for βKL, FGFR4 and IGF-1R may play a role in the development of medullary thyroid cancers. However, further studies are required to confirm these findings and to use this knowledge in clinical practice.


Transplantation of human menstrual blood progenitor cells improves hyperglycemia by promoting endogenous progenitor differentiation in type 1 diabetic mice.
Recently, a unique population of progenitor cells was isolated from human menstrual blood. The human menstrual blood progenitor cells (MBPCs) possess many advantages, such as the noninvasive acquisition procedure, broad multipotency, a higher proliferative rate, and low immunogenicity, and have attracted extensive attention in regenerative medicine. Preclinical studies to test the safety and efficacy of MBPCs have been underway in several animal models. However, relevant studies in type 1 diabetes mellitus (T1DM) have not yet been proceeded. Herein, we studied the therapeutic effect of MBPCs and the mechanism of β-cell regeneration after MBPC transplantation in the T1DM model. Intravenous injection of MBPCs can reverse hyperglycemia and weight loss, prolong lifespan, and increase insulin production in diabetic mice. Histological and immunohistochemistry analyses indicated that T1DM mice with MBPC transplantation recovered islet structures and increased the β-cell number. We further analyzed in vivo distribution of MBPCs and discovered that a majority of MBPCs migrated into damaged pancreas and located at the islet, duct, and exocrine tissue. MBPCs did not differentiate into insulin-producing cells, but enhanced neurogenin3 (ngn3) expression, which represented endocrine progenitors that were activated. Ngn3(+) cells were not only in the ductal epithelium, but also in the islet and exocrine tissue. We analyzed a series of genes associated with the embryonic mode of β-cell development by real-time polymerase chain reaction and the results showed that the levels of those gene expressions all increased after cell transplantation. According to the results, we concluded that MBPCs stimulated β-cell regeneration through promoting differentiation of endogenous progenitor cells.


Protection against HEMA-Induced Mitochondrial Injury In Vitro by Nrf2 Activation.
Dental resin monomers such as 2-hydroxyethyl methacrylate (HEMA) disturb vital cell functions and induce mitochondrial intrinsic apoptosis via generation of oxidative stress. Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the gene expression of antioxidative enzymes and plays a crucial role in the maintenance of cellular redox equilibrium and mitochondrial homeostasis. The present study investigated the functional significance of Nrf2 in cellular response toward HEMA. It was found that HEMA stimulation promoted nuclear translocation of Nrf2 and increased Nrf2 and heme oxygenase-1 (HO-1) expression, which was further enhanced by Nrf2 activator tert-butylhydroquinone (tBHQ), but suppressed by Nrf2 inhibitor ML385. Pretreatment of primary human dental pulp cells (hDPCs) with tBHQ protected the cells from HEMA-induced oxidative injury (increased reactive oxygen species production and apoptosis) and mitochondrial impairment (morphological alterations, decreased ATP production, suppressed oxidative phosphorylation activity, depolarization of mitochondrial membrane potential, and disrupted electron transport chain). In contrast, pretreatment with ML385 increased cell sensitivity to these injurious processes. This protective effect on mitochondria could be related to peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α)/nuclear respiratory factor 1 (NRF1) pathway. These results contribute to the understanding of the function of Nrf2 and the development of novel therapies to counteract the adverse effects of dental resin monomers.


Temperature sensitive supramolecular self assembly of per-6-PEO-β-cyclodextrin and α,ω-di-(adamantylethyl)poly(N-isopropylacrylamide) in water.
The host/guest interactions in water of a star polymer consisting of a β-cyclodextrin (β-CD) core bearing six poly(ethylene oxide) arms linked to the C6 positions of β-CD (β-CD-PEO7, Mn 5000 g mol(-1)) and α,ω-di-(adamantylethyl)poly(N-isopropylacrylamide) (Ad-PNIPAM-12K, Mn 12,000 g mol(-1)) were studied by 1D and 2D (1)H and (13)C NMR spectroscopy, isothermal calorimetry (ITC), and light scattering (LS). In cold water (T < 26 °C) supramolecular "dumbbell" assemblies, consisting of PNIPAM chains with β-CD/Ad inclusion complexes at each end, formed viaβ-CD-insertion of the terminal Ads through the β-CD secondary face. Light scattering, microcalorimetry (DSC), and DOSY NMR studies indicated that mixed aqueous solutions of β-CD-PEO7 and Ad-PNIPAM-12K undergo a reversible heat-induced phase transition at ∼32 °C, accompanied by a release of a fraction of the Ad-bound β-CD-PEO7 into bulk solution and the formation of aggregated Ad-PNIPAM-12K stabilized by a β-CD-PEO7 shell.


When attractiveness demands longer looks: the effects of situation and gender.
We investigated how aesthetics guides our exploration of the environment. We embedded attractive and nonattractive faces into complex, real-world scenes and measured eye movements during scene viewing. We examined whether attractive faces would elicit longer looks, which would suggest that the aesthetic response orients people toward the rewarding and pleasing aspects of the environment. Experiment 1 showed that mean fixation, mean first fixation, and total fixation durations were longer to attractive faces, and fixations were longest to female faces and by female perceivers. In Experiment 2, we examined whether these effects of attractiveness are sensitive to situational factors. When perceivers were subjected to a threat or social approach manipulation prior to viewing the scenes, we confirmed specific hypotheses concerning the two manipulations. In accordance with the hypothesis that males have higher aggression potential than females, there were no differences in fixation durations between attractive and nonattractive male faces in the threat condition. On the other hand, in the social approach condition, both female and male attractive faces received longer looks. These results suggest that the aesthetic response orients people not only to the pleasing aspects of their environment, but also to those features that are adaptively relevant.


Phenotypic characterization of patient dengue virus isolates in BALB/c mice differentiates dengue fever and dengue hemorrhagic fever from dengue shock syndrome.
Dengue virus (DENV) infection is the most common arthropod-borne viral disease in man and there are approximately 100 million infections annually. Despite the global burden of DENV infections many important questions regarding DENV pathogenesis remain unaddressed due to the lack of appropriate animal models of infection and disease. A major problem is the fact that no non-human species naturally develop disease similar to human dengue fever (DF) or dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Apart from other risk factors for severe dengue such as host genetics and secondary infection with a heterologous DENV, virus virulence is a risk factor that is not well characterized. Three clinical DENV-1 isolates from Cambodian patients experiencing the various forms of dengue disease (DF, DHF, and DSS) were inoculated in BALB/c mice at three different concentrations. The DENV-1 isolates had different organ and cell tropism and replication kinetics. The DENV-1 isolate from a DSS patient infected the largest number of mice and was primarily neurotropic. In contrast, the DENV-1 isolates from milder clinical dengue cases infected predominantly lungs and liver, and to a lesser extent brain. In addition, infection with the DENV isolate derived from a DSS patient persisted for more than two weeks in a majority of mice compared to the other DENV-1 isolates that peaked during the first week. These results confirm the in vitro findings of the same DENV-1 isolates, that showed that the isolate derived from a DSS patient can be distinguished based on phenotypic characteristics that differ from the isolates derived from a DF and DHF case 1. We observed in this study that the DSS virus isolate persist longer in vivo with extensive neuroinvasion in contrast to the other DENV-1 isolates originating in milder human cases. Genomic characterization of the three clinical isolates identified six amino acid substitutions unique for the DSS isolates that were located both in structural genes (M and E) and in non-structural genes (NS1, NS3, and NS5). The characterization of these clinically distinct DENV-1 isolates highlight that DENVs within the same genotype may have different in vivo phenotypes. • Clinical DENV-1 isolates have different organ tropism in BALB/c mice.• The isolate from a DSS patient is primarily neurotropic compared to the other isolates.• The DENV-1 isolates have different in vivo replication kinetics.• The isolate from a DSS patient persists longer compared to the other isolates.• These phenotypic differences confirm our earlier in vitro findings with the same DENV-1 isolates. Thus, DENVs within the same serotype and genotype may differ enough to affect clinical conditions in vivo.


Glioma: Application of histogram analysis of pharmacokinetic parameters from T1-weighted dynamic contrast-enhanced MR imaging to tumor grading.
The usefulness of pharmacokinetic parameters for glioma grading has been reported based on the perfusion data from parts of entire-tumor volumes. However, the perfusion values may not reflect the entire-tumor characteristics. Our aim was to investigate the feasibility of glioma grading by using histogram analyses of pharmacokinetic parameters including the volume transfer constant, extravascular extracellular space volume per unit volume of tissue, and blood plasma volume per unit volume of tissue from T1-weighted dynamic contrast-enhanced perfusion MR imaging. Twenty-eight patients (14 men, 14 women; mean age, 49.75 years; age range, 25-72 years) with histopathologically confirmed gliomas (World Health Organization grade II, n = 7; grade III, n = 8; grade IV, n = 13) were examined before surgery or biopsy with conventional MR imaging and T1-weighted dynamic contrast-enhanced perfusion MR imaging at 3T. Volume transfer constant, extravascular extracellular space volume per unit volume of tissue, and blood plasma volume per unit volume of tissue were calculated from the entire-tumor volume. Histogram analyses from these parameters were correlated with glioma grades. The parameters with the best percentile from cumulative histograms were identified by analysis of the area under the curve of the receiver operating characteristic analysis and were compared by using multivariable stepwise logistic regression analysis for distinguishing high- from low-grade gliomas. All parametric values increased with increasing glioma grade. There were significant differences among the 3 grades in all parameters (P < .01). For the differentiation of high- and low-grade gliomas, the highest area under the curve values were found at the 98th percentile of the volume transfer constant (area under the curve, 0.912; cutoff value, 0.277), the 90th percentile of extravascular extracellular space volume per unit volume of tissue (area under the curve, 0.939; cutoff value, 19.70), and the 84th percentile of blood plasma volume per unit volume of tissue (area under the curve, 0.769; cutoff value, 11.71). The 98th percentile volume transfer constant value was the only variable that could be used to independently differentiate high- and low-grade gliomas in multivariable stepwise logistic regression analysis. Histogram analysis of pharmacokinetic parameters from whole-tumor volume data can be a useful method for glioma grading. The 98th percentile value of the volume transfer constant was the most significant measure.


Recontacting in clinical practice: an investigation of the views of healthcare professionals and clinical scientists in the United Kingdom.
This article explores the views and experiences of healthcare professionals and clinical scientists in genetics about the existence of a duty and/or responsibility to recontact former patients when the genetic information relevant to their health, or that of family members, changes in a potentially important manner. It is based on N=30 semi-structured interviews guided by vignettes of recontacting scenarios. The sample included healthcare professionals in the United Kingdom from different medical specialties (clinical genetics, other 'mainstream' specialties now offering genetic testing), and scientists from regional genetics laboratories. While viewing recontacting as desirable under certain circumstances, most respondents expressed concerns about its feasibility within the current constraints of the National Health Service (NHS). The main barriers identified were insufficient resources (time, staff, and suitable IT infrastructures) and lack of clarity about role boundaries and responsibilities. All of these are further complicated by genetic testing being increasingly offered by mainstream specialties. Reaching a consensus about roles and responsibilities of clinical specialties with regard to recontacting former patients in the light of evolving genetic information, and about what resources and infrastructures would be needed, was generally seen as a pre-requisite to developing guidelines about recontact.


A Smad transcriptional corepressor.
Following TGFbeta receptor-mediated phosphorylation and association with Smad4, Smad2 moves into the nucleus, binds to target promoters in association with DNA-binding cofactors, and recruits coactivators such as p300/CBP to activate transcription. We identified the homeodomain protein TGIF as a Smad2-binding protein and a repressor of transcription. A TGFbeta-activated Smad complex can recruit TGIF and histone deacetylases (HDACs) to a Smad target promoter, repressing transcription. Thus, upon entering the nucleus, a Smad2-Smad4 complex may interact with coactivators, forming a transcriptional activation complex, or with TGIF and HDACs, forming a transcriptional repressor complex. Formation of one of these two mutually exclusive complexes is determined by the relative levels of Smad corepressors and coactivators within the cell.


Reading the (functional) writing on the (structural) wall: Multimodal fusion of brain structure and function via a deep neural network based translation approach reveals novel impairments in schizophrenia.
This work presents a novel approach to finding linkage/association between multimodal brain imaging data, such as structural MRI (sMRI) and functional MRI (fMRI). Motivated by the machine translation domain, we employ a deep learning model, and consider two different imaging views of the same brain like two different languages conveying some common facts. That analogy enables finding linkages between two modalities. The proposed translation-based fusion model contains a computing layer that learns "alignments" (or links) between dynamic connectivity features from fMRI data and static gray matter patterns from sMRI data. The approach is evaluated on a multi-site dataset consisting of eyes-closed resting state imaging data collected from 298 subjects (age- and gender matched 154 healthy controls and 144 patients with schizophrenia). Results are further confirmed on an independent dataset consisting of eyes-open resting state imaging data from 189 subjects (age- and gender matched 91 healthy controls and 98 patients with schizophrenia). We used dynamic functional connectivity (dFNC) states as the functional features and ICA-based sources from gray matter densities as the structural features. The dFNC states characterized by weakly correlated intrinsic connectivity networks (ICNs) were found to have stronger association with putamen and insular gray matter pattern, while the dFNC states of profuse strongly correlated ICNs exhibited stronger links with the gray matter pattern in precuneus, posterior cingulate cortex (PCC), and temporal cortex. Further investigation with the estimated link strength (or alignment score) showed significant group differences between healthy controls and patients with schizophrenia in several key regions including temporal lobe, and linked these to connectivity states showing less occupancy in healthy controls. Moreover, this novel approach revealed significant correlation between a cognitive score (attention/vigilance) and the function/structure alignment score that was not detected when data modalities were considered separately.


High conopeptide diversity in Conus tribblei revealed through analysis of venom duct transcriptome using two high-throughput sequencing platforms.
The venom of each species of Conus contains different kinds of pharmacologically active peptides which are mostly unique to that species. Collectively, the ~500-700 species of Conus produce a large number of these peptides, perhaps exceeding 140,000 different types in total. To date, however, only a small fraction of this diversity has been characterized via transcriptome sequencing. In addition, the sampling of this chemical diversity has not been uniform across the different lineages in the genus. In this study, we used high-throughput transcriptome sequencing approach to further investigate the diversity of Conus venom peptides. We chose a species, Conus tribblei, as a representative of a poorly studied clade of Conus. Using the Roche 454 and Illumina platforms, we discovered 136 unique and novel putative conopeptides belonging to 30 known gene superfamilies and 6 new conopeptide groups, the greatest diversity so far observed from a transcriptome. Most of the identified peptides exhibited divergence from the known conopeptides, and some contained cysteine frameworks observed for the first time in cone snails. In addition, several enzymes involved in posttranslational modification of conopeptides and also some proteins involved in efficient delivery of the conopeptides to prey were identified as well. Interestingly, a number of conopeptides highly similar to the conopeptides identified in a phylogenetically distant species, the generalist feeder Conus californicus, were observed. The high diversity of conopeptides and the presence of conopeptides similar to those in C. californicus suggest that C. tribblei may have a broad range of prey preferences.


Antioxidant-mediated up-regulation of OGG1 via NRF2 induction is associated with inhibition of oxidative DNA damage in estrogen-induced breast cancer.
Estrogen metabolism-mediated oxidative stress is suggested to play an important role in estrogen-induced breast carcinogenesis. We have earlier demonstrated that antioxidants, vitamin C (Vit C) and butylated hydroxyanisole (BHA) inhibit 17β-estradiol (E2)-mediated oxidative stress and oxidative DNA damage, and breast carcinogenesis in female August Copenhagen Irish (ACI) rats. The objective of the present study was to characterize the mechanism by which above antioxidants prevent DNA damage during breast carcinogenesis. Female ACI rats were treated with E2; Vit C; Vit C + E2; BHA; and BHA + E2 for up to 240 days. mRNA and protein levels of a DNA repair enzyme 8-Oxoguanine DNA glycosylase (OGG1) and a transcription factor NRF2 were quantified in the mammary and mammary tumor tissues of rats after treatment with E2 and compared with that of rats treated with antioxidants either alone or in combination with E2. The expression of OGG1 was suppressed in mammary tissues and in mammary tumors of rats treated with E2. Expression of NRF2 was also significantly suppressed in E2-treated mammary tissues and in mammary tumors. Vitamin C or BHA treatment prevented E2-mediated decrease in OGG1 and NRF2 levels in the mammary tissues. Chromatin immunoprecipitation analysis confirmed that antioxidant-mediated induction of OGG1 was through increased direct binding of NRF2 to the promoter region of OGG1. Studies using silencer RNA confirmed the role of OGG1 in inhibition of oxidative DNA damage. Our studies suggest that antioxidants Vit C and BHA provide protection against oxidative DNA damage and E2-induced mammary carcinogenesis, at least in part, through NRF2-mediated induction of OGG1.


Age and gender as independent predictors of violence under the influence of alcohol in Zurich, Switzerland.
Violent behaviour associated with alcohol consumption is frequently reported by different media. Clinical data analysing the correlation between alcohol intoxication, age, gender and violence are scarce. The aim of this study was to evaluate the influence of age, gender and blood alcohol content on violent behaviour under the influence of alcohol under central European conditions. Three hundred patients admitted to the emergency department were included into this study in the time period from January 01. to December 31. 2009. The inclusion criteria were a blood alcohol content (BAC) of ≥10 mmol/l, any traumatic injury and an age ≥16 years. Violence was defined as an evitable act committed by others leading to patient's hospitalisation. The data were compared with Wilcoxon and χ2-test for proportions. The data were considered as significant if p<0,05. Predictive quality was evaluated by using receiver operating characteristic (ROC) curve. Independent predictors were analyzed by logistic regression analysis. The average age was 36,9±16,9 years (range: 16-84 years), 259 (86%) males and 41 (24%) females. There was a significant difference in gender (odds ratio for gender male 2,88; CI 95%: 1,24-6,67; p<0,001) and age dependent (odds ratio for each year of age 0,94; CI 95%: 0,93-0,96; p<0,0001) violence with no correlation to blood alcohol content found. Logistic regression analysis revealed male gender and young age as an independent predictor for violence. These results clarify the relationship between alcohol, age, gender and violence and have important implications for municipal-level alcohol policies.


The use of imagery to manipulate challenge and threat appraisal States in athletes.
The present study investigated whether imagery could manipulate athletes' appraisal of stress-evoking situations (i.e., challenge or threat) and whether psychological and cardiovascular responses and interpretations varied according to cognitive appraisal of three imagery scripts: challenge, neutral, and threat. Twenty athletes (M(age) = 20.85; SD = 1.76; 10 female, 10 male) imaged each script while heart rate, stroke volume, and cardiac output were obtained using Doppler echocardiography. State anxiety and self-confidence were assessed following each script using the Immediate Anxiety Measures Scale. During the imagery, a significant increase in heart rate, stroke volume, and cardiac output occurred for the challenge and threat scripts (p < .05). Although there were no differences in physiological response intensities for both stress-evoking scripts, these responses, along with anxiety symptoms, were interpreted as facilitative during the challenge script and debilitative during the threat script. Results support using imagery to facilitate adaptive stress appraisal.


Solubilization, molecular forms, purification and substrate specificity of two acetylcholinesterases in the medicinal leech (Hirudo medicinalis).
Two acetylcholinesterases (AChE) differing in substrate and inhibitor specificities have been characterized in the medical leech (Hirudo medicinalis). A 'spontaneously-soluble' portion of AChE activity (SS-AChE) was recovered from haemolymph and from tissues dilacerated in low-salt buffer. A second portion of AChE activity was obtained after extraction of tissues in low-salt buffer alone or containing 1% Triton X-100 [detergent-soluble (DS-) AChE). Both enzymes were purified to homogeneity by affinity chromatography on edrophonium- and concanavalin A-Sepharose columns. Denaturing SDS/PAGE under reducing conditions gave one band at 30 kDa for purified SS-AChE and 66 kDa for DS-AChE. Sephadex G-200 chromatography indicated a molecular mass of 66 kDa for native SS-AChE and of 130 kDa for DS-AChE. SS-AChE showed a single peak sedimenting at 5.0 S in sucrose gradients with or without Triton X-100, suggesting that it was a hydrophylic monomer (G1). DS-AChE sedimented as a single 6.1-6.5 S peak in the presence of Triton X-100 and aggregated in the absence of detergent. A treatment with phosphatidylinositol-specific phospholipase C suppressed aggregation and gave a 7 S peak. DS-AChE was thus an amphiphilic glycolipid-anchored dimer. Substrate specificities were studied using p-nitrophenyl esters (acetate, propionate and butyrate) and corresponding thiocholine esters as substrates. SS-AChE displayed only limited variations in Km values with charged and uncharged substrates, suggesting a reduced influence of electrostatic interactions in the enzyme substrate affinity. By contrast, DS-AChE displayed higher Km values with uncharged than with charged substrates. SS-AChE was more sensitive to eserine and di-isopropyl fluorophosphate (IC50 5 x 10(-8) and 10(-8) M respectively) than DS-AChE (5 x 10(-7) and 5 x 10(-5) M.


Achalasia after bariatric Roux-en-Y gastric bypass surgery reversal.
Achalasia is a rare esophageal motility disorder that is characterized by a loss of peristalsis in the distal esophagus and failure of lower esophageal sphincter relaxation. The risk of developing esophageal motility disorders, including achalasia, following bariatric surgery is controversial and differs based on the type of surgery. Most of the reported cases occurred with laparoscopic adjustable gastric banding. To our knowledge, there are only three reported cases of achalasia after Roux-en-Y gastric bypass and no reported cases after revision of the surgery. We present a case of a 70-year-old female who had a previous history of Roux-en-Y gastric bypass with revision. She presented with persistent nausea and regurgitation for one month. Esophagogastroduodenoscopy showed a dilated esophagus without strictures or stenosis. A barium study was performed after the endoscopy and was suggestive of achalasia. Those findings were confirmed by a manometry. The patient was referred for laparoscopic Heller's myotomy.


Cell survival and clinical outcome following intrastriatal transplantation in Parkinson disease.
Intrastriatal transplantation of embryonic dopaminergic neurons is currently explored as a restorative cell therapy for Parkinson disease (PD). Clinical results have varied, probably due to differences in transplantation methodology and patient selection. In this review, we assess clinical trials and autopsy findings in grafted PD patients and suggest that a minimum number of surviving dopaminergic neurons is required for a favorable outcome. Restoration of [18F]-fluorodopa uptake in the putamen to about 50% of the normal mean seems necessary for moderate to marked clinical benefit to occur. Some studies indicate that this may require mesencephalic tissue from 3-5 human embryos implanted into each hemisphere. The volume, density and pattern of fiber outgrowth and reinnervation, as well as functional integration and dopamine release. are postulated as additional important factors for an optimal clinical outcome. For neural transplantation to become a feasible therapeutic alternative in PD, graft survival must be increased and the need for multiple donors of human embryonic tissue substantially decreased or alternate sources of donor tissue developed. Donor cells derived from alternative sources should demonstrate features comparable to those associated with successful implantation of human embryonic tissue before clinical trials are considered.


Definition of blindness under National Programme for Control of Blindness: Do we need to revise it?
A review appropriateness of the current definition of blindness under National Programme for Control of Blindness (NPCB), Government of India. Online search of peer-reviewed scientific published literature and guidelines using PubMed, the World Health Organization (WHO) IRIS, and Google Scholar with keywords, namely blindness and visual impairment, along with offline examination of reports of national and international organizations, as well as their cross-references was done until December 2016, to identify relevant documents on the definition of blindness. The evidence for the historical and currently adopted definition of blindness under the NPCB, the WHO, and other countries was reviewed. Differences in the NPCB and WHO definitions were analyzed to assess the impact on the epidemiological status of blindness and visual impairment in India. The differences in the criteria for blindness under the NPCB and the WHO definitions cause an overestimation of the prevalence of blindness in India. These variations are also associated with an over-representation of refractive errors as a cause of blindness and an under-representation of other causes under the NPCB definition. The targets for achieving elimination of blindness also become much more difficult to achieve under the NPCB definition. Ignoring differences in definitions when comparing the global and Indian prevalence of blindness will cause erroneous interpretations. We recommend that the appropriate modifications should be made in the NPCB definition of blindness to make it consistent with the WHO definition.


Evaluation of the Learning Curve of Hand-Assisted Laparoscopic Donor Nephrectomy.
BACKGROUND In this study, we analyzed the learning curve of hand-assisted laparoscopic donor nephrectomy (HALDN). MATERIAL AND METHODS The first 198 consecutive donors (110 cases by expert surgeon and 88 cases by newbie surgeon) operated on using HALDN were included in this study. The primary outcome measures were warm ischemic time (WIT), total operation time and estimated blood loss (EBL). The secondary outcome measures included length of hospital stay (LOS), graft outcome, and surgery-related complications. We used the cumulative sum (CUSUM) method to generate learning curves. RESULTS Except for WIT, all operative and convalescence parameters of donors and graft outcomes were similar for the 2 groups, including the total operation time (174.13 minutes vs. 171.75 minutes, P=0.140), EBL (108.27 cc vs. 116.82 cc, P=0.494), LOS (4.80 days vs. 4.92 days, P=0.144), and overall rates of intraoperative and postoperative complications (P>0.05). A significant difference was observed in WIT between the 2 groups (140.59 sec vs. 106.85 sec, P=0.027). Upon visual assessment of the CUSUM plots, a downward inflexion point for decreasing WIT was observed in 4 cases, total operation time in 12 cases, and EBL in 15 cases. CONCLUSIONS HALDN has a relatively short learning curve and similar results may be expected from newbie urologists who are trained in minimally invasive surgery fellowship.


Failure of fertility therapy and subsequent adverse cardiovascular events.
Infertility may indicate an underlying predisposition toward premature cardiovascular disease, yet little is known about potential long-term cardiovascular events following fertility therapy. We investigated whether failure of fertility therapy is associated with subsequent adverse cardiovascular events. We performed a population-based cohort analysis of women who received gonadotropin-based fertility therapy between Apr. 1, 1993, and Mar. 31, 2011, distinguishing those who subsequently gave birth and those who did not. Using multivariable Poisson regression models, we estimated the relative rate ratio of adverse cardiovascular events associated with fertility therapy failure, accounting for age, year, baseline risk factors, health care history and number of fertility cycles. The primary outcome was subsequent treatment for nonfatal coronary ischemia, stroke, transient ischemic attack, heart failure or thromboembolism. Of 28 442 women who received fertility therapy, 9349 (32.9%) subsequently gave birth and 19 093 (67.1%) did not. The median number of fertility treatments was 3 (interquartile range 1-5). We identified 2686 cardiovascular events over a median 8.4 years of follow-up. The annual rate of cardiovascular events was 19% higher among women who did not give birth after fertility therapy than among those who did (1.08 v. 0.91 per 100 patient-years, p < 0.001), equivalent to a 21% relative increase in the annual rate (95% confidence interval 13%-30%). We observed no association between event rates and number of treatment cycles. Fertility therapy failure was associated with an increased risk of long-term adverse cardiovascular events. These women merit surveillance for subsequent cardiovascular events.


Small molecule-induced allosteric activation of the Vibrio cholerae RTX cysteine protease domain.
Vibrio cholerae RTX (repeats in toxin) is an actin-disrupting toxin that is autoprocessed by an internal cysteine protease domain (CPD). The RTX CPD is efficiently activated by the eukaryote-specific small molecule inositol hexakisphosphate (InsP6), and we present the 2.1 angstrom structure of the RTX CPD in complex with InsP6. InsP6 binds to a conserved basic cleft that is distant from the protease active site. Biochemical and kinetic analyses of CPD mutants indicate that InsP6 binding induces an allosteric switch that leads to the autoprocessing and intracellular release of toxin-effector domains.


64-slice computed tomography coronary angiography: diagnostic accuracy in the real world.
This study was done to evaluate the diagnostic accuracy of 64-slice computed tomography coronary angiography (CTCA) for the detection of significant coronary artery stenosis in the real clinical world. From the CTCA database of our institution, we enrolled 145 patients (92 men, 52 women, mean age 63.4 +/- 10.2 years) with suspected coronary artery disease. All patients presented with atypical or typical chest pain and underwent CTCA and conventional coronary angiography (CA). For the CTCA scan (Sensation 64, Siemens, Germany), we administered an IV bolus of 100 ml of iodinated contrast material (Iomeprol 400 mgI/ml, Bracco, Italy). The CTCA and CA reports used to evaluate diagnostic accuracy adopted > or =50% and > or =70%, respectively, as thresholds for significant stenosis. Eleven patients were excluded from the analysis because of the nondiagnostic quality of CTCA. The prevalence of disease demonstrated at CA was 63% (84/134). Sensitivity, specificity and positive and negative predictive values for CTCA on a per-segment, per-vessel, and per-patient basis were 75.6%, 85.1%, 97.6%; 86.9%, 81.8%, 58.0%; 48.2%, 68.1%, 79.6%; and 95.7%, 92.3%, 93.5%, respectively. Only two out of 134 eligible patients were false negative. Heart rate did not significantly influence diagnostic accuracy, whereas the absence or minimal presence of coronary calcification improved diagnostic accuracy. The positive and negative likelihood ratios at the per-patient level were 2.32 and 0.041, respectively. CTCA in the real clinical world shows a diagnostic performance lower than reported in previous validation studies. The excellent negative predictive value and negative likelihood ratio make CTCA a noninvasive gold standard for exclusion of significant coronary artery disease.


The effectiveness of sexual health interventions for people with severe mental illness: a systematic review.
Severe mental illnesses (SMIs), such as schizophrenia and bipolar disorder, persist over time and can cause extensive disability leading to impairments in social and occupational functioning. People with SMI have higher morbidity and mortality due to physical illness than the general population and may be more likely to engage in high-risk sexual behaviour (e.g. unprotected intercourse, having multiple partners, involvement in the sex trade and illicit drug use), putting them at risk of poorer sexual health outcomes including sexually transmitted infections. Sexual health promotion interventions, developed and implemented for people with SMI, may improve participants' knowledge, attitudes, beliefs or behavioural practices and could lead to a reduction in risky sexual behaviour. To evaluate the effectiveness of sexual health interventions for people with SMI compared with usual care and their applicability to the UK NHS setting. Thirteen electronic databases were searched from inception to December 2012. All controlled trials (randomised or non-randomised) that met the following criteria were included: any sexual health promotion intervention or combination of interventions intended to change the knowledge, attitudes, beliefs, behaviours or practices of individuals with SMI (defined as adults aged ≥ 18 years who have received a diagnosis of schizophrenia or bipolar disorder) living in the community. A systematic review of the clinical evidence was undertaken following recommended guidelines. Data were tabulated and discussed in a narrative review. Thirteen randomised controlled studies met the inclusion criteria. The methodological quality of the included studies varied considerably, with only a minority of studies (n = 2) being considered as having very few methodological limitations. Despite wide variations in the study populations, interventions, comparators and outcomes, four studies showed significant improvements in all measured sexual risk behaviour outcomes (e.g. human immunodeficiency virus knowledge and behaviour change) in the intervention groups compared with the control groups. In contrast, four studies found significant improvements in the intervention groups for some outcomes only and three studies found significant improvements in certain subgroups only, based on either gender or ethnicity. Finally, two studies reported no significant differences in any sexual risk behaviour outcomes between the intervention and control groups. Moreover, positive findings were not consistently sustained at follow-up in many studies. Little detail was provided in the studies regarding the content of interventions, how they were delivered and by whom, making replication or generalisability difficult. Owing to the large between-study variability (especially in the populations, interventions, comparators and reported outcomes) and mixed results, there is insufficient evidence to fully support or reject the identified sexual health interventions for people with SMI. In addition, there are considerable uncertainties around the generalisability of these findings to the UK setting. Further research recommendations include well-designed, UK-based trials of sexual health interventions for people with SMI as well as training and support for staff implementing sexual health interventions. PROSPERO number CRD42013003674. The National Institute for Health Research Health Technology Assessment Programme.


Tropical forests were the primary sources of new agricultural land in the 1980s and 1990s.
Global demand for agricultural products such as food, feed, and fuel is now a major driver of cropland and pasture expansion across much of the developing world. Whether these new agricultural lands replace forests, degraded forests, or grasslands greatly influences the environmental consequences of expansion. Although the general pattern is known, there still is no definitive quantification of these land-cover changes. Here we analyze the rich, pan-tropical database of classified Landsat scenes created by the Food and Agricultural Organization of the United Nations to examine pathways of agricultural expansion across the major tropical forest regions in the 1980s and 1990s and use this information to highlight the future land conversions that probably will be needed to meet mounting demand for agricultural products. Across the tropics, we find that between 1980 and 2000 more than 55% of new agricultural land came at the expense of intact forests, and another 28% came from disturbed forests. This study underscores the potential consequences of unabated agricultural expansion for forest conservation and carbon emissions.


Recursive Cluster Elimination based Rank Function (SVM-RCE-R) implemented in KNIME.
In our earlier study, we proposed a novel feature selection approach, Recursive Cluster Elimination with Support Vector Machines (SVM-RCE) and implemented this approach in Matlab. Interest in this approach has grown over time and several researchers have incorporated SVM-RCE into their studies, resulting in a substantial number of scientific publications. This increased interest encouraged us to reconsider how feature selection, particularly in biological datasets, can benefit from considering the relationships of those genes in the selection process, this led to our development of SVM-RCE-R. SVM-RCE-R, further enhances the capabilities of SVM-RCE by the addition of a novel user specified ranking function. This ranking function enables the user to stipulate the weights of the accuracy, sensitivity, specificity, f-measure, area under the curve and the precision in the ranking function This flexibility allows the user to select for greater sensitivity or greater specificity as needed for a specific project. The usefulness of SVM-RCE-R is further supported by development of the maTE tool which uses a similar approach to identify microRNA (miRNA) targets. We have also now implemented the SVM-RCE-R algorithm in Knime in order to make it easier to applyThe use of SVM-RCE-R in Knime is simple and intuitive and allows researchers to immediately begin their analysis without having to consult an information technology specialist. The input for the Knime implemented tool is an EXCEL file (or text or CSV) with a simple structure and the output is also an EXCEL file. The Knime version also incorporates new features not available in SVM-RCE. The results show that the inclusion of the ranking function has a significant impact on the performance of SVM-RCE-R. Some of the clusters that achieve high scores for a specified ranking can also have high scores in other metrics.


Autologous fat grafting: A comparative study of four current commercial protocols.
Autologous fat grafting is a widely used technique that gives natural results when treating soft tissue deficiencies. However, there is no consensus on which is the best procedure to use, leading to unpredictable results because of fat graft resorption. This study compared four commercial lipotransfer devices by analyzing the behavior of the processed adipose tissue and outcome of the adipose graft in an in vivo model. Four different protocols that used manual, power-assisted or water-assisted lipoaspiration and then decantation, centrifugation, or filtration were used on each of eight patients to process lipoaspirate. Harvested adipose tissue was assessed in vitro for tissue resorption, oil formation, and cytokine secretion. Graft resorption rate was calculated and histological analyses were performed after subcutaneously injecting the harvested adipose tissue in a murine model. All protocols resulted in very low oil formation and histologically healthy grafts. The tissue volume was significantly greater after 2 days in culture when using manual lipoaspiration and soft centrifugations/washing steps (Microfill®/Macrofill®) compared to Water-Assisted Lipoaspiration/Decantation (BodyJet®) and Power-Assisted Lipoaspiration/Filtration (PAL® + PureGraft®). These results were confirmed in mice 1 month after subcutaneous injection, with greater efficiency obtained with protocols that used (A) manual aspiration, (B) soft centrifugations, and (C) washing steps. We confirmed that the choice of technique used to process adipose tissue during lipotransfer surgery can highly influence fat grafting efficacy. In our study, the use of manual aspiration combined with soft centrifugations led to the best results in the selected models.


Reference values for cardiorespiratory fitness and incidence of type 2 diabetes.
In "Physical Activity Reference for Health Promotion 2013" the Japan Ministry of Health, Labour and Welfare publication gives reference values for cardiorespiratory fitness (CRF) required for good health. We examined the associations between the CRF reference values and incidence of type 2 diabetes. This prospective cohort study enrolled 4633 nondiabetic Japanese men aged 20 to 39 years at baseline. CRF was measured using the cycle ergometer test, and maximal oxygen uptake was estimated. On the basis of the CRF reference value, participants were classified into 2 groups: those with values less than the reference value (under-RV) and those with values equal to or greater than reference value (over-RV). Hazard ratios (HRs) and 95% CIs for incident type 2 diabetes were estimated using a Cox proportional hazards model. A total of 266 participants developed type 2 diabetes during the 14 years of follow-up. As compared with the under-RV group, the over-RV group had a significantly lower multivariable-adjusted HR for type 2 diabetes (HR 0.67; 95% CI, 0.51-0.89). In receiver operating characteristic analysis, the optimal CRF cut-off value for predicting incident type 2 diabetes was 10.8 metabolic equivalents (sensitivity, 0.64; specificity, 0.64), which was close to the CRF reference value of 11.0 metabolic equivalents. The reference CRF value appears to be reasonably valid for prevention of type 2 diabetes, especially among Japanese men younger than 40 years. Development of type 2 diabetes can be prevented by maintaining a CRF level above the reference value.


Just 2% of SARS-CoV-2-positive individuals carry 90% of the virus circulating in communities.
We analyze data from the fall 2020 pandemic response efforts at the University of Colorado Boulder, where more than 72,500 saliva samples were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using qRT-PCR. All samples were collected from individuals who reported no symptoms associated with COVID-19 on the day of collection. From these, 1,405 positive cases were identified. The distribution of viral loads within these asymptomatic individuals was indistinguishable from what has been previously observed in symptomatic individuals. Regardless of symptomatic status, ∼50% of individuals who test positive for SARS-CoV-2 seem to be in noninfectious phases of the disease, based on having low viral loads in a range from which live virus has rarely been isolated. We find that, at any given time, just 2% of individuals carry 90% of the virions circulating within communities, serving as viral "supercarriers" and possibly also superspreaders.


Striated myogenesis and peristalsis in the fetal murine esophagus occur without cell migration or interstitial cells of Cajal.
Esophageal striated myogenesis progresses differently from appendicular myogenesis, but the mechanism underlying this process is incompletely understood. Early theories of transdifferentiation of smooth muscle into striated muscle are not supported by transgenic fate-mapping experiments; however, the origin of esophageal striated muscle remains unknown. To better define the process of striated myogenesis, we examined myogenesis in murine fetal cultured esophageal whole-organ explants. Embryonic day 14.5 (E14.5) esophagi maintained a functional contractile phenotype for up to 7 days in culture. Striated myogenesis, as evidenced by myogenin expression, proceeded in a craniocaudal direction along the length of the esophagus. Esophageal length did not change during this process. Complete, but not partial, mechanical disruption of the rostral esophagus inhibited myogenesis distally. Addition of fibroblast growth factor-2 (FGF-2) to the culture media failed to inhibit striated myogenesis, but attenuated smooth muscle actin expression and reduced peristaltic activity. Inhibition of c-kit failed to inhibit peristalsis. These results suggest that striated myogenic precursors are resident along the entire length of the esophagus by day 14.5 and do not migrate along the esophagus after E14.5. Induction of myogenesis craniocaudally appears to require physical continuity of the esophagus and is not inhibited by FGF-2. Finally, peristalsis in E14.5 esophagi appears not to be regulated by interstitial cells of Cajal.


Continued versus Suspended Cardiac Resynchronization Therapy after Left Ventricular Assist Device Implantation.
Cardiac resynchronization therapy (CRT) improves outcomes in heart failure patients with wide QRS complex. However, CRT management following continuous flow Left Ventricular Assist Device (LVAD) implant vary: some centers continue CRT while others turn off the left ventricular (LV) lead at LVAD implant. We sought to study the effect of continued CRT versus turning off CRT pacing following continuous flow LVAD implantation. A comprehensive retrospective multicenter cohort of 295 patients with LVAD and pre-existing CRT was studied. CRT was programmed off after LVAD implant in 44 patients. We compared their outcomes to the rest of the cohort using univariate and multivariate models. Mean age was 60 ± 12 years, 83% were males, 52% had ischemic cardiomyopathy and 54% were destination therapy. Mean follow-up was 2.4 ± 2.0 years, and mean LVAD support time was 1.7 ± 1.4 years. Patients with CRT OFF had a higher Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) mean profile (3.9 vs 3.3, p = 0.01), more secondary prevention indication for a defibrillator (64.9% vs 44.5%, p = 0.023), and more pre-LVAD ventricular arrhythmias (VA) (77% vs 60%, p = 0.048). There were no differences between the CRT OFF and CRT ON groups in overall mortality (Log rank p = 0.32, adjusted HR = 1.14 [0.54-2.22], p = 0.71), heart transplantation, cardiac and noncardiac mortality, all cause hospitalizations, hospitalizations for ICD shocks, and number and frequency of ICD shocks or anti-tachycardia pacing therapy. There were no differences in post LVAD atrial arrhythmias (AA) (Adjusted OR = 0.45 [0.18-1.06], p = 0.31) and ventricular arrhythmias (OR = 0.65 [0.41-1.78], p = 0.41). There was no difference in change in LVEF, LV end diastolic and end systolic diameters between the 2 groups. Our study suggests that turning off CRT pacing after LVAD implantation in patients with previous CRT pacing did not affect mortality, heart transplantation, device therapies or arrhythmia burden. A prospective study is needed to confirm these findings.


Pain during walking and ascending stairs before hyaluronic acid injection was common in patients with knee osteoarthritis: a qualitative study
Knee osteoarthritis (OA) is a common pathology characterized by degeneration of the articular cartilage. The aim of the research was to ask patients how they decided to make the injection, what treatments they received, their complaints prior to and after the injection and how they feel at the moment, and whether they are currently exercising or not. Thus, to demonstrate the patients' outcomes with their own expression. A total of 92 knee OA patients completed semistructured interviews, which included six open-ended questions. A total of 92 patients (66 female, 26 male) aged between 36 and 95 years (mean 65.511.14) were included. Before the injection, the majority of the OA patients had pain complaints when walking (72.8%) and stair climbing (70.7%). One to four years after intraarticular injection, 45.2% of patients felt a decrease in their complaints. The majority of patients did not consider diet and exercise as a treatment option. In addition, almost all patients declared that they decided on hyaluronic acid injection treatment with the physician's recommendation. Pain during walking and stair climbing before hyaluronic acid injection was common in knee OA patients. Overall the patients felt a decrease in the symptoms after injection. Patients did not consider diet and exercise as a treatment option despite the recommendation by a physician.


Developing and Evaluating Collaborative Online-Based Instructional Designs in Health Information Management.
The number of students enrolled in online courses is increasing steadily. Distance education offers many advantages, but also has inherent challenges. Successful distance education needs a thoughtfully designed instructional strategy where students are supported to actively create knowledge. We present the design and evaluation of three online-based courses in health informatics. The courses were based on a collaborative instructional strategy. The evaluation comprised workload analysis, student evaluation, student interviews and student reflections. Students expressed high satisfaction with online learning, despite a high workload, and high perceived learning outcomes. Using the Community of Inquiry framework as reference, we found very high levels of teaching presence, social presence and cognitive presence. Summarizing, we found that the chosen instructional strategy supported student-centered, collaborative learning. We conclude by presenting lesson learned for online-based instructional design.


Catalysts from synthetic genetic polymers.
The emergence of catalysis in early genetic polymers such as RNA is considered a key transition in the origin of life, pre-dating the appearance of protein enzymes. DNA also demonstrates the capacity to fold into three-dimensional structures and form catalysts in vitro. However, to what degree these natural biopolymers comprise functionally privileged chemical scaffolds for folding or the evolution of catalysis is not known. The ability of synthetic genetic polymers (XNAs) with alternative backbone chemistries not found in nature to fold into defined structures and bind ligands raises the possibility that these too might be capable of forming catalysts (XNAzymes). Here we report the discovery of such XNAzymes, elaborated in four different chemistries (arabino nucleic acids, ANA; 2'-fluoroarabino nucleic acids, FANA; hexitol nucleic acids, HNA; and cyclohexene nucleic acids, CeNA) directly from random XNA oligomer pools, exhibiting in trans RNA endonuclease and ligase activities. We also describe an XNA-XNA ligase metalloenzyme in the FANA framework, establishing catalysis in an entirely synthetic system and enabling the synthesis of FANA oligomers and an active RNA endonuclease FANAzyme from its constituent parts. These results extend catalysis beyond biopolymers and establish technologies for the discovery of catalysts in a wide range of polymer scaffolds not found in nature. Evolution of catalysis independent of any natural polymer has implications for the definition of chemical boundary conditions for the emergence of life on Earth and elsewhere in the Universe.


PTF1 is an organ-specific and Notch-independent basic helix-loop-helix complex containing the mammalian Suppressor of Hairless (RBP-J) or its paralogue, RBP-L.
PTF1 is a trimeric transcription factor essential to the development of the pancreas and to the maintenance of the differentiated state of the adult exocrine pancreas. It comprises a dimer of P48/PTF1a (a pancreas and neural restricted basic helix-loop-helix [bHLH] protein) and a class A bHLH protein, together with a third protein that we show can be either the mammalian Suppressor of Hairless (RBP-J) or its paralogue, RBP-L. In mature acinar cells, PTF1 exclusively contains the RBP-L isoform and is bound to the promoters of acinar specific genes. P48 interacts with the RBP subunit primarily through two short conserved tryptophan-containing motifs, similar to the motif of the Notch intracellular domain (NotchIC) that interacts with RBP-J. The transcriptional activities of the J and L forms of PTF1 are independent of Notch signaling, because P48 occupies the NotchIC docking site on RBP-J and RBP-L does not bind the NotchIC. Mutations that delete one or both of the RBP-interacting motifs of P48 eliminate RBP-binding and are associated with a human genetic disorder characterized by pancreatic and cerebellar agenesis, which indicates that the association of P48 and RBPs is required for proper embryonic development. The presence of related peptide motifs in other transcription factors indicates a broader Notch-independent function for RBPJ/SU(H).


Genomic screening by 454 pyrosequencing identifies a new human IGHV gene and sixteen other new IGHV allelic variants.
Complete and accurate knowledge of the genes and allelic variants of the human immunoglobulin gene loci is critical for studies of B cell repertoire development and somatic point mutation, but evidence from studies of VDJ rearrangements suggests that our knowledge of the available immunoglobulin gene repertoire is far from complete. The reported repertoire has changed little over the last 15 years. This is, in part, a consequence of the inefficiencies involved in searching for new members of large, multigenic gene families by cloning and sequencing. The advent of high-throughput sequencing provides a new avenue by which the germline repertoire can be explored. In this report, we describe pyrosequencing studies of the heavy chain IGHV1, IGHV3 and IGHV4 gene subgroups in ten Papua New Guineans. Thousands of 454 reads aligned with complete identity to 51 previously reported functional IGHV genes and allelic variants. A new gene, IGHV3-NL1*01, was identified, which differs from the nearest previously reported gene by 15 nucleotides. Sixteen new IGHV alleles were also identified, 15 of which varied from previously reported functional IGHV genes by between one and four nucleotides, while one sequence appears to be a functional variant of the pseudogene IGHV3-25. BLAST searches suggest that at least six of these new genes are carried within the relatively well-studied populations of North America, Europe or Asia. This study substantially expands the known immunoglobulin gene repertoire and demonstrates that genetic variation of immunoglobulin genes can now be efficiently explored in different human populations using high-throughput pyrosequencing.


Analyzing Information Seeking and Drug-Safety Alert Response by Health Care Professionals as New Methods for Surveillance.
Patterns in general consumer online search logs have been used to monitor health conditions and to predict health-related activities, but the multiple contexts within which consumers perform online searches make significant associations difficult to interpret. Physician information-seeking behavior has typically been analyzed through survey-based approaches and literature reviews. Activity logs from health care professionals using online medical information resources are thus a valuable yet relatively untapped resource for large-scale medical surveillance. To analyze health care professionals' information-seeking behavior and assess the feasibility of measuring drug-safety alert response from the usage logs of an online medical information resource. Using two years (2011-2012) of usage logs from UpToDate, we measured the volume of searches related to medical conditions with significant burden in the United States, as well as the seasonal distribution of those searches. We quantified the relationship between searches and resulting page views. Using a large collection of online mainstream media articles and Web log posts we also characterized the uptake of a Food and Drug Administration (FDA) alert via changes in UpToDate search activity compared with general online media activity related to the subject of the alert. Diseases and symptoms dominate UpToDate searches. Some searches result in page views of only short duration, while others consistently result in longer-than-average page views. The response to an FDA alert for Celexa, characterized by a change in UpToDate search activity, differed considerably from general online media activity. Changes in search activity appeared later and persisted longer in UpToDate logs. The volume of searches and page view durations related to Celexa before the alert also differed from those after the alert. Understanding the information-seeking behavior associated with online evidence sources can offer insight into the information needs of health professionals and enable large-scale medical surveillance. Our Web log mining approach has the potential to monitor responses to FDA alerts at a national level. Our findings can also inform the design and content of evidence-based medical information resources such as UpToDate.


Online Adaptive Radiation Therapy.
The current paradigm of radiation therapy has the treatment planned on a snapshot dataset of the patient's anatomy taken at the time of simulation. Throughout the course of treatment, this snapshot may vary from initial simulation. Although there is the ability to image patients within the treatment room with technologies such as cone beam computed tomography, the current state of the art is largely limited to rigid-body matching and not accounting for any geometric deformations in the patient's anatomy. A plan that was once attuned to the initial simulation can become suboptimal as the treatment progresses unless improved technologies are brought to bear. Adaptive radiation therapy (ART) is an evolving paradigm that seeks to address this deficiency by accounting for ongoing changes in the patient's anatomy and/or physiology during the course of treatment, affording an increasingly more accurate targeting of disease. ART relies on several components working in concert, namely in-room treatment image guidance, deformable image registration, automatic recontouring, plan evaluation and reoptimization, dose calculation, and quality assurance. Various studies have explored how a putative ART solution would improve the current state of the art of radiation therapy-some centers have even clinically implemented online adaptation. These explorations are reviewed here for a variety of sites.


Selective Intracellular Delivery of Ganglioside GM3-Binding Peptide through Caveolae/Raft-Mediated Endocytosis.
Glycosphingolipids are major components of the membrane raft, and several kinds of viruses and bacterial toxins are known to bind to glycosphingolipids in the membrane raft. Since the viral genes and pathogenic proteins that are taken into cells are directly delivered to their target organelles, caveolae/raft-mediated endocytosis represents a promising pathway for specific delivery. In the present study, we demonstrated the ability of an artificial pentadecapeptide, which binds to ganglioside GM3, to deliver protein into cells by caveolae/raft-mediated endocytosis. The cellular uptake of a biotinylated GM3-binding peptide (GM3BP)-avidin complex into HeLa cells was observed, and the cellular uptake of this complex was inhibited by an incubation with sialic acid or endocytic inhibitors such as methyl-ß-cyclodextrin, and also by an incubation at 4 °C. These results indicate that the GM3BP-avidin complex bind to GM3 in membrane raft, and are taken into cell through caveolae/raft-mediated endocytosis. The GM3BP-avidin complex was transported into cells and localized around the nucleus more slowly than a human immunodeficiency virus type 1 TAT peptide. Furthermore, the uptake of a green fluorescent protein (GFP) linked with GM3BP into HeLa cells was similar to that of the GM3BP-avidin complex, and the localization of the GM3BP-GFP fusion protein was markedly different with that of the TAT-GFP fusion protein. The uptake and trafficking of GM3BP were distinguished from conventional cell-penetrating peptides. GM3BP has potential as a novel peptide for the selective delivery of therapeutic proteins and materials into cells in addition to being a cell-penetrating peptide.


Morphological and functional gastric cytoprotection by prostaglandin in rats receiving absolute ethanol orally.
Pretreatment with prostaglandins at non-antisecretory doses protects the gastric mucosa, including the parietal cells, from deep necrosis produced by intragastric administration of necrotising agents such as absolute ethanol. Whether the parietal cells also retained their ability to secrete acid when rats were pretreated with a prostaglandin, in spite of exposure to ethanol, was investigated. Gastric acid secretion was abolished 4 hours after ethanol, and secretion returned to control values only after 5-6 days. Pretreatment with a single, non-antisecretory dose of 16, 16-dimethyl prostaglandin E2 (dm PGE2) maintained acid secretion, in spite of exposure to absolute ethanol. Absolute ethanol caused histological changes - extensive gastric mucosal necrosis (through the muscularis mucosae), oedema, haemorrhages, polymorphonuclear infiltration, and formation of granulation tissue - that were maximal 24-48 hours after ethanol and persisted for 2 to 4 weeks. None of these changes were present in animals treated with the prostaglandin. It is concluded that a single oral pretreatment with dmPGE2 protects the gastric mucosa against not only the morphological damage of absolute ethanol (preventing necrosis, haemorrhages, and polymorphonuclear infiltration) but also the functional damage (maintaining the acid secretory function of parietal cells).


The Bin/amphiphysin/Rvs (BAR) domain protein endophilin B2 interacts with plectin and controls perinuclear cytoskeletal architecture.
Proteins of the Bin/amphiphysin/Rvs (BAR) domain superfamily are essential in controlling the shape and dynamics of intracellular membranes. Here, we present evidence for the unconventional function of a member of the endophilin family of BAR and Src homology 3 domain-containing proteins, namely endophilin B2, in the perinuclear organization of intermediate filaments. Using mass spectrometry analysis based on capturing endophilin B2 partners in in situ pre-established complexes in cells, we unravel the interaction of endophilin B2 with plectin 1, a variant of the cytoskeleton linker protein plectin as well as with vimentin. Endophilin B2 directly binds the N-terminal region of plectin 1 via Src homology 3-mediated interaction and vimentin indirectly via plectin-mediated interaction. The relevance of these interactions is strengthened by the selective and drastic reorganization of vimentin around nuclei upon overexpression of endophilin B2 and by the extensive colocalization of both proteins in a meshwork of perinuclear filamentous structures. By generating mutants of the endophilin B2 BAR domain, we show that this phenotype requires the BAR-mediated membrane binding activity of endophilin B2. Plectin 1 or endophilin B2 knockdown using RNA interference disturbed the perinuclear organization of vimentin. Altogether, these data suggest that the endophilin B2-plectin 1 complex functions as a membrane-anchoring device organizing and stabilizing the perinuclear network of vimentin filaments. Finally, we present evidence for the involvement of endophilin B2 and plectin 1 in nuclear positioning in individual cells. This points to the potential importance of the endophilin B2-plectin complex in the biological functions depending on nuclear migration and positioning.


Quantitative phosphoproteomic analysis of IL-33-mediated signaling.
Interleukin-33 (IL-33) is a novel member of the IL-1 family of cytokines that plays diverse roles in the regulation of immune responses. IL-33 exerts its effects through a heterodimeric receptor complex resulting in the production and release of proinflammatory cytokines. A detailed understanding of the signaling pathways activated by IL-33 is still unclear. To gain insights into the IL-33-mediated signaling mechanisms, we carried out a SILAC-based global quantitative phosphoproteomic analysis that resulted in the identification of 7191 phosphorylation sites derived from 2746 proteins. We observed alterations in the level of phosphorylation in 1050 sites corresponding to 672 proteins upon IL-33 stimulation. We report, for the first time, phosphorylation of multiple protein kinases, including mitogen-activated protein kinase activated protein kinase 2 (Mapkapk2), receptor (TNFRSF) interacting serine-threonine kinase 1 (Ripk1), and NAD kinase (Nadk) that are induced by IL-33. In addition, we observed IL-33-induced phosphorylation of several protein phosphatases including protein tyrosine phosphatase, nonreceptor-type 12 (Ptpn12), and inositol polyphosphate-5-phosphatase D (Inpp5d), which have not been reported previously. Network analysis revealed an enrichment of actin binding and cytoskeleton reorganization that could be important in macrophage activation induced by IL-33. Our study is the first quantitative analysis of IL-33-regulated phosphoproteome. Our findings significantly expand the understanding of IL-33-mediated signaling events and have the potential to provide novel therapeutic targets pertaining to immune-related diseases such as asthma where dysregulation of IL-33 is observed. All MS data have been deposited in the ProteomeXchange with identifier PXD000984 (http://proteomecentral.proteomexchange.org/dataset/PXD000984).


Thick Smear is a Good Substitute for the Thin Smear in Parasitological Confirmation of Canine Visceral Leishmaniasis.
Although direct examination methods are important for diagnosing leishmaniasis, such methods are often neglected because of their low sensitivity relative to other techniques. Our study aimed to evaluate the performance of bone marrow (BM) thick smears and cytocentrifugation tests as alternatives to direct examination for diagnosing canine visceral leishmaniasis (CVL). Ninety-two dogs exhibiting leishmaniasis seroreactivity were evaluated. The animals were euthanized; and healthy skin, spleen, popliteal lymph node, and BM puncture samples were cultured. BM cultures were used as the reference standard. Of the 92 dogs studied, 85.9% exhibited positive cultures, and Leishmania infantum (synonym Leishmania chagasi) was confirmed in all positive culture cases. The sensitivity rates for cytocentrifugation as well as thin and thick smears were 47.1%, 52.8%, and 77%, respectively. However, no association between the dogs' clinical status and culture or direct examination results was found. To our knowledge, this was the first study to use thick smears and cytocentrifugation for diagnosing CVL. Our results indicate that BM thick smears have a good sensitivity and their use reduces the time required to read slides. Therefore, thick smears can provide a rapid and safe alternative to parasitological confirmation of seroreactive dogs.