Green tea epigallocatechin 3-gallate alleviates hyperglycemia and reduces advanced glycation end products via nrf2 pathway in mice with high fat diet-induced obesity.
Epigallocatechin 3-gallate (EGCG) from green tea may reduce plasma glucose and alleviate complications of diabetes by attenuating advanced glycation end products (AGEs) formation. We hypothesized that EGCG would mitigate AGEs formation via activating the nuclear factor erythroid-2-related-factor-2 (Nrf2) pathway in a mouse model of high fat diet-induced obesity. Dietary EGCG was tested in C57BL/6 mice that were placed on a high-fat diet with or without ECGC for 17 weeks and compared to a control group placed on low-fat diet for the same period. Weight gain and fasting blood glucose were measured throughout the study duration. Supplementation of high fat diet with dietary EGCG significantly reduced weight gain, plasma glucose, insulin level, liver and kidney weight. EGCG administration also decreased the levels of AGEs in both plasma and liver while inhibiting the receptor for AGE (RAGE) expression of, activating Nrf2 and enhancing GSH/GSSG ratio compared to mice on high fat diet without added EGCG. This study demonstrated that EGCG has the potential to help control hyperglycemia, reduce weight, and alleviate diabetes complications.


Facial Identity and Self-Perception: An Examination of Psychosocial Outcomes in Cosmetic Surgery Patients.

The psychosocial health of patients undergoing cosmetic procedures has often been linked to a host of pre-existing conditions, including the type of procedure being performed. Age, gender, and the psychological state of the patients also contribute to the perceived outcome. Specifically, the presence or absence of Body Dysmorphic Disorder (BDD) has been identified as an independent marker for unhappiness following cosmetic procedures.1 However, no study has, to our knowledge, identified a more precise indicator that is associated with higher rates of patient dissatisfaction from cosmetic procedure. This review identifies facial identity and self-perception as potential identifiers of future patient dissatisfaction with cosmetic procedures. Specifically, we believe that patients with a realistic facial identity and self-perception are more likely to be satisfied than those whose self-perceptions are distorted. Patients undergoing restorative procedures, including blepharoplasty, rhytidectomy, and liposuction, are more likely to have an increased outcome favorability rating than those undergoing type change procedures, such as rhinoplasty and breast augmentation. Age, which typically is an independent variable for satisfaction, tends to be associated with increased favorability ratings following cosmetic procedures. Female gender is a second variable associated with higher satisfaction. The authors believe that negative facial identity and self-perception are risk factors for patient dissatisfaction with cosmetic procedural outcomes. Based on this assumption, clinicians may want to focus on the face as a particular area of psychosocial concern.

J Drugs Dermatol. 2017;16(6):617-620.
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Microcin-mediated interactions between Klebsiella pneumoniae and Escherichia coli strains.
Amensal indirect interactions between a Klebsiella pneumoniae microcin-producing strain and several Escherichia coli strains, all of intestinal origin, were studied. Mixed batch cultures of both microcin-producing and microcin-sensitive strains showed that microcin production and excretion into the medium allowed the producer strain to prevail over sensitive strains, even when initial competition conditions were highly unfavourable for the producer. Mixed cultures also showed the production of a microcin-antagonist by the same microcin-producing strain when the nutrients in the medium had been depleted. The antagonist apparently promoted the viability of sensitive cells already damaged by microcin. These results have likely ecological implications.


Inhibition of autophagic flux differently modulates cannabidiol-induced death in 2D and 3D glioblastoma cell cultures.
Radiotherapy combined with chemotherapy is the major treatment modality for human glioblastoma multiforme (GBM). GBMs eventually relapse after treatment and the average survival of GBM patients is less than two years. There is some evidence that cannabidiol (CBD) can induce cell death and increases the radiosensitivity of GBM by enhancing apoptosis. Beside initiation of death, CBD has been demonstrated as an inducer of autophagy. In the present study, we address the question whether CBD simultaneously induces a protective effect in GBM by upregulating autophagy. Addition of chloroquine that suppressed autophagic flux to 2D GBM cultures increased CBD-induced cell death, presenting proof for the protective autophagy. Blockage of autophagy upregulated radiation-induced cytotoxicity but only modestly affected the levels of cell death in CBD- or CBD/γ-irradiated 3D GBM cultures. Furthermore, CBD enhanced the pro-apoptotic activities of JNK1/2 and MAPK p38 signaling cascades while partially downregulated the pro-survival PI3K-AKT cascade, thereby changing a balance between cell death and survival. Suppression of JNK activation partially reduced CBD-induced cell death in 3D GBM cultures. In contrast, co-treatment of CBD-targeted cells with inhibitors of PI3K-AKT-NF-κB, IKK-NF-κB or JAK2-STAT3 pathways killed surviving GBM cells in both 2D and 3D cultures, potentially improving the therapeutic ratio of GBM.


Inappropriate use of the title 'chiropractor' and term 'chiropractic manipulation' in the peer-reviewed biomedical literature.
The misuse of the title 'chiropractor' and term 'chiropractic manipulation', in relation to injury associated with cervical spine manipulation, have previously been reported in the peer-reviewed literature. The objectives of this study were to--1) Prospectively monitor the peer-reviewed literature for papers reporting an association between chiropractic, or chiropractic manipulation, and injury; 2) Contact lead authors of papers that report such an association in order to determine the basis upon which the title 'chiropractor' and/or term 'chiropractic manipulation' was used; 3) Document the outcome of submission of letters to the editors of journals wherein the title 'chiropractor', and/or term 'chiropractic manipulation', had been misused and resulted in the over-reporting of chiropractic induced injury. One electronic database (PubMed) was monitored prospectively, via monthly PubMed searches, during a 12 month period (June 2003 to May 2004). Once relevant papers were located, they were reviewed. If the qualifications and/or profession of the care provider/s were not apparent, an attempt was made to confirm them via direct e-mail communication with the principal researcher of each respective paper. A letter was then sent to the editor of each involved journal. A total of twenty four different cases, spread across six separate publications, were located via the monthly PubMed searches. All twenty four cases took place in one of two European countries. The six publications consisted of four case reports, each containing one patient, one case series, involving twenty relevant cases, and a secondary report that pertained to one of the four case reports. In each of the six publications the authors suggest the care provider was a chiropractor and that each patient received chiropractic manipulation of the cervical spine prior to developing symptoms suggestive of traumatic injury. In two of the four case reports contact with the principal researcher revealed that the care provider was not a chiropractor, as defined by the World Federation of Chiropractic. The authors of the other two case reports did not respond to my communications. In the case series, which involved twenty relevant cases, the principal researcher conceded that the term chiropractor had been inappropriately used and that his case series did not relate to chiropractors who had undergone appropriate formal training. The author of the secondary report, a British Medical Journal editor, conceded that he had misused the title chiropractor. Letters to editors were accepted and published by all four journals to which they were sent. To date one of the four journals has published a correction. The results of this year-long prospective review suggests that the words 'chiropractor' and 'chiropractic manipulation' are often used inappropriately by European biomedical researchers when reporting apparent associations between cervical spine manipulation and symptoms suggestive of traumatic injury. Furthermore, in those cases reported here, the spurious use of terminology seems to have passed through the peer-review process without correction. Additionally, these findings provide further preliminary evidence, beyond that already provided by Terrett, that the inappropriate use of the title 'chiropractor' and term 'chiropractic manipulation' may be a significant source of over-reporting of the link between the care provided by chiropractors and injury. Finally, editors of peer-reviewed journals were amenable to publishing 'letters to editors', and to a lesser extent 'corrections', when authors had inappropriately used the title 'chiropractor' and/or term 'chiropractic manipulation'.


Relationship between resting medial gastrocnemius stiffness and drop jump performance.
Although the influence of the series elastic element of the muscle-tendon unit on jump performance has been investigated, the corresponding effect of the parallel elastic element remains unclear. This study examined the relationship between the resting calf muscle stiffness and drop jump performance. Twenty-four healthy men participated in this study. The shear moduli of the medial gastrocnemius and the soleus were measured at rest as an index of muscle stiffness using ultrasound shear wave elastography. The participants performed drop jumps from a 15 cm high box. The Spearman rank correlation coefficient was used to examine the relationships between shear moduli of the muscles and drop jump performance. The medial gastrocnemius shear modulus showed a significant correlation with the drop jump index (jump height/contact time) (r = 0.414, P = 0.044) and jump height (r = 0.411, P = 0.046), but not with contact time (P > 0.05). The soleus shear modulus did not correlate with these jump parameters (P > 0.05). These results suggest that the resting medial gastrocnemius stiffness can be considered as one of the factors that influence drop jump performance. Therefore, increase in resting muscle stiffness should enhance explosive athletic performance in training regimens.


Improving subspace constrained radial fast spin echo MRI using block matching driven non-local low rank regularization.
Subspace-constrained reconstruction methods restrict the relaxation signals (of size M) in the scene to a pre-determined subspace (of size K≪M) and allow multi-contrast imaging and parameter mapping from accelerated acquisitions. However, these constraints yield poor image quality at some imaging contrasts, which can impact the parameter mapping performance. Additional regularization such as the use of joint-sparse (JS) or locally-low-rank (LLR) constraints can help improve the recovery of these images but are not sufficient when operating at high acceleration rates. We propose a method, non-local rank 3D (NLR3D), that is built on block matching and transform domain low rank constraints to allow high quality recovery of subspace-coefficient images (SCI) and subsequent multi-contrast imaging and parameter mapping. The performance of NLR3D was evaluated using Monte-Carlo (MC) simulations and compared against the JS and LLR methods. In vivo T 2 mapping results are presented on brain and knee datasets. MC results demonstrate improved bias, variance, and MSE behavior in both the multi-contrast images and parameter maps when compared to the JS and LLR methods. In vivo brain and knee results at moderate and high acceleration rates demonstrate improved recovery of high SNR early TE images as well as parameter maps. No significant difference was found in the T2 values measured in ROIs between the NLR3D reconstructions and the reference images (Wilcoxon signed rank test). The proposed method, NLR3D, enables recovery of high-quality SCI and, consequently, the associated multi-contrast images and parameter maps.


Monitoring and evaluating capacity building activities in low and middle income countries: challenges and opportunities.
Lower and middle income countries (LMICs) are home to >80% of the global population, but mental health researchers and LMIC investigator led publications are concentrated in 10% of LMICs. Increasing research and research outputs, such as in the form of peer reviewed publications, require increased capacity building (CB) opportunities in LMICs. The National Institute of Mental Health (NIMH) initiative, Collaborative Hubs for International Research on Mental Health reaches across five regional 'hubs' established in LMICs, to provide training and support for emerging researchers through hub-specific CB activities. This paper describes the range of CB activities, the process of monitoring, and the early outcomes of CB activities conducted by the five research hubs. The indicators used to describe the nature, the monitoring, and the early outcomes of CB activities were developed collectively by the members of an inter-hub CB workgroup representing all five hubs. These indicators included but were not limited to courses, publications, and grants. Results for all indicators demonstrate a wide range of feasible CB activities. The five hubs were successful in providing at least one and the majority several courses; 13 CB recipient-led articles were accepted for publication; and nine grant applications were successful. The hubs were successful in providing CB recipients with a wide range of CB activities. The challenge remains to ensure ongoing CB of mental health researchers in LMICs, and in particular, to sustain the CB efforts of the five hubs after the termination of NIMH funding.


A model for training medical student innovators: the Harvard Medical School Center for Primary Care Abundance Agents of Change program.
In 2013, the Harvard Medical School Center for Primary Care established the Abundance Agents of Change (AoC) program to promote interprofessional learning and innovation, increase partnership between 15 academic and community health centers (CHCs) in Boston's most under-served communities, and increase medical student interest in primary care careers. The AoC is modeled in the form of a 'grants challenge', offering $20,000 to interprofessional student teams to develop an innovative solution that addresses a healthcare delivery need identified by CHCs. The program's initial two years were characterized by a four-stage process which included working with CHCs and crafting a request for proposals, forming interprofessional 20 student teams comprising students from across and outside of Harvard University, training students using a systems-based innovation curriculum, and performing program evaluation. Our evaluation data from cohorts 1 and 2 of the AoC program demonstrate that we succeeded in training students as innovators and members of interprofessional teams. We also learned valuable lessons regarding creating better alignment with CHC priorities, extending the program cycle from 12 to 18 months, and changing the way funding is disbursed to 25 students, which will be incorporated in later versions of the program. Based on our experience and evaluation data, we believe that this program is a replicable way to train students as innovators and members of interprofessional teams to address the current complex healthcare environment.


Fluoride metabolism and fluorosis.
This paper is primarily concerned with the only proven risk associated with water fluoridation: enamel fluorosis. Its purpose is to review current methods of measuring enamel fluorosis, its aetiology and metabolism. A further objective is to identify risk factors to reduce the prevalence of enamel fluorosis and employ methods to manage such risk factors. The prevalence of enamel fluorosis is increasing in Ireland and internationally. A critical period has been identified at which teeth are most at risk of developing enamel fluorosis: 15-24 months of age for males and 21-30 months of age for females. The data included took these two factors into account. A thorough narrative review of published literature was conducted to identify studies concerning the aetiology and metabolism of enamel fluorosis. Risk factors for fluorosis were identified from these studies. As it is the pre-eruptive phase of enamel development which represents the greatest risk to developing enamel fluorosis, studies examining sources of fluoride ingestion for young children were selected. These included studies on ingestion of fluoride toothpaste by young children, fluoride supplementation and infant formula reconstituted with fluoridated water. There is evidence that the age at which tooth brushing with fluoride toothpastes is commenced and the amount of fluoride placed on the brush are important risk factors in the incidence of dental fluorosis. It is recommended that brushing should not commence until the age of 2 and that a pea-sized amount (0.25 g) of toothpaste should be placed on the brush.


Influences of NOM composition and bacteriological characteristics on biological stability in a full-scale drinking water treatment plant.
The influences of natural organic matter (NOM) and bacteriological characteristics on the biological stability of water were investigated in a full-scale drinking water treatment plant. We found that prechlorination decreased the hydrophobicity of the organic matter and significantly increased the high-molecular-weight (MW) dissolved organic matter, such as biopolymers and humic substances. High-MW organic matter and structurally complex compounds are known to be relatively slowly biodegradable; however, because of the prechlorination step, the indigenous bacteria could readily utilise these fractions as assimilable organic carbon. Sequential coagulation and sedimentation resulted in the substantial removal of biopolymer (74%), humic substance (33%), bacterial cells (79%), and assimilable organic carbon (67%). Rapid sand and granular activated carbon filtration induced an increase in the low-nucleic-acid content bacteria; however, these bacteria were biologically less active in relation to enzymatic activity and ATP. The granular activated carbon step was essential to securing biological stability (the ability to prevent bacterial growth) by removing the residual assimilable organic carbon that had formed during the ozone treatment. The growth potential of Escherichia coli and indigenous bacteria were found to differ in respect to NOM characteristics. In comparison with E. coli, the indigenous bacteria utilised a broader range of NOM as a carbon source. Principal component analysis demonstrated that the measured biological stability of water could differ, depending on the NOM characteristics, as well as on the bacterial inoculum selected for the analysis.


Assessment and correlation between clinical patterns, disability and health-related quality of life in patients with low back pain.
Low back pain (LBP) is the main symptom of most lumbar spine diseases. This symptom, due to physical, psychological and social factors, is correlated to a real disability which can affect the quality of life (QoL.) The aim of this study is to evaluate the QoL and the disability in patients with LBP and to correlate them to clinical patterns. In this prospective multidimensional study, 108 patients underwent the following protocol: 1. collection of patient-oriented and disability data with the use of SF-36 and North American Spine Society (NASS); 2. collection of clinical, anamnestic and instrumental data (MRI, CT) with the use of DOVAC files; 3. assessment of disability with the use of Barthel index (BI) and deambulation index (DI). A significant correlation was found between the BI and the physical composite score (PCS), but no significant correlation was noticed between BI and the mental composite score (MCS). DI was not correlated to the SF-36. A positive Lasegue and the absence of the osteo-tendinous reflexes are significantly correlated to the disability and QoL. This study shows that there is no simple, linear correlation between QoL and disability.


Distribution and asymptotic behavior of the phylogenetic transfer distance.
The transfer distance (TD) was introduced in the classification framework and studied in the context of phylogenetic tree matching. Recently, Lemoine et al. (Nature 556(7702):452-456, 2018. https://doi.org/10.1038/s41586-018-0043-0 ) showed that TD can be a powerful tool to assess the branch support on large phylogenies, thus providing a relevant alternative to Felsenstein's bootstrap. This distance allows a reference branch[Formula: see text] in a reference tree [Formula: see text] to be compared to a branch b from another tree T (typically a bootstrap tree), both on the same set of n taxa. The TD between these branches is the number of taxa that must be transferred from one side of b to the other in order to obtain [Formula: see text]. By taking the minimum TD from [Formula: see text] to all branches in T we define the transfer index, denoted by [Formula: see text], measuring the degree of agreement of T with [Formula: see text]. Let us consider a reference branch [Formula: see text] having p tips on its light side and define the transfer support (TS) as [Formula: see text]. Lemoine et al. (2018) used computer simulations to show that the TS defined in this manner is close to 0 for random "bootstrap" trees. In this paper, we demonstrate that result mathematically: when T is randomly drawn, TS converges in probability to 0 when n tends to [Formula: see text]. Moreover, we fully characterize the distribution of [Formula: see text] on caterpillar trees, indicating that the convergence is fast, and that even when n is small, moderate levels of branch support cannot appear by chance.


Inhibition of adrenaline-forming enzyme in the brain prevents one-kidney, one-clip hypertension and deoxycorticosterone acetate-salt hypertension in the rabbit.
1. Phenylethanolamine N-methyltransferase (PNMT) converts noradrenaline into adrenaline and brain PNMT is elevated in spontaneously hypertensive and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. In view of the evidence for the involvement of central adrenergic neurons in renal hypertension, we measured the blood pressure response in one-clip, one-kidney Goldblatt hypertensive and DOCA-salt hypertensive rabbits to the PNMT inhibitor SK&F 64139, injected into the lateral cerebral ventricles. 2. Intracerebroventricular injection of SK&F 64139 (10 micrograms/kg) significantly attenuated the mean arterial blood pressure rise in one-clip, one-kidney and DOCA-salt rabbits, at 4 and 8 weeks. 3. These findings support the idea the hypertension in this animal model required an intact adrenaline biosynthetic process, and that central catecholaminergic neurons may be involved in the pathogenesis of low-renin dependent forms of hypertension.


The Effect of Vertically Yoked Prisms on Binocular Vision and Accommodation.
Vertically yoked prisms have been used in treatment of binocular vision dysfunction despite minimal supporting evidence. In people with normal binocular vision, the impact on phorias has been assessed but not the impact on accommodation, accommodation vergence interactions, or the horopter. We found that vertically yoked prisms have minor effects during short-term wear in young adults. The purpose of this study was to determine effects of vertically yoked prisms on accommodative response and several binocular vision tasks. There were 45 participants aged 18 to 24 years. The 23 myopes wore distance-corrected soft contact lenses. In a random arrangement, each person wore spectacles containing planopower lenses with either 8 Δ base-up, 4 Δ base-up, zero, 4 Δ base-down, and 8 Δ base-down prisms. Before spectacle wear, baseline measurements of near heterophoria, accommodation response, negative and positive relative accommodations, fusional vergence, and Nonius-horopter spatial perception were taken. Measurements were repeated after a 40-minute wear, spectacles were removed, and tests were performed 20 minutes later. On a 22-participant subset, on a separate occasion, measurements of heterophoria, accommodation response, and relative accommodation were made immediately after spectacles were fitted. Most changes relative to baseline were not significant. Where effects occurred, these were nearly all associated with prism presence rather than adaptation. There were significant effects on accommodation response, but these seem to be refraction effects produced by pantoscopic tilt-induced power changes rather than perceptual effects altering accommodation. There were statistically significant effects on negative relative accommodation (P < .01), with zero prism giving more negative relative accommodation than 8 Δ base-down prisms. Tendencies were noted for prisms to move horopter limits toward the observer. Effects were small and likely not of clinical relevance. Vertically yoked prisms have minor effects on accommodation and binocular vision, at least during short-term wear in young adults with normal binocular vision.


Elaborative encoding during REM dreaming as prospective emotion regulation.
Rapid eye movement (REM) dreaming results in "emotionally intelligent encoding," according to the target article. Building on this, we argue that elaborative encoding alters emotional processing of upcoming events and thereby functions as prospective emotion regulation. After elaborative encoding, future events are appraised differently and result in a redirected emotional response. Disturbed elaborative encoding might be relevant for emotional dysregulation in psychopathology.


Vertical nanocolumn-assisted pluripotent stem cell colony formation with minimal cell-penetration.
The biological applications of vertical nanostructures mostly rely on their intracellular accessibility through the cellular membrane by promoting cell-to-nanostructure interactions. Herein, we report a seemingly counter-intuitive approach for the spontaneous formation of mouse induced pluripotent stem cell (iPSC)-derived three-dimensional spherical colonies with unlimited self-renewal and differentiation potential. The comprehensive analyses of iPSCs cultured on vertical silicon nanocolumn arrays (vSNAs) with various nanocolumn geometries show reduced cell-to-substrate adhesion and enhanced cell-to-cell interactions under optimized vSNA conditions, successfully accommodating the spontaneous production of iPSC-derived spherical colonies. Remarkably, these colonies which were only minimally penetrated by and thereby easily harvested from wafer-sized vSNAs display a substantial increase in pluripotency marker expression and successfully differentiate into three germ layers. Our vSNAs capable of large-scale fabrication, efficient for spherical colony formation, and reusable for multiple iPSC culture could serve as a broad-impact culture platform for stem cell research.


Police Use of Force by Ethnicity, Sex, and Socioeconomic Class.
Disparities in police responses to Black and White people have received significant research and public attention in recent years. This study examines self-reported accounts of exposure to and perceptions of police use of force among Black and White ethnic groups by sex and income level. Using bivariate and multivariate approaches, we analyzed data from the Bureau of Justice Statistics 2011 Police-Public Contact Survey, a measure administered to a nationally representative sample of U.S. residents. Participants were asked a series of questions about their most recent contact with police during a 12-month period. Our analyses were limited to Black (n = 59; mean age 28.8 years) and White (n = 366; mean age 34.6 years) participants whose most recent involuntary contact with police included a street stop. For Black residents, being male and having an income under $20,000 significantly increased the risk for exposure to police use of force during a street stop. For White residents, being male, having an income under $20,000, or being age 35 or older significantly increased the risk for exposure to police use of force during a street stop. Future research will benefit from additional attention to the cumulative impact of police use of force and how experience with police use of force shapes U.S. residents' understanding of and expectations for procedural justice.


Mechanical Thrombectomy of Distal Occlusions in the Anterior Cerebral Artery: Recanalization Rates, Periprocedural Complications, and Clinical Outcome.
Patients with acute ischemic stroke in the anterior circulation are at risk for either primary or, following mechanical thrombectomy, secondary occlusion of the anterior cerebral artery. Because previous studies had only a limited informative value, we report our data concerning the frequency and location of distal anterior cerebral artery occlusions, recanalization rates, periprocedural complications, and clinical outcome. We performed a retrospective analysis of prospectively collected data of patients with acute ischemic stroke undergoing mechanical thrombectomy in the anterior circulation between June 2010 and April 2015. Of 368 patients included in this analysis, we identified 30 (8.1%) with either primary (n = 17, 4.6%) or secondary (n = 13, 3.5%) embolic occlusion of the distal anterior cerebral artery. The recanalization rate after placement of a stent retriever was 88%. Periprocedural complications were rare and included vasospasms (n = 3, 10%) and dissection (n = 1, 3.3%). However, 16 (53.5%) patients sustained an (at least partial) infarction of the anterior cerebral artery territory. Ninety days after the ictus, clinical outcome according to the modified Rankin Scale score was the following: 0-2, n = 11 (36.6%); 3-4, n = 9 (30%); 5-6, n = 10 (33.3%). Occlusions of the distal anterior cerebral artery affect approximately 8% of patients with acute ischemic stroke in the anterior circulation receiving mechanical thrombectomy. Despite a high recanalization rate and a low complication rate, subsequent (partial) infarction in the anterior cerebral artery territory occurs in approximately half of patients. Fortunately, clinical outcome appears not to be predominately unfavorable.


Distinct mutations led to inactivation of type 1 fimbriae expression in Shigella spp.
Shigella spp. are responsible for bacillary dysentery in humans. The acquisition or the modification of the virulence plasmid encoding factors promoting entry of bacteria into and dissemination within epithelial cells was a critical step in the evolution of these bacteria from their Escherichia coli ancestor(s). Incorporation of genomic islands (GI) and gene inactivation also shaped interactions between these pathogens and their human host. Sequence analysis of the GI inserted next to the leuX tRNA gene in S. boydii, S. dysenteriae, S. flexneri, S. sonnei and enteroinvasive E. coli (EIEC) suggests that this region initially carried the fec, yjhATS and fim gene clusters. The fim cluster encoding type I fimbriae is systematically inactivated in both reference strains and clinical isolates and distinct mutations are responsible for this inactivation in at least three phylogenetic groups. To investigate consequences of the presence of fimbriae on the outcome of the interaction of Shigella with host cells, we used a S. flexneri strain harboring a plasmid encoding the E. coli fim operon. Production of fimbriae by this recombinant strain increased the ability of bacteria to adhere to and enter into epithelial cells and had no effect on their ability to disseminate from cell to cell. The observations that production of type I fimbriae increases invasion of epithelial cells and that independent mutations abolish fimbriae production in Shigella suggest that these mutations correspond to pathoadaptive events.


Local anaesthetic dosage of peripheral nerve blocks in children: analysis of 40 121 blocks from the Pediatric Regional Anesthesia Network database.
The knowledge regarding appropriate dosage of local anaesthetics for peripheral nerve blocks in children is very scarce. The main objective of the current investigation was to evaluate dosing patterns of local anaesthetics in children receiving peripheral nerve blocks across multiple paediatric hospitals in the USA. We also sought to estimate the incidence of local anaesthetic systemic toxicity. This is an observational study using the Pediatric Regional Anesthesia Network (PRAN) database. Data on every peripheral nerve block in patients aged <18 years placed from April 1, 2007 to May 31, 2015 were examined as a subset of the PRAN protocol. Data were examined for the type and dose of local anaesthetic and for the presence of local anaesthetic systemic toxicity. In total, 40 121 peripheral nerve blocks in children were analysed. Individual analyses of block type demonstrated large local anaesthetic dose variability with a five- to 10-fold spread depending on the block type. Two patients developed local anaesthetic systemic toxicity, resulting in an estimated incidence (95% CI) per blocks performed of 0.005% (0.001-0.015%). None of the patients had any short- or long-term complications or sequelae. We detected a large variability in the local anaesthetic dosing practices for peripheral nerve blocks in children across multiple hospitals in the USA. Nonetheless, the risk of local anaesthetic systemic toxicity was very low. Due to the lack of dose findings studies, our results suggest the need to develop practice guidelines to minimize variability of regional anaesthesia practices in children.


Efficacy of Single-Dose Dexmedetomidine Combined with Low-Dose Remifentanil Infusion for Cough Suppression Compared to High-Dose Remifentanil Infusion: A Randomized, Controlled, Non-Inferiority Trial.
Background: Combination of dexmedetomidine and opioid may be an alternative to high-dose opioid in attenuating cough during emergence from anesthesia, while also reducing the adverse effects of high-dose opioid. We tested the hypothesis that a single-dose of dexmedetomidine combined with low-dose remifentanil infusion during emergence would not be inferior to high-dose remifentanil infusion alone in attenuating cough after thyroidectomy. Methods: One hundred sixty-nine patients undergoing thyroidectomy were enrolled and randomized in a 1:1 ratio into group DR or group R. Each patient received an infusion of dexmedetomidine (0.5 μg/kg) and low-dose remifentanil infusion of effect-site concentration (Ce) at 1 ng/mL or normal saline and high-dose remifentanil infusion of Ce at 2 ng/mL for 10 min at the end of surgery. Remifentanil was maintained until tracheal extubation. Primary endpoint was the severity of coughing, which was assessed for non-inferiority using a four-point scale at the time of extubation. For comparison of coughing incidence during emergence, coughing grade was also measured at three times: before extubation, at extubation, and after extubation. Time to awakening, hemodynamic and respiratory profile, pain, and postoperative nausea and vomiting were also evaluated for superiority. Results: The 95% confidence intervals for differences in cough grade during tracheal extubation were <0.9, indicating non-inferiority of the single dose of dexmedetomidine combined with low-dose remifentanil infusion. The incidence of coughing was similar in the two groups. Hemodynamic changes during tracheal extubation were attenuated, but emergence from anesthesia was delayed, in group DR. Use of rescue antiemetic was similar in both groups, but the incidence of vomiting was less in group DR. Conclusion: A single-dose of dexmedetomidine (0.5 μg/kg) combined with low-dose remifentanil infusion at 1 ng/mL of Ce during emergence from sevoflurane-remifentanil anesthesia was not inferior to high-dose remifentanil infusion alone at 2 ng/mL of Ce with regard to suppressing cough.


Morphological single cell profiling of the epithelial-mesenchymal transition.
Single cells respond heterogeneously to biochemical treatments, which can complicate the analysis of in vitro and in vivo experiments. In particular, stressful perturbations may induce the epithelial-mesenchymal transition (EMT), a transformation through which compact, sensitive cells adopt an elongated, resistant phenotype. However, classical biochemical measurements based on population averages over large numbers cannot resolve single cell heterogeneity and plasticity. Here, we use high content imaging of single cell morphology to classify distinct phenotypic subpopulations after EMT. We first characterize a well-defined EMT induction through the master regulator Snail in mammary epithelial cells over 72 h. We find that EMT is associated with increased vimentin area as well as elongation of the nucleus and cytoplasm. These morphological features were integrated into a Gaussian mixture model that classified epithelial and mesenchymal phenotypes with >92% accuracy. We then applied this analysis to heterogeneous populations generated from less controlled EMT-inducing stimuli, including growth factors (TGF-β1), cell density, and chemotherapeutics (Taxol). Our quantitative, single cell approach has the potential to screen large heterogeneous cell populations for many types of phenotypic variability, and may thus provide a predictive assay for the preclinical assessment of targeted therapeutics.


Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs.
For many years, Taq polymerase has served as the stalwart enzyme in the PCR amplification of DNA. However, a major limitation of Taq is its inability to amplify damaged DNA, thereby restricting its usefulness in forensic applications. In contrast, Y-family DNA polymerases, such as Dpo4 from Sulfolobus solfataricus, can traverse a wide variety of DNA lesions. Here, we report the identification and characterization of five novel thermostable Dpo4-like enzymes from Acidianus infernus, Sulfolobus shibatae, Sulfolobus tengchongensis, Stygiolobus azoricus and Sulfurisphaera ohwakuensis, as well as two recombinant chimeras that have enhanced enzymatic properties compared with the naturally occurring polymerases. The Dpo4-like polymerases are moderately processive, can substitute for Taq in PCR and can bypass DNA lesions that normally block Taq. Such properties make the Dpo4-like enzymes ideally suited for the PCR amplification of damaged DNA samples. Indeed, by using a blend of Taq and Dpo4-like enzymes, we obtained a PCR amplicon from ultraviolet-irradiated DNA that was largely unamplifyable with Taq alone. The inclusion of thermostable Dpo4-like polymerases in PCRs, therefore, augments the recovery and analysis of lesion-containing DNA samples, such as those commonly found in forensic or ancient DNA molecular applications.


Single-Domain Antibodies Represent Novel Alternatives to Monoclonal Antibodies as Targeting Agents against the Human Papillomavirus 16 E6 Protein.
Approximately one fifth of all malignancies worldwide are etiologically associated with a persistent viral or bacterial infection. Thus, there is a particular interest in therapeutic molecules which use components of a natural immune response to specifically inhibit oncogenic microbial proteins, as it is anticipated they will elicit fewer off-target effects than conventional treatments. This concept has been explored in the context of human papillomavirus 16 (HPV16)-related cancers, through the development of monoclonal antibodies and fragments thereof against the viral E6 oncoprotein. Challenges related to the biology of E6 as well as the functional properties of the antibodies themselves appear to have precluded their clinical translation. Here, we addressed these issues by exploring the utility of the variable domains of camelid heavy-chain-only antibodies (denoted as VHHs). Through construction and panning of two llama, immune VHH phage display libraries, a pool of potential VHHs was isolated. The interactions of these with recombinant E6 were further characterized using an enzyme-linked immunosorbent assay (ELISA), Western blotting under denaturing and native conditions, and surface plasmon resonance. Three VHHs were identified that bound recombinant E6 with nanomolar affinities. Our results lead the way for subsequent studies into the ability of these novel molecules to inhibit HPV16-infected cells in vitro and in vivo.


Characterizing breast conditions at an open-access breast clinic in South Africa: a model that is more than cancer care for a resource-limited setting.
While most breast-related research focuses on cancer, presentation of symptomatic persons in non-screened environments requires understanding the spectrum of breast diseases so as to plan services in resource-constrained settings. This study presents the variety of breast disease managed at a government, open-access breast clinic in South Africa. We performed a retrospective file review using a systematic random sample of patients 18 years and above presenting for breast care over a 14-month period. We collected demographics, clinical characteristics, management and final diagnoses from the first visit and twelve subsequent months. The final sample contained 365 individuals (97 · 5% women). Most were black, unmarried and South African citizens with a median age of 43 years (IQR 31-55) . Of those reporting their status (24 · 1%) 38 · 6% were HIV-positive. A mass (57 · 0%) and/or pain (28 · 5%) were the most common symptoms. Imaging and breast biopsies were required in 78 and 25% of individuals, respectively. Nearly half of biopsies identified breast cancer (44 · 1% of women ≤40 and 57 · 3% for women >40). Benign conditions (47 · 7%) and no abnormality (18 · 2%) were common final classifications among women. There was no difference between the final classifications of patients who self-referred versus those who were formally referred from another health care provider. Nearly half of the participants (46 · 6%) travelled 20 km or more to attend the clinic. Benign breast conditions far outweighed cancer diagnoses. As breast cancer awareness increases in resource-limited countries, facilities offering breast care require administrative and clinical preparation to manage a range of non-cancer related conditions.


A general protocol for determining the structures of molecularly ordered but noncrystalline silicate frameworks.
A general protocol is demonstrated for determining the structures of molecularly ordered but noncrystalline solids, which combines constraints provided by X-ray diffraction (XRD), one- and two-dimensional solid-state nuclear magnetic resonance (NMR) spectroscopy, and first-principles quantum chemical calculations. The approach is used to determine the structure(s) of a surfactant-directed layered silicate with short-range order in two dimensions but without long-range periodicity in three-dimensions (3D). The absence of long-range 3D molecular order and corresponding indexable XRD reflections precludes determination of a space group for this layered silicate. Nevertheless, by combining structural constraints obtained from solid-state (29)Si NMR analyses, including the types and relative populations of distinct (29)Si sites, their respective (29)Si-O-(29)Si connectivities and separation distances, with unit cell parameters (though not space group symmetry) provided by XRD, a comprehensive search of candidate framework structures leads to the identification of a small number of candidate structures that are each compatible with all of the experimental data. Subsequent refinement of the candidate structures using density functional theory calculations allows their evaluation and identification of "best" framework representations, based on their respective lattice energies and quantitative comparisons between experimental and calculated (29)Si isotropic chemical shifts and (2)J((29)Si-O-(29)Si) scalar couplings. The comprehensive analysis identifies three closely related and topologically equivalent framework configurations that are in close agreement with all experimental and theoretical structural constraints. The subtle differences among such similar structural models embody the complexity of the actual framework(s), which likely contain coexisting or subtle distributions of structural order that are intrinsic to the material.


Assessing atrophy measurement techniques in dementia: Results from the MIRIAD atrophy challenge.
Structural MRI is widely used for investigating brain atrophy in many neurodegenerative disorders, with several research groups developing and publishing techniques to provide quantitative assessments of this longitudinal change. Often techniques are compared through computation of required sample size estimates for future clinical trials. However interpretation of such comparisons is rendered complex because, despite using the same publicly available cohorts, the various techniques have been assessed with different data exclusions and different statistical analysis models. We created the MIRIAD atrophy challenge in order to test various capabilities of atrophy measurement techniques. The data consisted of 69 subjects (46 Alzheimer's disease, 23 control) who were scanned multiple (up to twelve) times at nine visits over a follow-up period of one to two years, resulting in 708 total image sets. Nine participating groups from 6 countries completed the challenge by providing volumetric measurements of key structures (whole brain, lateral ventricle, left and right hippocampi) for each dataset and atrophy measurements of these structures for each time point pair (both forward and backward) of a given subject. From these results, we formally compared techniques using exactly the same dataset. First, we assessed the repeatability of each technique using rates obtained from short intervals where no measurable atrophy is expected. For those measures that provided direct measures of atrophy between pairs of images, we also assessed symmetry and transitivity. Then, we performed a statistical analysis in a consistent manner using linear mixed effect models. The models, one for repeated measures of volume made at multiple time-points and a second for repeated "direct" measures of change in brain volume, appropriately allowed for the correlation between measures made on the same subject and were shown to fit the data well. From these models, we obtained estimates of the distribution of atrophy rates in the Alzheimer's disease (AD) and control groups and of required sample sizes to detect a 25% treatment effect, in relation to healthy ageing, with 95% significance and 80% power over follow-up periods of 6, 12, and 24months. Uncertainty in these estimates, and head-to-head comparisons between techniques, were carried out using the bootstrap. The lateral ventricles provided the most stable measurements, followed by the brain. The hippocampi had much more variability across participants, likely because of differences in segmentation protocol and less distinct boundaries. Most methods showed no indication of bias based on the short-term interval results, and direct measures provided good consistency in terms of symmetry and transitivity. The resulting annualized rates of change derived from the model ranged from, for whole brain: -1.4% to -2.2% (AD) and -0.35% to -0.67% (control), for ventricles: 4.6% to 10.2% (AD) and 1.2% to 3.4% (control), and for hippocampi: -1.5% to -7.0% (AD) and -0.4% to -1.4% (control). There were large and statistically significant differences in the sample size requirements between many of the techniques. The lowest sample sizes for each of these structures, for a trial with a 12month follow-up period, were 242 (95% CI: 154 to 422) for whole brain, 168 (95% CI: 112 to 282) for ventricles, 190 (95% CI: 146 to 268) for left hippocampi, and 158 (95% CI: 116 to 228) for right hippocampi. This analysis represents one of the most extensive statistical comparisons of a large number of different atrophy measurement techniques from around the globe. The challenge data will remain online and publicly available so that other groups can assess their methods.


Redefining Myeloid Cell Subsets in Murine Spleen.
Spleen is known to contain multiple dendritic and myeloid cell subsets, distinguishable on the basis of phenotype, function and anatomical location. As a result of recent intensive flow cytometric analyses, splenic dendritic cell (DC) subsets are now better characterized than other myeloid subsets. In order to identify and fully characterize a novel splenic subset termed "L-DC" in relation to other myeloid cells, it was necessary to investigate myeloid subsets in more detail. In terms of cell surface phenotype, L-DC were initially characterized as a CD11b(hi)CD11c(lo)MHCII(-)Ly6C(-)Ly6G(-) subset in murine spleen. Their expression of CD43, lack of MHCII, and a low level of CD11c was shown to best differentiate L-DC by phenotype from conventional DC subsets. A complete analysis of all subsets in spleen led to the classification of CD11b(hi)CD11c(lo)MHCII(-)Ly6C(lo)Ly6G(-) cells as monocytes expressing CX3CR1, CD43 and CD115. Siglec-F expression was used to identify a specific eosinophil population, distinguishable from both Ly6C(lo) and Ly6C(hi) monocytes, and other DC subsets. L-DC were characterized as a clear subset of CD11b(hi)CD11c(lo)MHCII(-)Ly6C(-)Ly6G(-) cells, which are CD43(+), Siglec-F(-) and CD115(-). Changes in the prevalence of L-DC compared to other subsets in spleens of mutant mice confirmed the phenotypic distinction between L-DC, cDC and monocyte subsets. L-DC development in vivo was shown to occur independently of the BATF3 transcription factor that regulates cDC development, and also independently of the FLT3L and GM-CSF growth factors which drive cDC and monocyte development, so distinguishing L-DC from these commonly defined cell types.


Onset of secondary progressive phase and long-term evolution of multiple sclerosis.
To assess factors affecting the rate of conversion to secondary progressive (SP) multiple sclerosis (MS) and its subsequent evolution. Among 806 patients with relapsing remitting (RR) onset MS from the London Ontario database, we used Kaplan-Meier, Cox regression and multiple logistic regression analyses to investigate the effect of baseline clinical and demographic features on (1) the probability of, and the time to, SP disease, (2) the time to bedbound status (Disability Status Scale (DSS 8)) from onset of progression. The risk of entering the SP phase increased proportionally with disease duration (OR=1.07 for each additional year; p<0.001). Shorter latency to SP was associated with shorter times to severe disability. The same association was found even when patients were grouped by number of total relapses before progression. However, the evolution of the SP phase was not influenced by the duration of the RR phase. Male sex (HR=1.41; p<0.001), older age at onset (age ≤20 and 21-30 vs >30 HR=0.52 (p<0.001), 0.65 (p<0.001), respectively) and high early relapse frequency (1-2 attacks vs ≥3 HR=0.63 (p<0.001), 0.75 (p=0.04), respectively) predicted significantly higher risk of SP MS and shorter latency to progression. Times to DSS 8 from onset of progression were significantly shorter among those with high early relapse frequency (≥3 attacks), and among those presenting with cerebellar and brainstem symptoms. The onset of SP MS is the dominant determinant of long-term prognosis, and its prevention is the most important target measure for treatment. Baseline clinical features of early relapse frequency and age at onset can be used to select groups at higher risk of developing severe disability based on the probability of their disease becoming progressive within a defined time period.


Anatomic parameters: direct intralaminar screw repair of spondylolysis.
Analysis via computed tomography imaging software. To determine parameters for ideal intralaminar screw trajectory and the feasibility of screw placement at L3, L4, and L5 laminae for pars defect fixation. To our knowledge, no studies provide anatomic parameters for ideal intralaminar screw trajectory for treating spondylolysis. We used advanced imaging software for 3-dimensional interactive viewing to analyze 20 randomly selected normal adolescent lumbar computed tomographic scans. The ideal intralaminar screw trajectory was drawn from the inferior lamina, a point chosen to maximize cortical diameter at the isthmus of the lamina and bisect the pedicle. We measured and evaluated ideal trajectory parameters for percutaneous screw placement for pars defect fixation at the L3 to L5 laminae. The ideal pathway was the thick portion of the lamina between the inferior edge of the lamina and the pedicle. This area was close to the inferior articular process (axial plane), becoming more so at progressively caudal levels. At the ideal trajectory, the mean (standard deviation) coronal angle slightly decreased (L3-L5): 7.3° (5.1°), 6.6° (3.7°), and 4.2° (2.5°), respectively. The trajectory distance increased from cranial to caudal. These parameters increased (L3-L5): mean distance (transverse plane) between the starting point and middle of the spinous process, 1.2 (0.18 cm), 1.3 (0.2 cm), and 1.6 (0.3 cm), respectively; mean screw sagittal angle with respect to the posterior skin, 15.5° (5.0°), 24.3° (6.5°), and 43° (5.8°), respectively; and mean distance for guide wire entry, 28.8 (10.6 cm), 20.1 (5.4 cm), and 11.9 (2.1 cm), respectively. At the ideal screw trajectory, pars fixation by intralaminar screw is uniformly feasible at L3 to L5 laminae, where most patients can accommodate a 4.5-mm screw. 2.


Survival in MS: a randomized cohort study 21 years after the start of the pivotal IFNβ-1b trial.
To examine the effects of interferon beta (IFNβ)-1b on all-cause mortality over 21 years in the cohort of 372 patients who participated in the pivotal randomized clinical trial (RCT), retaining (in the analysis) the original randomized treatment-assignments. For this randomized long-term cohort study, the primary outcome, defined before data collection, was the comparison of all-cause mortality between the IFNβ-1b 250 μg and placebo groups from the time of randomization through the entire 21-year follow-up interval (intention-to-treat, log-rank test for Kaplan-Meier survival curves). All other survival outcomes were secondary. After a median of 21.1 years from RCT enrollment, 98.4%(366 of 372) of patients were identified, and, of these, 81 deaths were recorded (22.1% [81 of 366]). Patients originally randomly assigned to IFNβ-1b 250 μg showed a significant reduction in all-cause mortality over the 21-year period compared with placebo (p = 0.0173), with a hazard ratio of 0.532 (95% confidence interval 0.314-0.902). The hazard rate of death at long-term follow-up by Kaplan-Meier estimates was reduced by 46.8% among IFNβ-1b 250 μg-treated patients (46.0% among IFNβ-1b 50 μg-treated patients) compared with placebo. Baseline variables did not influence the observed treatment effect. There was a significant survival advantage in this cohort of patients receiving early IFNβ-1b treatment at either dose compared with placebo. Near-complete ascertainment, together with confirmatory findings from both active treatment groups, strengthens the evidence for an IFNβ-1b benefit on all-cause mortality. This study provides Class III evidence that early treatment with IFNβ-1b is associated with prolonged survival in initially treatment-naive patients with relapsing-remitting multiple sclerosis.


Genetic counseling of patients with ovarian carcinoma: acceptance, timing, and psychological wellbeing.
The new Dutch guidelines on hereditary and familial ovarian carcinoma recommend genetic testing of all patients with epithelial ovarian cancer (EOC). With this study, we aimed to obtain insight into (1) the acceptance and timing of the offer of genetic counseling in women with EOC, (2) reasons for accepting or declining genetic counseling, and (3) psychological differences between women who did and did not have genetic counseling. A multicenter questionnaire survey was performed in patients with EOC in four Dutch oncology centers. The questionnaire addressed whether, how, and when genetic counseling was offered, women's arguments to accept or decline genetic counseling, and included the Cancer Worry Scale (CWS) and the Hospital Anxiety and Depression Scale (HADS). A total of 67 women completed the questionnaire, of which 43 had genetic counseling. Despite a wide variability in the timing of the offer of genetic counseling, 89% of the women were satisfied with the timing. No significant differences were found between the CWS and HADS scores for the timing of the offer of genetic counseling and whether or not women had genetic counseling. Taking the small sample size into account, the results tentatively suggest that genetic counseling may have limited impact on the psychosocial wellbeing of women with EOC. Therefore, we assume that implementation of the new guidelines offering genetic counseling to all patients with EOC will not cause considerable additional burden to these patients.


Monoclonal antibody that blocks phosphoinositide-dependent activation of mouse tumor DNA polymerase alpha.
A monoclonal antibody (MaB) against mouse sarcoma DNA polymerase alpha was isolated from the culture medium of an IgG-secreting hybridoma. The MaB demonstrated reactivity against two murine DNA polymerase alpha preparations and a calf thymus DNA polymerase alpha. Murine sarcoma polymerase was activated in vitro by phosphatidylinositol-4-monophosphate (PIP) showing increased deoxynucleotidyltransferase activity and enhanced binding affinity to activated DNA template. The MaB did not neutralize polymerase activity, but blocked further activation of the enzyme by PIP. Treatment of polymerase with MaB prior to treatment with PIP inhibited both increased enzyme activation and increased binding of the enzyme to DNA template. Treatment of polymerase with MaB subsequent to treatment with PIP did not block enzyme activation or increased DNA template binding. The data suggest that this anti-DNA polymerase alpha IgG is directed against a regulatory subunit of the polymerase rather than the catalytic subunit. The antibody may serve to distinguish between DNA polymerase alpha preparations with distinctly different regulatory subunits.


Differential expression of phospholipase D1 in the developing retina.
Expression patterns of phospholipase D1 (PLD1) in the developing rat retina were investigated using immunocytochemistry and Western blot analysis and compared with the expression patterns of glutamine synthetase. PLD1 immunoreactivity appeared first in a few neuroblasts in the middle of the mantle zone of the primitive retina by embryonic (E) day 13. PLD1-immunoreactive primitive ganglion cells were characterized in the ganglion cell layer by E17. Faint immunoreactivity at E17 profiled radially orientated cells and this pattern appeared up to postnatal (P) day 7. In the ganglion cell layer at P3, displaced amacrine cells and ganglion cells were classified. At P5, presumptive horizontal cells and amacrine cells were identified. By P7, a thin outermost layer of newly formed segments of the photoreceptor cells was also PLD1 immunoreactive. PLD1 immunoreactivity at P8 was limited to radial Müller cells and the outer segment layer of the photoreceptor cells, and the expression pattern was conserved to adulthood. Western blot analysis showed relatively high amounts of PLD1 protein at E17 and P3, a decrease at P7, and moderate amounts from P8 onward. Co-expression of PLD1 with glutamine synthetase in the retina appeared first after birth in differentiating neurons and in Müller cells by P8; thereafter the pattern was maintained. The expression pattern of the PLD1 during development of the retina suggests that PLD1 plays important roles in glutamate-associated differentiation of both specific neurons and radial glial cells, and in glutamate-mediated cellular signalling in Müller cells.


Does educational attainment contribute to risk for delirium? A potential role for cognitive reserve.
The objective of this study was to determine if level of educational attainment, a marker of cognitive reserve, was associated with the cumulative risk of delirium among hospitalized elders. We performed a secondary analysis of two hospital-based studies. The first (study 1) was an observational study involving 491 admissions. The second study (study 2) involved consecutive admissions assigned to the usual care condition in a controlled clinical trial, and included 461 persons. All participants were elderly (aged 70+) and free from delirium at admission. The outcome was the occurrence of delirium, as rated by the Confusion Assessment Method during hospitalization. In study 1 and 2, 22% and 14% of persons developed delirium (cumulative incidence), respectively. In both studies, risk of delirium was higher among persons with fewer years of education. Controlling for the effect of age, sex, dementia, comorbidity, and severity of illness, each year of completed education was associated with a 0.91 lower odds of delirium (95% confidence interval: 0.87, 0.95): compared to persons with 12 years of education, persons with 7 years of education had 1.6-fold increased odds of delirium (95% confidence interval: 1.4, 2.0). Hospitalized older persons with low educational attainment are at increased risk for delirium relative to persons with more education. This finding may have implications for the role of cognitive reserve in characterizing individual differences in risk for delirium.


Stromal fibroblast-derived miR-409 promotes epithelial-to-mesenchymal transition and prostate tumorigenesis.
Tumor-stromal interaction is a dynamic process that promotes tumor growth and metastasis via cell-cell interaction and extracellular vesicles. Recent studies demonstrate that stromal fibroblast-derived molecular signatures can be used to predict disease progression and drug resistance. To identify the epigenetic role of stromal noncoding RNAs in tumor-stromal interactions in the tumor microenvironment, we performed microRNA profiling of patient cancer-associated prostate stromal fibroblasts isolated by laser capture dissection microscopy and in bone-associated stromal models. We found specific upregulation of miR-409-3p and miR-409-5p located within the embryonically and developmentally regulated DLK1-DIO3 (delta-like 1 homolog-deiodinase, iodothyronine 3) cluster on human chromosome 14. The findings in cell lines were further validated in human prostate cancer tissues. Strikingly, ectopic expression of miR-409 in normal prostate fibroblasts conferred a cancer-associated stroma-like phenotype and led to the release of miR-409 via extracellular vesicles to promote tumor induction and epithelial-to-mesenchymal transition in vitro and in vivo. miR-409 promoted tumorigenesis through repression of tumor suppressor genes such as Ras suppressor 1 and stromal antigen 2. Thus, stromal fibroblasts derived miR-409-induced tumorigenesis, epithelial-to-mesenchymal transition and stemness of the epithelial cancer cells in vivo. Therefore, miR-409 appears to be an attractive therapeutic target to block the vicious cycle of tumor-stromal interactions that plagues prostate cancer patients.


Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum.
A novel member of the serine/threonine protein kinase gene family, designated sgk, for serum and glucocorticoid-regulated kinase, was identified in a differential screen for glucocorticoid-inducible transcripts expressed in the Con8.hd6 rat mammary tumor cell line. sgk encodes a protein of 49 kDa which has significant sequence homology (45 to 55% identity) throughout its catalytic domain with rac protein kinase, the protein kinase C family, ribosomal protein S6 kinase, and cyclic AMP-dependent protein kinase. sgk mRNA is expressed in most adult rat tissues, with the highest levels in the thymus, ovary, and lung, as well as in several rodent and human cell lines. sgk mRNA was stimulated by glucocorticoids and by serum within 30 min, and both inductions were independent of de novo protein synthesis. The transcriptional regulation by glucocorticoids is a primary response, since the promoter of sgk contains a glucocorticoid response element consensus sequence 1.0 kb upstream of the start of transcription which is able to stimulate chloramphenicol acetyltransferase reporter gene activity in a dexamethasone-dependent manner. Antibodies that specifically recognize sgk-encoded protein on an immunoblot were generated. This protein was shown to increase in abundance with glucocorticoid treatment in a manner which paralleled the mRNA accumulation. This is the first report of a presumed serine/threonine protein kinase that is highly regulated at the transcriptional level by glucocorticoid hormones and suggests a novel interplay between glucocorticoid receptor signalling and a protein kinase of the second messenger family.


The genetics and biomechanics of thoracic aortic diseases.
Thoracic aortic aneurysms and aortic dissections (TAAD) are highly fatal emergencies within cardiothoracic surgery. With increasing age, thoracic aneurysms become more prevalent and pose an even greater threat when they develop into aortic dissections. Both diseases are multifactorial and are influenced by a multitude of physiological and biomechanical processes. Structural stability of aorta can be disrupted by genes, such as those for extracellular matrix and contractile protein, as well as telomere dysfunction, which leads to senescence of smooth muscle and endothelial cells. Biomechanical changes such as increased luminal pressure imposed by hypertension are also very prevalent and lead to structural instability. Furthermore, ageing is associated with a pro-inflammatory state that exacerbates degeneration of vessel wall, facilitating the development of both aortic aneurysms and aortic dissection. This literature review provides an overview of the aetiology and pathophysiology of both thoracic aneurysms and aortic dissections. With an improved understanding, new therapeutic targets may eventually be identified to facilitate treatment and prevention of these diseases.


Route-based travel and shared routes in sympatric spider and woolly monkeys: cognitive and evolutionary implications.
Many wild primates occupy large home ranges and travel long distances each day. Navigating these ranges to find sufficient food presents a substantial cognitive challenge, but we are still far from understanding either how primates represent spatial information mentally or how they use this information to navigate under natural conditions. In the course of a long-term socioecological study, we investigated and compared the travel paths of sympatric spider monkeys (Ateles belzebuth) and woolly monkeys (Lagothrix poeppigii) in Amazonian Ecuador. During several field seasons spanning an 8-year period, we followed focal individuals or groups of both species continuously for periods of multiple days and mapped their travel paths in detail. We found that both primates typically traveled through their home ranges following repeatedly used paths, or "routes". Many of these routes were common to both species and were stable across study years. Several important routes appeared to be associated with distinct topographic features (e.g., ridgetops), which may constitute easily recognized landmarks useful for spatial navigation. The majority of all location records for both species fell along or near identified routes, as did most of the trees used for fruit feeding. Our results provide strong support for the idea that both woolly and spider monkey use route-based mental maps similar to those proposed by Poucet (Psychol Rev 100:163-182, 1993). We suggest that rather than remembering the specific locations of thousands of individual feeding trees and their phenological schedules, spider and woolly monkeys could nonetheless forage efficiently by committing to memory a series of route segments that, when followed, bring them into contact with many potential feeding sources for monitoring or visitation. Furthermore, because swallowed and defecated seeds are deposited in greater frequency along routes, the repeated use of particular travel paths over generations could profoundly influence the structure and composition of tropical forests, raising the intriguing possibility that these and other primate frugivores are active participants in constructing their own ecological niches. Building upon the insights of Byrne (Q J Exp Psychol 31:147-154, 1979, Normality and pathology in cognitive functions. Academic, London, pp 239-264, 1982) and Milton (The foraging strategy of howler monkeys: a study in primate economics. Columbia University Press, New York, 1980, On the move: how and why animals travel in groups. University of Chicago Press, Chicago, pp 375-417, 2000), our results highlight the likely general importance of route-based travel in the memory and foraging strategies of nonhuman primates.


Metabolic regulation of renal gluconeogenesis in response to sepsis in the rat.
1. The regulation of renal gluconeogenesis was studied in rats made septic by a caecal ligation and puncture technique. 2. Blood glucose concentrations were not markedly different in septic rats, but lactate, pyruvate and alanine concentrations were markedly increased, compared with sham-operated rats. Conversely, blood ketone body concentrations were significantly decreased in septic rats. Both plasma insulin and glucagon concentrations were markedly elevated in response to sepsis. 3. The maximal activities of glucose-6-phosphatase (EC 184.108.40.206), fructose-1,6-bisphosphatase (EC 184.108.40.206), pyruvate carboxylase (EC 22.214.171.124) and phosphoenolpyruvate carboxykinase (EC 18.104.22.168) were markedly decreased in kidneys obtained from septic rats, suggesting diminished renal gluconeogenesis. 4. Renal concentrations of lactate, pyruvate and other gluconeogenetic intermediates were markedly elevated in septic rats, whereas those of acetyl-CoA and fructose 2,6-bisphosphate were decreased and unchanged, respectively. 5. The rate of gluconeogenesis from added lactate, pyruvate and glycerol was decreased in isolated incubated renal tubules from septic rats. 6. Sepsis decreased the arteriovenous concentration difference for glucose, lactate, and alanine. Septic rats showed decreased net rates of glucose production and net rates of removal of lactate and alanine as compared with sham-operated controls. 7. It is concluded that the diminished capacity for renal gluconeogenesis in septic rats could be the result of changes in the maximal activities or regulation of key non-equilibrium gluconeogenic enzymes or both, but the effect of other factors (e.g. toxins) has not been excluded.


SRSF2 Mutations in Uveal Melanoma: A Preference for In-Frame Deletions?
Background: Uveal melanoma (UM) is the most common primary ocular malignancy in adults in the Western world. UM with a mutation in SF3B1, a spliceosome gene, is characterized by three or more structural changes of chromosome 1, 6, 8, 9, or 11. Also UM without a mutation in SF3B1 harbors similar chromosomal aberrations. Since, in addition to SF3B1, mutations in U2AF1 and SRSF2 have also been observed in hematological malignancies, UM without a SF3B1 mutation-but with the characteristic chromosomal pattern-might harbor mutations in one of these genes. Methods: 42 UMs were selected based on their chromosomal profile and wildtype SF3B1 status. Sanger sequencing covering the U2AF1 (exon 2 and 7) hotspots and SRSF2 (exon 1 and 2) was performed on DNA extracted from tumor tissue. Data of three UM with an SRSF2 mutation was extracted from the The Cancer Genome Atlas (TCGA). Results: Heterozygous in-frame SRSF2 deletions affecting amino acids 92-100 were detected in two UMs (5%) of 42 selected tumors and in three TGCA UM specimens. Both the UM with an SRSF2 mutation from our cohort and the UM samples from the TCGA showed more than four structural chromosomal aberrations including (partial) gain of chromosome 6 and 8, although in two TCGA UMs monosomy 3 was observed. Conclusions: Whereas in myelodysplastic syndrome predominantly missense SRSF2 mutations are described, the observed SRSF2 mutations in UM are all in-frame deletions of 8-9 amino acids. This suggests that the R625 missense SF3B1 mutations and SRSF2 mutations in UM are different compared to the spliceosome gene mutations in hematological cancers, and probably target a different, as yet unknown, set of genes involved in uveal melanoma etiology.


Induction of inflammation in vascular endothelial cells by metal oxide nanoparticles: effect of particle composition.
The mechanisms governing the correlation between exposure to ultrafine particles and the increased incidence of cardiovascular disease remain unknown. Ultrafine particles appear to cross the pulmonary epithelial barrier into the bloodstream, raising the possibility of direct contact with the vascular endothelium. Because endothelial inflammation is critical for the development of cardiovascular pathology, we hypothesized that direct exposure of human aortic endothelial cells (HAECs) to ultrafine particles induces an inflammatory response and that this response depends on particle composition. To test the hypothesis, we incubated HAECs for 1-8 hr with different concentrations (0.001-50 mug/mL) of iron oxide (Fe(2)O(3)), yttrium oxide (Y(2)O(3)), and zinc oxide (ZnO) nanoparticles and subsequently measured mRNA and protein levels of the three inflammatory markers intra-cellular cell adhesion molecule-1, interleukin-8, and monocyte chemotactic protein-1. We also determined nanoparticle interactions with HAECs using inductively coupled plasma mass spectrometry and transmission electron microscopy. Our data indicate that nanoparticle delivery to the HAEC surface and uptake within the cells correlate directly with particle concentration in the cell culture medium. All three types of nanoparticles are internalized into HAECs and are often found within intracellular vesicles. Fe(2)O(3) nanoparticles fail to provoke an inflammatory response in HAECs at any of the concentrations tested; however, Y(2)O(3) and ZnO nanoparticles elicit a pronounced inflammatory response above a threshold concentration of 10 mug/mL. At the highest concentration, ZnO nanoparticles are cytotoxic and lead to considerable cell death. These results demonstrate that inflammation in HAECs following acute exposure to metal oxide nanoparticles depends on particle composition.


Improving the clinical understanding of hypertrophic cardiomyopathy by combining patient data, machine learning and computer simulations: A case study.
Most patients with hypertrophic cardiomyopathy (HCM), the most common genetic cardiac disease, remain asymptomatic, but others may suffer from sudden cardiac death. A better identification of those patients at risk, together with a better understanding of the mechanisms leading to arrhythmia, are crucial to target high-risk patients and provide them with appropriate treatment. However, this currently remains a challenge. In this paper, we present a successful example of implementing computational techniques for clinically-relevant applications. By combining electrocardiogram and imaging data, machine learning and high performance computing simulations, we identified four phenotypes in HCM, with differences in arrhythmic risk, and provided two distinct possible mechanisms that may explain the heterogeneity of HCM manifestation. This led to a better HCM patient stratification and understanding of the underlying disease mechanisms, providing a step further towards tailored HCM patient management and treatment.


Vitiligo and the melanocyte reservoir.
Repigmentation of vitiligo depends on available melanocytes from three possible sources: from the hair follicle unit which is the main provider of pigment cells, from the border of vitiligo lesions, and from unaffected melanocytes within depigmented areas; pigment cells at these locations originate a perifollicular, border spreading and a diffuse repigmentation pattern. In order for repigmentation to take place under stimulation with diverse therapies, melanocytes should be present in appropriate numbers. Melanocyte tissue stem cells located in the niche at the bulge region of the hair follicle are the most important sources for providing immature pigment cells that undergo terminal differentiation and originate repigmentation, but cytokines, UVR and other molecules acting in melanogenesis with adequate regulation mechanisms contribute to successful recovery in vitiligo. The presence of keratinocyte stem cells in the interfollicular epidermis raises the question on the possibility of melanocyte stem cells in a similar location and the development of future strategies for therapeutic purposes.


An evolutionary perspective on gastrointestinal nematodes of sheep.
The purpose of this paper was to discuss from an evolutionary perspective the interaction between domestic sheep (Ovis aries) and their gastrointestinal nematodes. Although evolution is the central theme of biology, there has been little attempt to consider how evolutionary forces have shaped and continue to shape the relationships between domestic animals and their parasite community. Mathematical modelling of the host-parasite relationship indicated that the system is remarkably robust to perturbations in its parameters. This robustness may be a consequence of the long coevolution of host and parasites. Although nematodes can potentially evolve faster than the host, coevolution is not dominated by the parasite and there are several examples where breeds of cattle or sheep have evolved high levels of resistance to disease. Coevolution is a more equal partnership between host and nematode than is commonly assumed. Coevolution between parasites and the host immune system is often described as an arms race where both host immune response genes and parasite proteins evolve rapidly in response to each other. However, initial results indicate that nematode antigens are not evolving rapidly; the arms race between the immune system and nematodes, if it exists, is happening very slowly. Fisher's fundamental theorem of natural selection states that genes with positive effects on fitness will be fixed by natural selection. Consequently, heritable variation in fitness traits is expected to be low. Contrary to this argument, there is considerable genetic variation in resistance to nematode infection. In particular, the heritabilities of nematode-specific IgA and IgE activity are moderate to high. The reasons for this apparent violation of the fundamental theorem of natural selection are not clear but several possible explanations are explored. Faecal nematode egg counts increase at the beginning of the grazing season - a phenomenon known as the periparturient rise. This increase benefits host and parasite and appears to be a consequence of coevolution. In conclusion, an evolutionary perspective can shed light on many aspects of the host-parasite relationship in domestic animals.


Views on the use of the term 'elder orphans': A qualitative study.
Older adults living alone in the community with no immediate family network or support are referred in the literature as 'elder orphans'. The grey literature has a growing emphasis on the importance of supporting this vulnerable group, particularly with health and social care. However, there is a dearth of empirical research on 'elder orphans', and definitions remained semantically complicated and unknown to the public, healthcare professionals and those being referred as elder orphans. This research explored the views of older adults on the use of the terminology 'elder orphans', and the implications of using the terminology in health and social care systems. A descriptive qualitative approach through face-to-face interviews of 11 older adults was undertaken in Auckland, New Zealand in October 2018. Narratives were analysed using thematic analysis. Two themes were identified from the analysis of interview data. The first theme was 'realistic and practical term', which emphasised the participants' impression of the term 'elder orphan' resonating to themselves with great relevance to their current and future situations. The second theme is 'visibility and vulnerability', which highlighted participants' both positive and ambivalent views on the use of the terminology in healthcare settings. The participants viewed this terminology as beneficial to alerting care services, and in promoting awareness among healthcare providers. Older adults from the study, who are living alone without immediate family networks and support self-identified themselves as 'elder orphans' through a gradual realisation of their current and anticipated social situations. The use of the term within healthcare was considered and preferred by the participants to be used contextually, and targeted towards appropriate health and social care services within and outside hospital-care settings.


Investigating the asymmetry in the EDL response of C60/graphene supercapacitors.
Development of efficient electrodes is one of the main ways to increase the performance of an electrochemical energy storage device. It is known that such performance is associated with the electrode specific area, which allows a much larger interfacial interaction with the electrolyte. In this work, molecular dynamics is employed to model C60/graphene composite electrodes that can expand the effective area by approximately 70% relative to a pure graphene electrode. Our simulations indicate that the performance of supercapacitors of C60/graphene electrodes is superior to those made of planar graphene, in some cases up to 150%. The inherent electrolyte asymmetry in the investigated supercapacitors has a negative effect on the total capacitance, indicating that even better results could be obtained after rational design of the fullerene density on the surface of the graphene as well as the choice of the ions in the liquid ionic composition.


Local control with reduced-dose radiotherapy for low-risk rhabdomyosarcoma: a report from the Children's Oncology Group D9602 study.
To analyze the effect of reduced-dose radiotherapy on local control in children with low-risk rhabdomyosarcoma (RMS) treated in the Children's Oncology Group D9602 study. Patients with low-risk RMS were nonrandomly assigned to receive radiotherapy doses dependent on the completeness of surgical resection of the primary tumor (clinical group) and the presence of involved regional lymph nodes. After resection, most patients with microscopic residual and uninvolved nodes received 36 Gy, those with involved nodes received 41.4 to 50.4 Gy, and those with orbital primary tumors received 45 Gy. All patients received vincristine and dactinomycin, with cyclophosphamide added for patient subsets with a higher risk of relapse in Intergroup Rhabdomyosarcoma Study Group III and IV studies. Three hundred forty-two patients were eligible for analysis; 172 received radiotherapy as part of their treatment. The cumulative incidence of local/regional failure was 15% in patients with microscopic involved margins when cyclophosphamide was not part of the treatment regimen and 0% when cyclophosphamide was included. The cumulative incidence of local/regional failure was 14% in patients with orbital tumors. Protocol-specified omission of radiotherapy in girls with Group IIA vaginal tumors (n = 5) resulted in three failures for this group. In comparison with Intergroup Rhabdomyosarcoma Study Group III and IV results, reduced-dose radiotherapy does not compromise local control for patients with microscopic tumor after surgical resection or with orbital primary tumors when cyclophosphamide is added to the treatment program. Girls with unresected nonbladder genitourinary tumors require radiotherapy for postsurgical residual tumor for optimal local control to be achieved.


Decreasing the effects of horizontal gene transfer on bacterial phylogeny: the Escherichia coli case study.
Phylogenetic reconstructions of bacterial species from DNA sequences are hampered by the existence of horizontal gene transfer. One possible way to overcome the confounding influence of such movement of genes is to identify and remove sequences which are responsible for significant character incongruence when compared to a reference dataset free of horizontal transfer (e.g., multilocus enzyme electrophoresis, restriction fragment length polymorphism, or random amplified polymorphic DNA) using the incongruence length difference (ILD) test of Farris et al. [Cladistics 10 (1995) 315]. As obtaining this "whole genome dataset" prior to the reconstruction of a phylogeny is clearly troublesome, we have tested alternative approaches allowing the release from such reference dataset, designed for a species with modest level of horizontal gene transfer, i.e., Escherichia coli. Eleven different genes available or sequenced in this work were studied in a set of 30 E. coli reference (ECOR) strains. Either using ILD to test incongruence between each gene against the all remaining (in this case 10) genes in order to remove sequences responsible for significant incongruence, or using just a simultaneous analysis without removals, gave robust phylogenies with slight topological differences. The use of the ILD test remains a suitable method for estimating the level of horizontal gene transfer in bacterial species. Supertrees also had suitable properties to extract the phylogeny of strains, because the way they summarize taxonomic congruence clearly limits the impact of individual gene transfers on the global topology. Furthermore, this work allowed a significant improvement of the accuracy of the phylogeny within E. coli.


Alcohol in moderation, premorbid intelligence and cognition in older adults: results from the Psychiatric Morbidity Survey.
To test the hypothesis that the association previously reported between moderate alcohol use and better cognition is an artefact of confounding by (a) higher premorbid education and socioeconomic status; (b) a lifestyle of moderation (using smoking as a risk marker); and (c) decreased alcohol consumption in people with physical illnesses. Data were analysed from people aged 60-74 years interviewed for the 2000 British National Psychiatric Morbidity Survey, representative of people living in private homes. Alcohol use information was available for 1985 (98.9%) of the eligible participants, of whom 1735 (87.4%) who drank moderately or abstained were included in the analyses. Our main outcome measures were the Alcohol Use Disorders Identification Test (AUDIT), the Telephone Interview for Cognitive Status Screen for Cognitive Impairment and the National Adult Reading Test to measure crystallised (premorbid) intelligence. Our physical health measures were the number of prescribed medications and physical illness reported, and the 12 item Short Form Health Survey's Physical Component Score. The relationship between current cognition and alcohol use was reduced and no longer significant after considering premorbid intelligence or physical health. In our final model, the significant predictors of current cognition among non-problem drinkers were: age (B = -0.13, -0.18 to -0.08; p<0.001) and crystallised intelligence (B = 0.14, 0.12 to 0.17; p<0.001). Smoking was not associated with cognition. In people who were not problem drinkers, higher alcohol intake was not associated with improved current cognition after controlling for premorbid intelligence and physical health. Our findings suggest that, despite previous suggestions, moderate alcohol consumption does not protect older people from cognitive decline.


The conformation of calsequestrin determines its ability to regulate skeletal ryanodine receptors.
Ca2+ efflux from the sarcoplasmic reticulum decreases when store Ca2+ concentration falls, particularly in skinned fibers and isolated vesicles where luminal Ca2+ can be reduced to very low levels. However ryanodine receptor activity in many single channel studies is higher when the luminal free Ca2+ concentration is reduced. We investigated the hypothesis that prolonged exposure to low luminal Ca2+ causes conformational changes in calsequestrin and deregulation of ryanodine receptors, allowing channel activity to increase. Lowering of luminal Ca2+ from 1 mM to 100 microM for several minutes resulted in conformational changes with dissociation of 65-75% of calsequestrin from the junctional face membrane. The calsequestrin remaining associated no longer regulated channels. In the absence of this regulation, ryanodine receptors were more active when luminal Ca2+ was lowered from 1 mM to 100 microM. In contrast, when ryanodine receptors were calsequestrin regulated, lowering luminal Ca2+ either did not alter or decreased activity. Ryanodine receptors are regulated by calsequestrin under physiological conditions where calsequestrin is polymerized. Since depolymerization occurs slowly, calsequestrin can regulate the ryanodine receptor and prevent excess Ca2+ release when the store is transiently depleted, for example, during high frequency activity or early stages of muscle fatigue.


Retromer binding to FAM21 and the WASH complex is perturbed by the Parkinson disease-linked VPS35(D620N) mutation.
Retromer is a protein assembly that plays a central role in orchestrating export of transmembrane-spanning cargo proteins from endosomes into retrieval pathways destined for the Golgi apparatus and the plasma membrane [1]. Recently, a specific mutation in the retromer component VPS35, VPS35(D620N), has linked retromer dysfunction to familial autosomal dominant and sporadic Parkinson disease [2, 3]. However, the effect of this mutation on retromer function remains poorly characterized. Here we established that in cells expressing VPS35(D620N) there is a perturbation in endosome-to-TGN transport but not endosome-to-plasma membrane recycling, which we confirm in patient cells harboring the VPS35(D620N) mutation. Through comparative stable isotope labeling by amino acids in cell culture (SILAC)-based analysis of wild-type VPS35 versus the VPS35(D620N) mutant interactomes, we establish that the major defect of the D620N mutation lies in the association to the actin-nucleating Wiskott-Aldrich syndrome and SCAR homolog (WASH) complex. Moreover, using isothermal calorimetry, we establish that the primary defect of the VPS35(D620N) mutant is a 2.2 ± 0.5-fold decrease in affinity for the WASH complex component FAM21. These data define the primary molecular defect in retromer assembly that arises from the VPS35(D620N) mutation and, by revealing functional effects on retromer-mediated endosome-to-TGN transport, provide new insight into retromer deregulation in Parkinson disease.


Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009.
Different types of cell death are often defined by morphological criteria, without a clear reference to precise biochemical mechanisms. The Nomenclature Committee on Cell Death (NCCD) proposes unified criteria for the definition of cell death and of its different morphologies, while formulating several caveats against the misuse of words and concepts that slow down progress in the area of cell death research. Authors, reviewers and editors of scientific periodicals are invited to abandon expressions like 'percentage apoptosis' and to replace them with more accurate descriptions of the biochemical and cellular parameters that are actually measured. Moreover, at the present stage, it should be accepted that caspase-independent mechanisms can cooperate with (or substitute for) caspases in the execution of lethal signaling pathways and that 'autophagic cell death' is a type of cell death occurring together with (but not necessarily by) autophagic vacuolization. This study details the 2009 recommendations of the NCCD on the use of cell death-related terminology including 'entosis', 'mitotic catastrophe', 'necrosis', 'necroptosis' and 'pyroptosis'.


Exploring the link between human embryonic stem cell organization and fate using tension-calibrated extracellular matrix functionalized polyacrylamide gels.
Human embryonic stem cell (hESc) lines are likely the in vitro equivalent of the pluripotent epiblast. hESc express high levels of the extracellular matrix (ECM) laminin integrin receptor α6β1 and consequently can adhere robustly and be propagated in an undifferentiated state on tissue culture plastic coated with the laminin rich basement membrane preparation, Matrigel, even in the absence of supporting fibroblasts. Such cultures represent a critical step in the development of more defined feeder free cultures of hESc; a goal deemed necessary for regenerative medical applications and have been used as the starting point in some differentiation protocols. However, on standard non-deformable tissue culture plastic hESc either fail or inadequately develop the structural/morphological organization of the epiblast in vivo. By contrast, growth of hESc on appropriately defined mechanically deformable polyacrylamide substrates permits recapitulation of many of these in vivo features. These likely herald differences in the precise nature of the integration of signal transduction pathways from soluble morphogens and represent an unexplored variable in hESc (fate) state space. In this chapter we describe how to establish viable hESc colonies on these functionalized polyacrylamide gels. We suggest this strategy as a prospective in vitro model of the genetics, biochemistry, and cell biology of pre- and early-gastrulation stage human embryos and the permissive and instructive roles that cellular and substrate mechanics might play in early embryonic cell fate decisions. Such knowledge should inform regenerative medical applications aimed at enabling or improving the differentiation of specific cell types from embryonic or induced embryonic stem cells.


High-speed 2D and 3D fluorescence microscopy of cardiac myocytes.
Oblique plane microscopy (OPM) is a light sheet microscopy technique that uses a single high numerical aperture microscope objective to both illuminate a tilted plane within the specimen and to obtain an image of the tilted illuminated plane. In this paper, we present a new OPM configuration that enables both the illumination and detection focal planes to be swept simultaneously and remotely through the sample volume, enabling high speed volumetric imaging. We demonstrate the high speed imaging capabilities of the system by imaging calcium dynamics in cardiac myocytes in 2D at 926 frames per second and in 3D at 21 volumes per second. In the future, higher frame rate CCD cameras will enable volumetric imaging at much greater rates, leading to new capabilities to study dynamic events in cells at high speeds in two and three dimensions.


Control of directionality of chromatin folding for the inter- and intra-domain contacts at the Tfap2c-Bmp7 locus.
Contact domains of chromatin serve as a fundamental unit to regulate action of enhancers for target genes. Looping between a pair of CCCTC-binding factor (CTCF)-binding sites in convergent orientations underlies the formation of contact domains, while those in divergent orientations establish domain boundaries. However, every CTCF site is not necessarily engaged in loop or boundary structures, leaving functions of CTCF in varied genomic contexts still elusive. The locus containing Tfap2c and Bmp7 encompasses two contact domains separated by a region between the two genes, termed transition zone (TZ), characterized by two arrays of CTCF sites in divergent configuration. In this study, we created deletion and inversion alleles of these and other regions across the locus and investigated how they impinge on the conformation. Deletion of the whole two CTCF arrays with the CRISPR/Cas9 system resulted in impairment of blocking of chromatin contacts by the TZ, as assessed by the circular chromatin conformation capture assay (4C-seq). Deletion and inversion of either of the two arrays similarly, but less pronouncedly, led to reduction in the blocking activity. Thus, the divergent configuration provides the TZ with the strong boundary activity. Uniquely, we show the TZ harbors a 50-kb region within one of the two arrays that contacts broadly with the both flanking intervals, regardless of the presence or orientation of the other CTCF array. Further, we show the boundary CTCF array has little impact on intra-domain folding; instead, locally associating CTCF sites greatly affect it. Our results show that the TZ not only separates the two domains, but also bears a wide interval that shows isotropic behavior of chromatin folding, indicating a potentially complex nature of actual boundaries in the genome. We also show that CTCF-binding sites inside a domain greatly contribute to the intra-domain folding of chromatin. Thus, the study reveals diverse and context-dependent roles of CTCF in organizing chromatin conformation at different levels.