Feasibility and cost-effectiveness of stroke prevention through community screening for atrial fibrillation using iPhone ECG in pharmacies. The SEARCH-AF study.
Atrial fibrillation (AF) causes a third of all strokes, but often goes undetected before stroke. Identification of unknown AF in the community and subsequent anti-thrombotic treatment could reduce stroke burden. We investigated community screening for unknown AF using an iPhone electrocardiogram (iECG) in pharmacies, and determined the cost-effectiveness of this strategy.Pharmacists performedpulse palpation and iECG recordings, with cardiologist iECG over-reading. General practitioner review/12-lead ECG was facilitated for suspected new AF. An automated AF algorithm was retrospectively applied to collected iECGs. Cost-effectiveness analysis incorporated costs of iECG screening, and treatment/outcome data from a United Kingdom cohort of 5,555 patients with incidentally detected asymptomatic AF. A total of 1,000 pharmacy customers aged ≥65 years (mean 76 ± 7 years; 44% male) were screened. Newly identified AF was found in 1.5% (95% CI, 0.8-2.5%); mean age 79 ± 6 years; all had CHA2DS2-VASc score ≥2. AF prevalence was 6.7% (67/1,000). The automated iECG algorithm showed 98.5% (CI, 92-100%) sensitivity for AF detection and 91.4% (CI, 89-93%) specificity. The incremental cost-effectiveness ratio of extending iECG screening into the community, based on 55% warfarin prescription adherence, would be $AUD5,988 (€3,142; $USD4,066) per Quality Adjusted Life Year gained and $AUD30,481 (€15,993; $USD20,695) for preventing one stroke. Sensitivity analysis indicated cost-effectiveness improved with increased treatment adherence.Screening with iECG in pharmacies with an automated algorithm is both feasible and cost-effective. The high and largely preventable stroke/thromboembolism risk of those with newly identified AF highlights the likely benefits of community AF screening. Guideline recommendation of community iECG AF screening should be considered.


Photodynamic treatment: a novel method for sanitation of food handling and food processing surfaces.
The photodynamic bactericidal effect of the photoactive dyes acriflavine neutral, rose bengal, phloxine B, and malachite green (oxalate salt) at concentrations of 5 to 5,000 microg/ml against two gram-negative strains (Escherichia coli LJH 128 and Salmonella Typhimurium C1058), two gram-positive strains (Bacillus sp. C578 and Listeria monocytogenes LJH 375), and yeast (Saccharomyces cerevisiae C1172) was investigated. Incubation of the investigated bacteria with acriflavine neutral under illumination resulted in a significant reduction in cell numbers compared with dark incubation. Rose bengal caused a significant killing effect for bacteria incubated both in the dark and under illumination. Malachite green was active against gram-positive bacteria under illumination and did not affect gram-negative bacteria or yeasts. Incubation with phloxine B resulted in a significant decline in cell numbers for gram-positive bacteria, both in the dark and under illumination; gram-negative bacteria and yeasts were unaffected. Conjugation of rose bengal and phloxine B with poly(vinyl amine) resulted in an enhanced bactericidal effect during both dark and light incubation. This was explained by electrostatic interaction of the polymer with the cell surface, which resulted in closer contact of the photoactive dye and cell. No killing effect was observed for yeasts incubated with dye conjugates. Filter paper treated with dye-poly(vinyl amine) conjugates showed high photodynamic bactericidal activity against the bacterial strains, but not against the yeasts. The extent of bacterial killing depended on the nature and concentration of the dye conjugate and the type of microorganism. The presented data suggest that a photodynamic approach for constructing "self-decontaminating" materials has potential.


The evolution in the prioritization for liver transplantation.
Policies for organ allocation can be based on medical urgency, utility or transplant benefit. With an urgency policy, patients with worse outcomes on the waiting list are given higher priority for transplantation [based on the Child-Turcotte-Pugh score or the Model for End-stage Liver Disease (MELD) score, or United Kingdom model for End-stage Liver Disease (UKELD) score]. The MELD and UKELD scores have statistical validation and use objective and widely available laboratory tests. However, both scores have important limitations. Adjustments to the original MELD equation and new scoring systems have been proposed to overcome these limitations; incorporation of serum sodium improves its predictive accuracy and is part of the UKELD score. The utility-based systems are based on post-transplant outcome taking into account donor and recipient characteristics. MELD and UKELD scores poorly predict outcomes after liver transplantation due to the absence of donor factors. The transplant benefit models rank patients according to the net survival benefit that they would derive from transplantation. These models would be based on the maximization of the lifetime gained through liver transplantation. Well-designed prospective studies and simulation models are necessary to establish the optimal allocation system in liver transplantation, as no current model has all the best characteristics.


Structure of a SMG8-SMG9 complex identifies a G-domain heterodimer in the NMD effector proteins.
Nonsense-mediated mRNA decay (NMD) is a eukaryotic mRNA degradation pathway involved in surveillance and post-transcriptional regulation, and executed by the concerted action of several trans-acting factors. The SMG1 kinase is an essential NMD factor in metazoans and is associated with two recently identified and yet poorly characterized proteins, SMG8 and SMG9. We determined the 2.5 Å resolution crystal structure of a SMG8-SMG9 core complex from C. elegans We found that SMG8-SMG9 is a G-domain heterodimer with architectural similarities to the dynamin-like family of GTPases such as Atlastin and GBP1. The SMG8-SMG9 heterodimer forms in the absence of nucleotides, with interactions conserved from worms to humans. Nucleotide binding occurs at the G domain of SMG9 but not of SMG8. Fitting the GDP-bound SMG8-SMG9 structure in EM densities of the human SMG1-SMG8-SMG9 complex raises the possibility that the nucleotide site of SMG9 faces SMG1 and could impact the kinase conformation and/or regulation.


Biomechanical evaluation of transosseous rotator cuff repair: do anchors really matter?
Suture anchor fixation has become the preferred method for arthroscopic repairs of rotator cuff tears. Recently, newer arthroscopic repair techniques including transosseous-equivalent repairs with anchors or arthroscopic transosseous suture passage have been developed. To compare the initial biomechanical performance including ultimate load to failure and localized cyclic elongation between transosseous-equivalent repair with anchors (TOE), traditional transosseous repair with a curved bone tunnel (TO), and an arthroscopic transosseous repair technique utilizing a simple (AT) or X-box suture configuration (ATX). Controlled laboratory study. Twenty-eight human cadaveric shoulders were dissected to create an isolated supraspinatus tear and randomized into 1 of 4 repair groups (TOE, TO, AT, ATX). Tensile testing was conducted to simulate the anatomic position of the supraspinatus with the arm in 60° of abduction and involved an initial preload, cyclic loading, and pull to failure. Localized elongation during testing was measured using optical tracking. Data were statistically assessed using analysis of variance with a Tukey post hoc test for multiple comparisons. The TOE repair demonstrated a significantly higher mean ± SD failure load (558.4 ± 122.9 N) compared with the TO (325.3 ± 79.9 N), AT (291.7 ± 57.9 N), and ATX (388.5 ± 92.6 N) repairs (P < .05). There was also a significantly larger amount of first-cycle excursion in the AT group (8.19 ± 1.85 mm) compared with the TOE group (5.10 ± 0.89 mm). There was no significant difference between repair groups in stiffness during maximum load to failure or in normalized cyclic elongation. Failure modes were as follows: TOE, tendon (n = 4) and bone (n = 3); TO, suture (n = 6) and bone (n = 1); AT, tendon (n = 2) and bone (n = 3) and suture (n = 1); ATX, tendon (n = 7). This study demonstrates that anchorless repair techniques using transosseous sutures result in significantly lower failure loads than a repair model utilizing anchors in a TOE construct. Suture anchor repair appears to offer superior biomechanical properties to transosseous repairs regardless of tunnel or suture configuration.


Beyond the Failure of Direct-Matching in Keyword Evaluation: A Sketch of a Graph Based Solution.
The starting point of this paper is the observation that methods based on the direct match of keywords are inadequate because they do not consider the cognitive ability of concept formation and abstraction. We argue that keyword evaluation needs to be based on a semantic model of language capturing the semantic relatedness of words to satisfy the claim of the human-like ability of concept formation and abstraction and achieve better evaluation results. Evaluation of keywords is difficult since semantic informedness is required for this purpose. This model must be capable of identifying semantic relationships such as synonymy, hypernymy, hyponymy, and location-based abstraction. For example, when gathering texts from online sources, one usually finds a few keywords with each text. Still, these keyword sets are neither complete for the text nor are they in themselves closed, i.e., in most cases, the keywords are a random subset of all possible keywords and not that informative w.r.t. the complete keyword set. Therefore all algorithms based on this cannot achieve good evaluation results and provide good/better keywords or even a complete keyword set for a text. As a solution, we propose a word graph that captures all these semantic relationships for a given language. The problem with the hyponym/hyperonym relationship is that, unlike synonyms, it is not bidirectional. Thus the space of keyword sets requires a metric that is non-symmetric, in other words, a quasi-metric. We sketch such a metric that works on our graph. Since it is nearly impossible to obtain such a complete word graph for a language, we propose for the keyword task a simpler graph based on the base text upon which the keyword sets should be evaluated. This reduction is usually sufficient for evaluating keyword sets.


Social genes are selection hotspots in kin groups of a soil microbe.
The composition of cooperative systems, including animal societies, organismal bodies, and microbial groups, reflects their past and shapes their future evolution. However, genomic diversity within many multiunit systems remains uncharacterized, limiting our ability to understand and compare their evolutionary character. We have analyzed genomic and social-phenotype variation among 120 natural isolates of the cooperative bacterium Myxococcus xanthus derived from six multicellular fruiting bodies. Each fruiting body was composed of multiple lineages radiating from a unique recent ancestor. Genomic evolution was concentrated in selection hotspots associated with evolutionary change in social phenotypes. Synonymous mutations indicated that kin lineages within the same fruiting body often first diverged from a common ancestor more than 100 generations ago. Thus, selection appears to promote endemic diversification of kin lineages that remain together over long histories of local interaction, thereby potentiating social coevolution.


The 5A structure of heterologously expressed plant aquaporin SoPIP2;1.
SoPIP2;1 is one of the major integral proteins in spinach leaf plasma membranes. In the Xenopus oocyte expression system its water channel activity is regulated by phosphorylation at the C terminus and in the first cytosolic loop. To assess its structure, SoPIP2;1 was heterologously expressed in Pichia pastoris as a His-tagged protein and in the non-tagged form. Both forms were reconstituted into 2D crystals in the presence of lipids. Tubular crystals and double-layered crystalline sheets of non-tagged SoPIP2;1 were observed and analyzed by cryo-electron microscopy. Crystalline sheets were highly ordered and diffracted electrons to a resolution of 2.96A. High-resolution projection maps of tilted specimens provided a 3D structure at 5A resolution. Superposition of the SoPIP2;1 potential map with the atomic model of AQP1 demonstrates the generally well conserved overall structure of water channels. Differences concerning the extracellular loop A explain the particular crystal contacts between oppositely oriented membrane sheets of SoPIP2;1 2D crystals, and may have a function in rapid volume changes observed in stomatal guard cells or mesophyll protoplasts. This crystal packing arrangement provides access to the phosphorylated C terminus as well as the loop B phosphorylation site for studies of channel gating.


Early Versus Delayed Feeding in Patients With Acute Pancreatitis: A Systematic Review.
Acute pancreatitis is among the most common and costly reasons for hospitalization in the United States. Bowel rest, pain control, and intravenous fluids are the cornerstones of treatment, but early feeding might also be beneficial. To compare length of hospital stay, mortality, and readmission in adults hospitalized with pancreatitis who received early versus delayed feeding. MEDLINE via Ovid, EMBASE, the Cochrane Library, CINAHL, and Web of Science through January 2017. Two authors independently reviewed and selected studies if they were randomized clinical trials, included adults hospitalized with acute pancreatitis, and compared early versus delayed feeding (≤48 vs. >48 hours after hospitalization). Two investigators independently extracted study data and rated risk of bias using the Cochrane Collaboration tool. Eleven randomized trials (8 peer-reviewed publications, 3 abstract-only presentations) that included 948 patients were eligible. Seven trials (3 with low risk of bias) enrolled patients with mild to moderate pancreatitis. Four trials (1 with low risk of bias) included patients with predicted severe pancreatitis. Routes used for early feeding included oral (4 studies), nasogastric (2 studies), nasojejunal (4 studies), and oral or nasoenteric (1 study). Among patients with mild to moderate pancreatitis, early feeding was associated with reduced length of stay in 4 of 7 studies (including 2 of 3 with low risk of bias). Other outcomes were heterogeneous and variably reported, but no study showed an increase in adverse events with early feeding. Among patients with severe pancreatitis, limited evidence revealed no statistically significant difference in outcomes between early and delayed feeding. Heterogeneity of feeding protocols and outcomes, scant data, and unclear or high risk of bias in several studies. Limited data suggest that early feeding in patients with acute pancreatitis does not seem to increase adverse events and, for patients with mild to moderate pancreatitis, may reduce length of hospital stay. None. (PROSPERO: CRD42015016193).


Falsibacillus pallidus to replace the homonym Bacillus pallidus Zhou et al. 2008.
The prokaryotic species name Bacillus pallidus Zhou et al. 2008 is illegitimate because it is a later homonym of the name Bacillus pallidus Scholz et al. 1988 (Bacteria, Firmicutes, Bacillales, Bacillaceae, Bacillus). Although Bacillus pallidus Scholz et al. 1988 was transferred to the genus Geobacillus as Geobacillus pallidus comb. nov. in 2004, the species name Bacillus pallidus Scholz et al. 1988 remains validly published and has priority at that position [Principle 8 of the Bacteriological Code (1990 Revision)]. In order to solve this problem, a new name was sought for this taxon, represented by the type strain CW 7(T). In addition, the placement of this species in the genus Bacillus was reassessed. The data collected were re-examined as part of a taxonomic study using a polyphasic approach. It was concluded, primarily based on the chemotaxonomic data, that strain CW 7(T) did not belong to the genus Bacillus, but should be placed in a new genus, for which the name Falsibacillus gen. nov. is proposed. The genus currently comprises a single species, Falsibacillus pallidus comb. nov. represented by the type strain CW 7(T) (=KCTC 13200(T)=CCTCC AB 207188(T)=LMG 24451(T)).


Histopathological characteristics and artificial intelligence for predicting tumor mutational burden-high colorectal cancer.
Tumor mutational burden-high (TMB-H), which is detected with gene panel testing, is a promising biomarker for immune checkpoint inhibitors (ICIs) in colorectal cancer (CRC). However, in clinical practice, not every patient is tested for TMB-H using gene panel testing. We aimed to identify the histopathological characteristics of TMB-H CRC for efficient selection of patients who should undergo gene panel testing. Moreover, we attempted to develop a convolutional neural network (CNN)-based algorithm to predict TMB-H CRC directly from hematoxylin and eosin (H&E) slides. We used two CRC cohorts tested for TMB-H, and whole-slide H&E digital images were obtained from the cohorts. The Japanese CRC (JP-CRC) cohort (N = 201) was evaluated to detect the histopathological characteristics of TMB-H using H&E slides. The JP-CRC cohort and The Cancer Genome Atlas (TCGA) CRC cohort (N = 77) were used to develop a CNN-based TMB-H prediction model from the H&E digital images. Tumor-infiltrating lymphocytes (TILs) were significantly associated with TMB-H CRC (P < 0.001). The area under the curve (AUC) for predicting TMB-H CRC was 0.910. We developed a CNN-based TMB-H prediction model. Validation tests were conducted 10 times using randomly selected slides, and the average AUC for predicting TMB-H slides was 0.934. TILs, a histopathological characteristic detected with H&E slides, are associated with TMB-H CRC. Our CNN-based model has the potential to predict TMB-H CRC directly from H&E slides, thereby reducing the burden on pathologists. These approaches will provide clinicians with important information about the applications of ICIs at low cost.


Drosophila homologues of adenomatous polyposis coli (APC) and the formin diaphanous collaborate by a conserved mechanism to stimulate actin filament assembly.
Vertebrate APC collaborates with Dia through its Basic domain to assemble actin filaments. Despite limited sequence homology between the vertebrate and Drosophila APC Basic domains, Drosophila APC1 collaborates with Dia to stimulate actin assembly in vitro. The mechanism of actin assembly is highly conserved over evolution. APC-Dia collaborations may be crucial in a wide range of animal cells. Adenomatous polyposis coli (APC) is a large multidomain protein that regulates the cytoskeleton. Recently, it was shown that vertebrate APC through its Basic domain directly collaborates with the formin mDia1 to stimulate actin filament assembly in the presence of nucleation barriers. However, it has been unclear whether these activities extend to homologues of APC and Dia in other organisms. Drosophila APC and Dia are each required to promote actin furrow formation in the syncytial embryo, suggesting a potential collaboration in actin assembly, but low sequence homology between the Basic domains of Drosophila and vertebrate APC has left their functional and mechanistic parallels uncertain. To address this question, we purified Drosophila APC1 and Dia and determined their individual and combined effects on actin assembly using both bulk fluorescence assays and total internal reflection fluorescence microscopy. Our data show that APC1, similar to its vertebrate homologue, bound to actin monomers and nucleated and bundled filaments. Further, Drosophila Dia nucleated actin assembly and protected growing filament barbed ends from capping protein. Drosophila APC1 and Dia directly interacted and collaborated to promote actin assembly in the combined presence of profilin and capping protein. Thus, despite limited sequence homology, Drosophila and vertebrate APCs exhibit highly related activities and mechanisms and directly collaborate with formins. These results suggest that APC-Dia interactions in actin assembly are conserved and may underlie important in vivo functions in a broad range of animal phyla.


Recognizing animal personhood in compassionate conservation.
Compassionate conservation is based on the ethical position that actions taken to protect biodiversity should be guided by compassion for all sentient beings. Critics argue that there are 3 core reasons harming animals is acceptable in conservation programs: the primary purpose of conservation is biodiversity protection; conservation is already compassionate to animals; and conservation should prioritize compassion to humans. We used argument analysis to clarify the values and logics underlying the debate around compassionate conservation. We found that objections to compassionate conservation are expressions of human exceptionalism, the view that humans are of a categorically separate and higher moral status than all other species. In contrast, compassionate conservationists believe that conservation should expand its moral community by recognizing all sentient beings as persons. Personhood, in an ethical sense, implies the individual is owed respect and should not be treated merely as a means to other ends. On scientific and ethical grounds, there are good reasons to extend personhood to sentient animals, particularly in conservation. The moral exclusion or subordination of members of other species legitimates the ongoing manipulation and exploitation of the living worlds, the very reason conservation was needed in the first place. Embracing compassion can help dismantle human exceptionalism, recognize nonhuman personhood, and navigate a more expansive moral space.


[Fall-related factors in a cohort of elderly community residents].
To identify factors associated to fall and recurrent fall episodes among elderly living in a community, and to determine the relative risk of each factor as a fall predictor. A two-year follow-up study with two segments of multi-dimensional household surveys (1991-1992 and 1994-1995) was carried out in a cohort of 1,667 elderly, aged 65 or older, living in a community in the city of São Paulo, Brazil. The instrument used for data collection was the BOMFAQ, the Brazilian version of the Multidimensional Functional Assessment Questionnaire (OARS). A stepwise logistic regression analysis was conducted with p<0.05 and 95% CI. About 31% of the elderly referred a fall episode and around 11% referred two or more fall episodes in the previous year of the first survey. After follow-up, 53.4% didn't refer any fall episodes, 32.7% referred a fall episode either in the first survey or in the second one and almost 14% referred fall episodes in both surveys. The predictive model of recurrent falls was composed by the following variables: absence of a spouse (OR=1.59 95% CI 1.00-2.52), not having a reading habit (OR= 1.56 95% CI 1.03-2.37), history of fractures (OR=4.6 95% CI 2.23-9.69 difficulty to perform one to three activities of daily life (OR=2.37 95% CI 1.49-3.78), difficulty to perform four or more activities of daily life (OR=3.31 95%CI 1.58-6.93) and among those whose sight is most impaired (OR=1.53 95%CI 1.00-2.34). Population ageing and increase in life expectancy demand preventive and rehabilitation actions in order to reduce risk factors for falls, such as impaired functional capacity, impaired eye sight and lack of cognitive stimulation.


Costs of implementing and sustaining enhanced collaborative care programs involving community partners.
Collaborative care is an evidence-based program for treating depression in primary care. We sought to expand this model by recruiting clinics interested in incorporating community partners (i.e., community-based organizations (CBO) and/or family members) in the care team. Seven sites implemented evidence-based collaborative care programs with community partners while collecting information on costs of implementing and sustaining programs. Sites retrospectively collected data on planning and implementation costs with technical assistance from study researchers. Sites also prospectively collected cost of care activities over a 1-month period once the program was implemented to determine resources needed to sustain programs. Personnel salary costs were adjusted, adding 30% for benefits and 30% for administrative overhead. The programs implemented varied considerably in staffing, involvement of care partners, and allocation of costs. Total planning and implementation costs varied from $39,280 to $60,575. The largest implementation cost category involved workflow development and ranged from $16,325 to $31,375 with the highest costs in this category attributed to the most successful implementation among clinic-CBO programs. Following implementation, cost per patient over the 1-month period ranged from $154 to $544. Ongoing strategic decision-making and administrative costs, which were included in cost of care, ranged from $284 to $2328 for the month. Sites implemented collaborative care through differing partnerships, staffing, and related costs. Costs to implement and sustain programs developed in partnership are often not collected but are crucial to understanding financial aspects of developing sustainable partnerships. Assessing such costs is feasible and can inform future partnership efforts.


Predictors of smoking cessation after percutaneous coronary revascularization.
To identify factors predictive of smoking cessation after successful percutaneous coronary revascularization. We undertook a case-control study of the smoking status of all patients at Mayo Clinic Rochester from September 1979 through December 1995 who were smokers at the time of an index percutaneous coronary revascularization procedure in the non-peri-infarction setting (no myocardial infarction within 24 hours). Maximal duration of prospective follow-up was 16 years. Patients were classified into those who permanently quit smoking immediately after the procedure (N = 435; mean follow-up, 5.1 +/- 3.7 years) or those who continued to smoke at some time during follow-up (N = 734; mean follow-up, 5.3 +/- 3.7 years). Logistics regression models were formulated to determine independent predictors of smoking cessation. Predictors of continued smoking were greater prior cigarette consumption (odds ratio [OR] = 1.009 for each pack-year; 95% confidence interval [CI] = 1.004 to 1.014) and having one or more risk factors for coronary artery disease other than cigarette smoking (OR = 1.49; 95% CI = 1.15 to 1.93). Older age (OR = 0.98 for each additional year; 95% CI = 0.97 to 0.99) and unstable angina at time of initial assessment (OR = 0.69; 95% CI = 0.52 to 0.91) were associated with less likelihood of continued smoking. Younger patients with a worse risk profile and greater prior cigarette consumption were more likely than other patients to continue smoking after percutaneous coronary revascularization in the non-peri-infarction setting. Patients who had unstable angina were more likely to quit smoking than those who had stable angina. Despite the proven benefits of smoking cessation after percutaneous coronary revascularization, a substantial proportion of smokers (63%) continue to smoke; thus, smoking-cessation counseling should be addressed in this population.


Avoiding monocular artifacts in clinical stereotests presented on column-interleaved digital stereoscopic displays.
New forms of stereoscopic 3-D technology offer vision scientists new opportunities for research, but also come with distinct problems. Here we consider autostereo displays where the two eyes' images are spatially interleaved in alternating columns of pixels and no glasses or special optics are required. Column-interleaved displays produce an excellent stereoscopic effect, but subtle changes in the angle of view can increase cross talk or even interchange the left and right eyes' images. This creates several challenges to the presentation of cyclopean stereograms (containing structure which is only detectable by binocular vision). We discuss the potential artifacts, including one that is unique to column-interleaved displays, whereby scene elements such as dots in a random-dot stereogram appear wider or narrower depending on the sign of their disparity. We derive an algorithm for creating stimuli which are free from this artifact. We show that this and other artifacts can be avoided by (a) using a task which is robust to disparity-sign inversion-for example, a disparity-detection rather than discrimination task-(b) using our proposed algorithm to ensure that parallax is applied symmetrically on the column-interleaved display, and (c) using a dynamic stimulus to avoid monocular artifacts from motion parallax. In order to test our recommendations, we performed two experiments using a stereoacuity task implemented with a parallax-barrier tablet. Our results confirm that these recommendations eliminate the artifacts. We believe that these recommendations will be useful to vision scientists interested in running stereo psychophysics experiments using parallax-barrier and other column-interleaved digital displays.


Internalization and observation of fluorescent biomolecules in living microorganisms via electroporation.
The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisiæ, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.


Identification of master regulator candidates in conjunction with network screening and inference.
We developed a procedure for identifying transcriptional Master Regulators (MRs) related to special biological phenomena, such as diseases, in conjunction with network screening and inference. Network screening is a system for detecting activated transcriptional regulatory networks under particular conditions, based on the estimation of graph structure consistency with the measured data. Since network screening utilises the known Transcriptional Factor (TF)-gene relationships as the experimental evidence for molecular relationships, its performance depends on the ensemble of known TF networks used for its analysis. To compensate for its restrictions, a network inference method, the path consistency algorithm, is concomitantly utilised to identify MRs. The performance is illustrated by means of the known MRs in brain tumours that were computationally inferred and experimentally verified. As a result, the present procedure worked well for identifying MRs, in comparison to the previous computational selection for experimental verification.


A tissue-like platform for studying engineered quiescent human T-cells' interactions with dendritic cells.
Research in the field of human immunology is restricted by the lack of a system that reconstitutes the in-situactivation dynamics of quiescent human antigen-specific T-cells interacting with dendritic cells. Here we report a tissue-like system that recapitulates the dynamics of engineered primary human immune cell. Our approach facilitates real-time single-cell manipulations, tracking of interactions and functional responses complemented by population-based measurements of cytokines, activation status and proliferation. As a proof of concept, we recapitulate immunological phenomenon such as CD4 T-cells' help to CD8 T-cells through enhanced maturation of DCs and the effect of PD-1 checkpoint blockades. In addition, we characterise unique dynamics of T-cell/DC interactions as a function of antigen affinity.


The xanthate route to organofluorine derivatives. A brief account.
The present account summarises routes to organofluorine derivatives based on the chemistry of xanthates developed in the author's laboratory. It concerns mostly radical pathways, but a few ionic transformations are also discussed. The degenerative reversible transfer of xanthates allows radical addition even to unactivated alkenes and tolerates numerous functional groups, in particular common polar groups such as ketones, esters, amides, carbamates, nitriles etc. Since the fluorine atoms can be placed on the alkene or the xanthate partner or on both, this opens a convergent approach to a vast array of otherwise inaccessible organofluorine structures that could be of interest to the pharmaceutical and agrochemical industries and to materials science.


The Economic Costs of Type 2 Diabetes: A Global Systematic Review.
There has been a widely documented and recognized increase in diabetes prevalence, not only in high-income countries (HICs) but also in low- and middle-income countries (LMICs), over recent decades. The economic burden associated with diabetes, especially in LMICs, is less clear. We provide a systematic review of the global evidence on the costs of type 2 diabetes. Our review seeks to update and considerably expand the previous major review of the costs of diabetes by capturing the evidence on overall, direct and indirect costs of type 2 diabetes worldwide that has been published since 2001. In addition, we include a body of economic evidence that has hitherto been distinct from the cost-of-illness (COI) work, i.e. studies on the labour market impact of diabetes. We searched PubMed, EMBASE, EconLit and IBSS (without language restrictions) for studies assessing the economic burden of type 2 diabetes published from January 2001 to October 2014. Costs reported in the included studies were converted to international dollars ($) adjusted for 2011 values. Alongside the narrative synthesis and methodological review of the studies, we conduct an exploratory linear regression analysis, examining the factors behind the considerable heterogeneity in existing cost estimates between and within countries. We identified 86 COI and 23 labour market studies. COI studies varied considerably both in methods and in cost estimates, with most studies not using a control group, though the use of either regression analysis or matching has increased. Direct costs were generally found to be higher than indirect costs. Direct costs ranged from $242 for a study on out-of-pocket expenditures in Mexico to $11,917 for a study on the cost of diabetes in the USA, while indirect costs ranged from $45 for Pakistan to $16,914 for the Bahamas. In LMICs-in stark contrast to HICs-a substantial part of the cost burden was attributed to patients via out-of-pocket treatment costs. Our regression analysis revealed that direct diabetes costs are closely and positively associated with a country's gross domestic product (GDP) per capita, and that the USA stood out as having particularly high costs, even after controlling for GDP per capita. Studies on the labour market impact of diabetes were almost exclusively confined to HICs and found strong adverse effects, particularly for male employment chances. Many of these studies also took into account the possible endogeneity of diabetes, which was not the case for COI studies. The reviewed studies indicate a large economic burden of diabetes, most directly affecting patients in LMICs. The magnitude of the cost estimates differs considerably between and within countries, calling for the contextualization of the study results. Scope remains large for adding to the evidence base on labour market effects of diabetes in LMICs. Further, there is a need for future COI studies to incorporate more advanced statistical methods in their analysis to account for possible biases in the estimated costs.


Concentration and origin of lead (Pb) in liver and bone of Eurasian buzzards (Buteo buteo) in the United Kingdom.
Ingestion of lead (Pb) derived from ammunition used in the hunting of game animals is recognised to be a significant potential source of Pb exposure of wild birds, including birds of prey. However, there are only limited data for birds of prey in Europe regarding tissue concentrations and origins of Pb. Eurasian buzzards (Buteo buteo) found dead in the United Kingdom during an 11-year period were collected and the concentrations of Pb in the liver and femur were measured. Concentrations in the liver consistent with acute exposure to Pb were found in 2.7% of birds and concentration in the femur consistent with exposure to lethal levels were found in 4.0% of individuals. Pb concentration in the femur showed no evidence of consistent variation among or within years, but was greater for old than for young birds. The Pb concentration in the liver showed no effect of the birds' age, but varied markedly among years and showed a consistent tendency to increase substantially within years throughout the UK hunting season for gamebirds. The resemblance of the stable isotope composition of Pb from buzzard livers to that of Pb from the types of shotgun ammunition most widely-used in the UK increased markedly with increasing Pb concentration in the liver. Stable isotope results were consistent with 57% of the mass of Pb in livers of all of the buzzards sampled being derived from shotgun pellets, with this proportion being 89% for the birds with concentrations indicating acute exposure to Pb. Hence, most of the Pb acquired by Eurasian buzzards which have liver concentrations likely to be associated with lethal and sublethal effects is probably obtained when they prey upon or scavenge gamebirds and mammals shot using Pb shotgun pellets.


Adolescent transport and unintentional injuries: a systematic analysis using the Global Burden of Disease Study 2019.
Globally, transport and unintentional injuries persist as leading preventable causes of mortality and morbidity for adolescents. We sought to report comprehensive trends in injury-related mortality and morbidity for adolescents aged 10-24 years during the past three decades. Using the Global Burden of Disease, Injuries, and Risk Factors 2019 Study, we analysed mortality and disability-adjusted life-years (DALYs) attributed to transport and unintentional injuries for adolescents in 204 countries. Burden is reported in absolute numbers and age-standardised rates per 100 000 population by sex, age group (10-14, 15-19, and 20-24 years), and sociodemographic index (SDI) with 95% uncertainty intervals (UIs). We report percentage changes in deaths and DALYs between 1990 and 2019. In 2019, 369 061 deaths (of which 214 337 [58%] were transport related) and 31·1 million DALYs (of which 16·2 million [52%] were transport related) among adolescents aged 10-24 years were caused by transport and unintentional injuries combined. If compared with other causes, transport and unintentional injuries combined accounted for 25% of deaths and 14% of DALYs in 2019, and showed little improvement from 1990 when such injuries accounted for 26% of adolescent deaths and 17% of adolescent DALYs. Throughout adolescence, transport and unintentional injury fatality rates increased by age group. The unintentional injury burden was higher among males than females for all injury types, except for injuries related to fire, heat, and hot substances, or to adverse effects of medical treatment. From 1990 to 2019, global mortality rates declined by 34·4% (from 17·5 to 11·5 per 100 000) for transport injuries, and by 47·7% (from 15·9 to 8·3 per 100 000) for unintentional injuries. However, in low-SDI nations the absolute number of deaths increased (by 80·5% to 42 774 for transport injuries and by 39·4% to 31 961 for unintentional injuries). In the high-SDI quintile in 2010-19, the rate per 100 000 of transport injury DALYs was reduced by 16·7%, from 838 in 2010 to 699 in 2019. This was a substantially slower pace of reduction compared with the 48·5% reduction between 1990 and 2010, from 1626 per 100 000 in 1990 to 838 per 100 000 in 2010. Between 2010 and 2019, the rate of unintentional injury DALYs per 100 000 also remained largely unchanged in high-SDI countries (555 in 2010 vs 554 in 2019; 0·2% reduction). The number and rate of adolescent deaths and DALYs owing to environmental heat and cold exposure increased for the high-SDI quintile during 2010-19. As other causes of mortality are addressed, inadequate progress in reducing transport and unintentional injury mortality as a proportion of adolescent deaths becomes apparent. The relative shift in the burden of injury from high-SDI countries to low and low-middle-SDI countries necessitates focused action, including global donor, government, and industry investment in injury prevention. The persisting burden of DALYs related to transport and unintentional injuries indicates a need to prioritise innovative measures for the primary prevention of adolescent injury. Bill & Melinda Gates Foundation.


Linked insurance-tumor registry database for health services research.
Breast cancer screening and treatment data are often limited to restricted populations, including women older than 65 years old. The goal of this project was to develop procedures to link tumor registry and insurance claims databases on women younger than 65 years old with breast cancer and to assess the accuracy and validity of the linked dataset. Iowa Cancer Registry (ICR) and Wellmark Blue Cross/Blue Shield of Iowa (BC/BS) membership files of women with incident in situ or invasive breast cancer from 1989 to 1996 were linked. An automated deterministic match was followed with visual inspection from three independent reviewers applying a matching protocol. Matched and overall registry data were compared to assess population representativeness. Claims from BC/BS for incident cases during 1994 were examined for coding of a recent breast cancer diagnosis or treatment. The final dataset included 4,397 matched cases of patients aged 21 years and older from 1989 to 1996. The sociodemographic and tumor characteristics of the ICR population younger than 65 years old (n = 7,469) with breast cancer or carcinoma in situ were nearly identical with those of the matched patients younger than 65 years old (n = 3,449). Nearly all (96%) of the 445 matched incident cases in 1994 had claims data (CPT, DRG, or ICD-9 code) indicative of breast cancer. Treatment patterns varied by data source, with agreement ranging from 76% to 82%. The validity and generalizability of these data demonstrate their potential for further health services research among younger insured women with breast cancer. Additionally, the process outlined may be useful for developing other datasets to study other cancers in the population younger than 65 years old.


Prediction of venous thromboembolism using semantic and sentiment analyses of clinical narratives.
Venous thromboembolism (VTE) is the third most common cardiovascular disorder. It affects people of both genders at ages as young as 20 years. The increased number of VTE cases with a high fatality rate of 25% at first occurrence makes preventive measures essential. Clinical narratives are a rich source of knowledge and should be included in the diagnosis and treatment processes, as they may contain critical information on risk factors. It is very important to make such narrative blocks of information usable for searching, health analytics, and decision-making. This paper proposes a Semantic Extraction and Sentiment Assessment of Risk Factors (SESARF) framework. Unlike traditional machine-learning approaches, SESARF, which consists of two main algorithms, namely, ExtractRiskFactor and FindSeverity, prepares a feature vector as the input to a support vector machine (SVM) classifier to make a diagnosis. SESARF matches and maps the concepts of VTE risk factors and finds adjectives and adverbs that reflect their levels of severity. SESARF uses a semantic- and sentiment-based approach to analyze clinical narratives of electronic health records (EHR) and then predict a diagnosis of VTE. We use a dataset of 150 clinical narratives, 80% of which are used to train our prediction classifier support vector machine, with the remaining 20% used for testing. Semantic extraction and sentiment analysis results yielded precisions of 81% and 70%, respectively. Using a support vector machine, prediction of patients with VTE yielded precision and recall values of 54.5% and 85.7%, respectively.


Risk of Readmission Among HIV Patients in Public Portuguese Hospitals: Longitudinal Multilevel Population-Based Study.
Background: Thirty-day hospital readmission is receiving growing attention as an indicator of the quality of hospital care. Understanding factors associated with 30-day hospital readmission among HIV patients in Portugal is essential given the high burden cost of HIV hospitalizations in Portugal, a country suffering from financial constrains for almost 10 years. Objectives: We aimed to estimate the 30-day hospital readmission rates among HIV patients in Portugal and to identify its determinants using population-based data for Portuguese public hospitals. Study Design: A multilevel longitudinal population-based study. Methods: Between January 2009 and December 2014, a total of 37,134 registered discharges in the Portuguese National Health Service (NHS) facilities with HIV/AIDS as a main or secondary cause of admission were analyzed. Logistic regression was used to compare 30-day hospital readmission categories by computing odds ratio (OR) and corresponding 95% confidence intervals (95% CIs). A normal random effects model was used to determine unmeasured factors specific to each hospital. Results: A total of 4914 (13.2%, 95% CI: 12.9%-13.6%) hospitalizations had a subsequent 30-day readmission. Hospitalizations that included exit against medical opinion (OR = 1.18, 95% CI: 1.01-1.39), scheduled admissions (OR = 1.71, 95% CI: 1.58-1.85), and tuberculosis infection (OR = 1.20, 95% CI: 1.05-1.38) exhibited a higher risk of hospitalizations with subsequent 30-day readmission. In contrast, hospitalizations that included females (OR = 0.87, 95% CI: 0.81-0.94), a transfer to another facility (OR = 0.78, 95% CI: 0.67-0.91), and having a responsible financial institution (OR = 0.63, 95% CI: 0.55-0.72) exhibited a lower risk of hospitalizations with subsequent 30-day readmission. Hospitalizations associated with higher number of diagnosis, older ages, or hospitalizations during the economic crisis showed an increasing trend of 30-day readmission, whereas an opposite trend was observed for hospitalizations with higher number of procedures. Significant differences exist between hospital quality, adjusting for other factors. Conclusion: This study analyzes the indicators of 30-day hospital readmission among HIV patients in Portugal and provides useful information for enlightening policymakers and health care providers for developing health policies that can reduce costs associated with HIV hospitalizations.


Intravenous immunoglobulin therapy for children with Stevens-Johnson syndrome.
To review the experience with intravenous immunoglobulin (IVIG) therapy in patients with Stevens-Johnson syndrome (SJS) in our institution. All charts of patients with SJS admitted to Children's Hospital between November 1988 and June 1998 were reviewed. Twelve patients with SJS were detected. There were 8 males and 4 females, with a mean age 6 years (range 10 mo to 17 yrs). All patients presented with high fever and cutaneous and mucous membrane changes, and the diagnosis SJS was confirmed by a dermatologist. Of the 12 patients with SJS, 7 were treated with IVIG, 2 with corticosteroids, and 3 with supportive care. IVIG was administered in a single infusion at 1.5-2 g/kg, and was given on an average of hospital day 3 (range 1-8 days). The average duration of fever was 8 days (range 3-14) in the IVIG treated patients compared to 14 days (range 6-20) in the non-IVIG treated group (p = 0.06). The mean hospital stay was 12 days (range 4-22) for the patients treated with IVIG and 15 days (range 6-25) for the non-IVIG treated group (p = 0.5). No toxicity was observed with IVIG therapy. Duration of fever was shortened in patients treated with IVIG, although statistical significance was marginal. The hospital stay was slightly shortened in patients treated with IVIG; however, statistical significance was not reached. Prospective and controlled, multicenter studies are needed to further investigate these preliminary findings.


The phenotype of a germline mutation in PIGA: the gene somatically mutated in paroxysmal nocturnal hemoglobinuria.
Phosphatidylinositol glycan class A (PIGA) is involved in the first step of glycosylphosphatidylinositol (GPI) biosynthesis. Many proteins, including CD55 and CD59, are anchored to the cell by GPI. Loss of CD55 and CD59 on erythrocytes causes complement-mediated lysis in paroxysmal nocturnal hemoglobinuria (PNH), a disease that manifests after clonal expansion of hematopoietic cells with somatic PIGA mutations. Although somatic PIGA mutations have been identified in many PNH patients, it has been proposed that germline mutations are lethal. We report a family with an X-linked lethal disorder involving cleft palate, neonatal seizures, contractures, central nervous system (CNS) structural malformations, and other anomalies. An X chromosome exome next-generation sequencing screen identified a single nonsense PIGA mutation, c.1234C>T, which predicts p.Arg412(∗). This variant segregated with disease and carrier status in the family, is similar to mutations known to cause PNH as a result of PIGA dysfunction, and was absent in 409 controls. PIGA-null mutations are thought to be embryonic lethal, suggesting that p.Arg412(∗) PIGA has residual function. Transfection of a mutant p.Arg412(∗) PIGA construct into PIGA-null cells showed partial restoration of GPI-anchored proteins. The genetic data show that the c.1234C>T (p.Arg412(∗)) mutation is present in an affected child, is linked to the affected chromosome in this family, is rare in the population, and results in reduced, but not absent, biosynthesis of GPI anchors. We conclude that c.1234C>T in PIGA results in the lethal X-linked phenotype recognized in the reported family.


Peginterferon alfa-2a and ribavirin in patients with chronic hepatitis C who have failed prior treatment.
The most effective therapy currently available for treatment of chronic hepatitis C virus (HCV) is the combination of peginterferon and ribavirin. This study evaluated the effectiveness of this treatment in patients who were nonresponders to previous interferon-based therapy. The first 604 patients enrolled in the Hepatitis C Antiviral Long-Term Treatment Against Cirrhosis (HALT-C) Trial were evaluated. All were HCV RNA positive, previous nonresponders to interferon, with or without ribavirin, and had bridging fibrosis or cirrhosis on liver biopsy (Ishak fibrosis stage 3-6). Patients were retreated with peginterferon alfa-2a 180 microg/wk plus ribavirin 1000-1200 mg/day. Those with no detectable HCV RNA in serum at week 20 continued treatment for a total of 48 weeks and were then followed for an additional 24 weeks. Thirty-five percent of patients had no detectable HCV RNA in serum at treatment week 20, and 18% achieved sustained virologic response (SVR). Factors associated with an SVR included previous treatment with interferon monotherapy, infection with genotypes 2 or 3, a lower AST:ALT ratio, and absence of cirrhosis. Reducing the dose of ribavirin from > or =80% to < or =60% of the starting dose during the first 20 weeks of treatment was associated with a decline in SVR from 21% to 11% (P < or = 0.05). In contrast, reducing the dose of peginterferon or reducing ribavirin after week 20, when HCV RNA was already undetectable, did not significantly affect SVR. Selected nonresponders to previous interferon-based therapy can achieve SVR following retreatment with peginterferon alfa-2a and ribavirin.


Extra-articular disease manifestations in rheumatoid arthritis: incidence trends and risk factors over 46 years.
To investigate the trends in incidence of extra-articular rheumatoid arthritis (ExRA) in a well defined community based cohort of patients with rheumatoid arthritis (RA), and to examine possible predictors of ExRA occurrence. Using the resources of the Rochester Epidemiology Project, a retrospective medical record review was conducted of a cohort of 609 cases of RA in Olmsted County, MN, diagnosed during 1955-94. These cases had been previously classified using the ACR 1987 criteria for RA. Patients were followed up from 1955 to 2000 (median follow up 11.8 years; range 0.1-42.8), and incident ExRA manifestations were recorded according to predefined criteria. Time to first presentation of ExRA was compared in patients with RA by decade of diagnosis. Possible ExRA risk factors were identified in case record reviews. ExRA occurred in 247 patients (40.6%). A subgroup of 78 patients (12.8%) had ExRA manifestations considered to be severe in a previous study from Malmö, Sweden. The incidence of severe ExRA did not change significantly over the decades (p=0.165). In a multivariate analysis the main predictors of severe ExRA were smoking at RA diagnosis (risk ratio (RR)=2.94; 95% confidence interval (95% CI) 1.68 to 5.13) and early disability (Steinbrocker class III-IV at diagnosis) (RR=2.45; 95% CI 1.51 to 4.00). The effect of smoking overwhelmed the weaker effect of rheumatoid factor seropositivity. There was no decrease in the incidence of extra-articular manifestations in patients with RA diagnosed up to 1995. Smoking and early disability are independent risk factors for extra-articular RA.


Does divergence in female mate choice affect male size distributions in two cave fish populations?
Sexual selection by female choice can maintain male traits that are counter selected by natural selection. Alteration of the potential for sexual selection can thus lead to shifts in the expression of male traits. We investigated female mate choice for large male body size in a fish (Poecilia mexicana) that, besides surface streams, also inhabits two caves. All four populations investigated, exhibited an ancestral visual preference for large males. However, only one of the cave populations also expressed this female preference in darkness. Hence, the lack of expression of female preference in darkness in the other cave population leads to relaxation of sexual selection for large male body size. While P. mexicana populations with size-specific female mate choice are characterized by a pronounced male size variation, the absence of female choice in one cave coincides with the absence of large bodied males in that population. Our results suggest that population differences in the potential for sexual selection may affect male trait variation.


Babesiosis in free-ranging chamois (Rupicapra r. rupicapra) from Switzerland.
Pathological examination of five adult chamois (Rupicapra r. rupicapra) found dead in two different regions from the Swiss Alps revealed pale mucous membranes and musculature, swollen spleen and haemoglobinuria. Histologically, haemosiderosis in the spleen and centrilobular hepatic necrosis were the predominant findings. On blood smears, small (approximately 0.84-1.47 microm), round to pyriform, peripherally located inclusions were present in the erythrocytes. PCR followed by sequencing of DNA extracted from blood or spleen of the infected animals revealed 99-100% identity of the amplified part of the 18S rRNA gene with GenBank entries attributed to Babesia divergens/Babesia capreoli. This is the first report of fatal Babesia infections in chamois raising the question of an emerging disease in this species.


Evidence for facultative protocarnivory in Capsella bursa-pastoris seeds.
Many plants derive nutrients by attracting, ensnaring and killing invertebrates, a process that is described as "protocarnivory". This has been observed in seeds of the weed Capsella bursa-pastoris, but it is unclear as to whether it confers any material benefit in terms of germination, establishment and development. In the present study, seeds were germinated in zero, low, medium and high nutrient soils in both the presence and absence of nematodes (Steinernema feltiae). Nematodes were attracted to the seeds, with many dying within three days. Germination rates and seedling fresh masses were higher at all nutrient levels, and seedling fresh lengths were higher in all but the zero nutrient treatment, in the presence of nematodes. After transplantation, young plant fresh root lengths and dried leaf and root masses were generally higher in plants that had been germinated in the presence of nematodes across all nutrient levels, with the majority of significant differences being observed in the low-nutrient treatment. Our findings suggest that protocarnivory may play a role in the germination, establishment and early development of C. bursa-pastoris, and that this process may be facultative, since differences between nematode and non-nematode treatments were generally more pronounced in soils with low nutrient levels.


Salivary cortisol and pathogen disgust predict men's preferences for feminine shape cues in women's faces.
Recent studies suggest that individuals who are particularly concerned about infectious diseases show stronger preferences for exaggerated sex-typical characteristics in potential mates' faces. However, these studies have generally investigated individual differences in women's mate preferences and relied on questionnaires to assess disease-related concerns. Here we show that men's scores on the pathogen disgust subscale of the Three Domains of Disgust Scale are positively correlated with their preferences for femininity in women's faces and that this relationship is independent of the possible effects of both sexual and moral disgust. We then show that men with higher trait (i.e., average) salivary cortisol, a biomarker for immunosuppression, have stronger preferences for femininity in women's faces. Finally, we show that pathogen disgust is correlated with partnered men's femininity ratings of both their actual and ideal romantic partner. Together, these findings suggest that disease-related factors are important for individual differences in men's mate preferences.


Scenario planning for community development in Vietnam: a new tool for integrated health approaches?
Like many countries in Southeast Asia, Vietnam's rapid population and economic growth has met challenges in infrastructure development, especially sanitation in rural areas. As an entry point, we developed scenario planning as an action-research tool in a peri-urban community to identify first steps towards improving their complex sanitation problem and to, systemically, address emerging/re-emerging infectious diseases, as these are commonly linked to unsafe water and inadequate sanitation conditions. As an integrated approach, the process of constructing scenarios allowed us to work across sectors and stakeholders to incorporate this knowledge into a common vision. We conducted focus group discussions to identify and rank driving forces, orally constructed scenarios for the most uncertain drivers, discussed scenario implications and options, and examined the overall process for usefulness and sustainability. During a one-month scoping phase and in between focus group meetings, we carried out household visits which helped us understand the context of data and gather feedback from participants outside of the formal data collection process. Recorded results from these activities were used to develop subsequent tools. The research process gave us insights into how to adapt the scenario planning tool to identify alternative options. This involved choosing boundary partners, negotiating priorities, drawing out participant learning through self-assessment of our process (a prerequisite for changing mental models and thus achieving outcomes), and understanding how conveyed messages may reinforce the status quo. These insights showed the importance of examining research results beyond outputs and outcomes, namely through process.


Blind phase shift estimation in phase-shifting interferometry.
A blind phase shift estimation algorithm that allows simultaneous calculation of phases and phase shifts from three or more interferograms is presented. In phase-shifting interferometry, the phase shift errors introduce specific correlations between the calculated background intensity distribution and the fringe component. These correlations can be measured with a cross-power spectrum. By minimization of an objective function based on this cross-power spectrum, the actual phase shifts are estimated and used for phase recovery. The validity of this algorithm is verified by both the numerical simulation and the experiment results.


Assessing decision-making capacity: a primer for the development of hospital practice guidelines.
Decision making capacity (DMC) is a fundamental concept grounding the principle of respect for autonomy and the practice of obtaining informed consent. DMC must be determined and documented every time a patient undergoes a hospital procedure and for routine care when there is reason to believe decision making ability is compromised. In this paper we explore a path toward ethically informed development and implementation of a hospital policy related to DMC assessment. We begin with a review of the context of DMC assessment before discussing some considerations relevant to policy creation by healthcare ethics committees. The discussion concludes in a presentation of a typology of capacity assessment policies, which draws upon a sampling of currently used hospital policies to illustrate relevant ethical considerations.


The Relationship between Dietary Patterns and Metabolic Health in a Representative Sample of Adult Australians.
Studies assessing dietary intake and its relationship to metabolic phenotype are emerging, but limited. The aims of the study are to identify dietary patterns in Australian adults, and to determine whether these dietary patterns are associated with metabolic phenotype and obesity. Cross-sectional data from the Australian Bureau of Statistics 2011 Australian Health Survey was analysed. Subjects included adults aged 45 years and over (n = 2415). Metabolic phenotype was determined according to criteria used to define metabolic syndrome (0-2 abnormalities vs. 3-7 abnormalities), and additionally categorized for obesity (body mass index (BMI) ≥30 kg/m2 vs. BMI <30 kg/m2). Dietary patterns were derived using factor analysis. Multivariable models were used to assess the relationship between dietary patterns and metabolic phenotype, with adjustment for age, sex, smoking status, socio-economic indexes for areas, physical activity and daily energy intake. Twenty percent of the population was metabolically unhealthy and obese. In the fully adjusted model, for every one standard deviation increase in the Healthy dietary pattern, the odds of having a more metabolically healthy profile increased by 16% (odds ratio (OR) 1.16; 95% confidence interval (CI): 1.04, 1.29). Poor metabolic profile and obesity are prevalent in Australian adults and a healthier dietary pattern plays a role in a metabolic and BMI phenotypes. Nutritional strategies addressing metabolic syndrome criteria and targeting obesity are recommended in order to improve metabolic phenotype and potential disease burden.


Scyphophyllidium uruguayense n. sp. (Eucestoda: Tetraphyllidea) in Mustelus mento (Cope, 1877) (Chondrichthyes: Carcharhiniformes: Triakidae) from La Paloma, Uruguay.
A new species of Scyphophyllidium inhabits Mustelus mento near La Paloma, Uruguay. It resembles Scyphophyllidium giganteum from the Atlantic Ocean and specimens identified as S. giganteum from California by having anapolytic strobilae 155-258 mm long, 250-300 craspedote proglottids, scoleces 1.2-1.4 mm wide, necks 34-41 mm long, immature and mature proglottids wider than long, gravid proglottids wider than long to longer than wide, genital pores averaging 28% of proglottid length from the anterior end, relatively flat ovaries with digitiform lobes reaching the lateralmost extent of the testicular field, vitellaria in 2 fields converging toward the proglottid midline, straight and short cirrus sacs, and postvaginal vas deferens. The bothridia of the new species have accessory bothridial suckers that are smaller than those of California specimens; European specimens reportedly lack accessory bothridial suckers. The new species possesses a uterine duct that joins the uterus at the level of the genital atrium and ventral osmoregulatory ducts medial rather than lateral to the dorsal ducts, an arrangement described for Californian but not European specimens. It differs from both European and Californian specimens by having longer cirri, more testes per proglottid, prominent scales covering the neck, and vaginae and uterine ducts coiled immediately preovarially. Pithophorus, Marsupiobothrium, and Scyphophyllidium may form a clade.


Suppression of B-cell activation and IgE, IgA, IgG1 and IgG4 production by mammalian telomeric oligonucleotides.
The fine balance of immunoglobulins (Ig) E, IgG1, IgG4 and IgA in healthy production is maintained by the interaction of B cells with adaptive and innate immune response. The regulation of toll-like receptors (TLRs)-driven innate and adaptive immune effector B-cell response and the role of mammalian telomeric TTAGGG repeat elements represent an important research area. Human PBMC and purified naive and memory B cells were stimulated with specific ligands for TLR2, TLR3, TLR4, TLR5, TLR7, TLR8 and TLR9 in the presence or absence of telomeric oligonucleotides. B-cell proliferation, differentiation and antibody production were determined. TLR9 ligand directly activates naive and memory B cells, whereas TLR7 can stimulate them in the presence of plasmacytoid dendritic cells. Human B cells proliferate and turn into antibody-secreting cells in response to TLR3, TLR7 and TLR9, but not to TLR2, TLR4, TLR5 and TLR8 ligands. Stimulation of B cells with intracellular TLR3, TLR7 and TLR9 induced an activation cascade leading to memory B-cell generation and particularly IgG1, but also IgA, IgG4 and very low levels of IgE production. Mammalian telomeric oligodeoxynucleotide (ODN) significantly inhibited all features of TLR ligand-induced events in B cells including B-cell proliferation, IgE, IgG1, IgG4, IgA production, class switch recombination, plasma cell differentiation induced by TLR3, TLR7 and TLR9 ligands. B cells require specific TLR stimulation, T-cell and plasmacytoid dendritic cell help for distinct activation and Ig production profiles. Host-derived telomeric ODN suppress B-cell activation and antibody production demonstrating a natural mechanism for the control of overexuberant B-cell activation, antibody production and generation of memory.


Deoxyribonucleic acid microarray analysis of gene expression pattern in the arcuate nucleus/ventromedial nucleus of hypothalamus during lactation.
Lactation is characterized by extreme hyperphagia and negative energy balance resulting from a large energy drain due to milk production and by a suppression of cyclic ovarian function. Increases in neuropeptide Y and agouti-related protein and a decrease in proopiomelanocortin expression in the arcuate nucleus of hypothalamus (ARH) may contribute to the hyperphagia to maintain energy balance and to the suppression of LH secretion associated with lactation. However, little is known about the full extent of neuroendocrine changes in the ARH that may contribute to the various adaptations occurring during lactation. To address this issue, we used Affymetrix microarray to acquire a reliable profile of the lactation-induced transcriptional changes in micropunches containing the ARH and a portion of the ventromedial nucleus of the hypothalamus. Using high stringency criteria, 12 genes were identified as being differentially regulated during lactation, and an additional 10 genes and three transcribed sequences were identified using moderate stringency criteria. Changes in neuropeptide Y, enkephalin, tyrosine hydroxylase, and dynorphin, genes previously shown to be differentially regulated during lactation, provide validation for the microarray analysis. New genes identified as being differentially expressed include those related to neurotransmission, growth factors, signal transduction, and structure remodeling. These data identify new genes in ARH/ventromedial nucleus of the hypothalamus that may play an important role in the adaptations of lactation related to hyperphagia, milk production, and the suppression of cyclic reproductive function and may contribute to elucidating a framework for integrating changes in energy intake with the regulation of reproductive function during lactation.


Flash Joule heating for ductilization of metallic glasses.
Metallic glasses (MGs) inherit their amorphous structure from the liquid state, which predetermines their ability to withstand high loads approaching the theoretical limit. However, the absence of slip systems makes them very sensitive to the type of loading and extremely brittle in tension. The latter can be improved by precipitation of ductile crystals, which suppress a catastrophic propagation of shear bands in a glassy matrix. Here we report a novel approach to obtain MG-matrix composites with tensile ductility by flash Joule heating applied to Cu47.5Zr47.5Al5 (at.%) metallic glass. This homogeneous, volumetric and controllable rapid heat treatment allows achieving uniformly distributed metastable B2 CuZr crystals in the glassy matrix. It results in a significant tensile strain of 6.8±0.5%. Moreover, optimized adjustment of the heat-treatment conditions enables tuning of microstructure to achieve desired mechanical properties.


What Should Be the Primary Target of "Treat to Target" in Psoriatic Arthritis?
Recommendations regarding "treat to target" in psoriatic arthritis (PsA) have stated that the target should be remission or inactive disease. Potential definitions include very low disease activity (VLDA), PsA Disease Activity Score (PASDAS) near remission, Disease Activity Index for PsA (DAPSA) or clinical DAPSA (cDAPSA) remission. Our aim was to investigate the proportion of patients who fulfill these definitions and how much residual active disease remained. This analysis used 2 datasets: first, trial data from the Tight Control of PsA (TICOPA) study, which included 206 patients with recent-onset (< 2 yrs) PsA receiving standard and biological disease-modifying antirheumatic drugs (DMARD); and second, an observational clinical dataset from Italy of patients receiving biological DMARD. Proportions achieving each of the 4 potential targets were calculated in each dataset and comparisons between treatment groups were performed in the TICOPA dataset. Levels of residual disease were established for key clinical domains of PsA. All measures could differentiate the TICOPA trial treatment groups (p < 0.03). Lower proportions of patients fulfilled the VLDA criteria compared to DAPSA or cDAPSA remission. PASDAS results were different between the cohorts. Residual active disease was low across all definitions although higher levels were seen in DAPSA and cDAPSA compared to VLDA, particularly for psoriasis. In all measures, the proportion with elevated C-reactive protein was similar and low. VLDA appears the most stringent measure. It ensures that significant active arthritis, enthesitis, and psoriasis are not present, in contrast with DAPSA and PASDAS, in which composite scores can "hide" active disease in some domains.


Universal Features in Phonological Neighbor Networks.
Human speech perception involves transforming a countinuous acoustic signal into discrete linguistically meaningful units (phonemes) while simultaneously causing a listener to activate words that are similar to the spoken utterance and to each other. The Neighborhood Activation Model posits that phonological neighbors (two forms [words] that differ by one phoneme) compete significantly for recognition as a spoken word is heard. This definition of phonological similarity can be extended to an entire corpus of forms to produce a phonological neighbor network (PNN). We study PNNs for five languages: English, Spanish, French, Dutch, and German. Consistent with previous work, we find that the PNNs share a consistent set of topological features. Using an approach that generates random lexicons with increasing levels of phonological realism, we show that even random forms with minimal relationship to any real language, combined with only the empirical distribution of language-specific phonological form lengths, are sufficient to produce the topological properties observed in the real language PNNs. The resulting pseudo-PNNs are insensitive to the level of lingustic realism in the random lexicons but quite sensitive to the shape of the form length distribution. We therefore conclude that "universal" features seen across multiple languages are really string universals, not language universals, and arise primarily due to limitations in the kinds of networks generated by the one-step neighbor definition. Taken together, our results indicate that caution is warranted when linking the dynamics of human spoken word recognition to the topological properties of PNNs, and that the investigation of alternative similarity metrics for phonological forms should be a priority.


Decline in lung function related to exposure and selection processes among workers in the grain processing and animal feed industry.
To follow up workers in the grain processing and animal feed industry five years after an initial survey, and to monitor exposures to organic dust and endotoxin and changes in prevalence of respiratory symptoms and lung function. Outcome measures in the present survey were decline in lung function over five years, rapid annual decline in forced expiratory volume in one second (FEV1) above 90 ml.s-1, and loss to follow up. Among 140 workers included in the longitudinal analysis, annual decline in FEV1 and maximal mid-expiratory flow (MMEF) were significantly related to occupational exposure to dust and endotoxin in the grain processing and animal feed industry. Assuming a cumulative exposure over a working life of 40 years with an exposure of 5 mg.m-3, the estimated effect on the FEV1 would be a decline of 157 ml.s-1 (95% CI 13 to 300)--that is, about 4% of the group mean FEV1 and 473 ml.s-1 (95% CI 127 to 800) of the MMEF (about 12%). Workers with a dust exposure > 4 mg.m-3 or endotoxin concentrations > 20 ng.m-3 at the 1986-8 survey had significantly higher risk of rapid decline in FEV1 (odds ratio (OR) 3.3, 95% CI 1.02 to 10.3). The relations between occupational exposure and decline in lung function in this study occurred, despite the selection through the healthy worker effect that occurred as well. Increasing working years was related to decreasing annual decline in FEV1 and fewer people with rapid decline in FEV1 (OR 0.04, 95% CI 0 to 0.61 for over 20 v < 5 working years in the grain processing and animal feed industry). The presence of respiratory symptoms at baseline was a strong predictor of subsequent loss to follow up. Baseline lung function was not found to be predictive of subsequent loss to follow up. However, among workers lost to follow up the number of working years was more strongly negatively related to baseline lung function than among the workers who were studied longitudinally. The existence of the healthy worker effect implies that an exposure-response relation in the grain processing and animal feed industry may well be underestimated. This should be taken into account when health based recommended limit values are to be developed.


Predicting the extent of prostate cancer using the combination of systematic biopsy and serum prostate-specific antigen in Japanese men.
To determine the utility of systematic biopsy alone or combined with an assay of serum prostate-specific antigen (PSA) level to predict the extent of prostate cancer in Japanese men. Thirty-two patients who were diagnosed as having clinically organ-confined prostate cancer and who underwent prostatectomy were evaluated retrospectively for the results of systematic biopsy (percentage of positive biopsy cores and cancer location), serum PSA and the pathological stage of whole-mount sections of the prostatectomy specimens. The incidence of extraprostatic disease (pT3N0M0 or N+) in patients with >/= 8 ng/mL of serum PSA and cancer in bilateral lobes was significantly higher than in those with <8 ng/mL PSA and cancer in one lobe (83% vs 30%, P=0.020). In those with more than half the biopsy cores positive, extraprostatic disease was significantly more common than in those with less than half positive (93% vs 44%, P=0.0075); moreover, in patients with more than half the cores positive and >/= 8 ng/mL serum PSA, it was significantly more common than in those with less than half positive and <8 ng/mL of serum PSA (93% vs 27%, P=0.0021). However, the incidence of extraprostatic disease predicted by three variables (cancer location, percentage positive biopsy cores and serum PSA) was not significantly better than that predicted by two variables (percentage positive cores and serum PSA). The combination of systematic biopsy and serum PSA may be useful in predicting extraprostatic cancer. Patients with >/= 8 ng/mL serum PSA and more than half the biopsy cores positive could avoid a prostatectomy because there is a high probability that they have extraprostatic disease.


Vitamin D Promotes Pneumococcal Killing and Modulates Inflammatory Responses in Primary Human Neutrophils.
Streptococcus pneumoniae is a major human pathogen and a leading cause of pneumonia, septicemia, and meningitis worldwide. Despite clinical studies linking vitamin D deficiency and pneumonia, molecular mechanisms behind these observations remain unclear. In particular, the effects of vitamin D on neutrophil responses remain unknown. Using pneumococcal strains, primary neutrophils isolated from human blood, and sera from patients with frequent respiratory tract infections (RTIs), we investigated the effects of vitamin D on neutrophil bactericidal and inflammatory responses, including pattern recognition receptors, antimicrobial peptides, and cytokine regulation. We found that vitamin D upregulated pattern recognition receptors, TLR2, and NOD2, and induced the antimicrobial human neutrophil peptides (HNP1-3) and LL-37, resulting in increased killing of pneumococci in a vitamin D receptor-dependent manner. Antibodies targeting HNP1-3 inhibited bacterial killing. Vitamin D supplementation of serum from patients with bacterial RTIs enhanced neutrophil killing. Moreover, vitamin D lowered inflammatory cytokine production by infected neutrophils via IL-4 production and the induction of suppressor of cytokine signaling (SOCS) proteins SOCS-1 and SOCS-3, leading to the suppression of NF-κB signaling. Thus, vitamin D enhances neutrophil killing of S. pneumoniae while dampening excessive inflammatory responses and apoptosis, suggesting that vitamin D could be used alongside antibiotics when treating pneumococcal infections.


Beyond ABO and human histocompatibility antigen: other histocompatibility antigens with a role in transplantation.
In the past few years, there has been an increasing interest in nonhuman histocompatibility antigens as targets of injury in organ-transplant recipients. This increased interest has been spurred by the fact that human-histocompatibility-antigen-identical kidney transplants also undergo immunological rejections. Polymorphisms within nonhuman histocompatibility antigen genes associated with evoking an immune response to alloantigens are currently being studied for their association with transplant outcome. Studies identify the polymorphic major histocompatibility complex class I-related chain A as one of the important nonhuman histocompatibility antigen antibody targets in kidney allograft rejections. Use of endothelial cells as targets may clarify the specificities of other clinically relevant nonhuman histocompatibility antigen antibodies in graft rejections. This review summarizes current knowledge of the specificities and associations of nonhuman histocompatibility antigens with graft outcome after transplantation. The aims of current research into the role of nonhuman histocompatibility antigens and their genetics in predicting outcome are to develop an improved insight into the basic science of transplantation and to develop a risk or prognostic index for use in the clinical setting. Undoubtedly, this will continue to be an area of interest in terms of fully understanding the role of nonhuman histocompatibility antigens as targets of immune-mediated injury and the potential for clinical intervention.


Topography of the lingual foramen using micro-computed tomography for improving safety during implant placement of anterior mandibular region.
The aim of this study was to determine the location and size of the lingual foramina and the course of their canals using micro-computed tomography. Twenty Korean mandibles were scanned using a micro-computed tomography system and reconstructed three-dimensionally to enable observation of the lingual foramina and their canals. Four mandibles (20%) had a single foramen at the lingual side of the mandibular midline, 8 mandibles (40%) had 2 foramina, and 5 mandibles (25%) had 3 foramina. Three mandibles (15%) had 4 small foramina with short canals. The foramina were classified as either superior lingual foramina or inferior lingual foramina according to their vertical location relative to the mental spine. The diameters of superior lingual foramina and inferior lingual foramina were 0.75 ± 0.36 and 0.73 ± 0.38 mm (mean ± SD), respectively. The distances from the inferior border of the mandible to superior lingual foramina and inferior lingual foramina were 12.58 ± 2.49 and 6.43 ± 3.08 mm, respectively. Ten canals (21%) traveled upward to the labial side, 24 canals (51%) downward, and 13 canals (28%) parallel to it. Ten mandibles (50%) had lateral foramina.With regard to implant surgery of the anterior mandibular region, the most hazardous zones for lingual foramina are 0 to 2 mm from the midline, the upper 3 to 17 mm from the inferior border of the mandible, and the anterior 0 to 7 mm from the lingual side. Careful preoperative planning taking into account the anatomical location of these foramina might help to avoid complications due to damage to the foramina, their canals, and their contents.


Synthesis and characterization of antigenic influenza A M2e protein peptide-poly(acrylic) acid bioconjugate and determination of toxicity in vitro.
The influenza A virus is a critical public health problem that causes epidemics and pandemics, and occurs widely all over the world. Various vaccines against the virus have not provided a solution to the problem. Different approaches, particularly M2e peptide-based vaccines, are available for developing universal vaccines against influenza A. However, it is important to select a suitable carrier to obtain an effective vaccine. Accordingly, studies on the usage of various carriers are ongoing. Particularly, polymer-based carriers have gained importance due to both drug delivery and adjuvant effects. Therefore, bioconjugate of the M2e protein peptide from the influenza A virus covalent bonded with poly(acrylic) acid was synthesized in our study for the first time. The characterization was performed using size-exclusion chromatography and fluorescence spectroscopy; subsequently, it was found that the bioconjugate of the examined lower doses (0.05 and 0.5 mg/ml) have no toxic effects on human cell lines. These results suggest that, in the future, the poly(acrylic) acid bioconjugate of the M2e peptide should be studied in vivo for universal vaccine development against the influenza A virus.


Immunological cross reactivity between Candida albicans and human tissue.
An old concept to account for autoimmunity is the existence of immunologic cross reactions, or shared determinants between an exogenous agent and self antigen. To study molecular mimicry between the Candida antigen and an autoantigen, sera from clinical specimens were screened, based on seronegativity or positivity for thyroid, ovary and adrenal antibodies. Compared with tissue antibody negative sera and sera from healthy controls, samples from positive tissue antibody subjects exhibited significantly higher levels of Candida IgG (P < 0.001) IgM (P < 0.001) and IgA (P < 0.01) antibodies. While Candida antibodies were elevated in 60% of tissue antibody positive samples, these antibodies were present in only 7.5% of tissue antibody negative subjects and in 10% of healthy controls. Since PAGE electrophoresis showed similar bands mobility in Candida and different tissues, these positive antibodies and rabbit anti Candida antibodies were reacted in immunodiffusion and Western Blot Assay against Candida and tissue antigens, simultaneously. The results of immunodiffusion showed a clear precipitation line against tissue antigens when rabbit anti Candida or human positive Candida serum was used. Similarly, Western Blot Assays with rabbit or human anti Candida serum showed several positive bands with Candida and one or two positive bands with different tissues. The common antigens were located in the regions of 72 and 36 KD. The 72 KD was detected in capsule antigens, placenta, ovary, adrenal, thymus, liver, pancreas, spleen, brain and kidney, but not in sperm or epithelial cell antigen. The 36 KD antigen was positive in placenta, spleen adrenal, pancreas and capsule tissues. Absorbtion of sera containing high levels of Candida antibodies with tissue antigens caused 10-15% reduction in antibody titers. Moreover, treatment of thyroid antibody positive sera with C. Albicans caused a similar reduction in thyroid antibody levels. These reductions in antibody levels are an additional support for cross reactivity between C. Albicans and mammalian tissues. A demonstration of immunological cross reactivity between Candida and human tissues may be associated with the possible pathogenic role of Candida Albicans in the development of autoimmune diseases which warrants further investigation.


A comparison of the moment arms of pelvic limb muscles in horses bred for acceleration (Quarter Horse) and endurance (Arab).
Selective breeding for performance has resulted in distinct breeds of horse, such as the Quarter Horse (bred for acceleration) and the Arab (bred for endurance). Rapid acceleration, seen during Quarter Horse racing, requires fast powerful muscular contraction and the generation of large joint torques, particularly by the hind limb muscles. This study compared hind limb moment arm lengths in the Quarter Horse and Arab. We hypothesized that Quarter Horse hind limb extensor muscles would have longer moment arms when compared to the Arab, conferring a greater potential for torque generation at the hip, stifle and tarsus during limb extension. Six Quarter Horse and six Arab hind limbs were dissected to determine muscle moment arm lengths for the following muscles: gluteus medius, biceps femoris, semitendinosus, vastus lateralis, gastrocnemius (medialis and lateralis) and tibialis cranialis. The moment arms of biceps femoris (acting at the hip) and gastrocnemius lateralis (acting at the stifle) were significantly longer in the Quarter Horse, although the length of the remaining muscle moment arms were similar in both breeds of horse. All the Quarter Horse muscles were capable of generating greater muscle moments owing to their greater physiological cross-sectional area (PCSA) and therefore greater isometric force potential, which suggests that PCSA is a better determinant of muscle torque than moment arm length in these two breeds of horse. With the exception of gastrocnemius and tibialis cranialis, the observed muscle fascicle length to moment arm ratio (MFL : MA ratio) was greater for the Arab horse muscles. It appears that the Arab muscles have the potential to operate at slower velocities of contraction and hence generate greater force outputs when compared to the Quarter Horse muscles working over a similar range of joint motion; this would indicate that Arab hind limb muscles are optimized to function at maximum economy rather than maximum power output.


Social learning through associative processes: a computational theory.
Social transmission of information is a key phenomenon in the evolution of behaviour and in the establishment of traditions and culture. The diversity of social learning phenomena has engendered a diverse terminology and numerous ideas about underlying learning mechanisms, at the same time that some researchers have called for a unitary analysis of social learning in terms of associative processes. Leveraging previous attempts and a recent computational formulation of associative learning, we analyse the following learning scenarios in some generality: learning responses to social stimuli, including learning to imitate; learning responses to non-social stimuli; learning sequences of actions; learning to avoid danger. We conceptualize social learning as situations in which stimuli that arise from other individuals have an important role in learning. This role is supported by genetic predispositions that either cause responses to social stimuli or enable social stimuli to reinforce specific responses. Simulations were performed using a new learning simulator program. The simulator is publicly available and can be used for further theoretical investigations and to guide empirical research of learning and behaviour. Our explorations show that, when guided by genetic predispositions, associative processes can give rise to a wide variety of social learning phenomena, such as stimulus and local enhancement, contextual imitation and simple production imitation, observational conditioning, and social and response facilitation. In addition, we clarify how associative mechanisms can result in transfer of information and behaviour from experienced to naive individuals.


Optimized CRISPR-Cas9 Genome Editing for Leishmania and Its Use To Target a Multigene Family, Induce Chromosomal Translocation, and Study DNA Break Repair Mechanisms.
CRISPR-Cas9-mediated genome editing has recently been adapted for Leishmania spp. parasites, the causative agents of human leishmaniasis. We have optimized this genome-editing tool by selecting for cells with CRISPR-Cas9 activity through cotargeting the miltefosine transporter gene; mutation of this gene leads to miltefosine resistance. This cotargeting strategy integrated into a triple guide RNA (gRNA) expression vector was used to delete all 11 copies of the A2 multigene family; this was not previously possible with the traditional gene-targeting method. We found that the Leishmania donovani rRNA promoter is more efficient than the U6 promoter in driving gRNA expression, and sequential transfections of the oligonucleotide donor significantly eased the isolation of edited mutants. A gRNA and Cas9 coexpression vector was developed that was functional in all tested Leishmania species, including L. donovani, L. major, and L. mexicana. By simultaneously targeting sites from two different chromosomes, all four types of targeted chromosomal translocations were generated, regardless of the polycistronic transcription direction from the parent chromosomes. It was possible to use this CRISPR system to create a single conserved amino acid substitution (A189G) mutation for both alleles of RAD51, a DNA recombinase involved in homology-directed repair. We found that RAD51 is essential for L. donovani survival based on direct observation of the death of mutants with both RAD51 alleles disrupted, further confirming that this CRISPR system can reveal gene essentiality. Evidence is also provided that microhomology-mediated end joining (MMEJ) plays a major role in double-strand DNA break repair in L. donovani. IMPORTANCELeishmania parasites cause human leishmaniasis. To accelerate characterization of Leishmania genes for new drug and vaccine development, we optimized and simplified the CRISPR-Cas9 genome-editing tool for Leishmania. We show that co-CRISPR targeting of the miltefosine transporter gene and serial transfections of an oligonucleotide donor significantly eased isolation of edited mutants. This cotargeting strategy was efficiently used to delete all 11 members of the A2 virulence gene family. This technical advancement is valuable, since there are many gene clusters and supernumerary chromosomes in the various Leishmania species and isolates. We simplified this CRISPR system by developing a gRNA and Cas9 coexpression vector which could be used to delete genes in various Leishmania species. This CRISPR system could also be used to generate specific chromosomal translocations, which will help in the study of Leishmania gene expression and transcription control. This study also provides new information about double-strand DNA break repair mechanisms in Leishmania.


Multiplexed proteomic reactor for the processing of proteomic samples.
We report the development of a 96-well plate proteomic reactor for gel-free processing of minute amounts of complex proteomic samples. The device performs multiplexed trapping, enrichment, and biochemical processing of proteins, resulting in concentrated peptide solutions ready for mass spectrometric analysis. Individual wells on the reactor can process up to 2 microg of protein. We also report the coupling of the plate proteomic reactor with protein fractionation using size-exclusion chromatography for large-scale identification of proteins. To illustrate the potential of this approach, we separated 400 microg of MCF7 cell lysate using size-exclusion chromatography and processed 35 protein fractions on the reactor plate. Using stringent criteria when searching the data, a total of 875 unique proteins were identified. More relaxed searching conditions associated with a 1% false positive rate led to the identification of 2683 unique proteins, meaning that one protein was identified per 3-10 ng of total protein lysate loaded on the reactor plate.


Targeting dendritic cells with antigen-containing liposomes: a highly effective procedure for induction of antitumor immunity and for tumor immunotherapy.
Dendritic cells (DCs) are potent stimulators of immunity, and DCs pulsed with tumor antigen ex vivo have applications in tumor immunotherapy. However, DCs are a small population of cells, and their isolation and pulsing with antigen can be impractical. Here we show that a crude preparation of plasma membrane vesicles (PMV) from the highly metastatic murine melanoma (B16-OVA) and a surrogate tumor antigen (OVA) can be targeted directly to DCs in vivo to elicit functional effects. A novel metal-chelating lipid, 3(nitrilotriacetic acid)-ditetradecylamine, was incorporated into B16-OVA-derived PMV, allowing recombinant hexahistidine-tagged forms of single chain antibody fragments to the DC surface molecules CD11c and DEC-205, to be conveniently "engrafted" onto the vesicle surface by metal-chelating linkage. The modified PMV, or similarly engrafted synthetic stealth liposomes containing OVA or OVA peptide antigen, were found to target DCs in vitro and in vivo, in experiments using flow cytometry and fluorescence confocal microscopy. When used as vaccines in syngeneic mice, the preparations stimulated strong B16-OVA-specific CTL responses in splenic T cells and a marked protection against tumor growth. Protection was dependent on the simultaneous delivery of both antigen and a DC maturation or "danger signal" signal (IFN-gamma or lipopolysaccharide). Administration of the DC-targeting vaccine to mice challenged with B16-OVA cells induced a dramatic immunotherapeutic effect and prolonged disease-free survival. The results show that the targeting of antigen to DCs in this way is highly effective at inducing immunity and protection against the tumor, with protection being at least partially dependent on the eosinophil chemokine eotaxin.


Variants in GCNA, X-linked germ-cell genome integrity gene, identified in men with primary spermatogenic failure.
Male infertility impacts millions of couples yet, the etiology of primary infertility remains largely unknown. A critical element of successful spermatogenesis is maintenance of genome integrity. Here, we present a genomic study of spermatogenic failure (SPGF). Our initial analysis (n = 176) did not reveal known gene-candidates but identified a potentially significant single-nucleotide variant (SNV) in X-linked germ-cell nuclear antigen (GCNA). Together with a larger follow-up study (n = 2049), 7 likely clinically relevant GCNA variants were identified. GCNA is critical for genome integrity in male meiosis and knockout models exhibit impaired spermatogenesis and infertility. Single-cell RNA-seq and immunohistochemistry confirm human GCNA expression from spermatogonia to elongated spermatids. Five identified SNVs were located in key functional regions, including N-terminal SUMO-interacting motif and C-terminal Spartan-like protease domain. Notably, variant p.Ala115ProfsTer7 results in an early frameshift, while Spartan-like domain missense variants p.Ser659Trp and p.Arg664Cys change conserved residues, likely affecting 3D structure. For variants within GCNA's intrinsically disordered region, we performed computational modeling for consensus motifs. Two SNVs were predicted to impact the structure of these consensus motifs. All identified variants have an extremely low minor allele frequency in the general population and 6 of 7 were not detected in > 5000 biological fathers. Considering evidence from animal models, germ-cell-specific expression, 3D modeling, and computational predictions for SNVs, we propose that identified GCNA variants disrupt structure and function of the respective protein domains, ultimately arresting germ-cell division. To our knowledge, this is the first study implicating GCNA, a key genome integrity factor, in human male infertility.