Clinical relevance of in vitro propagation of activated lymphocytes from endomyocardial biopsy samples of pediatric heart transplant recipients.
In vivo activated T-lymphocytes can be cultured from endomyocardial biopsy samples of human cardiac allografts, sometimes even in the absence of histological rejection. We investigated the clinical relevance of this "lymphocyte growth assay" in pediatric heart transplant recipients. Specifically, we wished to determine if: (i) positive lymphocyte growth from EMB samples in the absence of significant rejection identifies a patient as being at increased risk for the development of acute rejection; (ii) withdrawal or major dose reduction of corticosteroids in the presence of lymphocyte growth results in high risk of rebound rejection; and (iii) presence of lymphocyte growth during acute rejection helps predict the response to treatment. Cultures were performed on 789 consecutive EMB samples from 65 pediatric heart transplant recipients in media containing 30 U/ml of recombinant IL-2. T-lymphocytes were cultured from 16% of EMB samples with low grade rejection (grade 0-1b) and from 34% of EMB samples with grade 2-4 rejection. EMB samples obtained early post-transplant (<180 days) were significantly more likely to yield positive lymphocyte growth compared to biopsies obtained late for any given rejection grade. Lymphocyte growth was comparable between patients managed with cyclosporine or tacrolimus based immunosuppression. For 227 EMB samples without rejection, a subsequent EMB sample was obtained within 12 weeks. Lymphocyte cultures were positive in 47 of these 227 EMB samples (21%), and in 19 out of 47 (40%) cases acute rejection (grade 2-4) was present on the follow-up EMB sample. By contrast, of 180 biopsies without growth, only 29 (16%) showed rejection at the next EMB (p<0.0001). When a follow-up biopsy was performed within 12 weeks of corticosteroid withdrawal, "rebound rejection" was observed in 3 out of 10 (30%) cases where the previous EMB sample yielded positive lymphocyte growth and in 4 out of 38 (11%) cases when it did not (p=0.29). The presence of lymphocyte growth in association with rejection was also predictive of whether rejection would resolve following high dose intravenous corticosteroid therapy (persistent rejection in 33 out of 50 (66%) cases with positive growth, versus 25 out of 80 (31%) cases without growth (p<0.0001)). Thus, positive lymphocyte growth is strongly associated with higher grade of rejection and earlier time from transplantation. Lymphocyte growth in the absence of rejection indicates high risk for rejection within the next 12 weeks. Growth in association with acute rejection indicates high probability of persistence of rejection following treatment with high dose corticosteroids.


Gene expression variability and the analysis of large-scale RNA-seq studies with the MDSeq.
Rapidly decreasing cost of next-generation sequencing has led to the recent availability of large-scale RNA-seq data, that empowers the analysis of gene expression variability, in addition to gene expression means. In this paper, we present the MDSeq, based on the coefficient of dispersion, to provide robust and computationally efficient analysis of both gene expression means and variability on RNA-seq counts. The MDSeq utilizes a novel reparametrization of the negative binomial to provide flexible generalized linear models (GLMs) on both the mean and dispersion. We address challenges of analyzing large-scale RNA-seq data via several new developments to provide a comprehensive toolset that models technical excess zeros, identifies outliers efficiently, and evaluates differential expressions at biologically interesting levels. We evaluated performances of the MDSeq using simulated data when the ground truths are known. Results suggest that the MDSeq often outperforms current methods for the analysis of gene expression mean and variability. Moreover, the MDSeq is applied in two real RNA-seq studies, in which we identified functionally relevant genes and gene pathways. Specifically, the analysis of gene expression variability with the MDSeq on the GTEx human brain tissue data has identified pathways associated with common neurodegenerative disorders when gene expression means were conserved.


A comparison of several algorithms for the single individual SNP haplotyping reconstruction problem.
Single nucleotide polymorphisms are the most common form of variation in human DNA, and are involved in many research fields, from molecular biology to medical therapy. The technological opportunity to deal with long DNA sequences using shotgun sequencing has raised the problem of fragment recombination. In this regard, Single Individual Haplotyping (SIH) problem has received considerable attention over the past few years. In this article, we survey seven recent approaches to the SIH problem and evaluate them extensively using real human haplotype data from the HapMap project. We also implemented a data generator tailored to the current shotgun sequencing technology that uses haplotypes from the HapMap project. The data we used to compare the algorithms are available on demand, since we think they represent an important benchmark that can be used to easily compare novel algorithmic ideas with the state of the art. Moreover, we had to re-implement six of the algorithms surveyed because the original code was not available to us. Five of these algorithms and the data generator used in this article endowed with a Web interface are available at http://bioalgo.iit.cnr.it/rehap.


Plant-soil microorganism interactions: heritable relationship between plant genotype and associated soil microorganisms.
Although soil microbial communities are known to play crucial roles in the cycling of nutrients in forest ecosystems and can vary by plant species, how microorganisms respond to the subtle gradients of plant genetic variation is just beginning to be appreciated. Using a model Populus system in a common garden with replicated clones of known genotypes, we evaluated microbial biomass and community composition as quantitative traits. Two main patterns emerged. (1) Plant genotype influenced microbial biomass nitrogen in soils under replicated genotypes of Populus angustifolia, F1, and backcross hybrids, but not P. fremontii. Genotype explained up to 78% of the variation in microbial biomass as indicated by broad-sense heritability estimates (i.e., clonal repeatability). A second estimate of microbial biomass (total phospholipid fatty acid) was more conservative and showed significant genotype effects in P. angustifolia and backcross hybrids. (2) Plant genotype significantly influenced microbial community composition, explaining up to 70% of the variation in community composition within P. angustifolia genotypes alone. These findings suggest that variation in above- and belowground traits of individual plant genotypes can alter soil microbial dynamics, and suggests that further investigations of the evolutionary implications of genetic feedbacks are warranted.


Pilot and full scale applications of sulfur-based autotrophic denitrification process for nitrate removal from activated sludge process effluent.
Sulfur-based autotrophic denitrification of nitrified activated sludge process effluent was studied in pilot and full scale column bioreactors. Three identical pilot scale column bioreactors packed with varying sulfur/lime-stone ratios (1/1-3/1) were setup in a local wastewater treatment plant and the performances were compared under varying loading conditions for long-term operation. Complete denitrification was obtained in all pilot bioreactors even at nitrate loading of 10 mg NO3(-)-N/(L.h). When the temperature decreased to 10 °C during the winter time at loading of 18 mg NO3(-)-N/(L.h), denitrification efficiency decreased to 60-70% and the bioreactor with S/L ratio of 1/1 gave slightly better performance. A full scale sulfur-based autotrophic denitrification process with a S/L ratio of 1/1 was set up for the denitrification of an activated sludge process effluent with a flow rate of 40 m(3)/d. Almost complete denitrification was attained with a nitrate loading rate of 6.25 mg NO3(-)-N/(L.h).


Three members of the iodothyronine deiodinase family, dio1, dio2 and dio3, are expressed in spatially and temporally specific patterns during metamorphosis of the flounder, Paralichthys olivaceus.
Flounder metamorphosis, marked by eye migration, lateralized pigmentation, and tissue differentiation in the stomach and skeletal muscle, is stimulated by thyroid hormone (TH). It is known that tri-iodothyronine (T3) produced by iodothyronine deiodinase type-1 (Dio1) from thyroxine (T4) enters the blood, whereas T3 produced by Dio2 penetrates into the nucleus of the Dio2-expressing cells, and then Dio3 inactivates both T4 and T3. To better understand the distinct functions of these three deiodinases in T3 regulation during flounder metamorphosis, we examined the tissue expression patterns of dio1, dio2, and dio3 in larvae of the Japanese flounder, Paralichthys olivaceus, by section in situ hybridization (SISH). We found that each deiodinase is expressed in a spatially and temporally specific pattern. dio1 is expressed in liver parenchymal cells from pro-metamorphosis to early climax, while dio2 is expressed in limited regions of the eyes, tectum, and skeletal muscles from pro-metamorphosis to post-climax. Considering these findings together with reports on other vertebrates, we predict that the liver cells expressing dio1 supply T3 to the blood, and that this systemic T3 synchronizes metamorphosis of differentiating tissues throughout the larval body, whereas the eyes, tectum, and skeletal muscles autonomously produce additional T3 for local tissue differentiation. Finally, dio3 expression is detected in skeletal muscle and gastric gland blastemas, which both undergo marked tissue differentiation at metamorphic climax. We hypothesize that dio3 expression protects these tissues from basal T3 levels early in metamorphosis, ensuring, together with the T3 surge from the liver, the synchronization of tissue differentiation at metamorphic climax.


Contraceptive counseling for continuation and satisfaction.
Quality contraceptive counseling has been identified as a potential means to reduce unintended pregnancy and to increase contraceptive continuation and satisfaction. Past approaches that focused on autonomous decision making and directive counseling have not been shown to meet these goals consistently. Women's health organizations globally are calling for improved counseling through more thorough discussion of side-effects and bleeding changes, and renewed focus on shared decision making and patient-centered care. Reproductive life planning can help initiate contraceptive counseling but does not resonate with all patients. A client-centered approach using shared decision making, building trust, and eliciting client preferences has been shown to increase satisfaction and continuation. Patient preferences vary widely and may or may not prioritize extremely high effectiveness. Decision support tools have mixed results, but generally can help improve the method choice process when they are well designed. Clinicians should strive for good interpersonal relationships with patients, and elicit patient experiences and preferences to tailor their counseling to each individual's needs. Shared decision making with input from both the patient and clinician is preferred by many women, and clinicians should be cognizant of perceptions of pushing any method too strongly, especially among marginalized populations. More research on long-term satisfaction and continuation linked to different counseling practices is needed.


Fraction of organic carbon predicts labile desorption rates of chlorinated organic pollutants in laboratory-spiked geosorbents.
The resuspension of large volumes of sediments that are contaminated with chlorinated pollutants continues to threaten environmental quality and human health. Whereas kinetic models are more accurate for estimating the environmental impact of these events, their widespread use is substantially hampered by the need for costly, time-consuming, site-specific kinetics experiments. The present study investigated the development of a predictive model for desorption rates from easily measurable sorbent and pollutant properties by examining the relationship between the fraction of organic carbon (fOC) and labile release rates. Duplicate desorption measurements were performed on 46 unique combinations of pollutants and sorbents with fOC values ranging from 0.001 to 0.150. Labile desorption rate constants indicate that release rates predominantly depend upon the fOC in the geosorbent. Previous theoretical models, such as the macro-mesopore and organic matter (MOM) diffusion model, have predicted such a relationship but could not accurately predict the experimental rate constants collected in the present study. An empirical model was successfully developed to correlate the labile desorption rate constant (krap) to the fraction of organic material where log(krap)=0.291-0.785 . log(fOC). These results provide the first experimental evidence that kinetic pollution releases during resuspension events are governed by the fOC content in natural geosorbents.


CNV Association of Diverse Clinical Phenotypes from eMERGE reveals novel disease biology underlying cardiovascular disease.
Cardiovascular disease is the leading cause of death in the United States. Consequently, individuals who are genetically predisposed for high risk of cardiovascular disease would benefit most from prevention and early intervention approaches. Among common health risk factors affecting adult populations, we evaluated 23 cardiovascular disease-related traits, including BMI, glucose levels and lipid profiling to determine their associations with low-frequency recurrent copy number variations (CNV) (population frequency < 5%). We examined 10,619 unrelated subjects of European ancestry from the Electronic Medical Records and Genomics (eMERGE) Network who were genotyped with 657,366 markers genome-wide on the Illumina Infinium Quad 660 array. We performed CNV calling based on array marker intensity and evaluated data quality, ancestry stratification, and relatedness to ensure unbiased association discovery. Using a segment-based scoring approach, we assessed the association of all CNVs with each trait. In this large genome-wide analysis of low-frequency CNVs, we observed 11 novel genome-wide significant associations of low-frequency CNVs with major cardiovascular disease traits. In one of the largest genome-wide studies for low-frequency recurrent CNVs, we identified 11 loci associated with cardiovascular disease and related traits at the genome-wide significance level that may serve as biomarkers for prevention and early intervention studies in subjects who are at elevated risk. Our study further supports the role of low-frequency recurrent CNVs in the pathogenesis of common complex disease traits.


Long-read sequencing and haplotype linkage analysis enabled preimplantation genetic testing for patients carrying pathogenic inversions.
Preimplantation genetic testing (PGT) has already been applied in patients known to carry chromosomal structural variants to improve the clinical outcome of assisted reproduction. However, conventional molecular techniques are not capable of reliably distinguishing embryos that carry balanced inversion from those with a normal karyotype. We aim to evaluate the use of long-read sequencing in combination with haplotype linkage analysis to address this challenge. Long-read sequencing on Oxford Nanopore platform was employed to identify the precise positions of inversion break points in four patients. Comprehensive chromosomal screening and genome-wide haplotype linkage analysis were performed based on SNP microarray. The haplotypes, including the break point regions, the whole chromosomes involved in the inversion and the corresponding homologous chromosomes, were established using informative SNPs. All the inversion break points were successfully identified by long-read sequencing and validated by Sanger sequencing, and on average only 13 bp differences were observed between break points inferred by long-read sequencing and Sanger sequencing. Eighteen blastocysts were biopsied and tested, in which 10 were aneuploid or unbalanced and eight were diploid with normal or balanced inversion karyotypes. Diploid embryos were transferred back to patients, the predictive results of the current methodology were consistent with fetal karyotypes of amniotic fluid or cord blood. Nanopore long-read sequencing is a powerful method to assay chromosomal inversions and identify exact break points. Identification of inversion break points combined with haplotype linkage analysis is an efficient strategy to distinguish embryos with normal or balanced inversion karyotypes, facilitating PGT applications.


Cognitive function and behavior of children with adenotonsillar hypertrophy suspected of having obstructive sleep-disordered breathing.
The purpose of this study was to determine whether risks of impaired cognitive function could be predicted for children or groups of children with adenotonsillar hypertrophy who were suspected of having obstructive sleep-disordered breathing, from historical and polysomnographic variables used separately or in combination. We studied 114 consecutive 6- to 12-year-old children with adenotonsillar hypertrophy, who were referred because of suspected obstructive sleep-disordered breathing, with questionnaires, assessment of tonsil size, general and memory cognitive tests, and attended polysomnography with the use of nasal pressure recording to detect flow. There were important significant relationships between snore group (snored every night versus less often), sleep efficiency, and race and 2 of 3 general cognitive tests (vocabulary and similarities). Significant but weaker relationships were observed between sleep latency and 2 memory indices (verbal memory and general memory) and between sleep efficiency and 2 behavior indices (attention-deficit/hyperactivity disorder summary and hyperactive-impulsive summary). The number of episodes of apnea and hypopnea per 1 hour of sleep predicted the vocabulary score as well as did the snore group, but it did not predict other tests as well as other variables. Tonsil size did not predict any cognitive or behavior score. Confidence intervals for group means were small, whereas prediction intervals for individual children were large. Risk of impaired cognitive function and behavior can be predicted from snoring history, sleep efficiency, sleep latency, and race but not tonsil size. The combination of snoring history and polysomnographic variables predicted impaired cognitive scores better than did either alone. The snoring history predicted more test scores than the number of episodes of apnea and hypopnea per 1 hour of sleep.


Innervation sites of dog hindpaw series and parallel vascular circuits.
The neural control of the distribution of blood flow between the parallel nutritional and nonnutritional vascular circuits in the dog hindpaw has been assessed. The right hindpaw of anesthetized dogs was vascularly and neurally isolated and placed in a volume recorder sealed by the skin flap. The animals were heparinized. The autoperfused preparation was isovolumetric during the control period. The inflow tubing had side arms for measurement of perfusion pressure and for injections of indicators (131I-albumin, 86Rb and 85Sr-microspheres). Vascular volume changes were determined from venous time-concentration curves and by plethysmography. The peripheral cut ends of the superficial and deep fibular nerves and the tibial nerve were individually stimulated at 5 and 11 Hz. The permeability surface area product of 86Rb and the capillary filtration coefficient were determined and A-V shunt patency was assessed by the venous recovery of 85Sr 15 microns microspheres. Each nerve stimulation increased blood flow resistance. Superficial fibular nerve innervation included essentially uniform effects on the arterioles and A-V shunts, the upstream arteries and also the veins. Deep fibular nerve stimulation indicated that its innervation included the arteries and the A-V shunt nonexchange vessels. Tibial nerve stimulations indicated that its innervation included the A-V shunt vessels and veins with minimal influences on the arteries and arterioles.


Field and Laboratory Studies of Resistance to Bt Corn by Western Corn Rootworm (Coleoptera: Chrysomelidae).
Western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), has developed resistance to transgenic corn that produces the insecticidal toxin Cry3Bb1 derived from the bacterium Bacillus thuringiensis (Bacillales: Bacillaceae) (Bt), with cross-resistance extending to corn with Bt toxins mCry3A and eCry3.1Ab. Additionally, some populations of western corn rootworm have evolved resistance to Cry34/35Ab1 corn. We conducted a 2-yr field and laboratory study that included three field locations: 1) Bt-susceptible population, 2) field with a recent history of Cry3Bb1 resistance, and 3) field with a long-term history of Cry3Bb1 resistance. The population with recently evolved Cry3Bb1 resistance showed resistance to Cry3Bb1 corn in both laboratory bioassays and field evaluations; by contrast, the population with a long-term history of Cry3Bb1 resistance showed resistance, in both laboratory and field experiments to Cry3Bb1 corn and corn with a pyramid of mCry3A plus eCry3.1Ab corn. Field-based evaluations also showed that the field population with a long-term history of Cry3Bb1 resistance imposed higher root injury to Cry3Bb1 corn and the pyramid of mCry3A plus eCry3.1Ab compared with the susceptible control. The results of this study are discussed in the context of developing strategies to manage western corn rootworm in areas where populations have evolved resistance to Cry3Bb1 corn.


Sickle cell anemia: a potential nutritional approach for a molecular disease.
A certain population of red blood cells in patients with sickle cell anemia has an elevated density and possesses an abnormal membrane. These "dense cells" have a tendency to adhere to neutrophils, platelets, and vascular endothelial cells, and, thus, they could trigger vasoocclusion and the subsequent painful crisis from which these patients suffer. We developed a laboratory method of preparing such dense cells and found that nutritional antioxidant supplements, hydroxyl radical scavengers, and iron-binding agents could inhibit the formation of dense cells in vitro. The concentrations at which effective nutritional supplements could inhibit dense cell formation by 50% were 4.0 mg/mL for aged garlic extract, 0.38 mg/mL for black tea extract, 0.13 mg/mL for green tea extract, 0.07 mg/mL for Pycnogenol, 930 microM for alpha-lipoic acid, 270 microM for vitamin E, 45 microM for coenzyme Q(10), and 32 microM for beta-carotene. Both an ex vivo study and a pilot clinical trial demonstrated that a cocktail consisting of daily doses of 6 g of aged garlic extract, 4-6 g of vitamin C, and 800 to 1200 IU of vitamin E may indeed be beneficial to the patients.


Bladder dose estimation during intracavitary brachytherapy for carcinoma of the cervix using a single line source system.
The estimation of maximum bladder doses from orthogonal radiographs is unreliable when triple source systems are used for intracavitary brachytherapy (BT) for gynaecological cancers. For single line source systems, the estimation of maximum bladder doses from radiographs should be more reliable due to the radial symmetry of the isodose distribution. A pilot study has been carried out to compare the estimated maximum bladder doses from standard radiographs with data obtained from CT for single line source BT treatments. 12 patients undergoing treatment for carcinoma of the cervix were selected for CT assessment of their bladder doses. For each patient, the dose rates at the International Commission of Radiation Units and Measurements (ICRU) bladder reference point B1 and a second reference point B2 2.5 cm cranially were computed from orthogonal radiographs and were compared with the maximum bladder dose rate as determined by CT scanning. Dose rates were computed for two different source loading patterns: (1) a 6 cm line source with uniformly distributed linear activity along its length; (2) a 6 cm line source with increased activity in the central 2 cm segment. The mean ratio of the maximum CT bladder dose rate to the dose rate at the ICRU reference point B1 on orthogonal radiographs for the line source with uniform linear activity was 1.32 (range 0.62-2.43, SD = 0.54). When the dose rates at both reference points B1 and B2 were considered, the mean ratio of the maximum CT dose rate to the maximum dose rate from radiographs was only 1.05 (range 0.72-1.72, SD = 0.24). For the line source with increased activity in the central segment, the mean ratio of the maximum CT bladder dose rate to the dose rate at B1 was 1.38 (range 0.60-2.63, SD = 0.64). When both B1 and B2 were considered, the variation in the ratio of the maximum CT dose rate to the maximum dose rate from radiographs was considerably smaller (mean ratio = 1.07, range 0.69-1.76, SD = 0.26). For single line source systems, single point dose estimation using the ICRU reference point on orthogonal radiographs will underestimate the maximum bladder dose although the discrepancy is less than for triple source systems. If the ICRU reference point is used in conjunction with a second reference point 2.5 cm cranially, then underestimation of the maximum bladder dose is unlikely to occur as at least one of the points is likely to be a reasonable estimate of the maximum bladder dose.


Intratumoral delivery of a Tim-3 antibody-encoding oncolytic adenovirus engages an effective antitumor immune response in liver cancer.
The use of oncolytic viruses as a gene therapy vector is an area of active biomedical research, particularly in the context of cancer treatment. However, the actual therapeutic success of this approach to tumor elimination remains limited. As such, the present study was developed with the goal of simultaneously enhancing the antitumor efficacy of oncolytic viruses and the local immune response by combining the Ad-GD55 oncolytic adenovirus and an antibody specific for the TIM-3 immune checkpoint molecule (α-TIM-3). The results of Virus and cell-mediated cytotoxicity assay, qPCR, and Western immunoblotting showed that Ad-GD55-α-Tim-3 oncolytic adenovirus is capable of inducing α-TIM-3 expression within hepatoma cells upon infection, and Ad-GD55-α-TIM-3 exhibited inhibitory efficacy superior to that of Ad-GD55 when used to treat these tumor cells together with the induction of enhanced intracellular immunity. In vivo experiments revealed that Ad-GD55-α-TIM-3 administration was sufficient to inhibit tumor growth and engage in a more robust local immune response within the simulated tumor immune microenvironment. These results highlighted the promising therapeutic effects of Ad-GD55-α-TIM-3 oncolytic adenovirus against HCC in vitro and in vivo. As such, this Ad-GD55-α-TIM-3 oncolytic adenovirus may represent a viable approach to the treatment of hepatocellular carcinoma.


Effects of gear modifications in a North Atlantic pelagic longline fishery: A multiyear study.
The threat of population declines caused by pelagic longline fisheries in the Atlantic has increased the concern to find strategies that minimize the bycatch and mortality of non-target marine animals. Gear modification, such as the use of circle hooks instead of conventional J-hooks, has been identified as an effective bycatch reduction strategy in different pelagic longline fisheries around the world. This study aimed to verify the effectiveness of the use of circle hooks by quantifying catch rates, relative size selectivity, and anatomical hooking position for the most common target species (swordfish, Xiphias gladius, and blue shark, Prionace glauca), and some bycatch species (loggerhead sea turtles, Caretta caretta, and shortfin mako, Isurus oxyrinchus) caught by the Azorean longline fishing fleet. The trial was conducted for five consecutive years (2000-2004) using eight different types of hooks. In general, the blue shark catches using circle hooks were significantly higher compared to J (Mustad 9/0). The circle hooks also showed high probabilities of catching juvenile blue sharks. Conversely, the circle hooks were efficient in reducing the loggerhead sea turtle bycatch and were related to fewer catches of small sea turtle individuals. The use of circle hooks was also associated with reduced swordfish catches compared to J (Mustad 9/0), and the effect of hook types on length at capture was only significant for Circle (L. & P. 18/0-CLP18) and Ringed Tuna (RT). No significant differences were observed comparing hook type to either catch rates or size selectivity for shortfin mako. Additionally, circle hooks were more likely to lodge in the mouth than in deeper anatomical positions, when compared to J (Mustad 9/0), for the four species analysed. The present study demonstrated that the use of circle hooks could mitigate the impact of the pelagic longline fisheries in the Azores by decreasing the bycatch of sea turtles and reducing animal injuries caused by deep hooking.


Enantioselective catalytic β-amination through proton-coupled electron transfer followed by stereocontrolled radical-radical coupling.
A new mechanistic approach for the catalytic, enantioselective conjugate addition of nitrogen-based nucleophiles to acceptor-substituted alkenes is reported, which is based on a visible light induced and phosphate base promoted transfer of a single electron from a nitrogen nucleophile to a catalyst-bound acceptor-substituted alkene, followed by a stereocontrolled C-N bond formation through stereocontrolled radical-radical coupling. Specifically, N-aryl carbamates are added to the β-position of α,β-unsaturated 2-acyl imidazoles using a visible light activated photoredox mediator in combination with a chiral-at-rhodium Lewis acid catalyst and a weak phosphate base, affording new C-N bonds in a highly enantioselective fashion with enantioselectivities reaching up to 99% ee and >99 : 1 dr for a menthol-derived carbamate. As an application, the straightforward synthesis of a chiral β-amino acid ester derivative is demonstrated.


Structural equilibrium in new nitroxide-capped cyclodextrins: CW and pulse EPR study.
Design of the new spin-labeled cyclodextrins can significantly extend the functionality of nitroxides. A series of new complexes based on fully methylated cyclodextrin (TRIMEB) covalently bound to the piperidine, pyrroline, pyrrolidine, and pH-sensitive imidazoline type nitroxides has been synthesized and studied using pulse and continuous wave electron paramagnetic resonance (EPR). The influence of the radical and linker properties on the structure of complexes formed has been investigated. Using the electron spin echo envelope modulation technique, we have analyzed quantitatively the accessibility of radicals to solvent molecules in studied complexes depending on the structure and length of the linkers. In all studied systems we observed different types of equilibria between conformations with radical fragment being outside the TRIMEB cavity and radical fragment capping the cavity of TRIMEB. The observed guest-induced shift of equilibrium toward the complex with radical capping TRIMEB cavity was explained by a change of macrocyclic configuration of TRIMEB. Complex with the -NH-CO- linker has been found most perspective for the applications requiring close location of nitroxide to the inclusion complex of TRIMEB. Using continuous wave EPR, we have shown that the pH-sensitive radical covalently bound to TRIMEB maintains its pH-sensitivity, but this complexation does not reduce radical reduction rate in the reaction with ascorbic acid.


Ectopic acanthosis nigricans occurring in a child after syndactyly repair.
Skin grafts from the groin area were used to repair syndactyly of the fourth and fifth fingers in an 8-month-old infant with oculodentodigital dysplasia (ODD). At 12 years of age, he developed hyperpigmented velvety plaques at the repair sites. This patient is the first reported case of acanthosis nigricans (AN) occurring in a graft site after syndactyly repair. We propose the term ectopic acanthosis nigricans to describe the phenomenon of AN occurring in transplanted skin away from the original donor site.


Interleukin-1beta upregulates MMP-9 expression in stromal cells of human giant cell tumor of bone.
Giant cell tumor (GCT) of bone is a progressive, potentially malignant process that destroys skeletal tissue. It consists of multinucleated giant cells, which are hypothesized to be derived from a monocyte/macrophage lineage and mononuclear stromal cells, and the precise relationship of these cells is not fully understood. Recently, we demonstrated that the production of matrix metalloproteinase-9 (MMP-9) in GCT stromal cells is regulated by certain factor(s) secreted by the multinucleated giant cells. In the present study, we evaluated for the presence of interleukin-1beta (IL-1beta) and attempted to establish its possible role for the induction of MMP-9 in GCT stromal cells. Using enzyme-linked immunosorbent assay (ELISA), we have demonstrated that the primary GCT cultures secrete both IL-1beta and MMP-9. The addition of monoclonal antibody (mAb) against IL-1beta partially abrogated, but did not abolish, MMP-9 expression. Our results on gelatin zymography, reverse transcriptase-polymerase chain reaction (RT-PCR), and immunofluorescence showed that GCT stromal cells did not express MMP-9, although treatment with IL-1beta induced MMP-9 expression in a dose-dependent manner, and the secretion peaked 24 h after stimulation and then plateaued. Studies with cycloheximide, a protein synthesis inhibitor, demonstrated that de novo protein synthesis is required for IL-1beta induced MMP-9 expression. Moreover, nuclear run-on analysis has revealed that IL-1beta significantly increased MMP-9 gene transcription in GCT stromal cells. The data suggest that IL-1beta secreted by the multinucleated giant cells in GCT may be one of the factors responsible for the induction of MMP-9 at the transcriptional level in GCT stromal cells in vivo. We conclude that GCT has a self-stimulatory system for the production of MMP-9, and the ability of stromal cells to produce MMP-9 with appropriate stimuli, such as IL-1beta, and possibly in concert with other cytokines may contribute to the aggressive and potentially malignant behavior of GCT.


Xenografting of human fetal testis tissue: a new approach to study fetal testis development and germ cell differentiation.
Abnormal fetal testis development can result in disorders of sex development (DSDs) and predispose to later testicular dysgenesis syndrome (TDS) disorders such as testicular germ cell tumours. Studies of human fetal testis development are hampered by the lack of appropriate model, and intervention systems. We hypothesized that human fetal testis xenografts can recapitulate normal development. Human fetal testes (at 9 weeks, n = 4 and 14-18 weeks gestation, n = 6) were xenografted into male nude mice for 6 weeks, with or without hCG treatment of the host, and evaluated for normal cellular development and function using immunohistochemistry, triple immunofluorescence and testosterone assay. The differentiation and proliferation status of germ cells within xenografts was quantified and compared with age-matched controls. Xenografts showed >75% survival with normal morphology. In the first-trimester xenografts seminiferous cord formation was initiated and in first- and second-trimester grafts normal functional development of Sertoli, Leydig and peritubular myoid cells was demonstrated using cell-specific protein markers. Grafts produced testosterone when hosts were treated with hCG (P = 0.004 versus control). Proliferation of germ cells and differentiation from gonocytes (OCT4(+)) into pre-spermatogonia (VASA(+)) occurred in grafts and quantification showed this progressed comparably with age-matched ungrafted controls. Human fetal testis tissue xenografts demonstrate normal structure, function and development after xenografting, including normal germ cell differentiation. This provides an in vivo system to study normal human fetal testis development and its susceptibility to disruption by exogenous factors (e.g. environmental chemicals). This should provide mechanistic insight into the fetal origins of DSDs and TDS disorders.


[Formation of compressive bandage after sclerotherapy for lower limb varices].
Invention concerns compressive sclerotherapy as a treatment modality for lower limb varices. Technical result of investigation is the development of compressive bandage that creates and maintains adequate level of limb compression both in supine position (during bed rest) and standing or walking. Technical result is achieved by formation of two compressive layers of elastic bandage. Highly expansible elastic bandage is used for the first layer aimed for fixation and compression of latex or foam pads at injection sites to create local compression of variceal nodes. Open toe elastic stocking (I compression class) is placed over the bandage to maintain adequate compression during bed rest. The second external layer consists of elastic bandage with moderate expansion (II compression class). It is placed over the first one from toes to thigh upper third and creates optimal compression in patient's vertical position. The patient is permitted to take it off or loose exclusively in supine position, to wash or refresh foot with wet towel, to change it with a new one.


Electrohydrodynamic patterning of ultra-thin ionic liquid films.
In the electrohydrodynamic (EHD) patterning process, electrostatic destabilization of the air-polymer interface results in micro- and nano-size patterns in the form of raised formations called pillars. The polymer film in this process is typically assumed to behave like a perfect dielectric (PD) or leaky dielectric (LD). In this study, an electrostatic model is developed for the patterning of an ionic liquid (IL) polymer film. The IL model has a finite diffuse electric layer which overcomes the shortcoming of assuming infinitesimally large and small electric diffuse layers inherent in the PD and LD models respectively. The process of pattern formation is then numerically simulated by solving the weakly nonlinear thin film equation using finite difference with pseudo-staggered discretization and an adaptive time step. Initially, the pillar formation process in IL films is observed to be the same as that in PD films. Pillars initially form at random locations and their cross-section increases with time as the contact line expands on the top electrode. After the initial growth, for the same applied voltage and initial film thickness, the number of pillars on IL films is found to be significantly higher than that in PD films. The total number of pillars formed in 1 μm(2) area of the domain in an IL film is almost 5 times more than that in a similar PD film for the conditions simulated. In addition, the pillar structure size in IL films is observed to be more sensitive to initial film thickness compared to PD films.


Rapid tilt-series method for cryo-electron tomography: Characterizing stage behavior during FISE acquisition.
We and others recently developed rapid tilt-series acquisition methods for cryo-electron tomography on a Titan Krios G3i equipped with a single axis holder and a K-series direct electron detector and showed that one of these, the fast-incremental single exposure (FISE) method, significantly accelerates tilt-series acquisition when compared to traditional methods while preserving the quality of the images. Here, we characterize the behavior of our single axis holder in detail during a FISE experiment to optimally balance data quality with speed. We explain our methodology in detail so others can characterize their own stages, and conclude with recommendations for projects with different resolution goals.


Unexpected Acetylation of Endogenous Aliphatic Amines by Arylamine N-Acetyltransferase NAT2.
N-Acetyltransferases play critical roles in the deactivation and clearance of xenobiotics, including clinical drugs. NAT2 has been classified as an arylamine N-acetyltransferase that mainly converts aromatic amines, hydroxylamines, and hydrazines. Herein, we demonstrate that the human arylamine N-acetyltransferase NAT2 also acetylates aliphatic endogenous amines. Metabolomic analysis and chemical synthesis revealed increased intracellular concentrations of mono- and diacetylated spermidine in human cell lines expressing the rapid compared to the slow acetylator NAT2 phenotype. The regioselective N8 -acetylation of monoacetylated spermidine by NAT2 answers the long-standing question of the source of diacetylspermidine. We also identified selective acetylation of structurally diverse alkylamine-containing drugs by NAT2, which may contribute to variations in patient responses. The results demonstrate a previously unknown functionality and potential regulatory role for NAT2, and we suggest that this enzyme should be considered for re-classification.


Actibacterium pelagium sp. nov., a novel alphaproteobacterium, and emended description of the genus Actibacterium.
A Gram-stain-negative, non-motile, rod-shaped bacterium, designated JN33T, was isolated from seawater collected from the western Pacific Ocean. Strain JN33T was positive for hydrolysis of aesculin and gelatin. On the basis of 16S rRNA gene sequence analysis, strain JN33T showed high 16S rRNA gene sequence similarity to Actibacterium atlanticum 22II-S11-z10T (97.3 %), A. mucosum KCTC 23349T (96.6 %) and A. ureilyticum LS-811T (95.7 %) and exhibited less than 97.0 % 16S rRNA gene sequence similarity with respect to the other type strains within the family Rhodobacteraceae. Phylogenetic analysis revealed that strain JN33T fell within the cluster of the genus Actibacterium. The average nucleotide identity and in silico DNA-DNA hybridization values between strain JN33T and the type strains of Actibacterium species were 73.1-73.8 % and 19.8-20.1 %, respectively. The sole respiratory quinone was ubiquinone 10 (Q-10). The principal fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and C16 : 0. The major polar lipids were phosphatidylglycerol, one unidentified phospholipid and two unidentified aminolipids. The DNA G+C content was 57.8 mol%. Distinctly different phylogenetic characteristics, chemotaxonomic differences, as well as phenotypic properties, revealed that strain JN33T could be differentiated from the Actibacterium species with validly published names. Therefore, it is proposed that strain JN33T represents a novel species of the genus Actibacterium, for which the name Actibacterium pelagium sp. nov. (type strain, JN33T=CGMCC 1.16012T=KCTC 52653T) is proposed.


Survival and division fate programs are preserved but retuned during the naïve to memory CD8+ T-cell transition.
Memory T cells are generated from naïve precursors undergoing proliferation during the initial immune response. Both naïve and memory T cells are maintained in a resting, quiescent state and respond to activation with a controlled proliferative burst and differentiation into effector cells. This similarity in the maintenance and response dynamics points to the preservation of key cellular fate programs; however, whether memory T cells have acquired intrinsic changes in these programs that may contribute to the enhanced immune protection in a recall response is not fully understood. Here we used a quantitative model-based analysis of proliferation and survival kinetics of in vitro-stimulated murine naïve and memory CD8+ T cells in response to homeostatic and activating signals to establish intrinsic similarities or differences within these cell types. We show that resting memory T cells display heightened sensitivity to homeostatic cytokines, responding to interleukin (IL)-2 in addition to IL-7 and IL-15. The proliferative response to αCD3 was equal in size and kinetics, demonstrating that memory T cells undergo the same controlled division burst and automated return to quiescence as naïve T cells. However, perhaps surprisingly, we observed reduced expansion of αCD3-stimulated memory T cells in response to activating signals αCD28 and IL-2 compared with naïve T cells. Overall, we demonstrate that although sensitivities to cytokine and costimulatory signals have shifted, fate programs regulating the scale of the division burst are conserved in memory T cells.


The function of multiple extracellular matrix receptors in mediating cell adhesion to extracellular matrix: preparation of monoclonal antibodies to the fibronectin receptor that specifically inhibit cell adhesion to fibronectin and react with platelet glycoproteins Ic-IIa.
We have identified monoclonal antibodies that inhibit human cell adhesion to collagen (P1H5), fibronectin (P1F8 or P1D6), and collagen and fibronectin (P1B5) that react with a family of structurally similar glycoproteins referred to as extracellular matrix receptors (ECMRs) II, VI, and I, respectively. Each member of this family contains a unique alpha subunit, recognized by the antibodies, and a common beta subunit, each of approximately 140 kD. We show here that ECMR VI is identical to the fibronectin receptor (FNR), very late antigen (VLA) 5, and platelet glycoproteins Ic-IIa and shall be referred to as FNR. Monoclonal antibodies to FNR inhibit lymphocyte, fibroblast, and platelet adhesion to fibronectin-coated surfaces. ECMRs I, II, and FNR were differentially expressed in platelets, resting or activated lymphocytes, and myeloid, epithelial, endothelial, and fibroblast cell populations, suggesting a functional role for the receptors in vascular emigration and selective tissue localization. Tissue staining of human fetal skin localized ECMRs I and II to the basal epidermis primarily, while monoclonal antibodies to the FNR stained both the dermis and epidermis. Experiments carried out to investigate the functional roles of these receptors in mediating cell adhesion to complex extracellular matrix (ECM) produced by cells in culture revealed that complete inhibition of cell adhesion to ECM required antibodies to both the FNR and ECMR II, the collagen adhesion receptor. These results show that multiple ECMRs function in combination to mediate cell adhesion to complex EMC templates and predicts that variation in ECM composition and ECMR expression may direct cell localization to specific tissue domains.


Implementing Effective Noise Reduction Techniques in Implantable NIRS Sensors.
Near-Infrared Spectroscopy (NIRS) is a noninvasive optical method widely used for evaluating tissue hemodynamics and various physiological characteristics. Despite its advantages, NIRS faces limitations in light sampling depth and spatial resolution, which has led to the development of implantable NIRS sensors. However, these implantable sensors are prone to Common-Mode Voltage (CMV) interference due to their increased sensor-to-tissue capacitance, which can compromise the signal-to-noise ratio and accuracy of measurements.In this paper, we present a novel active CMV reduction technique that enhances the signal-to-noise ratio of NIRS signals. We propose an electrical model of a patient's body and NIRS sensor to characterize the CMV interference and the active CMV cancellation (ACC) electronic circuit. The ACC circuit measures CMV through a common-mode amplifier, which then inverts and introduces the amplified signal to the patient's body via an additional surface electrode. This technique effectively attenuates the CMV (50 and 60 Hz) by 80 to 90 dB, significantly improving the signal quality without causing system instability.The method has been validated through both analytical simulations and experimental measurements, demonstrating the circuit's ability to suppress CMV within a bandwidth of 0.1 to 100 Hz. Experimental verification of the active noise cancellation method was conducted by recording data from the fingertip and palm, showing effective suppression of the CMV. The proposed method has substantial clinical relevance as it enhances the reliability and accuracy of implantable NIRS sensors, enabling more precise monitoring of internal organs and improved patient care.


Spanish and Swedish eldercare managers' influence on employees.
Hierarchical and flat organizational types are predominant in Spain and Sweden, respectively. To study how managers' commitment and work overcommitment (WOC) affect employee well-being, and job perception in these different countries can shed insight on how to improve eldercare organization. The purpose of this paper was to study the association between eldercare employee exposure to managers' commitment and WOC, and employee mental well-being and job perception and how these associations differed between Spain and Sweden. A questionnaire with validated questions on commitment, WOC, mental well-being and job perception, operationalized as the perception of quality of care and turnover intent, was sent out to eldercare managers and employees in Spain and Sweden. t-Tests, χ 2 and linear regression were applied to study the associations and differences between the countries. Interaction analyses revealed that Spanish employees' mental well-being and job perception were influenced by their managers' commitment and WOC in that manager commitment improved and WOC impaired well-being and job perception. However, the Swedish eldercare employees were not influenced by their managers on these parameters. The impact of managerial commitment and WOC differed between employees in Spain and Sweden, possibly because the preconditions for leadership varied due to differences in organizational type. This study compares the managers' impact on employee health and job perception in two countries with different organizational prerequisites. Moreover, managers' commitment and WOC were estimated by the managers themselves and did not rely on the employees' perception, which improved ecological validity.


Comparison of detergent-based sample preparation workflows for LTQ-Orbitrap analysis of the Escherichia coli proteome.
This work presents a comparative evaluation of several detergent-based sample preparation workflows for the MS-based analysis of bacterial proteomes, performed using the model organism Escherichia coli. Initially, RapiGest- and SDS-based buffers were compared for their protein extraction efficiency and quality of the MS data generated. As a result, SDS performed best in terms of total protein yields and overall number of MS identifications, mainly due to a higher efficiency in extracting high molecular weight (MW) and membrane proteins, while RapiGest led to an enrichment in periplasmic and fimbrial proteins. Then, SDS extracts underwent five different MS sample preparation workflows, including: detergent removal by spin columns followed by in-solution digestion (SC), protein precipitation followed by in-solution digestion in ammonium bicarbonate or urea buffer, filter-aided sample preparation (FASP), and 1DE separation followed by in-gel digestion. On the whole, about 1000 proteins were identified upon LC-MS/MS analysis of all preparations (>1100 with the SC workflow), with FASP producing more identified peptides and a higher mean sequence coverage. Each protocol exhibited specific behaviors in terms of MW, hydrophobicity, and subcellular localization distribution of the identified proteins; a comparative assessment of the different outputs is presented.


Effect of a hospital-based oral health-education program on Iranian staff: evaluating a theory-driven intervention.
Tooth decay and periodontitis are among the most prevalent dental diseases globally with adverse effects on an individual's general health. Recently the prevalence of dental caries has decreased significantly, but caries epidemiology remains a major problem in dental public health. This study investigated the impact of an oral health education intervention on Theory of Planned Behavior (TPB) variables, and whether changes in these variables persisted and were associated with changes in identified oral health behaviors at 2-month follow-up. This descriptive pre/post test study was conducted with 160 staff in the Baqiyatallah Hospital in Tehran. Six hospital wards were selected using a randomized multi-stratified sampling frame. The size for each cluster was calculated as 22 with each ward being allocated to either the intervention or the control arm of the study. Self-report questionnaires were used to evaluate socio-demographic factors, dental attendance as well as the constructs of the augmented TPB model (attitudes, subjective norms, perceived behavioral control intentions). The intervention was an educational program based on TPB constructs delivered via direct training to half the participants. The control group was provided with usual training only. The independent-samples T-test, Repeated-Measures one-way ANOVA, and matched T-test with the significance level set at p < 0.05 were applied. Findings revealed significant variations between the two groups immediately after the educational intervention concerning the attitudes, subjective norms, perceived behavioral control, intentions to seek treatment, oral health behavior as well as decayed, missing, and filled teeth and bleeding on probing (p<0.001). Two months after the intervention, except for the brushing construct (p = 0.18), the differences between the two groups were all statistically significant (p<0.001). Our findings affirm the positive effect an oral health education program has on enhancing the attitudes, subjective norms, perceived behavioral control, intentions and behavior of staff in this hospital. The results of our study confirm that developing and applying an educational intervention in accordance with the theory of planned behavior can lead to significant changes in the knowledge, attitudes, and behavior of hospital staff regarding preventing tooth decay.


Dexamethasone therapy for bacterial meningitis. Results of two double-blind, placebo-controlled trials.
We enrolled 200 infants and older children with bacterial meningitis in two prospective double-blind, placebo-controlled trials to evaluate the efficacy of dexamethasone therapy in addition to either cefuroxime (Study 1) or ceftriaxone (Study 2). Altogether, 98 patients received placebo and 102 received dexamethasone (0.15 mg per kilogram of body weight every six hours for four days). At the beginning of therapy, the clinical and demographic characteristics of the patients in the treatment groups were comparable. The mean increase in the cerebrospinal fluid concentration of glucose and the decreases in lactate and protein levels after 24 hours of therapy were significantly greater in those who received dexamethasone than in those who received placebo (glucose, 2.0 vs. 0.4 mmol per liter [36.0 vs. 6.9 mg per deciliter], P less than 0.001; lactate, 4.0 vs. 2.1 mmol per liter [38.3 vs. 19.8 mg per deciliter], P less than 0.001; and protein, 0.64 vs. 0.25 g per liter [64.0 vs. 25.3 mg per deciliter], P less than 0.05). One patient in the placebo group in Study 1 died. As compared with those who received placebo, the patients who received dexamethasone became afebrile earlier (1.6 vs. 5.0 days; P less than 0.001) and were less likely to acquire moderate or more severe bilateral sensorineural hearing loss (15.5 vs. 3.3 percent; P less than 0.01). Twelve patients in the two placebo groups (14 percent) had severe or profound bilateral hearing loss requiring the use of a hearing aid, as compared with 1 (1 percent) in the two dexamethasone groups (P less than 0.001). We conclude that dexamethasone is beneficial in the treatment of infants and children with bacterial meningitis, particularly in preventing deafness.


Evidence for two distinct modalities of CA2+ influx into pancreatic B cell.
The pathways through which glucose stimulates Ca2+ inflow into islet cells were investigated by comparing the inhibitory effect of verapamil, a selective blocker of voltage-sensitive Ca2+ channels, on glucose- and K+-stimulated insulin release and 45Ca efflux from perifused rat pancreatic islets. The islets stimulated by K+ (20 mM) were more sensitive to verapamil than those exposed to glucose (27.8 mM). The stimulation of 45Ca efflux by a low concentration of glucose (8.3 mM) was extremely resistant to verapamil, whereas that induced by a rise in the glucose concentration from 8.3 to 27.8 mM displayed the same sensitivity towards verapamil as that characterizing the response to K+. Because the increase in 45Ca efflux evoked by glucose or K+. Because the increase in 45Ca efflux evoked by glucose or K+ reflects a stimulation of Ca2+ entry into islet cells, it is proposed that the B cell may be equipped with two populations of Ca2+ channels that differ in their sensitivity towards verapamil and possibly their voltage-dependency. Glucose apparently stimulates Ca2+ inflow through both types of channels. At low concentrations, glucose may stimulate Ca2+ inflow, in part, by gating the voltage-insensitive pathway.


Studies on isolated cell components.
1. The addition of heparin to rat liver, kidney, or brain nuclei has been found to bring about the release of a gel. Chemical analysis and histochemical studies on whole homogenates and isolated nuclei demonstrated that the material released by heparin contained desoxyribonucleic acid (DNA) and protein. The action of heparin on nuclei is interpreted as the result of a combination with the basic proteins of the nucleus with a consequent displacement of DNA. 2. The addition of heparin to a finely divided dilute liver homogenate prepared in a phosphate-sucrose solution at pH 7.1 brings about a marked increase in viscosity which reaches a maximum in 6 to 8 minutes at 23 degrees and then declines. 3. The concentration threshold for the viscosity effect was 0.1 mg. per 100 mg. fresh rat liver, with further increases in viscosity at higher heparin concentrations. Over a period of several hours a marked decrease in response to heparin was observed in homogenates stored at 0 degrees . 4. Fractionation of the homogenate demonstrated that the viscosity increase was due to the presence of the nuclei alone, other components showing no effect. Microscopic observation showed that the increase in viscosity was associated with the appearance of a clear gel around nuclei treated with heparin. 5. Heparin brought about the release of DNA from the nuclei of incubated rat liver, kidney, and brain homogenates. In some instances over half the DNA is found in the supernatant after high speed centrifugation (20 minutes, 21,000 x g). 6. No correlation was found between anticoagulant activity of heparin preparations and their effectiveness in causing an increase in the viscosity of liver homogenates. Desulfated heparin produced none of the results described here for heparin.


[DNA methylation characteristics of different fetal and maternal body tissues in rats administered 1,2-dimethylhydrazine].
A high rate of DNA methylation in the liver and large colon mucosa was registered 6 hrs after dimethylhydrazine (SDMH) treatment of rats with 19-day gestations. However, in fetal liver, a low concentration of methylated guanine (7-methyl guanine) derivatives was found, while in large colon and brain, these substances were not detected at all. It is suggested that the resistance of embryonal tissues to the transplacental effect of SDMH is due to the underdeveloped enzymatic system of the fetus.


Advances in comparative genetics: influence of genetics on obesity.
Obesity has reached epidemic proportions and is recognised as a significant global health problem. Increased food intake and decreased physical activity are traditionally to blame for the development of obesity; however, many variables such as behaviour, diet, environment, social structures and genetics also contribute to this multifactorial disease. Complex interactions among these variables (for example, gene-environment, gene-diet and gene-gene) contribute not only to individual differences in the development of obesity, but also in treatment response. Mouse models have historically played valuable roles in understanding the genetics of traits related to energy balance and obesity. In the present review, we survey past use and examine new advances in mouse models designed to uncover the genetic architecture of obesity and its component traits. We discuss traditional models such as inbred strains and selectively bred lines and their contributions and shortcomings. We consider the evolution of mouse models into more informative resources such as outbred crosses and the Hybrid Mouse Diversity Panel, as well as novel next-generation approaches such as the Collaborative Cross. Moreover, the genetic architecture of voluntary exercise and the interactive relationship between host genetics and the gut microbiome are presented as novel phenotypes that augment studies using body weight and body fat percentage as endpoints. Understanding the intricate network of phenotypic, genotypic and environmental variables that predispose individuals to obesity will elucidate biological networks involved in the development of obesity. Knowledge obtained from advances in mouse models will inform human health and provide insight into inter-individual variability in the aetiology of obesity-related diseases.


Oncogenic microRNAs: miR-155, miR-19a, miR-181b, and miR-24 enable monitoring of early breast cancer in serum.
MicroRNAs (miRs) represent a distinct class of posttranscriptional modulators of gene expression with remarkable stability in sera. Several miRs are oncogenic (oncomiRs) and are deregulated in the pathogenesis of breast cancer and function to inhibit tumor suppressors. Routine blood monitoring of these circulating tumor-derived products could be of significant benefit to the diagnosis and relapse detection of early-stage breast cancer (EBC) patients. Aim of this project was to determine expression of miR-155, miR-19a, miR-181b, miR-24, relative to let-7a in sera of 63 patients with EBC and 21 healthy controls. Longitudinal multivariate data analysis was performed to stochastically model the serum levels of each of the oncomiRs during disease phases: from diagnosis, after surgery, and following chemo/radiotherapy. Moreover, this analysis was utilized to evaluate oncomiR levels in EBC patients subgrouped using current clinical prognostic factors including HER2, Ki-67, and grade III. EBC patients significantly over-express the oncomiRs at the time of diagnosis. Following surgical resection the serum levels of miR-155, miR-181b, and miR-24 significantly decreased (p = 1.89e-05, 5.41e-06, and 0.00638, respectively) whereas the miR-19a decreased significantly after the therapy (p = 0.00869). Furthermore, in case of high-risk patients serum levels of miR-155, miR-19a, miR-181b, and miR-24 are significantly more abundant in comparison to low-risk group (p = 0.026, 0.02567, 0.0250, and 0.00990) and show a decreasing trend upon therapy. OncomiRs are significantly more abundant in the sera of EBC patients compared to controls at diagnosis. Differences in oncomiR levels reflecting EBC risk were also observed. Testing the oncomiRs may be useful for diagnostic purpose and possibly also for relapse detection in follow-up studies of EBC.


Short lingual frenulum: From diagnosis to laser and speech-language therapy.
To evaluate the effects of tongue frenulectomy performed with two therapeutic approaches: Laser frenulectomy and combined laser and speech-language therapy. The study involved 180 patients (90 males and 90 females) aged between 6 and 12 years. After examination and data collection, the patients were stratified according to three degrees of severity: mild, moderate and severe. After treatment, the test group (laser frenulectomy and combined laser and speech-language therapy) was compared with the control group (laser frenulectomy) in the pre-surgical phase, at one week, 1 month, 3 months, 6 months and 12 months after surgery. Statistical analysis showed statistically significant differences between the pre-surgical and post-surgical values at 3 months, 6 months and 12 months after surgery (p<0,05). It is essential to establish diagnosis criteria to which the clinician should refer in order to decide the treatment plan. This study shows that combined laser and speech-language therapy leads to better results than the resection treatment of the frenulum with laser technique alone.


Evaluation of the agency for healthcare research and quality pediatric quality indicators.
Pediatric quality indicators were developed in 2006 by the Agency for Healthcare Research and Quality to identify potentially preventable complications in hospitalized children. Our objectives for this study were to (1) apply these algorithms to an aggregate children's hospital's discharge abstract database, (2) establish rates for each of the pediatric quality indicator events in the children's hospitals, (3) use direct chart review to investigate the accuracy of the pediatric quality indicators, (4) calculate the number of complications that were already present on admission and, therefore, not attributable to the specific hospitalization, and (5) evaluate preventability and calculate positive predictive value for each of the indicators. In addition, we wanted to use the data to set priorities for ongoing clinical investigation. The Agency for Healthcare Research and Quality pediatric quality indicator algorithms were applied to 76 children's hospital's discharge abstract data (1794675 discharges) from 2003 to 2005. Rates were calculated for 11 of the pediatric quality indicators from all 3 years of discharge data: accidental puncture or laceration, decubitus ulcer, foreign body left in during a procedure, iatrogenic pneumothorax in neonates at risk, iatrogenic pneumothorax in nonneonates, postoperative hemorrhage or hematoma, postoperative respiratory failure, postoperative sepsis, postoperative wound dehiscence, selected infections caused by medical care, and transfusion reaction. Subsequently, clinicians from 28 children's hospitals reviewed 1703 charts in which complications had been identified. They answered questions as to correctness of secondary diagnoses that were associated with the indicator, whether a complication was already present on admission, and whether that complication was preventable, nonpreventable, or uncertain. Across 3 years of data the rates of pediatric quality indicators ranged from a low of 0.01/1000 discharges for transfusion reaction to a high of 35/1000 for postoperative respiratory failure, with a median value of 1.85/1000 for the 11 pediatric quality indicators. Indicators were often already present on admission and ranged from 43% for infection caused by medical care to 0% for iatrogenic pneumothorax in neonates, with a median value of 16.9%. Positive predictive value for the subset of pediatric quality indicators occurring after admission was highest for decubitus ulcer (51%) and infection caused by medical care (40%). Because of the very large numbers of cases identified and its low preventability, the indicator postoperative respiratory failure is particularly problematic. The initial definition includes all children on ventilators postoperatively for >4 days with few exclusions. Being on a ventilator for 4 days would be a normal occurrence for many children with extensive surgery; therefore, the majority of the time does not indicate a complication and makes the indicator inappropriate. A subset of pediatric quality indicators derived from administrative data are reasonable screening tools to help hospitals prioritize chart review and subsequent improvement projects. However, in their present form, true preventability of these complications is relatively low; therefore, the indicators are not useful for public hospital comparison. Identifying which complications are present on admission versus those that occur within the hospitalization will be essential, along with adequate risk adjustment, for any valid comparison between institutions. Infection caused by medical care and decubitus ulcers are clinically important indicators once the present-on-admission status is determined. These complications cause significant morbidity in hospitalized children, and research has shown a high level of preventability. The pediatric quality indicator software can help children's hospitals objectively review their cases and target improvement activities appropriately. The postoperative-respiratory-failure indicator does not represent a complication in the majority of cases and, therefore, should not be included for hospital screening or public comparison. Chart review should become part of the development process for quality indicators to avoid inappropriate conclusions that misdirect quality-improvement resources.


Rhomboid flap principles and common variations.
The rhomboid flap was initially popularized by a Russian, Alexander Limberg, and modified by Claude Dufourmentel of Paris. Numerous variations on the flap have been proposed more recently by Webster and Gunter. This flap has found its way into the practice of most surgeons doing head and neck surgery, with its primary advantage being that it is an extremely simple flap operation to learn, with great versatility. The general principles and their clinical application are presented in order to increase proper utilization of the flap.


Quantitative and sensitive detection of rare mutations using droplet-based microfluidics.
Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan(®) assays and pyrosequencing, while quantitative, cannot detect less than ∼1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan(®) probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan(®) probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment.


Predicting the risk of diabetic retinopathy using explainable machine learning algorithms.
Diabetic retinopathy (DR) is a global health concern among diabetic patients. The objective of this study was to propose an explainable machine learning (ML)-based system for predicting the risk of DR. This study utilized publicly available cross-sectional data in a Chinese cohort of 6374 respondents. We employed boruta and least absolute shrinkage and selection operator (LASSO) based feature selection methods to identify the common predictors of DR. Using the identified predictors, we trained and optimized four widly applicable models (artificial neural network, support vector machine, random forest, and extreme gradient boosting (XGBoost) to predict patients with DR. Moreover, shapely additive explanation (SHAP) was adopted to show the contribution of each predictor of DR in the prediction. Combining Boruta and LASSO method revealed that community, TCTG, HDLC, BUN, FPG, HbAlc, weight, and duration were the most important predictors of DR. The XGBoost-based model outperformed the other models, with an accuracy of 90.01%, precision of 91.80%, recall of 97.91%, F1 score of 94.86%, and AUC of 0.850. Moreover, SHAP method showed that HbA1c, community, FPG, TCTG, duration, and UA1b were the influencing predictors of DR. The proposed integrating system will be helpful as a tool for selecting significant predictors, which can predict patients who are at high risk of DR at an early stage in China.


Alterations of domains in the plasmatic membrane due to damages of the perinuclear theca of pig preserved spermatozoa.
Samples of semen from 12 pigs, three from Yorkshire, Landrace, Duroc and Mexican Hairless each where obtained to study cryopreservation methods. Three stages of boar semen cryopreservation were evaluated: none (fresh stage), cooling at 5 degrees C and freezing at -196 degrees C then thawing to 56 degrees C for 12 sec. Perinuclear theca damage and domain alterations were selected as indices of seminal quality, as measured by electronic and fluorescence microcopy, respectively according to two lineal models considering by separately the effect of semen preservation and breed. Integrity and absence of perinuclear theca significantly (p < 0.001) decreased and increased, respectively according to a decrease in temperature of cryopreservation, from 87.4 to 58.8% and from 0.8 to 26.2%, respectively. This same significant (p < 0.001) effect was found for acrosomal and post-acrosomal membrane distribution of domains, from 92.1 to 76.8% and from 3.1 to 13.1% in this same order. Slight but highly significant (p < 0.001) differences were observed when theca integrity was evaluated as affected by breed, with highest and lowest values for Yorkshire and Pel6n Mexicano pigs, respectively. No breed effect was encountered for presence of acrosomal domains. A strong interdependence was found between perinuclear theca damage and domain distribution. In this connection, a highly significant (p < 0.001) positive, interdependence was observed between the theca damage and acrosomal domain (r = 0.87), while this same relationship was although highly significant (p < 0.001), negative in nature for equatorial and post-acrosonal domains (r = -0.77 and -0.85, respectively). This experiment confirmed that cryopreservation methods may severely affect semen quality of pigs and that genotype may further influence these same indices. More research is needed for improving methods of preservation of pig semen quality, from the point of view of perinuclear theca and domain characteristics of spermatozoa.


Evidence against observational spatial memory for cache locations of conspecifics in marsh tits Poecile palustris.
Many species in the family Paridae, such as marsh tits Poecile palustris, are large-scale scatter hoarders of food that make cryptic caches and disperse these in large year-round territories. The perhaps most well-known species in the family, the great tit Parus major, does not store food itself but is skilled in stealing caches from the other species. We have previously demonstrated that great tits are able to memorise positions of caches they have observed marsh tits make and later return and steal the food. As great tits are explorative in nature and unusually good learners, it is possible that such "memorisation of caches from a distance" is a unique ability of theirs. The other possibility is that this ability is general in the parid family. Here, we tested marsh tits in the same experimental set-up as where we previously have tested great tits. We allowed caged marsh tits to observe a caching conspecific in a specially designed indoor arena. After a retention interval of 1 or 24 h, we allowed the observer to enter the arena and search for the caches. The marsh tits showed no evidence of such observational memorization ability, and we believe that such ability is more useful for a non-hoarding species. Why should a marsh tit that memorises hundreds of their own caches in the field bother with the difficult task of memorising other individuals' caches? We argue that the close-up memorisation procedure that marsh tits use at their own caches may be a different type of observational learning than memorisation of caches made by others. For example, the latter must be done from a distance and hence may require the ability to adopt an allocentric perspective, i.e. the ability to visualise the cache from the hoarder's perspective. Members of the Paridae family are known to possess foraging techniques that are cognitively advanced. Previously, we have demonstrated that a non-hoarding parid species, the great tit P. major, is able to memorise positions of caches that they have observed marsh tits P. palustris make. However, it is unknown whether this cognitively advanced foraging strategy is unique to great tits or if it occurs also in other parids. Here, we demonstrated that "pilfering by observational memorization strategy" is not a general strategy in parids. We believe that such ability is important for a non-hoarding species such as the great tit and, most likely, birds owning many caches do not need this foraging strategy.


The spectral domain of multivariate harmonizable processes.
The spectral or frequency domain of a multivariate weakly harmonizable process is a vector space of vector functions that are integrable relative to a positive definite matrix bimeasure. It has a norm derived from a Gramian inner product. The completeness property of this space, which is needed for many applications, has been open for some years. An affirmative solution and an application to optimal least-squares estimation of a weakly harmonizable signal are presented here.


How can systems engineering inform the methods of programme evaluation in health professions education?
We evaluate programmes in health professions education (HPE) to determine their effectiveness and value. Programme evaluation has evolved from use of reductionist frameworks to those addressing the complex interactions between programme factors. Researchers in HPE have recently suggested a 'holistic programme evaluation' aiming to better describe and understand the implications of 'emergent processes and outcomes'. We propose a programme evaluation framework informed by principles and tools from systems engineering. Systems engineers conceptualise complexity and emergent elements in unique ways that may complement and extend contemporary programme evaluations in HPE. We demonstrate how the abstract decomposition space (ADS), an engineering knowledge elicitation tool, provides the foundation for a systems engineering informed programme evaluation designed to capture both planned and emergent programme elements. We translate the ADS tool to use education-oriented language, and describe how evaluators can use it to create a programme-specific ADS through iterative refinement. We provide a conceptualisation of emergent elements and an equation that evaluators can use to identify the emergent elements in their programme. Using our framework, evaluators can analyse programmes not as isolated units with planned processes and planned outcomes, but as unfolding, complex interactive systems that will exhibit emergent processes and emergent outcomes. Subsequent analysis of these emergent elements will inform the evaluator as they seek to optimise and improve the programme. Our proposed systems engineering informed programme evaluation framework provides principles and tools for analysing the implications of planned and emergent elements, as well as their potential interactions. We acknowledge that our framework is preliminary and will require application and constant refinement. We suggest that our framework will also advance our understanding of the construct of 'emergence' in HPE research.


Anti-inflammation activities of essential oil and its constituents from indigenous cinnamon (Cinnamomum osmophloeum) twigs.
In this study, chemical compositions of hydrodistilled essential oil and anti-inflammatory activities from the twigs of Cinnamomum osmophloeum Kaneh. were investigated for the first time. The chemical constituents of the twig essential oil were further analyzed by GC-MS and they were found to be L-bornyl acetate (15.89%), caryophyllene oxide (12.98%), gamma-eudesmol (8.03%), beta-caryophyllene (6.60%), T-cadinol (5.49%), delta-cadinene (4.79%), trans-beta-elemenone (4.25%), cadalene (4.19%), and trans-cinnamaldehyde (4.07%). The effects of essential oil on nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages were also examined. Results of nitric oxide tests indicated that twig essential oil and its major constituents such as trans-cinnamaldehyde, caryophyllene oxide, L-borneol, L-bornyl acetate, eugenol, beta-caryophyllene, E-nerolidol, and cinnamyl acetate have excellent activities. These findings demonstrated that essential oil of C. osmophloeum twigs have excellent anti-inflammatory activities and thus have great potential to be used as a source for natural health products.


An argument for a qualitative research approach to hypnotic experiencing and perceptually oriented hypnosis.
An argument for the significance of a qualitative research approach to hypnotic experiencing and a perceptually oriented view of hypnosis is presented with hypnosis framed in phenomenological, humanistic, and perceptual terms. An outline of threads of thought in Popper's writings are consistent with such a perspective. Qualitative approaches are noted and support for theoretical discussions leading to deeper understanding of issues of hypnotic experiencing, such as unconscious processes, nonlinear experiences, and researchers' countertransference are examined. Some limitations of current quantitative approaches to examining hypnotic experiencing and myths about qualitative research are discussed.


Methotrexate preconditioning allows sufficient engraftment to confer drug resistance in mice transplanted with marrow expressing drug-resistant dihydrofolate reductase activity.
Methotrexate (MTX) is an effective antitumor agent that has been demonstrated to be particularly useful in the treatment of hematopoietic neoplasms but causes substantial hematologic and gastrointestinal toxicity. We previously demonstrated that transplantation with transgenic marrow expressing drug-resistant dihydrofolate reductase (DHFR) into animals preconditioned by irradiation substantially protected recipient mice from the toxic side effects of methotrexate administration. Here we test the use of methotrexate itself as a preconditioning agent for engraftment of drug-resistant transgenic marrow, subsequently conferring drug resistance upon recipient animals. Administration of methotrexate beginning 1 or 2 weeks prior to or on the same day as transplantation with drug-resistant DHFR transgenic marrow did not allow sufficient engraftment to confer drug resistance to most unirradiated recipients. A small number of animals were curiously protected from lethal MTX toxicity but exhibited extremely low hematocrits and were not engrafted with stem cells, as indicated by low engraftment levels assessed in secondary transplant recipients. However, we subsequently found that MTX preconditioning allowed sufficient engraftment of DHFR transgenic marrow to confer drug resistance if MTX administration was withdrawn at the time of bone marrow transplantation (BMT) and withheld until 2 weeks post-transplant. Quantitative molecular analysis of primary and secondary recipients indicated a stem cell engraftment level of approximately 1%, consistent with previous studies demonstrating that a low level of DHFR transgenic cell engraftment was sufficient to confer drug resistance in recipient animals. We conclude that MTX can be used as a preconditioning agent for subsequent engraftment of hematopoietic stem cells, in this case conferring resistance to MTX.


Risk communication for nanobiotechnology: to whom, about what, and why?
Regulatory oversight and public communication are intimately intertwined. Oversight failures quickly galvanize media and public attention. In addition, regulations sometimes require that risks and uncertainties be included in communication efforts aimed at non-experts outside of the regulatory and policy communities - whether in obtaining informed consent for novel medical treatments; by including risk information on drug labels, in drug advertisements, or on chemicals used in the workplace; in providing nutritional information on food packages; or by opening environmental impact assessments to public comment. In recent decades, broad public input with respect to new technologies has also been sought "upstream" of hard policy decisions in the hope of ultimately gaining legitimacy for those decisions - and perhaps increasing their quality. When communication fails, oversight may also be seen as failing - rightly or wrongly. As part of a larger project organized by the University of Minnesota, this paper presents six models of public risk communication and uses those models to analyze the communication challenges facing nanotechnology and nanobiotechnology. Reviewing the communication dynamics associated with the historical cases of technology regulation with which this symposium issue is concerned (genetically engineered organisms [GEOs] in the food supply, pharmaceuticals and medical devices, chemicals in the workplace, and gene transfer research or "gene therapy") helps shed light on the communications challenges facing nanobiotechnology.


Evolution and virulence contributions of the autotransporter proteins YapJ and YapK of Yersinia pestis CO92 and their homologs in Y. pseudotuberculosis IP32953.
Yersinia pestis, the causative agent of plague, evolved from the gastrointestinal pathogen Yersinia pseudotuberculosis. Both species have numerous type Va autotransporters, most of which appear to be highly conserved. In Y. pestis CO92, the autotransporter genes yapK and yapJ share a high level of sequence identity. By comparing yapK and yapJ to three homologous genes in Y. pseudotuberculosis IP32953 (YPTB0365, YPTB3285, and YPTB3286), we show that yapK is conserved in Y. pseudotuberculosis, while yapJ is unique to Y. pestis. All of these autotransporters exhibit >96% identity in the C terminus of the protein and identities ranging from 58 to 72% in their N termini. By extending this analysis to include homologous sequences from numerous Y. pestis and Y. pseudotuberculosis strains, we determined that these autotransporters cluster into a YapK (YPTB3285) class and a YapJ (YPTB3286) class. The YPTB3286-like gene of most Y. pestis strains appears to be inactivated, perhaps in favor of maintaining yapJ. Since autotransporters are important for virulence in many bacterial pathogens, including Y. pestis, any change in autotransporter content should be considered for its impact on virulence. Using established mouse models of Y. pestis infection, we demonstrated that despite the high level of sequence identity, yapK is distinct from yapJ in its contribution to disseminated Y. pestis infection. In addition, a mutant lacking both of these genes exhibits an additive attenuation, suggesting nonredundant roles for yapJ and yapK in systemic Y. pestis infection. However, the deletion of the homologous genes in Y. pseudotuberculosis does not seem to impact the virulence of this organism in orogastric or systemic infection models.


Human Serum Albumin-Platinum(II) Agent Nanoparticles Inhibit Tumor Growth Through Multimodal Action Against the Tumor Microenvironment.
To overcome the limitations of traditional platinum (Pt)-based drugs and further improve the targeting ability and therapeutic efficacy in vivo, we proposed to design a human serum albumin (HSA)-Pt agent complex nanoparticle (NP) for cancer treatment by multimodal action against the tumor microenvironment. We not only synthesized a series of Pt(II) di-2-pyridone thiosemicarbazone compounds and obtained a Pt(II) agent [Pt(Dp44mT)Cl] with significant anticancer activity but also successfully constructed a novel HSA-Pt(Dp44mT) complex nanoparticle delivery system. The structure of the HSA-Pt(Dp44mT) complex revealed that Pt(Dp44mT)Cl binds to the IIA subdomain of HSA and coordinates with His-242. The HSA-His242-Pt-Dp44mT NPs had an obvious effect on the inhibition of tumor growth, which was superior to that of Dp44mT and Pt(Dp44mT)Cl, and they had almost no toxicity. In addition, the HSA-His242-Pt-Dp44mT NPs were found to kill cancer cells by inducing apoptosis, autophagy, and inhibiting angiogenesis.


Breast reconstruction after mastectomy. Some thoughts and experiences.
When faced with a patient with a lump or lumps in the breast, it is clear to the general surgeon how the situation should be handled. However, if the patient has already had multiple breast biopsies, then the surgeon is less certain. If the patient also has nodular or granular breasts, the choice of correct treatment becomes almost speculative. It is because of these uncertainties that this article has been written. A series of suggestions on the sequential steps to be taken in the treatment of women with precancerous or cancerous lesions, with emphasis on reconstruction of the breast, is listed.


Effect of commercial starter cultures on volatile compound profile and sensory characteristics of dry-cured foal sausage.
The present work deals with the evaluation of the effect of three different commercial starter cultures (Chr. Hansen, Hørsholm, Denmark) on the volatile compound profile and sensory properties, as well as some important physicochemical parameters, of dry-fermented foal sausages at the end of ripening in order to select the most suitable starter culture for this elaboration. The sausage batches were named as follows: CO (non-inoculated control), FS (Lactobacillus sakei + Staphylococcus carnosus), SM (L. sakei + S. carnosus + Staphylococcus xylosus + Pediococcus pentosaceus + Debaryomyces hansenii) and TR (L. sakei + S. carnosus +S. xylosus). The pH values differed significantly among batches, with the highest values corresponding to CO followed by TR, SM and FS. The highest amounts of volatile compounds were found in FS batch. Hexanal was the most abundant compound, especially in FS and SM batches. These batches also showed higher levels of compounds derived from carbohydrate fermentation and amino acid catabolism. Sensory results showed that acid taste was significantly lower in CO batch than in inoculated batches. According to most parameters, batches inoculated with FS and SM starters showed marked acidity compared with TR and CO batches, as expected from the manufacturer's indications. Therefore the most suitable starter culture for use in the manufacture of foal sausages in Mediterranean countries such as Spain with a preference for low-acidity products was found to be TR culture.


Improvements in the degree of understanding the treatment guidelines for schizophrenia and major depressive disorder in a nationwide dissemination and implementation study.
To implement clinical practice guidelines (CPGs), it is necessary for psychiatrists to deepen their understanding of the CPGs. The Effectiveness of Guidelines for Dissemination and Education in Psychiatric Treatment (EGUIDE) project is a nationwide dissemination and implementation study of two sets of CPGs for schizophrenia and major depressive disorder (MDD). A total of 413 psychiatrists (n = 212 in 2016; n = 201 in 2017) learned the two CPGs in the education program of the EGUIDE project, and clinical knowledge of these CPGs was evaluated at baseline and after the programs. To improve the correct answer rate for clinical knowledge after the programs, we revised the lecture materials associated with items that had a low correct answer rate in 2016 and used the revised lecture materials with the CPGs in 2017. The rates of correct answers after the programs between the 2016 and 2017 groups were compared. The correct answer rate of one item on the schizophrenia CPG and one item on the MDD CPG tended to be improved (S-D5 and D-C6) and that of one on the MDD CPG was significantly improved (D-D3, P = 0.0008) in the 2017 group compared to those in the 2016 group. We reported improvements in clinical knowledge of CPGs after the EGUIDE program in the 2017 group following revision of the lecture materials based on results from the 2016 group. These attempts to improve the degree of understanding of CPGs may facilitate the successful dissemination and implementation of psychiatric guidelines in everyday practice.


Associations between Oxytocin Receptor Gene Polymorphisms, Empathy towards Animals and Implicit Associations towards Animals.
Oxytocin has been well researched in association with psychological variables and is widely accepted as a key modulator of human social behaviour. Previous work indicates involvement of oxytocin receptor gene (OXTR) single nucleotide polymorphisms (SNPs) in human-human empathy, however little is known about associations of OXTR SNPs with empathy and affective reactions of humans towards animals. Five OXTR SNPs previously found to associate with human social behaviour were genotyped in 161 students. Empathy towards animals and implicit associations were evaluated. A General Linear Model was used to investigate the OXTR alleles and allelic combinations along with socio-demographic variables and their influence on empathy towards animals. Empathy towards animals showed a significant association with OXTR SNP rs2254298; homozygous G individuals reported higher levels of empathy towards animals than heterozygous (GA). Our preliminary findings show, for the first time, that between allelic variation in OXTR and animal directed empathy in humans maybe associated, suggesting that OXTRs social behaviour role crosses species boundaries, warranting independent replication.


An online interprofessional learning resource for physicians, pharmacists, nurse practitioners, and nurses in long-term care: benefits, barriers, and lessons learned.
The importance of collaborative practice in health care has been emphasized.1,21, 2 There is a critical need for convenient and flexible education opportunities that support the development of collaborative practice skills among the health care workforce. Consequently, the purpose of this project was to create and evaluate an online learning resource for physicians, nurses, nurse practitioners, and pharmacists working in long-term care that provided practitioners with the skills, knowledge, and motivation necessary to enhance their ability to act as an interprofessional team while providing clinical care. The Demand-Driven Learning Model 3 was used to guide the project. Findings revealed learners enjoyed the programme and acquired new skills and knowledge relating to collaborative practice that they transferred to their workplace resulting in higher levels of collaborative practice. The data did not reveal significant changes in the learners' attitudes towards collaborative practice; perhaps because the participants were early adopters and already had positive attitudes. Requests to change organizational structure to enhance collaborative practice were minimal, as was the impact of the resource on resident care. Given the short time frame from completion of the learning resource to the evaluation, this is perhaps not surprising as it is reasonable to expect that these types of changes will take time to take effect within the organization. Follow-ups at a later date are suggested.


Introduction of a temperature-sensitive phenotype into influenza A/WSN/33 virus by altering the basic amino acid domain of influenza virus matrix protein.
Our previous studies with influenza A viruses indicated that the association of M1 with viral RNA and nucleoprotein (NP) is required for the efficient formation of helical ribonucleoprotein (RNP) and for the nuclear export of RNPs. RNA-binding domains of M1 map to the following two independent regions: a zinc finger motif at amino acid positions 148 to 162 and a series of basic amino acids (RKLKR) at amino acid positions 101 to 105. Altering the zinc finger motif of M1 reduces viral growth slightly. A substitution of Ser for Arg at either position 101 or position 105 of the RKLKR domain partially reduces the nuclear export of RNP and viral replication. To further understand the role of the zinc finger motif and the RKLKR domain in viral assembly and replication, we introduced multiple mutations by using reverse genetics to modify these regions of the M gene of influenza virus A/WSN/33. Of multiple mutants analyzed, a double mutant, R101S-R105S, of RKLKR resulted in a temperature-sensitive phenotype. The R101S-R105S double mutant had a greatly reduced ratio of M1 to NP in viral particles and a weaker binding of M1 to RNPs. These results suggest that mutations can be introduced into the RKLKR domain to control viral replication.


Cutting edge: regulation of CD8+ T cell effector population size.
Naive CD8(+) T cells are activated on encounter with Ag presented on dendritic cells and proliferate rapidly. To investigate the regulation of naive CD8(+) T cells proliferation, we adoptively transferred TCR-transgenic CD8(+) T cells into intact mice together with Ag-pulsed dendritic cells. Regardless of the number of cells initially transferred, the expansion of activated Ag-specific CD8(+) T cells was limited to a ceiling of effector cells. This limit was reached from a wide range of T cell doses, including a physiological number of precursor cells, and was not altered by changing the amount of Ag or APCs. The total Ag-specific response was composed of similar numbers of host and donor transgenic cells regardless of donor cell input, suggesting that these populations were independently regulated. Regulation of the transgenic donor cell population was TCR specific. We hypothesize that a clone-specific regulatory mechanism controls the extent of CD8(+) T cell responses to Ag.