Identification of candidate lethal haplotypes and genomic association with post-natal mortality and reproductive traits in Nellore cattle.

The wide use of genomic information has enabled the identification of lethal recessive alleles that are the major genetic causes of reduced conception rates, longer calving intervals, or lower survival for live-born animals. This study was carried out to screen the Nellore cattle genome for lethal recessive haplotypes based on deviation from the expected population homozygosity, and to test SNP markers surrounding the lethal haplotypes region for association with heifer rebreeding (HR), post-natal mortality (PNM) and stayability (STAY). This approach requires genotypes only from apparently normal individuals and not from affected embryos. A total of 62,022 animals were genotyped and imputed to a high-density panel (777,962 SNP markers). Expected numbers of homozygous individuals were calculated, and the probabilities of observing 0 homozygotes was obtained. Deregressed genomic breeding values [(G)EBVs] were used in a GWAS to identify candidate genes and biological mechanisms affecting HR, STAY and PNM. In the functional analyses, genes within 100 kb down and upstream of each significant SNP marker, were researched. Thirty haplotypes had high expected frequency, while no homozygotes were observed. Most of the alleles present in these haplotypes had a negative mean effect for PNM, HR and STAY. The GWAS revealed significant SNP markers involved in different physiological mechanisms, leading to harmful effect on the three traits. The functional analysis revealed 26 genes enriched for 19 GO terms. Most of the GO terms found for biological processes, molecular functions and pathways were related to tissue development and the immune system. More phenotypes underlying these putative regions in this population could be the subject of future investigation. Tests to find putative lethal haplotype carriers could help breeders to eliminate them from the population or manage matings in order to avoid homozygous.

STRIVE: Stress Resilience In Virtual Environments: a pre-deployment VR system for training emotional coping skills and assessing chronic and acute stress responses.

The incidence of posttraumatic stress disorder (PTSD) in returning OEF/OIF military personnel is creating a significant healthcare challenge. This has served to motivate research on how to better develop and disseminate evidence-based treatments for PTSD. One emerging form of treatment for combat-related PTSD that has shown promise involves the delivery of exposure therapy using immersive Virtual Reality (VR). Initial outcomes from open clinical trials have been positive and fully randomized controlled trials are currently in progress to further validate this approach. Based on our research group's initial positive outcomes using VR to emotionally engage and successfully treat persons undergoing exposure therapy for PTSD, we have begun development in a similar VR-based approach to deliver stress resilience training with military service members prior to their initial deployment. The Stress Resilience In Virtual Environments (STRIVE) project aims to create a set of combat simulations (derived from our existing Virtual Iraq/Afghanistan exposure therapy system) that are part of a multi-episode narrative experience. Users can be immersed within challenging combat contexts and interact with virtual characters within these episodes as part of an experiential learning approach for training a range of psychoeducational and cognitive-behavioral emotional coping strategies believed to enhance stress resilience. The STRIVE project aims to present this approach to service members prior to deployment as part of a program designed to better prepare military personnel for the types of emotional challenges that are inherent in the combat environment. During these virtual training experiences users are monitored physiologically as part of a larger investigation into the biomarkers of the stress response. One such construct, Allostatic Load, is being directly investigated via physiological and neuro-hormonal analysis from specimen collections taken immediately before and after engagement in the STRIVE virtual experience.

Antimicrobial testing for surface-immobilized agents with a surface-separated live-dead staining method.

Modification of a traditional live-dead staining technique based on fluorescence microscopy has yielded an improved method capable of differentiating surface-immobilized antimicrobial agents from those agents acting via solution diffusion processes. By utilizing an inoculation chamber comprised of 50 µm polystyrene spheres as spacers between test substrate and coverslip control surfaces, three distinct bacterial cell populations can be probed by fluorescence microscopy for antimicrobial activity: (1) cells adhered to the coverslip, (2) cells adhered to the substrate, and (3) mobile cells in solution. Truly immobilized antimicrobial agents were found efficacious only at the substrate surface, while elutable agents were effective against all three populations. Glass surfaces derivatized with either quaternized poly dimethylaminoethylmethacrylate (pDMAEMA) or 3-(trimethoxysilyl) propyldimethyloctadecyl ammonium chloride (Si-QAC) were compared with bare glass control surfaces after contact and 4 h incubation with Staphylococcus aureus. pDMAEMA surfaces were both antimicrobial and immobilized, whereas the Si-QAC surfaces were only observed to be antimicrobial via active diffusion. In contrast to conventional thinking, Si-QAC surfaces showed no kill after removing all Si-QAC elutables via rinsing procedures. The semi-quantitative surface-separated live-dead staining (SSLDS) technique provides mechanistic insight and represents a significant improvement relative to current microbiological test methods for evaluating immobilized, antimicrobial agents.

Calcium/Phosphate Solubility Curves for Premasol and Trophamine Pediatric Parenteral Nutrition Formulations.

OBJECTIVES: Calcium and phosphate incompatibility in parenteral nutrition formulations remains a critical concern for patient safety. This study examined calcium phosphate solubility for 2-in-1 admixtures prepared using 2 commercially available pediatric amino acid solutions (Premasol, Baxter Healthcare Corp; or Trophamine, B. Braun Medical Inc), applying identical test methods, storage conditions, and acceptance criteria. METHODS: Parenteral 2-in-1 admixtures included amino acid; dextrose; static concentrations of sodium, potassium, and magnesium, and varying concentrations of calcium (0-60 mEq/L), phosphate (15-50 mmol/L), and cysteine. Three replicate samples were stored for 48 hours at 40°C ± 2°C and then visually inspected for particulate matter, evaluated for subvisible particulate matter, when particulate matter was noted, microscopic examination was performed to confirm the presence of calcium phosphate crystals. Pass criteria were: all replicates free of visible particulate matter related to calcium phosphate crystals and particle counts below US Pharmacopeia <788> limits. RESULTS: Premasol and Trophamine generated identical calcium phosphate curves for 2% amino acid formulations containing 20% dextrose with/without cysteine, and similar curves for the 1% or 3% amino acid formulations containing 10% or 20% dextrose with/without cysteine. Calcium phosphate particles were identified in failed samples by scanning electron microscopy/energy dispersive X-ray spectroscopy. Calcium phosphate solubility was higher in formulations containing cysteine 40 mg/g amino acid vs. cysteine 20 mg/g amino acid and in cysteine 20 mg/g amino acid vs. no cysteine. CONCLUSIONS: Admixtures made with 1%, 2%, or 3% Premasol or Trophamine have essentially equivalent calcium phosphate solubility curves when tested with identical methods, storage conditions, and acceptance criteria.

Single nucleotide variants and InDels identified from whole-genome re-sequencing of Guzerat, Gyr, Girolando and Holstein cattle breeds.

Whole-genome re-sequencing, alignment and annotation analyses were undertaken for 12 sires representing four important cattle breeds in Brazil: Guzerat (multi-purpose), Gyr, Girolando and Holstein (dairy production). A total of approximately 4.3 billion reads from an Illumina HiSeq 2000 sequencer generated for each animal 10.7 to 16.4-fold genome coverage. A total of 27,441,279 single nucleotide variations (SNVs) and 3,828,041 insertions/deletions (InDels) were detected in the samples, of which 2,557,670 SNVs and 883,219 InDels were novel. The submission of these genetic variants to the dbSNP database significantly increased the number of known variants, particularly for the indicine genome. The concordance rate between genotypes obtained using the Bovine HD BeadChip array and the same variants identified by sequencing was about 99.05%. The annotation of variants identified numerous non-synonymous SNVs and frameshift InDels which could affect phenotypic variation. Functional enrichment analysis was performed and revealed that variants in the olfactory transduction pathway was over represented in all four cattle breeds, while the ECM-receptor interaction pathway was over represented in Girolando and Guzerat breeds, the ABC transporters pathway was over represented only in Holstein breed, and the metabolic pathways was over represented only in Gyr breed. The genetic variants discovered here provide a rich resource to help identify potential genomic markers and their associated molecular mechanisms that impact economically important traits for Gyr, Girolando, Guzerat and Holstein breeding programs.

Allostatic Load as a Complex Clinical Construct: A Case-Based Computational Modeling Approach.

Allostatic load (AL) is a complex clinical construct, providing a unique window into the cumulative impact of stress. However, due to its inherent complexity, AL presents two major measurement challenges to conventional statistical modeling (the field's dominant methodology): it is comprised of a complex causal network of bioallostatic systems, represented by an even larger set of dynamic biomarkers; and, it is situated within a web of antecedent socioecological systems, linking AL to differences in health outcomes and disparities. To address these challenges, we employed case-based computational modeling (CBM), which allowed us to make four advances: (1) we developed a multisystem, 7-factor (20 biomarker) model of AL's network of allostatic systems; (2) used it to create a catalog of nine different clinical AL profiles (causal pathways); (3) linked each clinical profile to a typology of 23 health outcomes; and (4) explored our results (post hoc) as a function of gender, a key socioecological factor. In terms of highlights, (a) the Healthy clinical profile had few health risks; (b) the pro-inflammatory profile linked to high blood pressure and diabetes; (c) Low Stress Hormones linked to heart disease, TIA/Stroke, diabetes, and circulation problems; and (d) high stress hormones linked to heart disease and high blood pressure. Post hoc analyses also found that males were overrepresented on the High Blood Pressure (61.2%), Metabolic Syndrome (63.2%), High Stress Hormones (66.4%), and High Blood Sugar (57.1%); while females were overrepresented on the Healthy (81.9%), Low Stress Hormones (66.3%), and Low Stress Antagonists (stress buffers) (95.4%) profiles.

Developments for inverted atomic force microscopy.

Atomic force microscopy (AFM) has been used to study a wide range of systems. Chemically and biologically modified probes have extended AFM by coupling chemical and biological information with the physical measurements. In an effort to further expand the capabilities of modified AFM probes, previous studies investigated the use of an inverted AFM design (i-AFM), wherein a microfabricated tip array is used to image a cantilever-supported sample. This report details developments in cantilever and tip array fabrication which are aimed at improving the applicability and performance of this i-AFM design. Using an epoxy-based procedure, commercial cantilevers were modified with a series of standard substrates, including template-stripped gold, highly oriented pyrolytic graphite, and mica. The samples on these cantilevers were imaged with i-AFM, and lateral force images are obtained. This paper demonstrates the first use of i-AFM for measuring friction.

Self-Reported Differences in Personality, Emotion Control, and Presence Between Pre-Military and Non-Military Groups in a Pilot Study Using the Stress Resilience in Virtual Environments (STRIVE) System.

Mental health disorders are the signature wounds of war resulting from extended U.S. Military conflicts in the Middle East [1]. In an effort to abate the number of Service Members that develop mental health disorders in these conflicts, USC-ICT has created the Stress Resilience in Virtual Environments (STRIVE) project, a set of highly realistic virtual reality combat scenarios and resilience-building sessions designed for pre-deployed military personnel. This short-paper looks at self-reported differences in personality, emotion control, and presence between two different groups, pre-military and non-military, of pilot subjects that tested a prototype of the first four modules of STRIVE.

Automated system for kinetic analysis of particle size distributions for pharmaceutically relevant systems.

Detailing the kinetics of particle formation for pharmaceutically relevant solutions is challenging, especially when considering the combination of formulations, containers, and timescales of clinical importance. This paper describes a method for using commercial software Automate with a stream-selector valve capable of sampling container solutions from within an environmental chamber. The tool was built to monitor changes in particle size distributions via instrumental particle counters but can be adapted to other solution-based sensors. The tool and methodology were demonstrated to be highly effective for measuring dynamic changes in emulsion globule distributions as a function of storage and mixing conditions important for parenteral nutrition. Higher levels of agitation induced the fastest growth of large globules (≥5 µm) while the gentler conditions actually showed a decrease in the number of these large globules. The same methodology recorded calcium phosphate precipitation kinetics as a function of [Ca(2+)] and pH. This automated system is readily adaptable to a wide range of pharmaceutically relevant systems where the particle size is expected to vary with time. This instrumentation can dramatically reduce the time and resources needed to probe complex formulation issues while providing new insights for monitoring the kinetics as a function of key variables.

Probability-based compatibility curves for calcium and phosphates in parenteral nutrition formulations.

BACKGROUND: The information content of the calcium phosphate compatibility curves for adult parenteral nutrition (PN) solutions may benefit from a more sophisticated statistical treatment. Binary logistic regression analyses were evaluated as part of an alternate method for generating formulation compatibility curves. MATERIALS AND METHODS: A commercial PN solution was challenged with a systematic array of calcium and phosphate concentrations. These formulations were then characterized for particulates by visual inspection, light obscuration, and filtration followed by optical microscopy. Logistic regression analyses of the data were compared with traditional treatments for generating compatibility curves. RESULTS: Assay-dependent differences were observed in the compatibility curves and associated probability contours; the microscopic method of precipitate detection generated the most robust results. Calcium and phosphate compatibility data generated from small-volume glass containers reasonably predicted the observed compatibility of clinically relevant flexible containers. CONCLUSIONS: The published methods for creating calcium and phosphate compatibility curves via connecting the highest passing or lowest failing calcium concentrations should be augmented or replaced by probability contours of the entire experimental design to determine zones of formulation incompatibilities. We recommend researchers evaluate their data with logistic regression analysis to help build a more comprehensive probabilistic database of compatibility information.

Interactions between Parenteral Lipid Emulsions and Container Surfaces.

OBJECTIVE: To evaluate the relationship between changes in emulsion globule size distributions and container uptake of lipid emulsions in total nutrient admixtures. METHODS: A total nutrient admixture was prepared from a commercial lipid emulsion, 20% ClinOleic®, separated into glass (borosilicate) and ethylene vinyl acetate (EVA) plastic containers, and then stored at ambient conditions for approximately 24 h. The large globule size distribution was monitored continuously for both containers, and the quantity of triglycerides associated with both containers was measured by liquid chromatography. The changes in mass of the EVA containers were also measured gravimetrically. RESULTS: The volume percent of globules greater than 5 microns in diameter (PFAT5) levels for an emulsion admixture in EVA containers showed a 75% reduction compared to a marginal decrease of PFAT5 when in the glass container. Extraction of the containers showed that the quantity of triglycerides associated with the EVA surfaces steadily increased with emulsion exposure time, while the glass showed a significantly lower triglyceride content compared to the EVA. Gravimetric measurements confirmed that the EVA containers gained significant mass during exposure to the emulsion admixture. CONCLUSION: A time-dependent decrease in PFAT5 values for an emulsion admixture was associated with container triglyceride absorption where EVA containers had a greater uptake than glass containers. The larger globules appear to absorb preferentially, and the admixture globule size distribution fraction represented by PFAT5 accounts for 15-20% of the total triglyceride adsorption to the container. LAY ABSTRACT: The goal of this work is to evaluate how emulsions in total nutrition admixtures are affected by the containers within which they are stored. Specifically, the study examines how the emulsion globule size distribution in different containers is related to adsorption or absorption of the lipids onto or into the container. The admixtures were prepared from a commercial lipid emulsion, 20% ClinOleic®, and the containers were either glass (borosilicate) or plastic (ethylene vinyl acetate, EVA). The large globule size distribution was monitored continuously for both containers over the course of 24 h, and the quantity of triglycerides taken up by both containers was measured by liquid chromatography. The lipid uptake by the EVA containers was also monitored by gravimetric methods. Briefly, the percent of fat globules greater than 5 micrometers (PFAT5) in EVA containers showed a 75% reduction compared to a marginal decrease of PFAT5 when in the glass container. Extraction of the lipids from the containers showed that the quantity of triglycerides associated with the EVA surfaces steadily increased with admixture exposure time, while the glass showed a significantly lower triglyceride content. Gravimetric measurements confirmed that the EVA containers gained measurable mass during exposure to the emulsion admixture.

Review of immobilized antimicrobial agents and methods for testing.

Antimicrobial surfaces for food and medical applications have historically involved antimicrobial coatings that elute biocides for effective kill in solution or at surfaces. However, recent efforts have focused on immobilized antimicrobial agents in order to avoid toxicity and the compatibility and reservoir limitations common to elutable agents. This review critically examines the assorted antimicrobial agents reported to have been immobilized, with an emphasis on the interpretation of antimicrobial testing as it pertains to discriminating between eluting and immobilized agents. Immobilization techniques and modes of antimicrobial action are also discussed.

Forces between insulin microspheres and polymers surfaces for a dry powder inhaler.

Here we demonstrate the use of a colloidal probe atomic force microscope (AFM) to compare the interactions between a model protein microsphere (insulin) and a set of common device polymers (polytetrafluoroethylene, polyethylene and polypropylene) with and without antistatic additive. For inhalation-based delivery devices the solid protein microspheres will interact with the device surfaces under ambient atmospheric conditions, and as such we studied the particle device interaction at a range of relative humidities. The results clearly discriminate between the five different polymer choices, and the impact of the antistatic additive. Although the mechanistic understanding is incomplete, it is evident that the polypropylene with antistatic additive gives consistent and relatively small interaction forces over the entire humidity range. The other polymer surfaces have humidity ranges where the pull-off forces are substantially greater. At 80% relative humidity, the insulin-polymer adhesion forces were similar for all the polymers probably due to the dominance of static charge mitigation and surface hydration effects. Overall, direct measurement of adhesion forces between pharmaceutical microspheres and container substrates can help direct rational choice of plastics/coatings for medical devices.

Patterning of cantilevers with functionalized nanoparticles for combinatorial atomic force microscopy.

In this letter, we demonstrate a substantial improvement in the ease of attaching and recognizing chemical libraries on cantilevers for use with combinatorial atomic force microscopy. These experiments exploit chemically modified nanoparticles as a means of encoding various chemistries onto the cantilevers with structures that can be readily distinguished. For the first time, 16 combinations of interactions were measured in a single experiment and in the same solution. Furthermore, these experiments have not begun to push the limits of the technique; it is plausible that in the very near future hundreds of interactions could be rapidly measured, helping to transform AFM into a truly high throughput technique.

Tau and Kappa effects in physical space: the case of audition.

The organization of spatio-temporal information in an auditory memory task was studied in two experiments. Stimuli consisted of four different configurations of eight sequentially presented beeps. In two configurations, the stimuli were space-time congruent, with (constant or variable) inter-stimulus distances corresponding to (constant or variable) inter-stimulus time intervals. In the other two configurations, the stimuli were space-time incongruent, with (constant or variable) inter-stimulus distances not corresponding to (variable or constant) inter-stimulus time intervals. After a learning phase consisting of 20 presentations of the target configuration, participants reproduced the spatial (Experiment 1) or temporal (Experiment 2) characteristics of the target 60 times in succession without re-examining the target configuration. Accuracy (with respect to the target) and variability (between responses) were found to evolve independently. In the incongruent space-time conditions, effects of variable inter-stimulus time intervals or distances on the reproduction of, respectively, constant distances (Tau effect) or constant time intervals (Kappa effect) were observed, while the reverse was not the case. Thus, dimensional interference occurred when the dimension to be ignored was variable. The results are discussed in the light of the distinction between properties of the stabilized mental image and the process of stabilization.

Tapping mode imaging and measurements with an inverted atomic force microscope.

This report demonstrates the successful use of the inverted atomic force microscope (i-AFM) for tapping mode AFM imaging of cantilever-supported samples. i-AFM is a mode of AFM operation in which a sample supported on a tipless cantilever is imaged by one of many tips in a microfabricated tip array. Tapping mode is an intermittent contact mode whereby the cantilever is oscillated at or near its resonance frequency, and the amplitude and/or phase are used to image the sample. In the process of demonstrating that tapping mode images could be obtained in the i-AFM design, it was observed that the amplitude of the cantilever oscillation decreased markedly as the cantilever and tip array were approached. The source of this damping of the cantilever oscillations was identified to be the well-known "squeeze film damping", and the extent of damping was a direct consequence of the relatively shorter tip heights for the tip arrays, as compared to those of commercially available tapping mode cantilevers with integrated tips. The functional form for the distance dependence of the damping coefficient is in excellent agreement with previously published models for squeeze film damping, and the values for the fitting parameters make physical sense. Although the severe damping reduces the cantilever free amplitude substantially, we found that we were still able to access the low-amplitude regime of oscillation necessary for attractive tapping mode imaging of fragile molecules.

Patterning of cantilevers with inverted dip-pen nanolithography: efforts toward combinatorial AFM.

We report patterning of AFM cantilevers by inverted dip-pen nanolithography, thereby markedly enhancing the development of combinatorial AFM as a high-throughput force-measuring instrument capable of determining interactions between opposing libraries of biomolecules.