Traumatic brain injury heterogeneity affects cell death and autophagy.

Traumatic brain injury (TBI) mechanism and severity are heterogenous clinically, resulting in a multitude of physical, cognitive, and behavioral deficits. Impact variability influences the origin, spread, and classification of molecular dysfunction which limits strategies for comprehensive clinical intervention. Indeed, there are currently no clinically approved therapeutics for treating the secondary consequences associated with TBI. Thus, examining pathophysiological changes from heterogeneous impacts is imperative for improving clinical translation and evaluating the efficacy of potential therapeutic strategies. Here we utilized TBI models that varied in both injury mechanism and severity including severe traditional controlled cortical impact (CCI), modified mild CCI (MTBI), and multiple severities of closed-head diffuse TBI (DTBI), and assessed pathophysiological changes. Severe CCI induced cortical lesions and necrosis, while both MTBI and DTBI lacked lesions or significant necrotic damage. Autophagy was activated in the ipsilateral cortex following CCI, but acutely impaired in the ipsilateral hippocampus. Additionally, autophagy was activated in the cortex following DTBI, and autophagic impairment was observed in either the cortex or hippocampus following impact from each DTBI severity. Thus, we provide evidence that autophagy is a therapeutic target for both mild and severe TBI. However, dramatic increases in necrosis following CCI may negatively impact the clinical translatability of therapeutics designed to treat acute dysfunction in TBI. Overall, these results provide evidence that injury sequalae affiliated with TBI heterogeneity is linked through autophagy activation and/or impaired autophagic flux. Thus, therapeutic strategies designed to intervene in autophagy may alleviate pathophysiological consequences, in addition to the cognitive and behavioral deficits observed in TBI.

Identifying host-specific amino acid signatures for influenza A viruses using an adjusted entropy measure.

BACKGROUND: Influenza A viruses (IAV) exhibit vast genetic mutability and have great zoonotic potential to infect avian and mammalian hosts and are known to be responsible for a number of pandemics. A key computational issue in influenza prevention and control is the identification of molecular signatures with cross-species transmission potential. We propose an adjusted entropy-based host-specific signature identification method that uses a similarity coefficient to incorporate the amino acid substitution information and improve the identification performance. Mutations in the polymerase genes (e.g., PB2) are known to play a major role in avian influenza virus adaptation to mammalian hosts. We thus focus on the analysis of PB2 protein sequences and identify host specific PB2 amino acid signatures. RESULTS: Validation with a set of H5N1 PB2 sequences from 1996 to 2006 results in adjusted entropy having a 40% false negative discovery rate compared to a 60% false negative rate using unadjusted entropy. Simulations across different levels of sequence divergence show a false negative rate of no higher than 10% while unadjusted entropy ranged from 9 to 100%. In addition, under all levels of divergence adjusted entropy never had a false positive rate higher than 9%. Adjusted entropy also identifies important mutations in H1N1pdm PB2 previously identified in the literature that explain changes in divergence between 2008 and 2009 which unadjusted entropy could not identify. CONCLUSIONS: Based on these results, adjusted entropy provides a reliable and widely applicable host signature identification approach useful for IAV monitoring and vaccine development.

Variational Bayesian inference for association over phylogenetic trees for microorganisms.

With the advance of next generation sequencing technologies, researchers now routinely obtain a collection of microbial sequences with complex phylogenetic relationships. It is often of interest to analyze the association between certain environmental factors and characteristics of the microbial collection. Though methods have been developed to test for association between the microbial composition with environmental factors as well as between coevolving traits, a flexible model that can provide a comprehensive picture of the relationship between microbial community characteristics and environmental variables will be tremendously beneficial. We developed a Bayesian approach for association analysis while incorporating the phylogenetic structure to account for the dependence between observations. To overcome the computational difficulty related to the phylogenetic tree, a variational algorithm was developed to evaluate the posterior distribution. As the posterior distribution can be readily obtained for parameters of interest and any derived variables, the association relationship can be examined comprehensively. With two application examples, we demonstrated that the Bayesian approach can uncover nuanced details of the microbial assemblage with regard to the environmental factor. The proposed Bayesian approach and variational algorithm can be extended for other problems involving dependence over tree-like structures.

Using the heat generated from electrically conductive concrete slabs to reduce antibiotic resistance in beef cattle manure.

Proper treatment is necessary to reduce antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in livestock manure before land application. Conventional stockpiling suffers unreliable removal efficiency, while composting can be complicated and expensive. The objective of this study was to test the feasibility of a novel heat-based technology, i.e., stockpiling manure on conductive concrete slabs, to inactivate ARB and ARGs in beef cattle manure. In this study, two independent bench-scale trials were conducted. In both trials, samples were taken from manure piles on conductive concrete slabs and regular slabs (i.e., heated and unheated piles). In the heated pile of the first trial, 25.9% and 83.5% of the pile volume met the EPA Class A and Class B biosolids standards, respectively. For the heated pile of the second trial, the two values were 43.9% and 74.2%. In both trials, nearly all forms of the total and resistant Escherichia coli and enterococci were significantly lower in the heated piles than in the unheated piles. Besides, significant reduction of ARGs in heated piles was observed in the first trial. Through this proof-of-concept study, the new technology based on conductive concrete slabs offers an alternative manure storage method to conventional stockpiling and composting with respect to reduce ARB and ARGs in manure.

Antibiotic resistance genes in swine manure slurry as affected by pit additives and facility disinfectants.

Manure storage facilities are critical control points to reduce antibiotic resistance genes (ARGs) in swine manure slurry before the slurry is land applied. However, little is known about how exogenous chemicals entering the manure storage facilities may affect the fate of ARGs. The objective of this study was to analyze the impact of six commonly used pit additives and four facility disinfectants on the concentration of ARGs in swine manure slurry. Bench scale reactors, each containing approximately 50 L of liquid swine manure, were dosed with additives or disinfectants and were sampled for 40 days. Seven antibiotic resistance genes along with the intI1 gene and the 16S rRNA gene were monitored. Out of the six additives tested, Sludge Away significantly reduced the time-averaged absolute abundance of erm(C), erm(F), tet(Q), and the 16S rRNA gene as compared to the no additive control. Out of the four disinfectants tested, Tek-Trol significantly reduced the time-averaged absolute abundance of erm(B), erm(C), erm(F), intI1, tet(Q), and tet(X) than did the no-disinfectant control. According to Spearman's rank correlation, three genes erm(F), tet(Q), and tet(X) showed a strong to perfectly positive correlation and the two genes erm(B) and tet(O) showed a moderate to strong correlation in both the additive and disinfectant tests. Overall, the disinfectants were more effective in controlling the absolute abundance of ARGs than were the pit additives.

Corn stalk residue may add antibiotic-resistant bacteria to manure composting piles.

Manure is commonly used as a fertilizer or soil conditioner; however, land application of untreated manure may introduce pathogens and antibiotic-resistant bacteria (ARB) into the soil, with harmful implications for public health. Composting is a manure management practice wherein a carbon-rich bulking agent, such as corn (Zea mays L.) stalk residue, is added to manure to achieve desirable carbon/nitrogen ratios to facilitate microbial activities and generate enough heat to inactivate pathogens, including antibiotic-resistant pathogens. However, when comparing compost piles and stockpiles for ARB reduction, we noticed that bulking agents added ARB to composting piles and compromised the performance of composting in reducing ARB. We hypothesized that ARB could be prevalent in corn stalk residues, a commonly used bulking agent for composting. To test this hypothesis, corn stalk residue samples throughout Nebraska were surveyed for the presence of ARB. Of the samples tested, 54% were positive for antibiotic-resistant Escherichia coli or enterococci using direct plating or after enrichment. Although not statistically significant, there was a trend wherein the use of pesticides tended to result in a greater prevalence of some ARB. Results from this study suggest that bulking agents can be a source of ARB in manure composting piles and highlight the importance of screening bulking agents for effective ARB reduction in livestock manure during composting.

Most High-Intensity Transient Signals Are Not Associated With Specific Surgical Maneuvers.

BACKGROUND: Mortality after congenital heart defect surgery has dropped dramatically in the last few decades. Current research on long-term outcomes has focused on preventing secondary neurological sequelae, for which embolic burden is suspected. In children, little is known of the correlation between specific surgical maneuvers and embolic burden. Transcranial Doppler ultrasound is highly useful for detecting emboli but has not been widely used with infants and children. METHODS: Bilateral middle cerebral artery blood flow was continuously monitored from sternal incision to chest closure in 20 infants undergoing congenital heart defect repair or palliative surgery. Embolus counts for specific maneuvers were recorded using widely accepted criteria for identifying emboli via high-intensity transient signals (HITS). RESULTS: An average of only 13% of all HITS detected during an operation were correlated with any of the surgical maneuvers of interest. The highest mean number of HITS associated with a specific maneuver occurred during cross-clamp removal. Cross-clamp placement also had elevated HITS counts that significantly differed from other maneuvers. CONCLUSIONS: In this study of infants undergoing cardiac surgery with cardiopulmonary bypass, the great majority of HITS detected are not definitively associated with a specific subset of surgical maneuvers. Among the measured maneuvers, removal of the aortic cross-clamp was associated with the greatest occurrence of HITS. Future recommended research efforts include identifying and confirming other sources for emboli and longitudinal outcome studies to determine if limiting embolic burden affects long-term neurological outcomes.

Influence of Setback Distance on Antibiotics and Antibiotic Resistance Genes in Runoff and Soil Following the Land Application of Swine Manure Slurry.

The environmental spread of antibiotics and antibiotic resistance genes (ARGs) from the land application of livestock wastes can be a potential public health threat. The objective of this study was to assess the effects of setback distance, which determines how close manure may be applied in relation to surface water, on the transport of antibiotics and ARGs in runoff and soil following land application of swine manure slurry. Rainfall simulation tests were conducted on field plots covered with wheat residues, each of which contained an upslope manure region where slurry was applied and an adjacent downslope setback region that did not receive slurry. Results show that all three antibiotics (chlortetracycline, lincomycin, and tiamulin) and seven out of the ten genes tested (erm(B), erm(C), intI1, tet(O), tet(Q), tet(X), and the 16S rRNA gene) decreased significantly in runoff with increased setback distance. Only blaTEM, chlortetracycline, and tiamulin decreased significantly in surface soil with increased setback distance, while the other analytes did not exhibit statistically significant trends. By using linear regression models with field data, we estimate that a setback distance between 34-67 m may allow manure-borne antibiotics and ARGs in runoff to reach background levels under the experimental conditions tested.

Swine slurry characteristics as affected by selected additives and disinfectants.

Current swine industry practice is to house animals in confinement facilities which capture and store feces and urine as slurry in pits below the production area. Additives and disinfectants may be introduced into the manure pits. This study was conducted to measure the effects of additives and disinfectants on temporal changes in swine slurry characteristics. Slurry from a commercial swine production facility in southeast Nebraska, USA was collected and transferred to 57 L reactors located within a greenhouse. Selected additives and disinfectants were added to the reactors and physical properties, chemical characteristics, and antibiotic concentrations were monitored for 40 days. Concentrations of dry matter (DM), total nitrogen (TN), phosphorus pentoxide (P2O5), calcium (Ca), magnesium (Mg), zinc (Zn), iron (Fe), manganese (Mn), and copper (Cu) were significantly greater than the Control in each of the reactors containing additives. The reactors in which the additives MOC-7, More Than Manure®, Sludge Away, and Sulfi-Doxx were introduced had significantly greater values of chemical oxygen demand (COD), total volatile solids (TVS), total suspended solids (TSS), total solids (TS), dry matter (DM), TN, P2O5, Ca, Mg, Zn, Fe, Mn, Cu and chlortetracycline than the other additive treatments. Concentrations of TVS and TSS were significantly lower in the reactors containing Clorox® and Virkon™ than the other disinfectant treatments. The total dissolved solids (TDS) concentration of 26,500 mg L-1 and pH value of 7.27 obtained for the reactors containing Tek-Trol were significantly greater than measurements obtained for the other treatments. Concentrations of chlortetracycline and tiamulin of 8840 and 28.8 ng g-1, respectively, were significantly lower for the treatments containing Tek-Trol. The sodium (Na) concentration of 1070 mg L-1 measured in the reactors containing Clorox® was significantly greater than values for the other disinfectant treatments. The introduction of selected additives and disinfectants may influence certain physical properties, chemical characteristics, and antibiotic concentrations of swine slurry.

Dung Beetles Increase Greenhouse Gas Fluxes from Dung Pats in a North Temperate Grassland.

Soil fauna plays a critical role in various ecosystem processes, but empirical data measuring its impact on greenhouse gas (GHG) emissions from rangelands are limited. We quantified the effects of dung beetles on in situ CO, CH, and NO emissions from simulated cattle dung deposits. Soil in meadows of the semiarid Nebraska Sandhills was treated with three treatments (dung pats with exposure and without exposure to dung beetles, and a no dung control). A closed-chamber method was used to measure GHG fluxes at 0, 1, 2, 3, 7, 10, 14, 21, 28, and 56 d after dung placement in the early season (June-August) and late season (July-September) in 2014 and 2015. The greatest dung beetle abundance was 6 ± 2 beetles per quarter pat on Day 7; the abundance decreased to <2 ± 0.6 on Day 14 and 28 and zero on Day 56. Dung beetles increased fluxes of CO by 0.2 g C d m, NO by 0.4 mg N d m (only in late season 2015), and CH by 0.2 mg C d m. These increases were due to beetle-made macropores that facilitated gas transport in wet dung (initial moisture = 4.6 g g on a dry-weight basis) within 7 d after dung placement. Seasonal environmental differences resulted in greater CO, NO, and CH fluxes in the early season than in the late season. This study concluded that dung beetles increased GHG fluxes from early- and late-season dung deposits on meadows of the semiarid Nebraska Sandhills.

Optimization of polyhydroxybutyrate production by experimental design of combined ternary mixture (glucose, xylose and arabinose) and process variables (sugar concentration, molar C:N ratio).

Conversion of lignocellulosic feedstocks to polyhydroxybutyrate (PHB) could make lignocellulosic biorefineries more profitable and sustainable. Glucose, xylose and arabinose are the main sugars derived from pretreatment and hydrolysis of herbaceous feedstocks. Burkholderia sacchari DSM 17165 is a bacterium that can convert these sugars into PHB. However, the effects of sugar ratio, sugar concentration, and molar C:N ratio on PHB production have not been studied. In this study, a seven-run mixture design for sugar ratio combined with a 32 full factorial design for process variables was performed to optimize PHB production. A polynomial model was built based on experimental data, and optimum conditions for different sugar streams were derived and validated. The highest PHB production (3.81 g/L) was achieved with arabinose at a concentration of 25.54 g/L and molar C:N ratio of 74.35. Results provide references for manipulation of sugar mixture and process control to maximize PHB production.

Group screening for rare events based on incomplete block designs.

Fields such as, diagnostic testing, biotherapeutics, drug development, and toxicology among others, center on the premise of searching through many specimens for a rare event. Scientists in the business of "searching for a needle in a haystack" may greatly benefit from the use of group screening design strategies. Group screening, where specimens are composited into pools with each pool being tested for the presence of the event, can be much more cost-efficient than testing each individual specimen. A number of group screening designs have been proposed in the literature. Incomplete block screening designs are described here and compared with other group screening designs. It is shown under certain conditions, that incomplete block screening designs can provide nearly a 90% cost saving compared to other group screening designs such as when prevalence is 0.001 and screening 3876 specimens with an ICB-sequential design vs. a Dorfman design. In other cases, previous group screening designs are shown to be most efficient. Overall, when prevalence is small (≤0.05) group screening designs are shown to be quite cost effective at screening a large number of specimens and in general there is no one design that is best in all situations. © 2018 American Institute of Chemical Engineers Biotechnol Progress, 35: e2770, 2019.

Response Surface Methodology for Salmonella Inactivation during Extrusion Processing of Oat Flour.

An increase in the number of foodborne outbreaks and recalls due to Salmonella in low-moisture foods has resulted in the need for the development and validation of process controls to ensure their microbiological safety. Furthermore, the Food Safety Modernization Act Preventive Controls for Human Food final rule requires food processors to validate their process controls to ensure food safety. The objective of this study was to develop a response surface model to predict Salmonella inactivation in oat flour, as affected by moisture, fat content, screw speed, and temperature. Oat flour was adjusted to different moisture (14 to 26% wet basis) and fat (5 to 15% [w/w]) contents and was then inoculated with a five-strain cocktail of Salmonella. Inoculated material was extruded through a single-screw extruder running at different screw speeds (75 to 225 rpm) and temperatures (65 to 85°C), without a die. Once steady-state conditions were attained, extruded samples were collected, cooled, and stored under refrigeration, and Salmonella survivors were enumerated. A split-plot central composite second-order response surface design was used, with the central point replicated six times. Temperature showed a significant ( P < 0.0005) positive effect on microbial reduction. Moisture content showed significant linear ( P = 0.0014) and quadratic ( P = 0.0005) effects, whereas higher fat content showed a significant ( P < 0.0001) protective effect on Salmonella destruction. The screw speed did not play a major role in inactivating Salmonella, but it had a significant ( P = 0.0004) interactive effect with temperature. Results indicated that a >5.5-log reduction was achieved in oat flour extruded at a temperature above 85°C at all moisture and fat contents evaluated at a screw speed of 150 rpm. The developed response surface model can be used to identify the extrusion process conditions to achieve a desired reduction of Salmonella based on the moisture and fat contents of the product.

Evaluation of Enterococcus faecium NRRL B-2354 as a Surrogate for Salmonella During Extrusion of Low-Moisture Food.

Salmonella in low-moisture foods is an emerging challenge due to numerous food product recalls and foodborne illness outbreaks. Identification of suitable surrogate is critical for process validation at industry level due to implementation of new Food Safety Modernization Act of 2011. The objective of this study was to evaluate Enterococcus faecium NRRL B-2354 as a surrogate for Salmonella during the extrusion of low-moisture food. Oat flour, a low-moisture food, was adjusted to different moisture (14% to 26% wet basis) and fat (5% to 15% w/w) contents and was inoculated with E. faecium NRRL B-2354. Inoculated material was then extruded in a lab-scale single-screw extruder running at different screw speeds (75 to 225 rpm) and different temperatures (75, 85, and 95 °C). A split-plot central composite 2nd order response surface design was used, with the central point replicated six times. The data from the selective media (m-Enterococcus agar) was used to build the response surface model for inactivation of E. faecium NRRL B-2354. Results indicated that E. faecium NRRL B-2354 always had higher heat resistance compared to Salmonella at all conditions evaluated in this study. However, the patterns of contour plots showing the effect of various product and process parameters on inactivation of E. faecium NRRL B-2354 was different from that of Salmonella. Although E. faecium NRRL B-2354 may be an acceptable surrogate for extrusion of low-moisture products due to higher resistance than Salmonella, another surrogate with similar inactivation behavior may be preferred and needs to be identified. PRACTICAL APPLICATION: Food Safety Modernization Act requires the food industry to validate processing interventions. This study validated extrusion processing and demonstrated that E. faecium NRRL B-2354 is an acceptable surrogate for extrusion of low-moisture products. The developed response surface model allows the industry to identify process conditions to achieve a desired lethality for their products based on composition.

Extraction, identification, and quantification of antioxidant phenolics from hazelnut (Corylus avellana L.) shells.

Hazelnut shells are the major byproduct of the hazelnut industry. The objectives of this study were to optimize the conditions for extracting phenolics and to identify and quantify the phenolics in hazelnut shells. Preliminary optimization showed that a high recovery of phenolics could be achieved with shell particle size less than 0.5mm when extracted with acetone at 50°C. Response surface experiments showed that a 10g/l liquid to solid ratio, 58% acetone, and 12h extraction time yielded the highest amount of phenolics. Twenty-seven phenolic compounds were identified in hazelnut shells by mass spectrometry. Coumaroylquinic acid, epicatechin gallate, quercetin, and six other phenolics were identified in hazelnut shells for the first time. The most abundant phenolics in hazelnut shells were catechin, epicatechin gallate, and gallic acid, as quantified by high performance liquid chromatography (HPLC). These results can be useful for the development of industrial extraction processes of natural antioxidants from hazelnut shells.

Genotype, environment, seeding rate, and top-dressed nitrogen effects on end-use quality of modern Nebraska winter wheat.

BACKGROUND: Fine-tuning production inputs such as seeding rate, nitrogen (N), and genotype may improve end-use quality of hard red winter wheat (Triticum aestivium L.) when growing conditions are unpredictable. Studies were conducted at the Agronomy Research Farm (ARF; Lincoln, NE, USA) and the High Plains Agricultural Laboratory (HPAL; Sidney, NE, USA) in 2014 and 2015 in Nebraska, USA, to determine the effects of genotype (6), environment (4), seeding rate (3), and flag leaf top-dressed N (0 and 34 kg N ha-1 ) on the end-use quality of winter wheat. RESULTS: End-use quality traits were influenced by environment, genotype, seeding rate, top-dressed N, and their interactions. Mixograph parameters had a strong correlation with grain volume weight and flour yield. Doubling the recommended seeding rate and N at the flag leaf stage increased grain protein content by 8.1% in 2014 and 1.5% in 2015 at ARF and 4.2% in 2014 and 8.4% in 2015 at HPAL. CONCLUSION: The key finding of this research is that increasing seeding rates up to double the current recommendations with N at the flag leaf stage improved most of the end-use quality traits. This will have a significant effect on the premium for protein a farmer could receive when marketing wheat. © 2017 Society of Chemical Industry.

Type 2 BVDV Npro suppresses IFN-1 pathway signaling in bovine cells and augments BRSV replication.

Bovine viral diarrhea virus (BVDV) infection induces immunosuppression and in conjunction with bovine respiratory syncytial virus (BRSV) contributes to the bovine respiratory disease complex. Bovine turbinate cells were single or co-infected with type 2 BVDV wild-type (BVDV2-wt), its dysfunctional Npro mutant (BVDV2-E), and/or BRSV. BVDV2-E significantly up-regulated PKR, IRF-7, TBK-1, IRF-3, and IFN-ß mRNAs based on real-time Q-RT-PCR. BRSV-infected cells expressed significantly up-regulated PKR, IRF-3, IRF-7, and IFN-ß mRNAs, whereas BVDV2-wt, but not BVDV2-E, abolished this up-regulation in co-infection. No significant differences were observed in MAVS, NF-κB, and PIN-1 mRNAs. A dual-luciferase reporter assay showed that BVDV2-wt significantly increased NF-κB activity compared to BVDV2-E, while BVDV2-E significantly increased IFN-ß activity compared to BVDV2-wt. The BRSV titer and RNA levels significantly increased in cells co-infected with BRSV/BVDV2-wt compared to cells co-infected with BRSV/BVDV2-E or infected with BRSV alone. This data supports the synergistic action of BVDV2-wt and BRSV inhibition of IFN-1.

Experimental acute infection of alpacas with Bovine viral diarrhea virus 1 subgenotype b alters peripheral blood and GALT leukocyte subsets.

Bovine viral diarrhea virus (BVDV) is a pathogen in cattle and alpacas ( Vicugna pacos), causing acute and persistent BVDV infections. We characterized the effect of acute BVDV infection on the immune system of alpacas by determining lymphocyte subpopulations in peripheral blood and gut-associated lymphoid tissues (GALT) as well as serum interferon levels. Alpacas were experimentally infected with BVDV-1b (strain CO-06). Peripheral blood leukocytes were isolated at 0, 3, 6, and 9 d postinfection (dpi), and leukocytes of GALT at 9 dpi, and evaluated using flow cytometry. Serum interferon levels were determined daily. Flow cytometric analyses of peripheral blood leukocytes showed a significant decrease in CD4+, CD8+, and αß T-lymphocytes at 3 dpi. CD8+ lymphocytes were significantly increased, and activated lymphocytes were significantly decreased in the C3-stomach region in BVDV-infected alpacas. Serum interferon concentrations significantly increased in BVDV-infected alpacas at 3-6 dpi, peaking at 3 dpi. Our study confirms that BVDV can be a primary acute pathogen in alpacas and that it induces an interferon response and alters leukocyte subset populations. The changes in the proportion of T-lymphocytes during the early stages of BVDV infection may result in transient immunosuppression that may contribute to secondary bacterial and viral infections, similar to cattle.