Food and feed safety risk assessment uses multi-parameter models to evaluate the likelihood of adverse events associated with exposure to hazards in human health, plant health, animal health, animal ...welfare, and the environment. Systematic review and meta-analysis are established methods for answering questions in health care, and can be implemented to minimize biases in food and feed safety risk assessment. However, no methodological frameworks exist for refining risk assessment multi-parameter models into questions suitable for systematic review, and use of meta-analysis to estimate all parameters required by a risk model may not be always feasible. This paper describes novel approaches for determining question suitability and for prioritizing questions for systematic review in this area. Risk assessment questions that aim to estimate a parameter are likely to be suitable for systematic review. Such questions can be structured by their “key elements” e.g., for intervention questions, the population(s), intervention(s), comparator(s), and outcome(s). Prioritization of questions to be addressed by systematic review relies on the likely impact and related uncertainty of individual parameters in the risk model. This approach to planning and prioritizing systematic review seems to have useful implications for producing evidence-based food and feed safety risk assessment.
Structure-based grouping of chemicals for targeted testing and read-across is an efficient way to reduce resources and animal usage. For substances of unknown or variable composition, complex ...reaction products, or biological materials (UVCBs), structure-based grouping is virtually impossible. Biology-based approaches such as metabolomics could provide a solution. Here, 15 steam-cracked distillates, registered in the EU through the Lower Olefins Aromatics Reach Consortium (LOA), as well as six of the major substance constituents, were tested in a 14-day rat oral gavage study, in line with the fundamental elements of the OECD 407 guideline, in combination with plasma metabolomics. Beyond signs of clinical toxicity, reduced body weight (gain), and food consumption, pathological investigations demonstrated the liver, thyroid, kidneys (males only), and hematological system to be the target organs. These targets were confirmed by metabolome pattern recognition, with no additional targets being identified. While classical toxicological parameters did not allow for a clear distinction between the substances, univariate and multivariate statistical analysis of the respective metabolomes allowed for the identification of several subclusters of biologically most similar substances. These groups were partly associated with the dominant (> 50%) constituents of these UVCBs, i.e., indene and dicyclopentadiene. Despite minor differences in clustering results based on the two statistical analyses, a proposal can be made for the grouping of these UVCBs. Both analyses correctly clustered the chemically most similar compounds, increasing the confidence that this biological approach may provide a solution for the grouping of UVCBs.
Key points
Uterine artery vasodilatation is a key mechanism for increasing utero‐placental blood flow and fetal nutrient supply.
Since the pioneering work of Joseph Barcroft, the natural laboratory ...of high altitude has been used to study the mechanisms regulating uterine artery blood supply and fetal growth.
Genes near the metabolic sensor adenosine monophosphate‐activated protein kinase (AMPK) have been implicated in genetic protection from high altitude‐associated fetal growth restriction.
We show that AMPK is present in utero‐placental tissues and has vasodilator effects in murine uterine arteries, and that exposure to chronic hypoxia sufficient to decrease fetal growth increases the vasodilator actions of AMPK in opposing phenylephrine‐induced vasoconstriction.
These results point to AMPK as being a key link between maternal vascular responses to pregnancy and fetal growth. Manipulation of AMPK may be a novel mechanism for developing new therapies in pregnancies complicated by chronic hypoxia.
Genes near adenosine monophosphate‐activated protein kinase‐α1 (PRKAA1) have been implicated in the greater uterine artery (UtA) blood flow and relative protection from fetal growth restriction seen in altitude‐adapted Andean populations. Adenosine monophosphate‐activated protein kinase (AMPK) activation vasodilates multiple vessels but whether AMPK is present in UtA or placental tissue and influences UtA vasoreactivity during normal or hypoxic pregnancy remains unknown. We studied isolated UtA and placenta from near‐term C57BL/6J mice housed in normoxia (n = 8) or hypoxia (10% oxygen, n = 7–9) from day 14 to day 19, and placentas from non‐labouring sea level (n = 3) or 3100 m (n = 3) women. Hypoxia increased AMPK immunostaining in near‐term murine UtA and placental tissue. RT‐PCR products for AMPK‐α1 and ‐α2 isoforms and liver kinase B1 (LKB1; the upstream kinase activating AMPK) were present in murine and human placenta, and hypoxia increased LKB1 and AMPK‐α1 and ‐α2 expression in the high‐ compared with low‐altitude human placentas. Pharmacological AMPK activation by A769662 caused phenylephrine pre‐constricted UtA from normoxic or hypoxic pregnant mice to dilate and this dilatation was partially reversed by the NOS inhibitor l‐NAME. Hypoxic pregnancy sufficient to restrict fetal growth markedly augmented the UtA vasodilator effect of AMPK activation in opposition to PE constriction as the result of both NO‐dependent and NO‐independent mechanisms. We conclude that AMPK is activated during hypoxic pregnancy and that AMPK activation vasodilates the UtA, especially in hypoxic pregnancy. AMPK activation may be playing an adaptive role by limiting cellular energy depletion and helping to maintain utero‐placental blood flow in hypoxic pregnancy.
Key points
Uterine artery vasodilatation is a key mechanism for increasing utero‐placental blood flow and fetal nutrient supply.
Since the pioneering work of Joseph Barcroft, the natural laboratory of high altitude has been used to study the mechanisms regulating uterine artery blood supply and fetal growth.
Genes near the metabolic sensor adenosine monophosphate‐activated protein kinase (AMPK) have been implicated in genetic protection from high altitude‐associated fetal growth restriction.
We show that AMPK is present in utero‐placental tissues and has vasodilator effects in murine uterine arteries, and that exposure to chronic hypoxia sufficient to decrease fetal growth increases the vasodilator actions of AMPK in opposing phenylephrine‐induced vasoconstriction.
These results point to AMPK as being a key link between maternal vascular responses to pregnancy and fetal growth. Manipulation of AMPK may be a novel mechanism for developing new therapies in pregnancies complicated by chronic hypoxia.
Objectives
Describe the split staphylectomy procedure to address soft palate thickness and assess the complications and long‐term outcome of this procedure as a part of multi‐level surgery for ...brachycephalic obstructive airway syndrome. To consider whether same‐day discharge following this surgery can be recommended.
Materials and Methods
Medical records of dogs treated for brachycephalic obstructive airway syndrome using the split staphylectomy were reviewed. Owners were contacted to complete a questionnaire assessing initial postoperative concerns, the long‐term outcome and the effect of surgery on their dog's quality of life.
Results
Seventy‐five dogs underwent split staphylectomy during the study period. The overall complication rate was 8.3%, of which 2.7% were considered major. No life‐threatening complications occurred, and no complications were related to the staphylectomy. The questionnaire was completed by 66.7% of owners (median follow‐up 459 days), of which 88% felt that surgery had improved the quality of life for their dog. The majority (88%) of dogs were discharged from hospital on the day of surgery. Of the surveyed owners, 14% sought veterinary attention between their dog leaving the hospital and the scheduled postoperative reassessment 2 weeks after surgery. Four dogs were presented for veterinary intervention during this time period, but no intervention was related to the staphylectomy or for a life‐threatening condition.
Clinical Significance
The split staphylectomy offers a safe, straightforward method of addressing both excess thickness and length of soft palate in dogs with brachycephalic obstructive airway syndrome. Dogs can be discharged on the same day as brachycephalic obstructive airway syndrome surgery including split staphylectomy without an increased risk of complications.
Membrane proteins are traditionally extracted and purified in detergent for biochemical and structural characterisation. This process is often costly and laborious, and the stripping away of ...potentially stabilising lipids from the membrane protein of interest can have detrimental effects on protein integrity. Recently, styrene-maleic acid (SMA) co-polymers have offered a solution to this problem by extracting membrane proteins directly from their native membrane, while retaining their naturally associated lipids in the form of stable SMA lipid particles (SMALPs). However, the inherent nature and heterogeneity of the polymer renders their use challenging for some downstream applications – particularly mass spectrometry (MS). While advances in cryo-electron microscopy (cryo-EM) have enhanced our understanding of membrane protein:lipid interactions in both SMALPs and detergent, the resolution obtained with this technique is often insufficient to accurately identify closely associated lipids within the transmembrane annulus. Native-MS has the power to fill this knowledge gap, but the SMA polymer itself remains largely incompatible with this technique. To increase sample homogeneity and allow characterisation of membrane protein:lipid complexes by native-MS, we have developed a novel SMA-exchange method; whereby the membrane protein of interest is first solubilised and purified in SMA, then transferred into amphipols or detergents. This allows the membrane protein and endogenously associated lipids extracted by SMA co-polymer to be identified and examined by MS, thereby complementing results obtained by cryo-EM and creating a better understanding of how the lipid bilayer directly affects membrane protein structure and function.
•First reported exchange protocol for transferring membrane proteins solubilised in SMALPs, into detergent or amphipols.•Purification of protein:lipid complexes without detergent for mass spectrometry and subsequent lipid identification.•Cost effective membrane protein purification requiring only minimal amounts of detergents in the exchange process.
Regulators are considering reducing the nicotine content in cigarettes to a minimally addictive level. This could particularly benefit smokers from populations vulnerable to heavy smoking and ...difficulties quitting. We assessed predictors of adherence among adults from vulnerable populations assigned to use very low nicotine content cigarettes (VLNCs) in randomized clinical trials, to identify characteristics of those who require additional assistance if a nicotine reduction policy were implemented.
Data came from three populations of vulnerable adult smokers assigned to use VLNC cigarettes (0.4 mg/g nicotine) during 12-week randomized controlled trials (n = 286): Socioeconomically disadvantaged women of reproductive age, opioid-maintained adults, and adults with affective disorders. Logistic and linear regressions modeled predictors of adherence based on changes in cotinine at week-6 and week-12 assessments relative to baseline, and as a 90% reduction in cotinine relative to baseline (full adherence: yes/no). Predictors included satisfaction with study cigarettes, craving, nicotine dependence severity, withdrawal, population membership, baseline affective-disorder symptoms, and sociodemographic characteristics.
Dependence severity was negatively associated with both adherence measures at week 6 (p < .01), whereas increased satisfaction with study cigarettes and age were positively associated with both measures at weeks 6 and 12 (p < .01). Opioid-maintained adults exhibited reduced adherence and were less likely to reach full adherence at week 12 compared to disadvantaged women (p = .02).
Factors associated with VLNC adherence in vulnerable populations are similar to those in the general population of smokers. Furthermore, studies are indicated investigating nicotine supplements (e.g., e-cigarettes, NRT) to support highly dependent adults faced with using VLNCs.
This study identified factors predicting difficulty maintaining adherence to a regimen of very low nicotine content cigarettes (VLNC) among adults from vulnerable populations. Findings suggested that factors predicting difficulty maintaining adherence (greater nicotine dependence and low satisfaction with study-provided VLNC) were common across vulnerable smokers and the general population of adults who smoke. Furthermore, research should investigate alternatives to support highly dependent adults, such as pairing VLNC with supplemental, noncombusted nicotine. Some vulnerable populations (e.g., opioid-maintained adults) may be especially in need of supplemental, noncombusted nicotine.
Induction of CD8+ T cell responses against tumor cells and intracellular pathogens is an important goal of modern vaccinology. One approach of translational interest is the use of liposomes ...encapsulating pore-forming proteins (PFPs), such as Listeriolysin O (LLO), which has shown efficacy at priming strong and sustained CD8+ T cell responses. Recently, we have demonstrated that Sticholysin II (StII), a PFP from the sea anemone Stichodactyla helianthus, co-encapsulated into liposomes with ovalbumin (OVA) was able to stimulate, antigen presenting cells, antigen-specific CD8+ T cells and anti-tumor activity in mice. In the present study, we aimed to compare StII and LLO in terms of their abilities to stimulate dendritic cells and to induce major histocompatibility complex (MHC) class I restricted T cell responses against OVA. Interestingly, StII exhibited similar abilities to LLO in vitro of inducing dendritic cells maturation, as measured by increased expression of CD40, CD80, CD86 and MHC-class II molecules, and of stimulating OVA cross-presentation to a CD8+ T cell line. Remarkably, using an ex vivo Enzyme-Linked ImmunoSpot Assay (ELISPOT) to monitor gamma interferon (INF-γ) producing effector memory CD8+ T cells, liposomal formulations containing either StII or LLO induced comparable frequencies of OVA-specific INF-γ producing CD8+ T cells in mice that were sustained in time. However, StII-containing liposomes stimulated antigen-specific memory CD8+ T cells with a higher potential to secrete IFN-γ than liposomes encapsulating LLO. This StII immunostimulatory property further supports its use for the rational design of T cell vaccines against cancers and intracellular pathogens. In summary, this study indicates that StII has immunostimulatory properties similar to LLO, despite being evolutionarily distant PFPs.
•Sticholysin II (StII) and Listeriolysin O (LLO) induce comparable dendritic cell maturation and antigen cross-presentation.•Liposomes encapsulating ovalbumin (OVA) with either StII or LLO induce sustained and similar anti-OVA CD8+T response in vivo.•StII-liposomes stimulate memory CD8+T cells with a higher potential to secrete gamma interferon (IFN-γ) than LLO-liposomes.
Membrane proteins are essential for cellular growth, signalling and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them ...from the membrane creates challenges in their structural and functional study. Although amphipols have been very effective for single-particle electron cryo-microscopy (cryoEM) and mass spectrometry, they rely on initial detergent extraction before exchange into the amphipol environment. Therefore, circumventing this pre-requirement would be a big advantage. Here we use an alternative type of amphipol: a cycloalkane-modified amphiphile polymer (CyclAPol) to extract Escherichia coli AcrB directly from the membrane and demonstrate that the protein can be isolated in a one-step purification with the resultant cryoEM structure achieving 3.2 Å resolution. Together this work shows that cycloalkane amphipols provide a powerful approach for the study of membrane proteins, allowing native extraction and high-resolution structure determination by cryoEM.
Twelve ruminally cannulated crossbred Angus steers were used to evaluate ruminal fermentation characteristics and diet digestibility when 30% (DM) corn dried distillers grains with solubles (DDGS) ...containing 0.42 or 0.65% (DM) of dietary S was incorporated into finishing diets based on steam-flaked corn (SFC) or dry-rolled corn (DRC). The study was a replicated, balanced randomized incomplete block design with a 2 x 2 factorial arrangement of treatments. Factors consisted of dietary S concentration (0.42 and 0.65% of DM; 0.42S and 0.65S, respectively) and grain processing method (SFC or DRC). The 0.65S concentration was achieved by adding H2SO4 to DDGS before mixing rations. Steers were assigned randomly to diets and individual, slatted-floor pens, and fed once daily for ad libitum intake. Two 15-d experimental periods were used, each consisting of a 12-d diet adaptation phase and a 3-d sample collection phase. Samples were collected at 2-h intervals postfeeding during the collection phase. Ruminal pH was measured immediately after sampling, and concentrations of ruminal ammonia and VFA were determined. Fecal samples were composited by steer within period and used to determine apparent total tract digestibilities of DM, OM, NDF, CP, starch, and ether extract. Feeding 0.65S tended (P = 0.08) to decrease DMI but resulted in greater apparent total tract digestibilities of DM (P = 0.04) and ether extract (P = 0.03). Ruminal pH increased (P < 0.05) in steers fed 0.65S diets, which may be attributable, in part, to decreased (P = 0.05) VFA concentrations and greater (P < 0.01) ruminal ammonia concentrations when 0.65S was fed, compared with feeding 0.42S. These effects were more exaggerated in steers fed DRC (interaction, P < 0.01), compared with steers fed SFC. Steers fed DRC-0.65S had greater (P < 0.01) acetate concentration than steers fed DRC-0.42S, but acetate concentration was not affected by S concentration when SFC was fed. Propionate concentration was decreased (P < 0.01) in steers fed SFC-0.65S compared with steers fed SFC-0.42S, but dietary S concentration had no effect on propionate concentration when DRC was fed. Butyrate concentration was less (P < 0.01) in steers fed 0.65S diets than in steers fed 0.42S. Lactate concentrations tended (P = 0.06) to decrease in steers fed 0.65S diets. Feeding DDGS with increased S concentration may decrease feed intake and ruminal VFA concentration but increase ruminal ammonia concentration.
Two studies were conducted to evaluate effects of dried distillers grains with solubles (DDGS) and alfalfa hay (AH) or corn silage (CS) on feedlot performance, carcass characteristics, ruminal ...fermentation, and diet digestibility in cattle fed steam-flaked corn (SFC) diets. In trial 1, crossbred heifers (n = 358; BW = 353 ± 13 kg) were used in a finishing trial to evaluate interactions between corn-DDGS and roughage source (AH or CS) in terms of impact on feedlot performance and carcass characteristics. Experimental diets (DM basis) consisted of SFC and 11% CS without DDGS (SFC-CS), SFC and 11% CS with 25% DDGS (DDGS-CS), SFC and 6% AH without DDGS (SFC-AH), and SFC with 25% DDGS and 6% AH (DDGS-AH). Heifers were fed for ad libitum intake once daily for 97 d. Results indicated no interaction between DDGS and roughage source with respect to animal performance. Feeding DDGS did not affect ADG (P = 0.19), DMI (P = 0.14), or feed conversion (P = 0.67). Heifers fed CS had greater DMI than those fed AH (P = 0.05), but ADG (P = 0.56) and G:F (P = 0.63) were not different. There were no differences among treatments with respect to HCW, dressing percentage, subcutaneous fat thickness, quality grades, or yield grades (P > 0.20). Cattle fed CS tended (P = 0.10) to have greater marbling scores than those fed AH. There was an interaction (P = 0.02) between roughage and DDGS with respect to incidence of liver abscess. The greatest incidence was observed in cattle fed diets without DDGS when CS was fed, and the least was observed in cattle fed diets without DDGS when AH was used. In the second trial, ruminal fermentation characteristics and diet digestibility were examined in 12 cannulated Holstein steers fed similar diets to those fed in the finishing trial. Ruminal pH for all treatments was below 5.8 for 14 h after feeding. Acetate:propionate ratios were less (P = 0.02) in steers fed 25% DDGS but had greater (P = 0.02) ruminal lactate concentrations compared with cattle fed 0% DDGS. Feeding 25% DDGS decreased (P < 0.01) ruminal ammonia concentrations, and digestion of DM and OM was less (P < 0.01) compared with diets without DDGS. The decrease in digestibility was largely attributable to decreases in digestion of CP (P = 0.03) and NDF (P < 0.01). Feeding strategies aimed at increasing ruminal pH and ruminally available protein may improve digestion of DDGS in steam-flaked corn-based finishing diets.
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