Journal Articles

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    N-Methyl-D-Aspartate Receptor Hypofunction in Meg-01 Cells Reveals a Role for Intracellular Calcium Homeostasis in Balancing Megakaryocytic-Erythroid Differentiation
    (Thieme Gruppe, 2020-04-14) Hearn JI; Green TN; Chopra M; Nursalim YNS; Ladvanszky L; Knowlton N; Blenkiron C; Poulsen RC; Singleton DC; Bohlander SK; Kalev-Zylinska ML
    The release of calcium ions (Ca2+) from the endoplasmic reticulum (ER) and related store-operated calcium entry (SOCE) regulate maturation of normal megakaryocytes. The N-methyl-D-aspartate (NMDA) receptor (NMDAR) provides an additional mechanism for Ca2+ influx in megakaryocytic cells, but its role remains unclear. We created a model of NMDAR hypofunction in Meg-01 cells using CRISPR-Cas9 mediated knockout of the GRIN1 gene, which encodes an obligate, GluN1 subunit of the NMDAR. We found that compared with unmodified Meg-01 cells, Meg-01-GRIN1 -/- cells underwent atypical differentiation biased toward erythropoiesis, associated with increased basal ER stress and cell death. Resting cytoplasmic Ca2+ levels were higher in Meg-01-GRIN1 -/- cells, but ER Ca2+ release and SOCE were lower after activation. Lysosome-related organelles accumulated including immature dense granules that may have contributed an alternative source of intracellular Ca2+. Microarray analysis revealed that Meg-01-GRIN1 -/- cells had deregulated expression of transcripts involved in Ca2+ metabolism, together with a shift in the pattern of hematopoietic transcription factors toward erythropoiesis. In keeping with the observed pro-cell death phenotype induced by GRIN1 deletion, memantine (NMDAR inhibitor) increased cytotoxic effects of cytarabine in unmodified Meg-01 cells. In conclusion, NMDARs comprise an integral component of the Ca2+ regulatory network in Meg-01 cells that help balance ER stress and megakaryocytic-erythroid differentiation. We also provide the first evidence that megakaryocytic NMDARs regulate biogenesis of lysosome-related organelles, including dense granules. Our results argue that intracellular Ca2+ homeostasis may be more important for normal megakaryocytic and erythroid differentiation than currently recognized; thus, modulation may offer therapeutic opportunities.
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    Requirement of digestible calcium at different dietary concentrations of digestible phosphorus for broiler chickens 3. Broiler finishers (d 25 to 35 post-hatch)
    (y Elsevier Inc (USA), on behalf of Poultry Science Association Inc, 2023-04) David LS; Abdollahi MR; Bedford MR; Ravindran V
    An experiment was conducted to determine the digestible calcium (Ca) and digestible phosphorous (P) requirements of 25 to 35-day-old broiler chickens. Fifteen corn-soybean meal-based diets containing 2.0, 2.5, 3.0, 3.5, and 4.0 g/kg standardized ileal digestible (SID) Ca and 2.5, 3.5, and 4.5 g/kg SID P were fed to broilers from d 25 to 35 post-hatch. Each experimental diet was randomly allocated to 6 replicate cages (8 birds per cage). Body weight and feed intake were recorded, and the feed conversion ratio was calculated. On d 35, birds were euthanized to collect the ileal digesta, tibia, and carcass for the determination of ileal Ca, and P digestibility, concentrations of ash, Ca, and P in tibia and the retention of Ca and P in the carcass. Titanium dioxide (5.0 g/kg) was included in all diets as an indigestible indicator for the ileal digestibility measurement. Feed intake and total excreta output were measured during the last 4 d of the experimental period for the measurement of apparent total tract retention of Ca and P. Fixed effects of the experiment were dietary concentrations of SID Ca and SID P and their interaction. If the interaction or main effects were significant (P < 0.05), the parameter estimates for second-order response surface model (RSM) were determined using General Linear Model procedure of SAS. The maximum response was not predicted for most of the parameters (including growth performance and tibia) as the Ca effect was linear which indicated that the highest level of Ca employed in the study may have not been high enough. The requirement of dietary SID Ca for maximization of these parameters, therefore, depends on the dietary SID P concentration when the dietary SID Ca is within 2.0 to 4.0 g/kg. However, based on the factorial analysis, the highest weight gain was observed at 3.5 g/kg SID P and 3.5 g/kg SID Ca concentrations. Tibia ash was higher in birds fed 4.5 g/kg SID P and was unaffected by dietary SID Ca concentrations. However, based on overall findings, a combination of 3.5 g/kg SID P and 3.0-3.5 g/kg SID Ca may be recommended for the optimum tibia ash. The recommended SID Ca requirements (at 3.5 g/kg SID P) for weight gain (3.5 g/kg or 6.4 g/kg total Ca) and tibia ash (3.0-3.5 g/kg or 5.5-6.4 g/kg total Ca) are lower than the current Ca recommendations (7.8 g/kg total Ca equivalent to 4.25 g/kg SID Ca; Ross, 2019) for broiler finishers, suggesting possible excess of Ca in diets formulated based on the current recommendation.
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    pH-dependent sedimentation and protein interactions in ultra-high-temperature-treated sheep skim milk
    (Elsevier Inc and Fass Inc on behalf of the American Dairy Science Association, 2023-03) Pan Z; Ye A; Dave A; Fraser K; Singh H
    Sheep milk is considered unstable to UHT processing, but the instability mechanism has not been investigated. This study assessed the effect of UHT treatment (140°C/5 s) and milk pH values from 6.6 to 7.0 on the physical properties of sheep skim milk (SSM), including heat coagulation time, particle size, sedimentation, ionic calcium level, and changes in protein composition. Significant amounts of sediment were found in UHT-treated SSM at the natural pH (∼6.6) and pH 7.0, whereas lower amounts of sediment were observed at pH values of 6.7 to 6.9. The proteins in the sediment were mainly κ-casein (CN)-depleted casein micelles with low levels of whey proteins regardless of the pH. Both the pH and the ionic calcium level of the SSM at all pH values decreased after UHT treatment. The dissociation levels of κ-, β-, and αS2-CN increased with increasing pH of the SSM before and after heating. The protein content, ionic calcium level, and dissociation level of κ-CN were higher in the SSM than values reported previously in cow skim milk. These differences may contribute to the high amounts of sediment in the UHT-treated SSM at natural pH (∼6.6). Significantly higher levels of κ-, β-, and αS2-CN were detected in the serum phase after heating the SSM at pH 7.0, suggesting that less κ-CN was attached to the casein micelles and that more internal structures of the casein micelles may have been exposed during heating. This could, in turn, have destabilized the casein micelles, resulting in the formation of protein aggregates and high amounts of sediment after UHT treatment of the SSM at pH 7.0.
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    Requirement of digestible calcium at different dietary concentrations of digestible phosphorus for broiler chickens. 2. Broiler growers (d 11 to 24 post-hatch)
    (Elsevier Inc. on behalf of Poultry Science Association Inc., 2022-11) David LS; Abdollahi MR; Bedford MR; Ravindran V
    An experiment was conducted to determine the digestible calcium (Ca) and digestible phosphorous (P) requirements of 11 to 24 d old broiler chickens. Eighteen corn-soybean meal-based diets containing 1.80, 2.35, 2.90, 3.45, 4.00, and 4.55 g/kg standardized ileal digestible (SID) Ca and 3.5, 4.5, and 5.5 g/kg SID P were fed to broilers from d 11 to 24. Each experimental diet was randomly allocated to six replicate cages (8 birds per cage). Body weight and feed amount were recorded at the start and end of the experiment and the feed conversion ratio was calculated. On d 24, birds were euthanized to collect ileal digesta, tibia, and carcass for the determination of digestible Ca and P, the concentration of ash, Ca and P in tibia and the retention of Ca and P in the carcass, respectively. Titanium dioxide (5 g/kg) was included in all diets as an indigestible indicator for apparent ileal digestibility measurement. Total excreta output was measured during the last 4 d of the experimental period for the measurement of apparent total tract retention of Ca and P. Fixed effects of the experiment were dietary concentrations of SID Ca and SID P and their interaction. If the interaction or main effect was significant (P < 0.05), the parameter estimate for second-order response surface model was determined using General Linear Model procedure of SAS. The weight gain of broiler growers was optimized at the SID P concentration of 3.5 g/kg and SID Ca concentrations between 2.35 and 4.00 g/kg. At 3.5 g/kg SID P concentration, the required SID Ca for maximum weight gain was determined to be 3.05 g/kg, which corresponded to SID Ca to SID P ratios of 0.87. The concentration of SID Ca that maximized tibia ash at 3.5 g/kg SID P was 3.69 g/kg, which corresponded to SID Ca to SID P ratio of 1.05. Maximizing bone ash requires more Ca than maximizing weight gain. Carcass Ca and P retention were reflective of total tract Ca and P retention values. The estimated SID Ca requirements (at 3.5 g/kg SID P) for both maximized weight gain (3.05 g/kg or 6.11 g/kg total Ca) and bone ash (3.69 g/kg or 7.28 g/kg total Ca) are lower than the current Ca recommendation (8.70 g/kg total Ca equivalent to 4.03 g/kg SID Ca; Ross, 2019) for broiler growers, indicating possible oversupply of Ca in diets formulated based on the current recommendation.
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    50 Years of the steric-blocking mechanism in vertebrate skeletal muscle: a retrospective
    (Springer Nature Switzerland AG, 2023-09) Parry DAD
    Fifty years have now passed since Parry and Squire proposed a detailed structural model that explained how tropomyosin, mediated by troponin, played a steric-blocking role in the regulation of vertebrate skeletal muscle. In this Special Issue dedicated to the memory of John Squire it is an opportune time to look back on this research and to appreciate John’s key contributions. A review is also presented of a selection of the developments and insights into muscle regulation that have occurred in the years since this proposal was formulated.