The use of circulating cell-free DNA (cfDNA) as a biomarker in transplant recipients offers advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection and immunosuppression optimization. However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant recipient plasma is low and challenging to quantify. Previously reported methods to measure dd-cfDNA require donor and recipient genotyping, which is impractical in clinical settings and adds cost. We developed a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms to accurately quantify dd-cfDNA in transplant recipients without separate genotyping. Analytical performance of the assay was characterized and validated using 1117 samples comprising the National Institute for Standards and Technology Genome in a Bottle human reference genome, independently validated reference materials, and clinical samples. The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across the linear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision. Application of the assay to clinical samples from heart transplant recipients demonstrated increased levels of dd-cfDNA in patients with biopsy-confirmed rejection and decreased levels of dd-cfDNA after successful rejection treatment. This noninvasive clinical-grade sequencing assay can be completed within 3 days, providing the practical turnaround time preferred for transplanted organ surveillance.
The daily and within-day variation in udder temperature was monitored in dairy cows (n = 10) using infrared thermography (IRT). The initial assessment and prediction of udder surface temperature variation would hopefully form the basis for future development of an early detection method for mastitis. Our initial objective was to determine the magnitude and pattern of udder temperature variation. To accomplish this, we measured daily fluctuations in udder temperature and the influence of environmental factors upon these values in non-mastitic cows. Udder temperature rose significantly after an exercise period (P < 0.05). Withinday monitoring of udder temperature demonstrated there was a distinct circadian rhythm. Lag regression analysis showed that previous daily udder temperatures together with environmental temperature parameters could successfully predict current udder temperature with a high degree of accuracy. The variation between predicted and actual udder temperature was within the detectable range for an inflammatory response. Infrared thermography shows promise in its application if coupled with environmental temperature monitoring as an early detection method for mastitis.Key words: Thermography, dairy cattle, environment, temperature Berry, R. J., Kennedy, A. D., Scott, S. L., Kyle, B. L. et Schaefer, A. L. 2003. Détermination de la variation quotidienne de la température à la surface du pis des vaches laitières par thermographie infrarouge : utilité potentielle pour le dépistage de la mammite. Can. J. Anim. Sci. 83: 687-693. Les auteurs ont examiné la variation quotidienne et intra-quotidienne de la température du pis des vaches laitières (n = 10) par thermographie infrarouge (TI). En effet, une première évaluation et la prévision subséquente de la température à la surface du pis pourraient aboutir à l'élaboration d'une technique de dépistage précoce de la mammite. L'objectif initial consistait à établir l'ampleur d'une telle variation et ses particularités. Pour cela, les auteurs ont mesuré les fluctuations quotidiennes de la température du pis et l'influence de divers facteurs environnementaux chez des vaches ne souffrant pas de la mammite. La température du pis grimpe sensiblement (P < 0,05) après une période d'activité. Dans la journée, elle suit un rythme nettement circadien. L'analyse de régression avec retard indique qu'on pourrait utiliser la température du jour antérieur et les paramètres de la température ambiante pour prévoir la température courante avec une grande précision. L'écart entre la température prévue du pis et la température réelle est assez faible pour qu'on détecte une inflammation. La TI est une technique prometteuse pour le dépistage précoce de la mammite si on la combine à la surveillance des paramètres de la température ambiante.
Hooves of 16 lactating Holstein cows were examined twice for sole hemorrhages and underrun heels. Images of hooves were taken using infrared thermography to determine the temperatures of the coronary band and that of a control area above the coronary band. To adjust for skin (control) temperature, the difference (DeltaT) between the coronary band and the control area was calculated. Effects of stage of lactation, that is,
Smooth muscle cells convert between a motile, proliferative “synthetic” phenotype and a sessile, “contractile” phenotype. The ability to manipulate the phenotype of aortic smooth muscle cells with thin biocompatible polyelectrolyte multilayers (PEMUs) with common surface chemical characteristics but varying stiffness was investigated. The stiffness of (PAH/PAA) PEMUs was varied by heating to form covalent amide bond cross-links between the layers. Atomic force microscopy (AFM) showed that cross-linked PEMUs were thinner than those that were not cross-linked. AFM nanoindentation demonstrated that the Young’s modulus ranged from 6 MPa for hydrated native PEMUs to more than 8 GPa for maximally cross-linked PEMUs. Rat aortic A7r5 smooth muscle cells cultured on native PEMUs exhibited morphology and motility of synthetic cells and expression of the synthetic phenotype markers vimentin, tropomyosin 4, and nonmuscle myosin heavy chain IIB (nmMHCIIB). In comparison, cells cultured on maximally cross-linked PEMUs exhibited the phenotype markers calponin, smooth muscle myosin heavy chain (smMHC), myocardin, transgelin, and smooth muscle α-actin (smActin) that are characteristic of the smooth muscle “contractile” phenotype. Consistent with those cells being “contractile”, A7r5 cells grown on cross-linked PEMUs produced contractile force when stimulated with a Ca2+ ionophore.
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