Purification of MAP-kinase protein complexes and identification of candidate components by XL-TAP-MS.

The purification of low-abundance protein complexes and detection of in vivo protein-protein interactions in complex biological samples remains a challenging task. Here, we devised crosslinking and tandem affinity purification coupled to mass spectrometry (XL-TAP-MS), a quantitative proteomics approach for analyzing tandem affinity-purified, crosslinked protein complexes from plant tissues. We exemplarily applied XL-TAP-MS to study the MKK2-Mitogen-activated protein kinase (MPK4) signaling module in Arabidopsis thaliana. A tandem affinity tag consisting of an in vivo-biotinylated protein domain flanked by two hexahistidine sequences was adopted to allow for the affinity-based isolation of formaldehyde-crosslinked protein complexes under fully denaturing conditions. Combined with 15N stable isotopic labeling and tandem MS we captured and identified a total of 107 MKK2-MPK4 module-interacting proteins. Consistent with the role of the MPK signaling module in plant immunity, many of the module-interacting proteins are involved in the biotic and abiotic stress response of Arabidopsis. Validation of binary protein-protein interactions by in planta split-luciferase assays and in vitro kinase assays disclosed several direct phosphorylation targets of MPK4. Together, the XL-TAP-MS approach purifies low abundance protein complexes from biological samples and discovers previously unknown protein-protein interactions.

Dyadic Instruction for Middle School Students: Liking Promotes Learning.

This study examines whether friendship facilitates or hinders learning in a dyadic instructional setting. Working in 80 same-sex pairs, 160 (60 girls, 100 boys) middle school students (M = 12.13 years old) were taught a new computer programming language and programmed a game. Students spent 14 to 30 (M = 22.7) hours in a programming class. At the beginning and the end of the project, each participant separately completed (a) computer programming knowledge assessments and (b) questionnaires rating their affinity for their partner. Results support the proposition that liking promotes learning: Greater partner affinity predicted greater subsequent increases in computer programming knowledge for both partners. One partner's initial programming knowledge also positively predicted the other partner's subsequent partner affinity.

Compliance in Ambulatory Assessment Studies: Investigating Study and Sample Characteristics as Predictors.

Ambulatory assessment (AA) studies are becoming more and more popular. However, it can be challenging to motivate participants to comply with study protocols. The aim of the present study was to investigate possible predictors of compliance in AA studies with diverse samples and study designs. To do so, we extracted compliance information, study characteristics, and sample characteristics from 488 previously published studies. The average compliance across the studies was rather high. The total number of measurement occasions and the number of study days were negatively related to the compliance rate. Moreover, a higher percentage of healthy controls in clinical studies was associated with a higher compliance rate. By contrast, other study characteristics (e.g., the amount of financial compensation) and sample characteristics (clinical vs. healthy sample) were not related to compliance. The findings have implications for the design of future AA studies.

High-throughput Screening for Defense Priming-inducing Compounds in Parsley Cell Cultures.

Defense priming describes the enhanced potency of cells to activate defense responses. Priming accompanies local and systemic immune responses and can be triggered by microbial infection or upon treatment with certain chemicals. Thus, chemically activating defense priming is promising for biomedicine and agriculture. However, test systems for spotting priming-inducing chemicals are rare. Here, we describe a high-throughput screen for compounds that prime microbial pattern-spurred secretion of antimicrobial furanocoumarins in parsley culture cells. For the best possible throughput, we perform the assay with 1-ml aliquots of cell culture in 24-well microtiter plates. The advantages of the non-invasive test over competitive assays are its simplicity, remarkable reliability, and high sensitivity, which is based on furanocoumarin fluorescence in UV light.

No Increased Cardiac Mortality or Morbidity of Radiation Therapy in Breast Cancer Patients After Breast-Conserving Surgery: 20-Year Follow-up of the Randomized SweBCGRT Trial.

PURPOSE: Radiation therapy (RT) after breast-conserving surgery reduces locoregional recurrences and improves survival but may cause late side effects. The main purpose of this paper was to investigate long-term side effects after whole breast RT in a randomized clinical trial initiated in 1991 and to report dose-volume data based on individual 3-dimensional treatment plans for organs at risk. METHODS AND MATERIALS: The trial included 1187 patients with T1-2 N0 breast cancer randomized to postoperative tangential whole breast RT or no further treatment. The prescription dose to the clinical target volume was 48 to 54 Gy. We present 20-year follow-up on survival, cause of death, morbidity, and later malignancies. For a cohort of patients (n = 157) with accessible computed tomography-based 3-dimensional treatment plans in Dicom-RT format, dose-volume descriptors for organs at risk were derived. In addition, these were compared with dose-volume data for a cohort of patients treated with contemporary RT techniques. RESULTS: The cumulative incidence of cardiac mortality was 12.4% in the control group and 13.0% in the RT group (P = .8). There was an increase in stroke mortality: 3.4% in the control group versus 6.7% in the RT group (P = .018). Incidences of contralateral breast cancer and lung cancer were similar between groups. The median Dmean (range) heart dose for left-sided treatments was 3.0 Gy (1.1-8.1), and the corresponding value for patients treated in 2017 was 1.5 Gy (0.4-6.0). CONCLUSIONS: In this trial, serious late side effects of whole breast RT were limited and less than previously reported in large meta-analyses. We observed no increase in cardiac mortality in irradiated patients. Doses to the heart were a median Dmean of 3.0 Gy for left-sided RT. The observed increase in stroke mortality may partly be secondary to cardiac side effects, complications to anticoagulant treatment, or to chance, rather than a direct side effect of tangential whole breast irradiation.

Evaluation of surrogate markers for passive transfer of immunity in kittens.

OBJECTIVE: To identify surrogate markers of passive transfer of immunity in kittens. DESIGN: Prospective clinical trial. ANIMALS: 55 kittens from 12 specific-pathogen-free queens. PROCEDURE: Kittens were allocated at birth into colostrum-fed (n = 27) and colostrum-deprived (28) groups. Blood was collected at birth and on days 1, 2, 4, 7, 14, 28, and 56. Serum samples were analyzed for activities of alkaline phosphatase (ALP), alanine aminotransferase, aspartate aminotransferase, creatine kinase, lactate dehydrogenase, gamma-glutamyltransferase, amylase, and lipase and for concentrations of albumin, total protein, bilirubin, urea nitrogen, creatinine, cholesterol, glucose, calcium, phosphorus, and triglycerides by use of an automated analyzer. Total serum solids concentrations were estimated by use of refractometry. Serum IgG concentrations were quantified by use of radial immunodiffusion. RESULTS: All kittens were agammaglobulinemic at birth. Colostrum-fed kittens had significantly higher IgG concentrations than did colostrum-deprived kittens from 1 though 28 days of age. Transient significant differences in serum biochemical variables between the colostrum-deprived and colostrum-fed groups were substantially resolved by day 4. Passive transfer of immunity could be reliably determined at 1 day of age and to a lesser extent at 2 days of age only by measurement of serum activity of ALP. CONCLUSIONS AND CLINICAL RELEVANCE: Adequacy of passive transfer in kittens initially correlated with serum activity of ALP, but quantification of serum IgG concentration was necessary after 2 days of age.

Effect of age on reference intervals of serum biochemical values in kittens.

OBJECTIVE: To determine the effect of age on reference intervals of serum biochemical values in kittens. DESIGN: Prospective clinical trial. ANIMALS: 55 kittens from 12 specific-pathogen-free queens. PROCEDURE: Kittens were allocated at birth into colostrum-fed (n = 27) and colostrum-deprived (28) groups. Blood was collected at birth and on days 1, 2, 4, 7, 14, 28, and 56. Serum samples were analyzed for activities of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, creatine kinase, lactate dehydrogenase, gamma-glutamyltransferase, amylase, and lipase and for concentrations of albumin, total protein, bilirubin, urea nitrogen, creatinine, cholesterol, glucose, calcium, phosphorus, and triglycerides by use of an automated analyzer. Total serum solids concentrations were determined by use of refractometry. Serum IgG concentrations were quantified by use of radial immunodiffusion. RESULTS: For several analytes, reference intervals changed rapidly, most notably during the first few days of life. Reference intervals for alkaline phosphatase, creatine kinase, lactate dehydrogenase, and triglycerides were higher from birth to 8 weeks than adult reference intervals. Aspartate aminotransferase, bilirubin, urea nitrogen, and creatinine were higher than in adults at birth but were similar to or lower than adult reference intervals by 8 weeks. Compared with adult reference intervals, reference intervals for calcium and phosphorus concentrations were higher and for albumin and total protein concentrations were lower throughout the study period. CONCLUSIONS AND CLINICAL RELEVANCE: Important differences exist between reference intervals for serum biochemical values of neonatal and adult cats. Age-appropriate reference intervals should be used for accurate assessment of serum biochemical test results in cats.

Systemic lupus erythematosus in a cat: fulfillment of the American Rheumatism Association criteria with supportive skin histopathology.

Systemic lupus erythematosus (SLE) was diagnosed in a 9-year-old castrated male Persian cat. The cat described is the first to fulfil four of 11 American Rheumatism Association criteria for the diagnosis of SLE in humans (symmetrical facial dermatitis, thrombocytopenia, positive antinuclear antibodies, oral ulceration) with supportive skin histopathology. Haematological abnormalities included a mild anaemia and severe thrombocytopenia. Skin disease consisted of symmetrical multifocal alopecia with crusting, predominantly on the face. Histopathology of the skin revealed interface dermatitis and interface folliculitis with follicular atrophy. Complete remission was obtained with corticosteroid therapy.

Factor X deficiency in a Jack Russell terrier.

Deficiency in factor X (Stuart-Prower factor) was identified in a 7-month-old spayed female Jack Russell Terrier following recurrent bleeding episodes. Various relatives were screened for factor X deficiency and low and subnormal levels were identified in the father and paternal grandmother, respectively. Factor X deficiency has been previously documented in a family of American Cocker Spaniels, in which the inheritance pattern appeared to be an autosomal dominant trait with variable expression. This is the first report describing this coagulopathy in the Jack Russell Terrier.