Stimulation by mammalian insulin of glycogen metabolism in a wall-less strain of Neurospora crassa

Addition of bovine insulin to cells of the wall-less variant FGSC4761 of Neurospora crassa ("slime") produced several significant effects on glycogen metabolism. 1) Intracellular levels of the glycogen precursor UDP-glucose decreased 17-18% (P less than 0.01) within 30 min of insulin addition. 2) Cells grown with insulin possessed 40% more glycogen than did control cells. 3) The incorporation of 14C-labeled glucose into glycogen increased 41% after 30-min treatment with 100 nM bovine insulin (P less than 0.01). 4) Insulin treatment of the cells caused activation of the enzyme glycogen synthase from a glucose-6-phosphate-dependent form to an independent form. Half-maximum activation occurred with 2 nM insulin. These are similar to insulin-induced effects in some mammalian cells. In contrast, no insulin-induced effect on glucose transport could be demonstrated in these cells.     

Advances in allergen-specific immune cell measurements for improved detection of allergic sensitization and immunotherapy responses

Over the past two decades, precision medicine has advanced diagnostics and treatment of allergic diseases. Component-resolved analysis of allergen sensitization facilitates stratification of patients. Furthermore, new formulations of allergen immunotherapy (AIT) products can more effectively deliver the relevant components. Molecular insights from the identification of allergen component sensitization and clinical outcomes of treatment with new AIT formulations can now be utilized for a deeper understanding of the nature of the pathogenic immune response in allergy and how this can be corrected by AIT. Fundamental in these processes are the allergen-specific B and T cells. Within the large B- and T-cell compartments, only those that specifically recognize the allergen with their immunoglobulin (Ig) or T-cell receptor (TCR), respectively, are of clinical relevance. With peripheral blood allergen-specific B- and T-cell frequencies below 1%, bulk cell analysis is typically insufficiently sensitive. We here review the latest technologies to detect allergen-specific B and T cells, as well as new developments in utilizing these tools for diagnostics and therapy monitoring to advance precision medicine for allergic diseases.     

Effect of “Speak Up” educational tools to engage patients in advance care planning in outpatient healthcare settings: A prospective before-after study

BackgroundTools for advance care planning (ACP) are advocated to help ensure patient values guide healthcare decisions. Evaluation of the effect of tools introduced to patients in clinical settings is needed.ObjectiveTo evaluate the effect of the Canadian Speak Up Campaign tools on engagement in advance care planning (ACP), with patients attending outpatient clinics.Patient involvement: Patients were not involved in the problem definition or solution selection in this study but members of the public were involved in development of tools. The measurement of impacts involved patients.MethodsThis was a prospective pre-post study in 15 primary care and two outpatient cancer clinics. The outcome was scores on an Advance Care Planning Engagement Survey measuring Behavior Change Process on 5-point scales and Actions (0?21-point scale) administered before and six weeks after using a tool, with reminders at two or four weeks.Results177 of 220 patients (81%) completed the study (mean 68 years of age, 16% had cancer). Mean Behavior Change Process scores were 2.9 at baseline and 3.5 at follow-up (mean change 0.6, 95% confidence interval 0.5 to 0.7; large effect size of 0.8). Mean Action Measure score was 3.7 at baseline and 4.8 at follow-up (mean change 1.1, 95% confidence interval 0.6–1.5; small effect size of 0.2).Practical valuePublicly available ACP tools may have utility in clinical settings to initiate ACP among patients. More time and motivation may be required to stimulate changes in patient behaviors related to ACP.     

The Utility of Functional Data Analyses to Reveal Between-Limbs Asymmetries in Those With a History of Anterior Cruciate Ligament Reconstruction

ContextResearchers have traditionally used motion capture to quantify discrete data points (peak values) during hop testing. However, these analyses restrict the evaluation to a single time point (ie, certain percentage of stance) and provide only a narrow view of movement. Applying more comprehensive analyses may help investigators identify important characteristics that are masked by discrete analyses often used to screen patients for activity.ObjectiveTo examine the utility of functional data analyses to reveal asymmetries that are undetectable using discrete (ie, single time point) evaluations in participants with a history of anterior cruciate ligament reconstruction (ACLR) who achieved clinical hop symmetry.DesignCross-sectional study.SettingLaboratory.Patients or Other ParticipantsFifteen participants with unilateral ACLR (age = 21 ± 3 years, time from surgery = 4 ± 3 years) and 15 control participants without ACLR (age = 23 ± 2 years).Intervention(s)Lower extremity biomechanics during the triple–hop-for-distance task for the ACLR and contralateral limbs of patients and a representative limb of control participants were measured.Main Outcome Measure(s)Peak sagittal-plane joint power, joint work, and power profiles were determined.ResultsUsing discrete analyses, we identified lower peak knee power and work in the ACLR limb compared with the contralateral and control limbs (P < .05) but were unable to demonstrate differences at the ankle or hip. Using functional data analyses, we observed asymmetries at the ankle, knee, and hip between the ACLR and contralateral or control limbs throughout stance (P < .05), and it was revealed that these asymmetries stemmed from knee power deficits that were prominent during early loading.ConclusionsDespite achieving hop-distance symmetry, the ACLR knees absorbed less power. Although this information was revealed using discrete analyses, underlying asymmetries at the ankle and hip were masked. Using functional data analyses, we found interlimb asymmetries at the ankle, knee, and hip. Importantly, we found that functional data analyses more fully elucidated the extent and source of asymmetries, which can be used by clinicians and researchers alike to aid in clinical decision making.     

Kinematic pharyngeal transit times in myopathy: Evaluation for dysphagia

Measurement of kinematic pharyngeal transit times, a new videofluoroscopy technique, provides useful quantitative data to supplement the qualitative data previously available from videofluoroscopy swallowing studies. Kinematic pharyngeal transit times have not previously been reported for subjects with myopathy. This study demonstrates the use of quantitative kinematic pharyngeal transit times for dysphagia evaluation in 15 patients with myopathy. The successful treatment of dysphagia by cricopharyngeal myotomy is reported in two patients with limb-girdle syndrome.     

Identification of a Major Epitope Recognized by PLA2R Autoantibodies in Primary Membranous Nephropathy

Phospholipase A₂ receptor 1 (PLA2R) is a target autoantigen in 70% of patients with idiopathic membranous nephropathy. We describe the location of a major epitope in the N-terminal cysteine-rich ricin domain of PLA2R that is recognized by 90% of human anti-PLA2R autoantibodies. The epitope was sensitive to reduction and SDS denaturation in the isolated ricin domain and the larger fragment containing the ricin, fibronectin type II, first and second C-type lectin domains (CTLD). However, in nondenaturing conditions the epitope was protected against reduction in larger fragments, including the full-length extracellular region of PLA2R. To determine the composition of the epitope, we isolated immunoreactive tryptic fragments by Western blotting and analyzed them by mass spectrometry. The identified peptides were tested as inhibitors of autoantibody binding to PLA2R by surface plasmon resonance. Two peptides from the ricin domain showed strong inhibition, with a longer sequence covering both peptides (31-mer) producing 85% inhibition of autoantibody binding to PLA2R. Anti-PLA2R antibody directly bound this 31-mer peptide under nondenaturing conditions and binding was sensitive to reduction. Analysis of PLA2R and the PLA2R-anti-PLA2R complex using electron microscopy and homology-based representations allowed us to generate a structural model of this major epitope and its antibody binding site, which is independent of pH-induced conformational change in PLA2R. Identification of this major PLA2R epitope will enable further therapeutic advances for patients with idiopathic membranous nephropathy, including antibody inhibition therapy and immunoadsorption of circulating autoantibodies.     

Thrombopoietin following transfusion of platelets in preterm neonates

Thrombocytopenia is common in the neonatal intensive care unit. Transfusion of platelets is often required. The purpose of our study was to determine changes in thrombopoietin (Tpo) following transfusion of platelets in preterm neonates. Preterm neonates undergoing platelet transfusion were randomized to receive a transfusion volume of either 10 or 15 ml/kg. Blood was obtained for Tpo measurement pre-transfusion, one and 24 hours post-transfusion. Platelet Factor 4 (PF4) was also measured to quantify platelet activation. Statistical analysis was performed using repeated measures ANOVA, and Mann-Whitney U test as appropriate. Ten infants were enrolled in each group. Gestational age, birth weight, etiology of thrombocytopenia, and timing of transfusion did not differ between the 10 and 15 ml/kg groups. There were no differences between the groups in platelet count prior to and/or following transfusion. Both transfusion volumes were equally well tolerated. Tpo and PF4 did not differ between groups at any of the study time points. When both groups were analysed together, Tpo dropped 43% (95% confidence 37-49%, p = 0.01) 1-hour post compared to pre-transfusion. In conclusion the observed decrease in Tpo following platelet transfusion suggests that Tpo kinetics in neonates is similar to adults following transfusion. PF4 was not affected by transfusion. There was not an increase in platelet count following transfusion volume of 15 ml/kg compared to 10 ml/kg.