Role of band 3 tyrosine phosphorylation in the regulation of erythrocyte glycolysis.

Previous studies demonstrated that the in vitro tyrosine phosphorylation of the human erythrocyte anion transporter, band 3, prevented the binding of various glycolytic enzymes to the N terminus of the cytoplasmic tail. Since these enzymes are inhibited in their bound state, the functional consequences of band 3 tyrosine phosphorylation in the red cell should be to activate the enzymes and elevate glycolysis. We searched for various enhancers of band 3 tyrosine phosphorylation using a novel assay designed to measure the phosphotyrosine levels at the band 3 tyrosine phosphorylation/glycolytic enzyme-binding site. This assay measures the extent of phosphorylation of a synthetic band 3 peptide entrapped within resealed red cells. Using this assay, three distinct compounds, all mild oxidants, were found to stimulate the tyrosine phosphorylation of band 3. All three compounds were also found to elevate glycolytic rates in intact erythrocytes. Moreover, the antitumor drug adriamycin was found to coordinately prevent these agents from stimulating both band 3 tyrosine phosphorylation and erythrocyte glycolysis. These results suggest a possible function for a protein tyrosine kinase in human erythrocytes, to regulate glycolysis through the tyrosine phosphorylation of band 3

Elevated Phospholipase A2 Activities in Plasma Samples from Multiple Cancers.

Only in recent years have phospholipase A2 enzymes (PLA2s) emerged as cancer targets. In this work, we report the first detection of elevated PLA2 activities in plasma from patients with colorectal, lung, pancreatic, and bladder cancers as compared to healthy controls. Independent sets of clinical plasma samples were obtained from two different sites. The first set was from patients with colorectal cancer (CRC; n = 38) and healthy controls (n = 77). The second set was from patients with lung (n = 95), bladder (n = 31), or pancreatic cancers (n = 38), and healthy controls (n = 79). PLA2 activities were analyzed by a validated quantitative fluorescent assay method and subtype PLA2 activities were defined in the presence of selective inhibitors. The natural PLA2 activity, as well as each subtype of PLA2 activity was elevated in each cancer group as compared to healthy controls. PLA2 activities were increased in late stage vs. early stage cases in CRC. PLA2 activities were not influenced by sex, smoking, alcohol consumption, or body-mass index (BMI). Samples from the two independent sites confirmed the results. Plasma PLA2 activities had approximately 70% specificity and sensitivity to detect cancer. The marker and targeting values of PLA2 activity have been suggested

Which exercise and behavioural interventions show most promise for treating fatigue in multiple sclerosis? A network meta-analysis

Fatigue is a common, debilitating symptom of multiple sclerosis (MS) without a current standardised treatment. The aim of this systematic review with network meta-analyses was to estimate the relative effectiveness of both fatigue-targeted and non-targeted exercise, behavioural and combined (behavioural and exercise) interventions. Nine electronic databases up to August 2018 were searched, and 113 trials (n = 6909) were included: 34 were fatigue-targeted and 79 non-fatigue-targeted trials. Intervention characteristics were extracted using the Template for Intervention Description and Replication guidelines. Certainty of evidence was assessed using GRADE. Pairwise meta-analyses showed that exercise interventions demonstrated moderate to large effects across subtypes regardless of treatment target, with the largest effect for balance exercise (SMD = 0.84). Cognitive behavioural therapies (CBTs) showed moderate to large effects (SMD = 0.60), with fatigue-targeted treatments showing larger effects than those targeting distress. Network meta-analysis showed that balance exercise performed significantly better compared to other exercise and behavioural intervention subtypes, except CBT. CBT was estimated to be superior to energy conservation and other behavioural interventions. Combined exercise also had a moderate to large effect. Treatment recommendations for balance and combined exercise are tentative as the certainty of the evidence was moderate. The certainty of the evidence for CBT was high.Multiple Sclerosis Societ

Relationship between molecular pathogen detection and clinical disease in febrile children across Europe: a multicentre, prospective observational study

BackgroundThe PERFORM study aimed to understand causes of febrile childhood illness by comparing molecular pathogen detection with current clinical practice.MethodsFebrile children and controls were recruited on presentation to hospital in 9 European countries 2016-2020. Each child was assigned a standardized diagnostic category based on retrospective review of local clinical and microbiological data. Subsequently, centralised molecular tests (CMTs) for 19 respiratory and 27 blood pathogens were performed.FindingsOf 4611 febrile children, 643 (14%) were classified as definite bacterial infection (DB), 491 (11%) as definite viral infection (DV), and 3477 (75%) had uncertain aetiology. 1061 controls without infection were recruited. CMTs detected blood bacteria more frequently in DB than DV cases for N. meningitidis (OR: 3.37, 95% CI: 1.92-5.99), S. pneumoniae (OR: 3.89, 95% CI: 2.07-7.59), Group A streptococcus (OR 2.73, 95% CI 1.13-6.09) and E. coli (OR 2.7, 95% CI 1.02-6.71). Respiratory viruses were more common in febrile children than controls, but only influenza A (OR 0.24, 95% CI 0.11-0.46), influenza B (OR 0.12, 95% CI 0.02-0.37) and RSV (OR 0.16, 95% CI: 0.06-0.36) were less common in DB than DV cases. Of 16 blood viruses, enterovirus (OR 0.43, 95% CI 0.23-0.72) and EBV (OR 0.71, 95% CI 0.56-0.90) were detected less often in DB than DV cases. Combined local diagnostics and CMTs respectively detected blood viruses and respiratory viruses in 360 (56%) and 161 (25%) of DB cases, and virus detection ruled-out bacterial infection poorly, with predictive values of 0.64 and 0.68 respectively.InterpretationMost febrile children cannot be conclusively defined as having bacterial or viral infection when molecular tests supplement conventional approaches. Viruses are detected in most patients with bacterial infections, and the clinical value of individual pathogen detection in determining treatment is low. New approaches are needed to help determine which febrile children require antibiotics.FundingEU Horizon 2020 grant 668303

Nucleocytoplasmic Trafficking of the Syk Protein Tyrosine Kinase

The protein tyrosine kinase Syk couples the B-cell receptor (BCR) for antigen to multiple intracellular signaling pathways and also modulates cellular responses to inducers of oxidative stress in a receptor-independent fashion. In B cells, Syk is found in both the nuclear and cytoplasmic compartments but contains no recognizable nuclear localization or export signals. Through the analysis of a series of deletion mutants, we identified the presence of an unconventional shuttling sequence near the junction of the catalytic domain and the linker B region that accounts for Syk's subcellular localization. This localization is altered following prolonged engagement of the BCR, which causes Syk to be excluded from the nucleus. Nuclear exclusion requires the receptor-mediated activation of protein kinase C and new protein synthesis. Both of these processes also potentiate the activation of caspase 3 in cells in response to oxidative stress in a manner that is dependent on the localization of Syk outside of the nucleus. In contrast, restriction of Syk to the nucleus greatly diminishes the stress-induced activation of caspase 3

The SH3 Domain of Lck Modulates T-Cell Receptor-Dependent Activation of Extracellular Signal-Regulated Kinase through Activation of Raf-1▿

Engagement of the T-cell antigen receptor (TCR) results in the proximal activation of the Src family tyrosine kinase Lck. The activation of Lck leads to the downstream activation of the Ras/Raf/MEK/ERK signaling pathway (where ERK is extracellular signal-related kinase). Under conditions of weak, but not strong, stimulation through the TCR, a version of Lck that contains a single point mutation in the SH3 (Src homology 3) domain (W97ALck) fails to support the activation of ERK, despite initiating signaling through the TCR, as demonstrated by the robust activation of ZAP-70, PLC-γ, and Ras. We determined that the signaling lesion in W97ALck-expressing cells lies at the level of Raf-1 activation and is dependent on the presence of tyrosines 340/341 in the Raf-1 sequence. These data demonstrate a second function for Lck in TCR-mediated signaling to ERK. Additionally, we found that a significant fraction of Lck is localized to the Golgi apparatus and that, compared with wild-type Lck, W97ALck displays aberrant Golgi membrane localization. Our results support a model where under conditions of weak stimulation through the TCR, in addition to activated Ras, Golgi apparatus-localized Lck is needed for the full activation of Raf-1

Multiple Model-Informed Open-Loop Control of Uncertain Intracellular Signaling Dynamics

<div><p>Computational approaches to tune the activation of intracellular signal transduction pathways both predictably and selectively will enable researchers to explore and interrogate cell biology with unprecedented precision. Techniques to control complex nonlinear systems typically involve the application of control theory to a descriptive mathematical model. For cellular processes, however, measurement assays tend to be too time consuming for real-time feedback control and models offer rough approximations of the biological reality, thus limiting their utility when considered in isolation. We overcome these problems by combining nonlinear model predictive control with a novel adaptive weighting algorithm that blends predictions from multiple models to derive a compromise open-loop control sequence. The proposed strategy uses weight maps to inform the controller of the tendency for models to differ in their ability to accurately reproduce the system dynamics under different experimental perturbations (i.e. control inputs). These maps, which characterize the changing model likelihoods over the admissible control input space, are constructed using preexisting experimental data and used to produce a model-based open-loop control framework. In effect, the proposed method designs a sequence of control inputs that force the signaling dynamics along a predefined temporal response without measurement feedback while mitigating the effects of model uncertainty. We demonstrate this technique on the well-known Erk/MAPK signaling pathway in T cells. <i>In silico</i> assessment demonstrates that this approach successfully reduces target tracking error by 52% or better when compared with single model-based controllers and non-adaptive multiple model-based controllers. <i>In vitro</i> implementation of the proposed approach in Jurkat cells confirms a 63% reduction in tracking error when compared with the best of the single-model controllers. This study provides an experimentally-corroborated control methodology that utilizes the knowledge encoded within multiple mathematical models of intracellular signaling to design control inputs that effectively direct cell behavior in open-loop.</p></div