Molecular Mechanisms of Mechanosensing at Cell-Cell and Cell-Matrix Adhesions

Ph.DPH.D. IN MECHANOBIOLOGY (FOS

Near-membrane ensemble elongation in the proline-rich LRP6 intracellular domain may explain the mysterious initiation of the Wnt signaling pathway

<p>Abstract</p> <p>Background</p> <p>LRP6 is a membrane protein crucial in the initiation of canonical Wnt/β-catenin signalling. Its function is dependent on its proline-serine rich intracellular domain. LRP6 has five PPP(S/T)P motifs that are phosphorylated during activation, starting with the site closest to the membrane. Like all long proline rich regions, there is no stable 3D structure for this isolated, contiguous region.</p> <p>Results</p> <p>In our study, we use a computational simulation tool to sample the conformational space of the LRP6 intracellular domain, under the spatial constraints imposed by (a) the membrane and (b) the close approach of the neighboring intracellular molecular complex, which is assembled on Frizzled when Wnt binds to both LRP6 and Frizzled on the opposite side of the membrane. We observe that an elongated form dominates in the LRP6 intracellular domain structure ensemble. This elongation could relieve conformational auto-inhibition of the PPP(S/T)PX(S/T) motif binding sites and allow GSK3 and CK1 to approach their phosphorylation sites, thereby activating LRP6 and the downstream pathway.</p> <p>Conclusions</p> <p>We propose a model in which the conformation of the LRP6 intracellular domain is elongated before activation. This is based on the intrusion of the Frizzled complex into the ensemble space of the proline rich region of LRP6, which alters the shape of its available ensemble space. To test whether this observed ensemble conformational change is sequence dependent, we did a control simulation with a hypothetical sequence with 50% proline and 50% serine in alternating residues. We confirm that this ensemble neighbourhood-based conformational change is independent of sequence and conclude that it is likely found in all proline rich sequences. These observations help us understand the nature of proline rich regions which are both unstructured and which seem to evolve at a higher rate of mutation, while maintaining sequence composition.</p

Calcium-Mediated Protein Folding and Stabilisation of Salmonella Biofilm-Associated Protein a

Biofilm-associated proteins (BAPs) are important for early biofilm formation (adhesion) by bacteria and are also found in mature biofilms. BapA from Salmonella is a ~386 kDa surface protein, comprised of 27 tandem repeats predicted to be bacterial Ig-like (BIg) domains. Such tandem repeats are conserved for BAPs across different bacterial species, but the function of these domains is not completely understood. In this work, we report the first study of the mechanical stability of the BapA protein. Using magnetic tweezers, we show that the folding of BapA BIg domains requires calcium- binding and the folded domains have differential mechanical stabilities. Importantly, we identify that >100 nM concentration of calcium is needed for folding of the BIg domains, and the stability of the folded BIg domains is regulated by calcium over a wide concentration range from sub-micromolar (?M) to millimolar (mM). Only at mM calcium concentrations, as found in the extracellular environment, do the BIg domains have the saturated mechanical stability. BapA has been suggested to be involved in Salmonella invasion, and it is likely a crucial mechanical component of biofilms. Therefore, our results provide new insights into the potential roles of BapA as a structural maintenance component of Salmonella biofilm and also Salmonella invasion

The mechanical response of vinculin

Vinculin is a mechanosensitive adapter protein that links the actin network to cell-extracellular matrix adhesions and cell-cell adhesions. It is perhaps the best characterized mechanoeffector, as it is recruited to sites of adhesion in response to force on the mechanotransducers talin and alpha-catenin. Here we examined the mechanical properties of vinculin to assess its potential role as a mechanotransducer. We find that at physiological loading rates, the structural domains of vinculin unfold at forces in the 5-15 pN range and rapidly refold when forces are reduced back to 1 pN. Thus, vinculin domains also have the potential to act as force dependent molecular switches, akin to those in talin and alpha-catenin. As with the force dependent switches in talin, the unfolding of these domains in vinculin introduces large extension changes in the vinculin cytoskeletal linkage up to 150 nm with 20-30 nm steps of unfolding. Modelling of the tension-dependent interactions of the unstructured vinculin linker region with a model protein containing two SH3 domains indicated that even unstructured protein regions can mediate force-dependent interactions with ligands, where the binding of a dual-SH3 model protein is predicted to be significantly suppressed by forces greater than 10 pN. Together, these findings suggest that vinculin has a complex mechanical response with force-dependent interaction sites, suggesting it also acts as a mechanotransducer, recruiting partners in response to force

Development and validation of novel immune-inflammation-based clinical predictive nomograms in HER2-negative advanced gastric cancer

PurposeTo explore the predictive value of multiple immune-inflammatory biomarkers including serum VEGFA and systemic immune-inflammation index (SII) in HER2-negative advanced gastric cancer (AGC) and establish nomograms for predicting the first-line chemotherapeutic efficacy, progression-free survival (PFS) and overall survival (OS) of patients with this fatal disease.MethodsFrom November 2017 to April 2022, 102 and 34 patients with a diagnosis of HER2-negative AGC at the First Affiliated Hospital of Bengbu Medical College were enrolled as development and validation cohorts, respectively. Univariate and multivariate analyses were performed to evaluate the clinical value of the candidate indicators. The variables were screened using LASSO regression analysis. Predictive models were developed using significant predictors and are displayed as nomograms.ResultsBaseline VEGFA expression was significantly higher in HER2-negative AGC patients than in nonneoplastic patients and was associated with malignant serous effusion and therapeutic efficacy (all p&lt;0.001). Multivariate analysis indicated that VEGFA was an independent predictor for first-line therapeutic efficacy and PFS (both p&lt;0.01) and SII was an independent predictor for first-line PFS and OS (both p&lt;0.05) in HER2-negative AGC patients. The therapeutic efficacy model had an R2 of 0.37, a Brier score of 0.15, and a Harrell’s C-index of 0.82 in the development cohort and 0.90 in the validation cohort. The decision curve analysis indicated that the model added more net benefits than VEGFA assessment alone. The PFS/OS models had Harrell’s C-indexes of 0.71/0.69 in the development cohort and 0.71/0.62 in the validation cohort.ConclusionThe established nomograms integrating serum VEGFA/SII and commonly available baseline characteristics provided satisfactory performance in predicting the therapeutic efficacy and prognosis of HER2-negative AGC patients

Pretreatment Serum Uric Acid as an Efficient Predictor of Prognosis in Men with Laryngeal Squamous Cell Cancer: A Retrospective Cohort Study

Purpose. Uric acid (UA) is a major antioxidant molecule that has been hypothesized to have a protective effect against cancer-induced oxidative damage. The aim of the present study was to investigate whether preoperative levels of serum UA are associated with the prognosis of laryngeal squamous cell cancer (LSCC). Methods. A total of 814 male LSCC patients (followed up for five years) and 814 normal control subjects were enrolled from January 2007 to December 2011. The rates of total mortality and cancer mortality were 23.46% and 21.36%, respectively. The prevalence of overall survival (OS), disease-free survival (DFS), and cancer-specific survival (CSS) was analysed using the Kaplan-Meier method. Univariate and multivariate Cox regression models were evaluated to identify UA as a prognostic factor. Results. The serum UA and UA/Cr (creatinine) ratio levels were significantly reduced (P0.310 mmol/l) and UA/Cr ratio (>3.97) experienced five more years of OS, DFS, and CSS than did patients with low UA (<0.310 mmol/l) and UA/Cr ratio (<3.97) levels. Conclusion. High preoperative UA serum levels were identified as an independent prognostic factor associated with improved clinical outcomes among LSCC patients

In situ single-molecule investigations of the impacts of biochemical perturbations on conformational intermediates of monomeric α-synuclein

α-Synuclein aggregation is a common trait in synucleinopathies, including Parkinson's disease. Being an unstructured protein, α-synuclein exists in several distinct conformational intermediates, contributing to both its function and pathogenesis. However, the regulation of these monomer conformations by biochemical factors and potential drugs has remained elusive. In this study, we devised an in situ single-molecule manipulation approach to pinpoint kinetically stable conformational intermediates of monomeric α-synuclein and explore the effects of various biochemical factors and drugs. We uncovered a partially folded conformation located in the non-amyloid-β component (NAC) region of monomeric α-synuclein, which is regulated by a preNAC region. This conformational intermediate is sensitive to biochemical perturbations and small-molecule drugs that influencing α-synuclein's aggregation tendency. Our findings reveal that this partially folded intermediate may play a role in α-synuclein aggregation, offering fresh perspectives for potential treatments aimed at the initial stage of higher-order α-synuclein aggregation. The single-molecule approach developed here can be broadly applied to the study of disease-related intrinsically disordered proteins

Characterization of complete mitochondrial genome of Nemania diffusa (Xylariaceae, Xylariales) and its phylogenetic analysis

Nemania diffusa is a parasitic fungus of many plant species and causes large economic losses to the forestry industry. In the present study, the complete mitochondrial genome of N. diffusa is first reported. The circular genome is 258,879 bp in length, containing 14 protein coding genes, 2 ribosomal RNA genes, 25 transfer RNA genes. The 258,879 bp long mtDNA of N. diffusa represents one of the largest sequenced fungal mitogenomes. The overall base composition is 34.4% A, 35.9% T, 14.0% G, 15.7% C and the content of GC is 29.7%. Phylogenetic analysis based on concatenated protein coding genes from 24 species in 8 orders was conducted using Bayesian inference (BI) method. Nemania diffusa is clustered in the order Xylariales and is more closely related to Annulohypoxylon stygium of Hypoxylaceae. This work facilitates the future study of molecular biology and evolution of xylariaceous fungi

Study on Safety Quality Training Index System of Air Force Aviation Crew

In the new era of Air Force equipment upgrading with times, in view of the air crew safety quality growing impact on the aviation flight safety, this paper, based on a large number of data, investigates the basic situation of the safety quality of the maintenance personnel by using the questionnaire survey method, analyzes the reliability of the questionnaire content by using SPSSAU system, summarizes the components of the safety quality of the maintenance personnel according to the feedback results of the questionnaire, and divides the training indicators; then the Delphi method and analytic hierarchy process are used to calculate the weight of each training index of the safety quality of the maintenance personnel. Finally, the training index system of the safety quality of the maintenance personnel is established, and some suggestions are given for the assessment and improvement of the safety quality of the maintenance personnel