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The Journal of Biological Chemistry May 2024Recent research has identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as a conserved direct effector of Ras proteins. While previous studies suggested...
Recent research has identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as a conserved direct effector of Ras proteins. While previous studies suggested the involvement of the Switch I (SWI) effector domain of Ras in binding mTORC2 components, the regulation of the Ras-mTORC2 pathway is not entirely understood. In Dictyostelium, mTORC2 is selectively activated by the Ras protein RasC, and the RasC-mTORC2 pathway then mediates chemotaxis to cAMP and cellular aggregation by regulating the actin cytoskeleton and promoting cAMP signal relay. Here, we investigated the role of specific residues in RasC's SWI, C-terminal allosteric domain, and hypervariable region (HVR) related to mTORC2 activation. Interestingly, our results suggest that RasC SWI residue A31, which was previously implicated in RasC-mediated aggregation, regulates RasC's specific activation by the Aimless RasGEF. On the other hand, our investigation identified a crucial role for RasC SWI residue T36, with secondary contributions from E38 and allosteric domain residues. Finally, we found that conserved basic residues and the adjacent prenylation site in the HVR, which are crucial for RasC's membrane localization, are essential for RasC-mTORC2 pathway activation by allowing for both RasC's own cAMP-induced activation and its subsequent activation of mTORC2. Therefore, our findings revealed new determinants of RasC-mTORC2 pathway specificity in Dictyostelium, contributing to a deeper understanding of Ras signaling regulation in eukaryotic cells.
PubMed: 38815864
DOI: 10.1016/j.jbc.2024.107423 -
The role of PALLD-STAT3 interaction in megakaryocyte differentiation and thrombocytopenia treatment.Haematologica May 2024Impaired differentiation of megakaryocytes constitutes the principal etiology of thrombocytopenia. The signal transducer and activator of transcription 3 (STAT3) is a...
Impaired differentiation of megakaryocytes constitutes the principal etiology of thrombocytopenia. The signal transducer and activator of transcription 3 (STAT3) is a crucial transcription factor in regulating megakaryocyte differentiation, yet the precise mechanism of its activation remains unclear. PALLD, an actin-associated protein, has been increasingly recognized for its essential functions in multiple biological processes. This study revealed that megakaryocyte/plateletspecific knockout of PALLD in mice exhibited thrombocytopenia due to diminished platelet biogenesis. In megakaryocytes, PALLD deficiency led to impaired proplatelet formation and polyploidization, ultimately weakening their differentiation for platelet production. Mechanistic studies demonstrated that PALLD bound to STAT3 and interacted with its DNA-binding domain (DBD) and Src homology 2 (SH2) domain via Immunoglobulin domain 3 (Ig3). Moreover, the absence of PALLD attenuated STAT3 Y705 phosphorylation and impeded STAT3 nuclear translocation. Based on the PALLD-STAT3 binding sequence, we designed a peptide C-P3, which can facilitate megakaryocyte differentiation and accelerate platelet production in vivo. In conclusion, this study highlights the pivotal role of PALLD in megakaryocyte differentiation and proposes a novel approach for treating thrombocytopenia by targeting the PALLD-STAT3 interaction.
PubMed: 38813732
DOI: 10.3324/haematol.2024.285242 -
Turkish Journal of Medical Sciences 2024Although high muscle strength worsens the sense of force, it is unknown whether there is a relationship between this deterioration and the underlying molecular...
BACKGROUND/AIM
Although high muscle strength worsens the sense of force, it is unknown whether there is a relationship between this deterioration and the underlying molecular mechanisms. This study examined the relationship between decreased force sense (FS) acuity and strength-related gene expressions.
MATERIALS AND METHODS
Maximal voluntary isometric contraction (MVIC) and FS (50% MVIC) tests were performed on the knee joints of twenty-two subjects. The expression analyses were evaluated by qRT-PCR in blood samples taken before, after MVIC, after 50% MVIC, and 15 min after the test.
RESULTS
MVIC and FS error values were significantly correlated with each other (r = .659, p = .001). The qRT-PCR analyses demonstrated that the expressed mRNAs of the interleukin 6 (IL-6), alpha-actinin 3 (ACTN3), angiotensin-converting enzyme (ACE), brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor receptor (CNTFR) genes dramatically increased until 50% MVIC and subsequently decreased 15 min after the exercise (p < .05). The muscle-specific creatine kinase (CKMM), myosin light chain kinase (MLCK), and G-protein β3 subunit (GNB3) genes reached their peak expression levels 30 min after MVIC (p < .05). ACE and ACTN3 gene expression increased significantly in parallel with the increased FS error (p < .05). These gene expression fluctuations observed at 50% MVIC and after the rest could be related to changes in cellular metabolism leading to fatigue.
CONCLUSION
The time points of gene expression levels during exercise need to be considered. The force acuity of those whose maximal force develops too much may deteriorate.
Topics: Humans; Male; Muscle Strength; Isometric Contraction; Adult; Young Adult; Gene Expression; Muscle, Skeletal; Interleukin-6; Female; Brain-Derived Neurotrophic Factor; Peptidyl-Dipeptidase A; Actinin; Knee Joint
PubMed: 38812641
DOI: 10.55730/1300-0144.5775 -
Turkish Journal of Medical Sciences 2024Anemia in the first week after birth, which could affect growth, development, and organ function, should be an important warning sign to clinicians. The aim of this...
BACKGROUND/AIM
Anemia in the first week after birth, which could affect growth, development, and organ function, should be an important warning sign to clinicians. The aim of this study was to assess the related risk factors of early neonatal anemia and to analyze the effect of anemia on the expression levels of myocardial markers in newborns.
MATERIALS AND METHODS
Clinical data from 122 confirmed cases of anemic newborns and 108 nonanemic newborns were collected to analyze the independent risk factors for early anemia using logistic regression analyses. Blood samples were collected from both groups for the detection of myocardial markers, including the protein marker cardiac troponin T (cTnT), as well as enzyme markers creatine kinase isoenzyme MB (CK-MB) and lactate dehydrogenase (LDH).
RESULTS
Multivariate logistic regression analysis revealed that preterm birth (OR: 3.589 [1.119-11.506], p < 0.05), multiple pregnancy (OR: 4.117 [1.021-16.611], p < 0.05), and abnormal placenta (OR: 4.712 [1.077-20.625], p < 0.05) were independent risk factors for early neonatal anemia. The levels of myocardial markers, including cTnT (303.1 ± 244.7 vs. 44.2 ± 55.41 ng/L), CK-MB (6.803 ± 8.971 vs. 2.5326 ± 2.927 μkat/L), and LDH (32.42 ± 35.26 vs. 19.73 ± 17.13 μkat/L), were significantly higher in the anemic group than in the nonanemic group.
CONCLUSION
Multiple pregnancy, preterm birth, and abnormal placenta were identified as risk factors for early neonatal anemia. The occurrence of early neonatal anemia was associated with increased levels of myocardial markers.
Topics: Humans; Infant, Newborn; Female; Risk Factors; Biomarkers; Anemia; Male; Troponin T; Creatine Kinase, MB Form; L-Lactate Dehydrogenase; Pregnancy; Myocardium; Logistic Models
PubMed: 38812621
DOI: 10.55730/1300-0144.5788 -
Frontiers in Immunology 2024Adipose tissue mesenchymal stem/stromal cells (ASC) can be used as advanced therapy medicinal product in regenerative and cancer medicine. We previously demonstrated...
INTRODUCTION
Adipose tissue mesenchymal stem/stromal cells (ASC) can be used as advanced therapy medicinal product in regenerative and cancer medicine. We previously demonstrated Supernatant Rich in Growth Factors (SRGF) can replace fetal bovine serum (FBS) to expand ASC by a clinical grade compliant protocol. The therapeutic potential of ASC is based also on their homing capacity toward inflammatory/cancer sites: oriented cell migration is a fundamental process in this scenario. We investigated the impact of SRGF on ASC migration properties.
METHODS
The motility/migration potential of ASC expanded in 5% SRGF was analyzed, in comparison to 10% FBS, by standard wound healing, bidimensional chemotaxis and transwell assays, and by millifluidic transwell tests. Mechanisms involved in the migration process were investigated by transient protein overexpression.
RESULTS
In comparison to standard 10% FBS, supplementation of the cell culture medium with 5% SRGF, strongly increased migration properties of ASC along the chemotactic gradient and toward cancer cell derived soluble factors, both in static and millifluidic conditions. We showed that, independently from applied migratory stimulus, SRGF expanded ASC were characterized by far lower expression of α-smooth muscle actin (αSMA), a protein involved in the cell migration machinery. Overexpression of αSMA induced a significant and marked decrease in migration capacity of SRGF expanded ASC.
DISCUSSION
In conclusion, 5% SRGF addition in the cell culture medium increases the migration potential of ASC, reasonably through appropriate downregulation of αSMA. Thus, SRGF could potentially improve the therapeutic impact of ASC, both as modulators of the immune microenviroment or as targeted drug delivery vehicles in oncology.
Topics: Humans; Cell Movement; Intercellular Signaling Peptides and Proteins; Adipose Tissue; Mesenchymal Stem Cells; Blood Platelets; Cells, Cultured; Culture Media; Actins; Female
PubMed: 38812519
DOI: 10.3389/fimmu.2024.1404228 -
Scientific Reports May 2024Mesotrypsin, encoded by the PRSS3 gene, is a distinctive trypsin isoform renowned for its exceptional resistance to traditional trypsin inhibitors and unique substrate...
Mesotrypsin, encoded by the PRSS3 gene, is a distinctive trypsin isoform renowned for its exceptional resistance to traditional trypsin inhibitors and unique substrate specificity. Within the skin epidermis, this protein primarily expresses in the upper layers of the stratified epidermis and plays a crucial role in processing pro-filaggrin (Pro-FLG). Although prior studies have partially elucidated its functions using primary cultured keratinocytes, challenges persist due to these cells' differentiation-activated cell death program. In the present study, HaCaT keratinocytes, characterized by minimal endogenous mesotrypsin expression and sustained proliferation in differentiated states, were utilized to further scrutinize the function of mesotrypsin. Despite the ready degradation of the intact form of active mesotrypsin in these cells, fusion with Venus, flanked by a peptide linker, enables evasion from the protein elimination machinery, thus facilitating activation of the Pro-FLG processing system. Inducing Venus-mesotrypsin expression in the cells resulted in a flattened phenotype and reduced proliferative capacity. Moreover, these cells displayed altered F-actin assembly, enhanced E-cadherin adhesive activity, and facilitated tight junction formation without overtly influencing epidermal differentiation. These findings underscore mesotrypsin's potentially pivotal role in shaping the characteristic cellular morphology of upper epidermal layers.
Topics: Keratinocytes; Humans; Trypsin; Cell Proliferation; Filaggrin Proteins; Cell Differentiation; Cadherins; Epidermis; Actins; HaCaT Cells; Tight Junctions; Cell Adhesion; Cell Line; Epidermal Cells
PubMed: 38811772
DOI: 10.1038/s41598-024-63271-w -
Circulation May 2024
Letter by Cardinale et al Regarding Article, "Multicenter, Prospective, Randomized Controlled Trial of High-Sensitivity Cardiac Troponin I-Guided Combination Angiotensin Receptor Blockade and Beta-Blocker Therapy to Prevent Anthracycline Cardiotoxicity: The Cardiac CARE Trial".
Topics: Humans; Troponin I; Cardiotoxicity; Anthracyclines; Adrenergic beta-Antagonists; Randomized Controlled Trials as Topic; Angiotensin Receptor Antagonists; Prospective Studies; Multicenter Studies as Topic; Drug Therapy, Combination
PubMed: 38805580
DOI: 10.1161/CIRCULATIONAHA.123.068069 -
Circulation May 2024
Response by Henriksen et al Regarding Article, "Multicenter, Prospective, Randomized Controlled Trial of High-Sensitivity Cardiac Troponin I-Guided Combination Angiotensin Receptor Blockade and Beta-Blocker Therapy to Prevent Anthracycline Cardiotoxicity: The Cardiac CARE Trial".
Topics: Humans; Troponin I; Cardiotoxicity; Anthracyclines; Adrenergic beta-Antagonists; Angiotensin Receptor Antagonists; Randomized Controlled Trials as Topic; Prospective Studies; Multicenter Studies as Topic; Drug Therapy, Combination
PubMed: 38805577
DOI: 10.1161/CIRCULATIONAHA.124.069375 -
Cancer Medicine May 2024Radiotherapy (RT) is a cornerstone of the glioblastoma (GBM) treatment. However, the resistance of tumour cells to radiation results in early recurrence. The mechanisms...
OBJECTIVE
Radiotherapy (RT) is a cornerstone of the glioblastoma (GBM) treatment. However, the resistance of tumour cells to radiation results in early recurrence. The mechanisms underlying GBM radioresistance remain unclear. Screening for differentially expressed genes (DEGs) related to radiation might be a potential solution to this problem.
METHOD
RT-associated DEGs were screened based on the RNA sequencing of 15 paired primary and recurrent GBMs. The mRNA and protein expression of candidate genes were validated in RNA sequencing of The Chinese Genome Atlas (CGGA) dataset and 18 cases of GBM samples. The relationship between the candidate gene and radiation was confirmed in irradiated GBM cells. The association of candidate gene with clinical characteristics and survival was investigated in the CGGA and TCGA dataset. Biological function and pathway analysis were explored by gene ontology analysis. The association of the candidate gene with radiosensitivity was verified using cell counting Kit-8, comet, and colony formation assays in vitro and subcutaneous tumour xenograft experiments in vivo.
RESULTS
Gelsolin (GSN) was selected for further study. GSN expression was significant elevated in recurrent GBM and up-regulated in irradiated GBM cell lines. High expression of GSN was enriched in malignant phenotype of glioma. Moreover, high expression of GSN was associated with poor prognosis. Further investigation demonstrated that GSN-knockdown (GSN-KD) combined with RT significantly inhibited cell proliferation and enhanced radiosensitivity in vivo and in vitro. Mechanistically, GSN-KD could lead to more serious DNA damage and promotes apoptosis after RT.
CONCLUSION
Radiation induced up-regulated of GSN. GSN-KD could enhance the radiosensitivity of GBM.
Topics: Humans; Glioblastoma; Radiation Tolerance; Gelsolin; Animals; Mice; Cell Line, Tumor; Brain Neoplasms; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Xenograft Model Antitumor Assays; Prognosis; Cell Proliferation; Apoptosis; Male; Female; Mice, Nude; Neoplasm Recurrence, Local
PubMed: 38803199
DOI: 10.1002/cam4.7286 -
Scientific Reports May 2024Hypertrophic cardiomyopathy (HCM) remains the most common cardiomyopathy in humans and cats with few preclinical pharmacologic interventional studies. Small-molecule...
Hypertrophic cardiomyopathy (HCM) remains the most common cardiomyopathy in humans and cats with few preclinical pharmacologic interventional studies. Small-molecule sarcomere inhibitors are promising novel therapeutics for the management of obstructive HCM (oHCM) patients and have shown efficacy in left ventricular outflow tract obstruction (LVOTO) relief. The objective of this study was to explore the 6-, 24-, and 48-hour (h) pharmacodynamic effects of the cardiac myosin inhibitor, CK-586, in six purpose-bred cats with naturally occurring oHCM. A blinded, randomized, five-treatment group, crossover preclinical trial was conducted to assess the pharmacodynamic effects of CK-586 in this oHCM model. Dose assessments and select echocardiographic variables were assessed five times over a 48-h period. Treatment with oral CK-586 safely ameliorated LVOTO in oHCM cats. CK-586 treatment dose-dependently eliminated obstruction (reduced LVOTOmaxPG), increased measures of systolic chamber size (LVIDs Sx), and decreased select measures of heart function (LV FS% and LV EF%) in the absence of impact on heart rate. At all tested doses, a single oral CK-586 dose resulted in improved or resolved LVOTO with well-tolerated, dose-dependent, reductions in LV systolic function. The results from this study pave the way for the potential use of CK-586 in both the veterinary and human clinical setting.
Topics: Animals; Cats; Cardiomyopathy, Hypertrophic; Cardiac Myosins; Cat Diseases; Male; Female; Ventricular Outflow Obstruction; Systole; Echocardiography; Cross-Over Studies
PubMed: 38802475
DOI: 10.1038/s41598-024-62840-3