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Molecules (Basel, Switzerland) Jun 2024The DEAD-box RNA helicase Ded1 is an essential yeast protein involved in translation initiation that belongs to the DDX3 subfamily. The purified Ded1 protein is an...
The DEAD-box RNA helicase Ded1 is an essential yeast protein involved in translation initiation that belongs to the DDX3 subfamily. The purified Ded1 protein is an ATP-dependent RNA-binding protein and an RNA-dependent ATPase, but it was previously found to lack substrate specificity and enzymatic regulation. Here we demonstrate through yeast genetics, yeast extract pull-down experiments, in situ localization, and in vitro biochemical approaches that Ded1 is associated with, and regulated by, the signal recognition particle (SRP), which is a universally conserved ribonucleoprotein complex required for the co-translational translocation of polypeptides into the endoplasmic reticulum lumen and membrane. Ded1 is physically associated with SRP components in vivo and in vitro. Ded1 is genetically linked with SRP proteins. Finally, the enzymatic activity of Ded1 is inhibited by SRP21 in the presence of SCR1 RNA. We propose a model where Ded1 actively participates in the translocation of proteins during translation. Our results provide a new understanding of the role of Ded1 during translation.
Topics: Signal Recognition Particle; DEAD-box RNA Helicases; Saccharomyces cerevisiae Proteins; Saccharomyces cerevisiae; Protein Binding; Protein Biosynthesis; Protein Transport
PubMed: 38931009
DOI: 10.3390/molecules29122944 -
Molecules (Basel, Switzerland) Jun 2024The CRISPR-Cas9 system has emerged as the most prevalent gene editing technology due to its simplicity, high efficiency, and low cost. However, the homology-directed...
The CRISPR-Cas9 system has emerged as the most prevalent gene editing technology due to its simplicity, high efficiency, and low cost. However, the homology-directed repair (HDR)-mediated gene knock-in in this system suffers from low efficiency, which limits its application in animal model preparation, gene therapy, and agricultural genetic improvement. Here, we report the design and optimization of a simple and efficient reporter-based assay to visualize and quantify HDR efficiency. Through random screening of a small molecule compound library, two groups of compounds, including the topoisomerase inhibitors and PIM1 kinase inhibitors, have been identified to promote HDR. Two representative compounds, etoposide and quercetagetin, also significantly enhance the efficiency of CRISPR-Cas9 and HDR-mediated gene knock-in in mouse embryos. Our study not only provides an assay to screen compounds that may facilitate HDR but also identifies useful tool compounds to facilitate the construction of genetically modified animal models with the CRISPR-Cas9 system.
Topics: Gene Editing; CRISPR-Cas Systems; Proto-Oncogene Proteins c-pim-1; Animals; Mice; Protein Kinase Inhibitors; Topoisomerase Inhibitors; Humans; Recombinational DNA Repair; Gene Knock-In Techniques
PubMed: 38930955
DOI: 10.3390/molecules29122890 -
Molecules (Basel, Switzerland) Jun 2024The epidermal growth factor receptor (EGFR) is a pivotal target in cancer therapy due to its significance within the tyrosine kinase family. EGFR inhibitors like AG-1478...
The epidermal growth factor receptor (EGFR) is a pivotal target in cancer therapy due to its significance within the tyrosine kinase family. EGFR inhibitors like AG-1478 and PD153035, featuring a 4-anilinoquinazoline moiety, have garnered global attention for their potent therapeutic activities. While pre-clinical studies have highlighted the significant impact of halogen substitution at the C3'-anilino position on drug potency, the underlying mechanism remains unclear. This study investigates the influence of halogen substitution (X = H, F, Cl, Br, I) on the structure, properties, and spectroscopy of halogen-substituted 4-anilinoquinazoline tyrosine kinase inhibitors (TKIs) using time-dependent density functional methods (TD-DFT) with the B3LYP functional. Our calculations revealed that halogen substitution did not induce significant changes in the three-dimensional conformation of the TKIs but led to noticeable alterations in electronic properties, such as dipole moment and spatial extent, impacting interactions at the EGFR binding site. The UV-visible spectra show that more potent TKI-X compounds typically have shorter wavelengths, with bromine's peak wavelength at 326.71 nm and hydrogen, with the lowest IC50 nM, shifting its lambda max to 333.17 nm, indicating a correlation between potency and spectral characteristics. Further analysis of the four lowest-lying conformers of each TKI-X, along with their crystal structures from the EGFR database, confirms that the most potent conformer is often not the global minimum structure but one of the low-lying conformers. The more potent TKI-Cl and TKI-Br exhibit larger deviations (RMSD > 0.65 Å) from their global minimum structures compared to other TKI-X (RMSD < 0.15 Å), indicating that potency is associated with greater flexibility. Dipole moments of TKI-X correlate with drug potency (ln(IC50 nM)), with TKI-Cl and TKI-Br showing significantly higher dipole moments (>8.0 Debye) in both their global minimum and crystal structures. Additionally, optical spectral shifts correlate with potency, as TKI-Cl and TKI-Br exhibit blue shifts from their global minimum structures, in contrast to other TKI-X. This suggests that optical reporting can effectively probe drug potency and conformation changes.
Topics: ErbB Receptors; Quinazolines; Protein Kinase Inhibitors; Halogens; Aniline Compounds; Humans; Binding Sites; Models, Molecular; Structure-Activity Relationship
PubMed: 38930865
DOI: 10.3390/molecules29122800 -
Medicina (Kaunas, Lithuania) Jun 2024Colorectal cancer is a major global health concern, with a significant increase in morbidity and mortality rates associated with metastatic stages. This study...
Influence of Biomarkers on Mortality among Patients with Hepatic Metastasis of Colorectal Cancer Treated with FOLFOX/CAPOX and FOLFIRI/CAPIRI, Including Anti-EGFR and Anti-VEGF Therapies.
Colorectal cancer is a major global health concern, with a significant increase in morbidity and mortality rates associated with metastatic stages. This study investigates the prognostic significance of various clinical and laboratory parameters in patients with metastatic CRC. A retrospective cohort of 188 CRC patients with hepatic metastasis from the OncoHelp Association in Timisoara was analyzed from January 2016 to March 2023. Data on demographics, clinical characteristics, and biomarkers, such as lymphocyte counts, as well as various inflammation indices, were examined. Statistical analyses included univariate and multivariate logistic regression, Kaplan-Meier survival analysis, and ROC curve assessments. Our findings indicate significant associations between survival outcomes and several biomarkers. Higher BMI and lymphocyte counts were linked with better survival rates, while higher values of Neutrophil-Hemoglobin-Lymphocyte (NHL) score, Neutrophil-Lymphocyte Ratio (NLR), Platelet-Lymphocyte Ratio (PLR), and Systemic Immune-Inflammation Index (SII) were predictors of poorer outcomes. Notably, the presence of hepatic metastasis at diagnosis was a critical factor, significantly reducing overall survival. The study has expanded the current understanding of prognostic factors in CRC, advocating for a multi-dimensional approach to prognostic evaluations. This approach should consider not only the traditional metrics such as tumor stage and histological grading but also incorporate a broader spectrum of biomarkers. Future studies should aim to validate these findings and explore the integration of these biomarkers into routine clinical practice, enhancing the precision of prognostic assessments and ultimately guiding more personalized treatment strategies for CRC patients.
Topics: Humans; Colorectal Neoplasms; Male; Female; Middle Aged; Liver Neoplasms; Retrospective Studies; Aged; Antineoplastic Combined Chemotherapy Protocols; Fluorouracil; Leucovorin; Organoplatinum Compounds; Camptothecin; Adult; Biomarkers, Tumor; Prognosis; ErbB Receptors; Kaplan-Meier Estimate
PubMed: 38929620
DOI: 10.3390/medicina60061003 -
Medicina (Kaunas, Lithuania) Jun 2024Chromophobe RCC (ChRCC) carries the best prognosis among all RCC subtypes, yet it lacks a proper grading system. Various systems have been suggested in the past, causing...
Chromophobe RCC (ChRCC) carries the best prognosis among all RCC subtypes, yet it lacks a proper grading system. Various systems have been suggested in the past, causing much controversy, and Avulova et al. recently proposed a promising four-tier grading system that takes into consideration tumor necrosis. Dysregulation of the mammalian target of the rapamycin (mTOR) pathway plays a key role in ChRCC pathogenesis, highlighting its molecular complexity. The present retrospective study aimed to evaluate the prognostic factors associated with a more aggressive ChRCC phenotype. Seventy-two patients diagnosed with ChRCC between 2004 and 2017 were included in our study. Pathology reports and tissue blocks were reviewed, and immunohistochemistry (IHC) was performed in order to assess the expressions of CYLD (tumor-suppressor gene) and mTOR, among other markers. Univariate analysis was performed, and OS was assessed using the Kaplan-Meier method. In our study, 74% of patients were male, with a mean age of 60 years, and the mean tumor size was 63 mm (±44). The majority (54%) were followed for more than 10 years at intervals ranging between 44 and 222 months. The risk of death was significantly higher for patients that were classified as Grade 4 in the Avulova system (HR: 5.83; 95% CI, 1.37-24.7; : = 0.017). As far as the IHC is concerned, mTOR expression was associated with an HR of 8.57 (95% CI, 1.91-38.5; = 0.005), and CYLD expression was associated with an HR of 17.3 (95% CI, 1.57-192; = 0.02). In our study, the Avulova grading system seems to be positively correlated with OS in patients diagnosed with ChRCC. Furthermore, an elevated mTOR expression also shows a negative correlation with OS, whereas an elevated CYLD expression does not seem to exert a protective role. However, because only a small proportion (4.2%) of our patients died due to ChRCC, despite the long follow-up period, the results must be interpreted with caution. Further research is needed to validate our findings.
Topics: Humans; Male; Carcinoma, Renal Cell; Female; Middle Aged; Retrospective Studies; Kidney Neoplasms; Prognosis; Aged; TOR Serine-Threonine Kinases; Neoplasm Grading; Adult; Immunohistochemistry; Deubiquitinating Enzyme CYLD; Kaplan-Meier Estimate; Biomarkers, Tumor
PubMed: 38929613
DOI: 10.3390/medicina60060996 -
Medicina (Kaunas, Lithuania) Jun 2024Patients with human epidermal growth factor receptor 2 (HER2) -positive, hormone receptor-positive (HR-positive) metastatic breast cancer (MBC) usually undergo...
Combining Endocrine Therapy with Trastuzumab Emtansine Improves Progression-Free Survival and Overall Survival in HER2-Positive, Hormone Receptor-Positive Metastatic Breast Cancer.
Patients with human epidermal growth factor receptor 2 (HER2) -positive, hormone receptor-positive (HR-positive) metastatic breast cancer (MBC) usually undergo trastuzumab emtansine (T-DM1) therapy in subsequent lines. Combining endocrine therapy (ET) with T-DM1 can improve treatment outcomes in this subtype. Therefore, this study aimed to investigate the benefits of using T-DM1 with ET in HER2-positive and HR-positive MBC. This study was the first to investigate the benefits of combining ET with T-DM1. This study analyzed the medical records of patients with HER2-positive and HR-positive MBC who were treated with T-DM1 from June 2010 to December 2021. The patients were divided into groups based on whether they received concomitant ET with T-DM1. The primary endpoint was to determine the progression-free survival (PFS), while the secondary endpoints were overall survival (OS), objective response rate, and safety of the treatment. Our analysis examined 88 patients, of whom 32 (36.4%) were treated with T-DM1 in combination with ET. The combination therapy showed a significant improvement in median PFS (15.4 vs. 6.4 months; = 0.00004) and median OS (35.0 vs. 23.1 months; = 0.026) compared to T-DM1 alone. The ORR was also higher in the combination group (65.6% vs. 29.3%; = 0.026). Patients treated with pertuzumab priorly had reduced median PFS on T-DM1 compared to those who were not treated with pertuzumab (11.7 vs. 5.4 months, respectively; < 0.01). T-DM1 demonstrated better median PFS in HER2 3+ patients compared to HER2 2+ patients, with an amplification ratio of >2.0 (10.8 vs 5.8 months, respectively; = 0.049). The safety profiles were consistent with previous T-DM1 studies. The combination of T-DM1 with ET can significantly improve PFS and OS in patients with HER2-positive and HR-positive MBC. Our study suggests that prior pertuzumab treatment plus trastuzumab treatment might decrease T-DM1 efficacy.
Topics: Humans; Breast Neoplasms; Female; Middle Aged; Receptor, ErbB-2; Ado-Trastuzumab Emtansine; Aged; Progression-Free Survival; Adult; Retrospective Studies; Antineoplastic Combined Chemotherapy Protocols; Antineoplastic Agents, Immunological; Neoplasm Metastasis; Aged, 80 and over; Trastuzumab; Receptors, Estrogen
PubMed: 38929568
DOI: 10.3390/medicina60060951 -
International Journal of Molecular... Jun 2024Tissue biopsy remains the standard for diagnosing gastrointestinal stromal tumors (GISTs), although liquid biopsy is emerging as a promising alternative in oncology. In...
Tissue biopsy remains the standard for diagnosing gastrointestinal stromal tumors (GISTs), although liquid biopsy is emerging as a promising alternative in oncology. In this pilot study, we advocate for droplet digital PCR (ddPCR) to diagnose GIST in tissue samples and explore its potential for early diagnosis via liquid biopsy, focusing on the D842V mutation and hypermethylated gene. We utilized ddPCR to analyze the predominant mutation (D842V) in surgical tissue samples from 15 GIST patients, correlating with pathologists' diagnoses. We expanded our analysis to plasma samples to compare DNA alterations between tumor tissue and plasma, also investigating gene hypermethylation. We successfully detected the D842V mutation in GIST tissues by ddPCR. Despite various protocols to enhance mutation detection in early-stage disease, it remained challenging, likely due to the low concentration of DNA in plasma samples. Additionally, the results of Area Under the Curve (AUC) for the hypermethylated gene, analyzing concentration, ratio, and abundance were 0.74 (95% Confidence Interval (CI): 0.52 to 0.97), 0.77 (95% CI: 0.56 to 0.98), and 0.79 (95% CI: 0.59 to 0.99), respectively. As a rare disease, the early detection of GIST through such biomarkers is particularly crucial, offering significant potential to improve patient outcomes.
Topics: Humans; Septins; Gastrointestinal Stromal Tumors; DNA Methylation; Liquid Biopsy; Pilot Projects; Receptor, Platelet-Derived Growth Factor alpha; Female; Male; Middle Aged; Mutation; Polymerase Chain Reaction; Aged; Gastrointestinal Neoplasms; Biomarkers, Tumor; Adult
PubMed: 38928487
DOI: 10.3390/ijms25126783 -
International Journal of Molecular... Jun 2024Psoriasis is a chronic inflammatory condition affecting 2% of the Western population. It includes diverse manifestations influenced by genetic predisposition,... (Review)
Review
Psoriasis is a chronic inflammatory condition affecting 2% of the Western population. It includes diverse manifestations influenced by genetic predisposition, environmental factors, and immune status. The sustained activation of mTOR is a key element in psoriasis pathogenesis, leading to an uncontrolled proliferation of cytokines. Furthermore, mTOR activation has been linked with the transition from psoriasis to non-skin manifestations such as psoriatic arthritis and cardiovascular events. While therapies targeting pro-inflammatory cytokines have shown efficacy, additional pathways may offer therapeutic potential. The PI3K/Akt/mTOR pathway, known for its role in cell growth, proliferation, and metabolism, has emerged as a potential therapeutic target in psoriasis. This review explores the relevance of mTOR in psoriasis pathophysiology, focusing on its involvement in cutaneous and atheromatous plaque proliferation, psoriatic arthritis, and cardiovascular disease. The activation of mTOR promotes keratinocyte and synovial cell proliferation, contributing to plaque formation and joint inflammation. Moreover, mTOR activation may exacerbate the cardiovascular risk by promoting pro-inflammatory cytokine production and dysregulation lipid and glucose metabolism. The inhibition of mTOR has shown promise in preclinical studies, reducing skin inflammation and plaque proliferation. Furthermore, mTOR inhibition may mitigate cardiovascular risk by modulating cholesterol metabolism and attenuating atherosclerosis progression. Understanding the role of mTOR in psoriasis, psoriatic arthritis, and cardiovascular disease provides insight into the potential treatment avenues and sheds light on the complex interplay of the immune and metabolic pathways in these conditions.
Topics: Humans; TOR Serine-Threonine Kinases; Psoriasis; Animals; Signal Transduction; Cardiovascular Diseases; Arthritis, Psoriatic
PubMed: 38928483
DOI: 10.3390/ijms25126778 -
International Journal of Molecular... Jun 2024Exposure to general anesthetics can adversely affect brain development, but there is little study of sedative agents used in intensive care that act via similar...
Exposure to general anesthetics can adversely affect brain development, but there is little study of sedative agents used in intensive care that act via similar pharmacologic mechanisms. Using quantitative immunohistochemistry and neurobehavioral testing and an established protocol for murine sedation, we tested the hypothesis that lengthy, repetitive exposure to midazolam, a commonly used sedative in pediatric intensive care, interferes with neuronal development and subsequent cognitive function via actions on the mechanistic target of rapamycin (mTOR) pathway. We found that mice in the midazolam sedation group exhibited a chronic, significant increase in the expression of mTOR activity pathway markers in comparison to controls. Furthermore, both neurobehavioral outcomes, deficits in Y-maze and fear-conditioning performance, and neuropathologic effects of midazolam sedation exposure, including disrupted dendritic arborization and synaptogenesis, were ameliorated via treatment with rapamycin, a pharmacologic mTOR pathway inhibitor. We conclude that prolonged, repetitive exposure to midazolam sedation interferes with the development of neural circuitry via a pathologic increase in mTOR pathway signaling during brain development that has lasting consequences for both brain structure and function.
Topics: Midazolam; Animals; TOR Serine-Threonine Kinases; Mice; Signal Transduction; Brain; Male; Hypnotics and Sedatives; Behavior, Animal; Female; Mice, Inbred C57BL; Maze Learning; Animals, Newborn
PubMed: 38928447
DOI: 10.3390/ijms25126743 -
International Journal of Molecular... Jun 2024Tumor cells reprogram their metabolism to meet the increased demand for nucleotides and other molecules necessary for growth and proliferation. In fact, cancer cells are... (Review)
Review
Tumor cells reprogram their metabolism to meet the increased demand for nucleotides and other molecules necessary for growth and proliferation. In fact, cancer cells are characterized by an increased "de novo" synthesis of purine nucleotides. Therefore, it is not surprising that specific enzymes of purine metabolism are the targets of drugs as antineoplastic agents, and a better knowledge of the mechanisms underlying their regulation would be of great help in finding new therapeutic approaches. The mammalian target of the rapamycin (mTOR) signaling pathway, which is often activated in cancer cells, promotes anabolic processes and is a major regulator of cell growth and division. Among the numerous effects exerted by mTOR, noteworthy is its empowerment of the "de novo" synthesis of nucleotides, accomplished by supporting the formation of purinosomes, and by increasing the availability of necessary precursors, such as one-carbon formyl group, bicarbonate and 5-phosphoribosyl-1-pyrophosphate. In this review, we highlight the connection between purine and mitochondrial metabolism, and the bidirectional relation between mTOR signaling and purine synthesis pathways.
Topics: Humans; Neoplasms; TOR Serine-Threonine Kinases; Purines; Signal Transduction; Animals; Mitochondria
PubMed: 38928439
DOI: 10.3390/ijms25126735