Blood leukocytes as a non-invasive diagnostic tool for thyroid nodules: a prospective cohort study.

BACKGROUND: Thyroid nodule (TN) patients in China are subject to overdiagnosis and overtreatment. The implementation of existing technologies such as thyroid ultrasonography has indeed contributed to the improved diagnostic accuracy of TNs. However, a significant issue persists, where many patients undergo unnecessary biopsies, and patients with malignant thyroid nodules (MTNs) are advised to undergo surgery therapy. METHODS: This study included a total of 293 patients diagnosed with TNs. Differential methylation haplotype blocks (MHBs) in blood leukocytes between MTNs and benign thyroid nodules (BTNs) were detected using reduced representation bisulfite sequencing (RRBS). Subsequently, an artificial intelligence blood leukocyte DNA methylation (BLDM) model was designed to optimize the management and treatment of patients with TNs for more effective outcomes. RESULTS: The DNA methylation profiles of peripheral blood leukocytes exhibited distinctions between MTNs and BTNs. The BLDM model we developed for diagnosing TNs achieved an area under the curve (AUC) of 0.858 in the validation cohort and 0.863 in the independent test cohort. Its specificity reached 90.91% and 88.68% in the validation and independent test cohorts, respectively, outperforming the specificity of ultrasonography (43.64% in the validation cohort and 47.17% in the independent test cohort), albeit with a slightly lower sensitivity (83.33% in the validation cohort and 82.86% in the independent test cohort) compared to ultrasonography (97.62% in the validation cohort and 100.00% in the independent test cohort). The BLDM model could correctly identify 89.83% patients whose nodules were suspected malignant by ultrasonography but finally histological benign. In micronodules, the model displayed higher specificity (93.33% in the validation cohort and 92.00% in the independent test cohort) and accuracy (88.24% in the validation cohort and 87.50% in the independent test cohort) for diagnosing TNs. This performance surpassed the specificity and accuracy observed with ultrasonography. A TN diagnostic and treatment framework that prioritizes patients is provided, with fine-needle aspiration (FNA) biopsy performed only on patients with indications of MTNs in both BLDM and ultrasonography results, thus avoiding unnecessary biopsies. CONCLUSIONS: This is the first study to demonstrate the potential of non-invasive blood leukocytes in diagnosing TNs, thereby making TN diagnosis and treatment more efficient in China.

Design, synthesis, and bioevaluation of SOS1 PROTACs derived from pyrido[2,3-d]pyrimidin-7-one-based SOS1 inhibitor.

Oncogenic KRAS mutations drive an approximately 25 % of all human cancers. Son of Sevenless 1 (SOS1), a critical guanine nucleotide exchange factor, catalyzes the activation of KRAS. Targeting SOS1 degradation has engaged as a promising therapeutic strategy for KRAS-mutant cancers. Herein, we designed and synthesized a series of novel CRBN-recruiting SOS1 PROTACs using the pyrido[2,3-d]pyrimidin-7-one-based SOS1 inhibitor as the warhead. One representative compound 11o effectively induced the degradation of SOS1 in three different KRAS-mutant cancer cell lines with DC50 values ranging from 1.85 to 7.53 nM. Mechanism studies demonstrated that 11o-induced SOS1 degradation was dependent on CRBN and proteasome. Moreover, 11o inhibited the phosphorylation of ERK and displayed potent anti-proliferative activities against SW620, A549 and DLD-1 cells. Further optimization of 11o may provide us promising SOS1 degraders with favorable drug-like properties for developing new chemotherapies targeting KRAS-driven cancers.

MiR-337-3p confers protective effect on facet joint osteoarthritis by targeting SKP2 to inhibit DUSP1 ubiquitination and inactivate MAPK pathway.

OBJECTIVE: To probe the performance of miR-337-3p on the facet joint osteoarthritis (FJOA) and its underlying mechanism. METHODS: qRT-PCR and Western blot were utilized to analyze the levels of miR-337-3p and DUSP1 in the synovial tissues from 36 FJOA patients and 10 healthy controls. The human synovial fibroblasts of FJOA were isolated and cultured followed by cell transfection. Then, cells were exposed to 10 ng/mL of IL-1ß to induce inflammatory response of synovial fibroblasts. The alternation on cell biological function in cell models was determined. The binding of miR-337-3p and SKP2 was predicted by StarBase, TargetScan, DIANA-microT and miRmap, and further verified by RIP assay and dual-luciferase reporter assay. Co-IP experiment and ubiquitination assay were used to display the binding of SKP2 and DUSP1 as well as the ubiquitination and degradation of DUSP1. After that, the FJOA rat model was established and miR-337-3p mimic or negative control was given to rats by tail vein injection. The pathological changes of synovial tissues, synovitis score, and inflammation level in rats were assessed. RESULTS: The low expressions of miR-337-3p and DUSP1 were noticed in the synovial tissues of FJOA patients and in IL-1ß-induced synovial fibroblasts, and highly expressed p-p38 MAPK was noticed. Upregulation of miR-337-3p/DUSP1 or downregulation of SKP2 inhibited IL-1ß-induced proliferation and inflammatory response of synovial fibroblasts. SKP2 was the target gene of miR-337-3p, and SKP2 induced the ubiquitination and degradation of DUSP1. MiR-337-3p exerted a protective effect on FJOA rats by alleviating damage of rat synovial tissues, promoting cell apoptosis and repressing inflammatory response. CONCLUSION: MiR-337-3p plays a protective role in FJOA by negatively targeting SKP2 to suppress DUSP1 ubiquitination and inactivate the p38 MAPK pathway.

MiR-99a alleviates apoptosis and extracellular matrix degradation in experimentally induced spine osteoarthritis by targeting FZD8.

BACKGROUND: Our previous study identified miR-99a as a negative regulator of early chondrogenic differentiation. However, the functional role of miR-99a in the pathogenesis of osteoarthritis (OA) remains unclear. METHODS: We examined the levels of miR-99a and Frizzled 8 (FZD8) expression in tissue specimens. Human SW1353 chondrosarcoma cells were stimulated with IL-6 and TNF-α to construct an in vitro OA environment. A luciferase reporter assay was performed to analyze the relationship between miR-99a and FZD8. CCK-8 assays, flow cytometry, and ELISA assays were used to assess cell viability, apoptosis, and inflammatory molecule expression, respectively. Percutaneous intra-spinal injections of papain mixed solution were performed to create an OA Sprague-Dawley rat model. Alcian Blue staining, Safranin O Fast Green staining, and Toluidine Blue O staining were performed to detect the degrees of cartilage injury. RESULTS: MiR-99a expression was downregulated in the severe spine OA patients when compared with the mild spine OA patients, and was also decreased in the experimentally induced in vitro OA environment when compared with the control environment. Functionally, overexpression of miR-99a significantly suppressed cell apoptosis and extracellular matrix degradation stimulated by IL-6 and TNF-α. FZD8 was identified as a target gene of miR-99a. Furthermore, the suppressive effects of miR-99a on cell injury induced by IL-6 and TNF-α were reversed by FZD8 overexpression. Moreover, the levels of miR-99a expression were also reduced in the induced OA model rats, and miR-99a agomir injection relieved the cartilage damage. At the molecular level, miR-99a overexpression downregulated the levels of MMP13, ß-catenin, Bax, and caspase-3 protein expression and upregulated the levels of COL2A1 and Bcl-2 protein expression in the in vitro OA-like chondrocyte model and also in the experimental OA model rats. CONCLUSIONS: Our data showed that miR-99a alleviated apoptosis and extracellular matrix degradation by targeting FZD8, and thereby suppressed the development and progression of experimentally induced spine osteoarthritis.

Iodine monitoring models contribute to avoid adverse birth outcomes related more than adequate iodine intake.

BACKGROUND: Iodine plays an important role in pregnancy. How to maintain adequate iodine intake amongst pregnant women in each trimester of pregnancy to prevent adverse birth outcomes in central China is a challenge for clinical practice. METHODS: 870 pregnant women and their infants were enrolled in the study. Urinary iodine concentration (UIC) was measured using an inductively coupled plasma mass spectrometry (ICP-MS). Maternal and newborn information were obtained during follow-up. Multinomial logistic regression models were established. RESULTS: Median UIC of pregnant women was 172 ± 135 µg/L which is currently considered to be sufficient. Multivitamin supplements containing iodine, iodized salt intake and frequent milk intake were significantly associated with higher UIC. Multivariate logistic regression analysis showed that multivitamin supplements containing iodine and milk consumption were risk factors for more than adequate iodine (UIC ≥ 250 µg/L). Iodine-rich diet was significantly related to heavier birthweight, larger head circumference and longer femur length of the newborns while more than adequate iodine intake (UIC ≥ 250 µg/L) was a risk factor for macrosomia. Logistic regression models based on potential risk factors involving iodine containing supplements and iodine-rich diet were established to predict and screen pregnant women with high risk of more than adequate iodine intake among local pregnant women in different trimesters and guide them to supplement iodine reasonably to prevent the risk. CONCLUSIONS: Multivitamin supplements containing iodine and milk consumption were risk factors for maternal UIC ≥ 250 µg/L which was a risk factor for macrosomia. Iodine monitoring models were established to provide guidance for pregnant women to reduce the risk of more than adequate iodine intake, thereby contributing to reduce the risk of having a macrosomia.

LncRNA DNM3OS regulates GREM2 via miR-127-5p to suppress early chondrogenic differentiation of rat mesenchymal stem cells under hypoxic conditions.

BACKGROUND: Improved chondrogenic differentiation of mesenchymal stem cells (MSCs) by genetic regulation is a potential method for regenerating articular cartilage. MiR-127-5p has been reported to promote cartilage differentiation of rat bone marrow MSCs (rMSCs); however, the regulatory mechanisms underlying hypoxia-stimulated chondrogenic differentiation remain unknown. METHODS: rMSCs were induced to undergo chondrogenic differentiation under normoxic or hypoxic conditions. Expression of lncRNA DNM3OS, miR-127-5p, and GREM2 was detected by quantitative real-time PCR. Proteoglycans were detected by Alcian blue staining. Western blot assays were performed to examine the relative levels of GREM2 and chondrogenic differentiation related proteins. Luciferase reporter assays were performed to assess the association among DNM3OS, miR-127-5p, and GREM2. RESULTS: MiR-127-5p levels were upregulated, while DNM3OS and GREM2 levels were downregulated in rMSCs induced to undergo chondrogenic differentiation, and those changes were attenuated by hypoxic conditions (1% O2). Further in vitro experiments revealed that downregulation of miR-127-5p reduced the production of proteoglycans and expression of chondrogenic differentiation markers (COL1A1, COL2A1, SOX9, and ACAN) and osteo/chondrogenic markers (BMP-2, p-SMAD1/2). MiR-127-5p overexpression produced the opposite results in rMSCs induced to undergo chondrogenic differentiation under hypoxic conditions. GREM2 was found to be a direct target of miR-127-5p, which was suppressed in rMSCs undergoing chondrogenic differentiation. Moreover, DNM3OS could directly bind to miR-127-5p and inhibit chondrogenic differentiation of rMSCs via regulating GREM2. CONCLUSIONS: Our study revealed a novel molecular pathway (DNM3OS/miR-127-5p/GREM2) that may be involved in hypoxic chondrogenic differentiation.

Association between carotid intima media thickness and small dense low-density lipoprotein cholesterol in acute ischaemic stroke.

BACKGROUND: Intima-media thickness (IMT) and small dense low-density lipoprotein cholesterol (sdLDL-C) have been reported to be related to atherosclerosis and stroke. This study is trying to explore the association between IMT and sdLDL-C in Chinese acute ischaemic stroke (AIS) subjects. METHODS: This study enrolled total 368 consecutive AIS patients and 165 non-AIS controls from November 2016 to February 2019. Mean IMT and carotid plaques were measured by using carotid ultrasonography method. Blood glucose and lipid parameters were measured by using an automatic biochemical instrument. SdLDL-C was detected by using the Lipoprint LDL system. IMT > 1.0 mm was defined as increased IMT. Plaque stability based on the nature of the echo was determined by ultrasound examination. Risk factors for IMT were identified by using multivariate logistic regression analysis. A logistic regression model was established to predict AIS risk. Python software (Version 3.6) was used for the statistical analysis of all data. RESULTS: The carotid IMT, proportion of plaques, and the sdLDL-C, triglycerides (TG) and glucose levels were obviously higher in AIS patients than those in controls. SdLDL-C level in the IMT thickening group was higher than that in the normal IMT group. SdLDL-C and total cholesterol (TC) were risk factors for IMT, while sdLDL-C was an independent risk factor. The IMT value of the unstable plaque group was markedly higher than that of the stable plaque group. The predictive value of IMT for AIS was better than that of low-density lipoprotein cholesterol (LDL-C) and non-high-density lipoprotein cholesterol (non-HDL-C) but not as good as that of sdLDL-C. A logistic regression model was established to predict AIS risk. Additionally, carotid IMT and sdLDL-C were closely related to AIS severity and outcomes. CONCLUSIONS: SdLDL-C and TC were risk factors for increased IMT, while sdLDL-C was an independent risk factor. A prediction model based on IMT and other variables was established to screen the population with high AIS risk.

Association between variants in vitamin D-binding protein gene and vitamin D deficiency among pregnant women in china.

BACKGROUND: The prevalence of vitamin D deficiency and insufficiency is extremely high in pregnant women worldwide. However, the association between single nucleotide polymorphisms (SNPs) in vitamin D metabolic pathway genes and 25-hydroxyvitamin D (25(OH)D) concentration among Chinese pregnant women is seldom reported. The risk of adverse neonatal outcomes due to maternal vitamin D deficiency has not been well investigated. METHODS: A total of 815 pregnant women and 407 infants were enrolled in this study. Serum 25(OH)D concentration was detected. DNA was extracted from the maternal blood for genotyping genetic SNPs in vitamin D pathway. An XGBoost model was established based on SNPs combined with external variables. RESULTS: Mean serum 25(OH)D level was 15.67 ± 7.98 ng/mL among the pregnant women. Seventy-five percent of pregnant women had 25(OH)D deficiency in China. SNPs of GC (rs17467825, rs4588, rs2282679, rs2298850, and rs1155563) were significantly associated with maternal 25(OH)D concentration. The influence of variants of rs17467825, rs4588, rs2282679, and rs2298850 on maternal 25(OH)D might be modified by vitamin D supplementation and sunshine exposure. An XGBoost model was established for monitoring 25(OH)D status in pregnant women and provided clinical advice to reduce the risk of 25(OH)D deficiency. Mothers with 25(OH)D deficiency hinted a risk for macrosomia. CONCLUSION: A high prevalence of vitamin D deficiency in China has been confirmed. A clinical model was established to guide pregnant women to supplement vitamin D according to genotype. Furthermore, we suggest the effect of maternal vitamin D status on the risk of macrosomia.

MiR-132-3p regulates ADAMTS-5 expression and promotes chondrogenic differentiation of rat mesenchymal stem cells.

Previous study showed that miRNA aberrant expression is involved in chondrogenic differentiation. In this study, we aimed to investigate the effects of miR-132-3p on chondrogenic differentiation and the underlying mechanisms. First, quantitative PCR were performed to determine the level of MiR-132-3p. Then, we used luciferase assay to examine the target of miR-132-3p. Proteoglycan was tested by Alcian blue staining assay. Moreover, the sex determining region Y-box 9 (SOX9), Collagen type II alpha 1 chain (COL2A1) and Aggrecan (ACAN) levels were analyzed by quantitative PCR, immunofluorescence and Western blotting. Our results showed that MiR-132-3p level was reduced in rat MSCs (rMSCs) during chondrogenic differentiation. Ectopic expression of miR-132-3p induced proteoglycan accumulation and the increase of ACAN, SOX9 and COL2A1 expression, which were involved in inducing chondrogenic differentiation of rMSCs. More importantly, ADAMTS-5 was identified as the target of MiR-132-3p. Knockdown of ADAMTS-5 increased proteoglycan level, but reduced the SOX9, ACAN, and COL2A1 levels during chondrogenic differentiation of rMSCs. Taken together, our results revels that MiR-132-3p promotes rMSCs chondrogenic differentiation, possibly mediated by targeting ADAMTS-5, which provided new perspective on the chondrogenic differentiation and pathology of osteoarthritis.

MicroRNA-99a regulates early chondrogenic differentiation of rat mesenchymal stem cells by targeting the BMPR2 gene.

Mesenchymal stem cells (MSCs) are candidates for the regeneration of articular cartilage as they possess the potential for chondrogenic differentiation. MSCs are easily obtained and expanded in vitro. Specific microRNAs (miRNAs) that regulate chondrogenesis have yet to be identified and the mechanisms involved remain to be defined. The miRNAs regulate biological processes by binding target mRNA to reduce protein synthesis. In this study, we show that expression of miR-99a and miR-125b-3p were increased during early chondrogenic differentiation of MSCs (rMSCs) derived from the Norwegian brown rat (Rattus norvegicus). MiR-99a knockdown promoted proteoglycan deposition and increased the expression of ACAN and COL2A1 during early chondrogenic differentiation. MiR-99a knockdown promoted early chondrogenic differentiation of rMSCs. A dual-luciferase reporter gene assay showed that miR-99a targeted a putative binding site in the 3'-UTR of bone morphogenetic protein (BMP) receptor type 2 (BMPR2). Overexpression of miR-99a reduced the expression levels of BMPR2 protein. The expression of total p38 and p-p38 increased at 7 and 14 days during early chondrogenic differentiation of rMSCs. Reduction in levels of total p38 and p-p38 protein followed miR-99a overexpression during early chondrogenic differentiation of rMSCs. BMPR2 silencing reversed the effects of miR-99a inhibition on proteoglycan deposition and protein expression of ACAN, COL2A1, total p38 and p-p38 during early chondrogenic differentiation of rMSCs. In conclusion, the findings of these in vitro studies in rat MSCs support a role for miR-99a as a negative regulator of early chondrogenic differentiation by directly targeting the BMPR2 gene at an early stage.

A nonsense mutation in DHTKD1 causes Charcot-Marie-Tooth disease type 2 in a large Chinese pedigree.

Charcot-Marie-Tooth (CMT) disease represents a clinically and genetically heterogeneous group of inherited neuropathies. Here, we report a five-generation family of eight affected individuals with CMT disease type 2, CMT2. Genome-wide linkage analysis showed that the disease phenotype is closely linked to chromosomal region 10p13-14, which spans 5.41 Mb between D10S585 and D10S1477. DNA-sequencing analysis revealed a nonsense mutation, c.1455T>G (p.Tyr485(∗)), in exon 8 of dehydrogenase E1 and transketolase domain-containing 1 (DHTKD1) in all eight affected individuals, but not in other unaffected individuals in this family or in 250 unrelated normal persons. DHTKD1 mRNA expression levels in peripheral blood of affected persons were observed to be half of those in unaffected individuals. In vitro studies have shown that, compared to wild-type mRNA and DHTKD1, mutant mRNA and truncated DHTKD1 are significantly decreased by rapid mRNA decay in transfected cells. Inhibition of nonsense-mediated mRNA decay by UPF1 silencing effectively rescued the decreased levels of mutant mRNA and protein. More importantly, DHTKD1 silencing was found to lead to impaired energy production, evidenced by decreased ATP, total NAD(+) and NADH, and NADH levels. In conclusion, our data demonstrate that the heterozygous nonsense mutation in DHTKD1 is one of CMT2-causative genetic alterations, implicating an important role for DHTKD1 in mitochondrial energy production and neurological development.

Genetic alterations predict poor efficacy, outcomes and resistance to second-line osimertinib treatment in non-small cell lung cancer.

The genetic heterogeneity of non-small cell lung cancer (NSCLC) may impact clinical response and outcomes to targeted therapies. In second-line osimertinib treatment for NSCLC, real-world data on genetic biomarkers for treatment efficacy and prognosis remain incomplete. This real-world study involved 68 NSCLC patients receiving first-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). All of these patients developed resistance, and 49 of them subsequently underwent second-line osimertinib treatment. A 639-gene DNA panel was employed to assess the impact of molecular alterations on treatment efficacy, clinical outcomes and resistance. The findings showed that the median progression-free survival (PFS) for second-line osimertinib therapy was 13.3 months. Genes alterations such as P21 (RAC1) activated kinase 5 (PAK5), RNA binding motif protein 10 (RBM10), and EPH receptor A3 (EPHA3) mutations were associated with significantly shorter PFS in osimertinib therapy. At multivariate analysis, they were all independent risk predictors of shorter PFS. Additionally, the median overall survival (OS) for osimertinib was 26.2 months. Glutamate ionotropic receptor NMDA type subunit 2A (GRIN2A), hepatocyte growth factor (HGF), and RBM10 mutations were significantly associated with poorer OS in osimertinib treatment. The multivariate analysis demonstrated that only RBM10 mutation emerged as an independent risk predictor of shorter OS. In vitro experiments showed that RBM10 mutations could promote the proliferation and migration ability of NSCLC cells and reduced cell apoptosis. The resistance mechanisms to osimertinib were heterogeneous. Histone cluster 1 H2B family member D (HIST1H2BD) acted as a novel resistance mechanism to osimertinib. Previously unreported HIST1H2BD mutations (p.K25Q and p.E36D) were detected in the NSCLC tissues. In vitro experiments confirmed that HIST1H2BD mutations led to resistance to osimertinib. In summary, we demonstrate that genetic biomarkers, such as PAK5, RBM10, and EPHA3, are independent predictors of PFS in second-line osimertinib treatment, with RBM10 emerging as an independent predictor of OS. Additionally, HIST1H2BD represents a novel resistance mutation to osimertinib. All of these findings offer valuable insights for making personalized treatment strategies for NSCLC patients.

DNM3OS/miR-127-5p/CDH11, activates Wnt3a/ß-catenin/LEF-1 pathway to form a positive feedback and aggravate spine facet joint osteoarthritis.

Spinal facet joint osteoarthritis (FJOA) is an OA disease with pathogenesis and progression uncovered. Our present study was performed to elucidate the role of DNM3OS on spinal FJOA. In this study, spine facet joint tissue of patients were collected. In vitro and in vivo models were constructed with SW1353 cells and rats. Hematoxylin and eosin (HE) staining, Safranin O-fast Green, Alcian blue staining, and Tolueine blue O (TBO) staining were employed for histology analyses. Quantitative PCR, western blotting, and Immunofluorescence were performed to evaluate the expression of genes. The levels of inflammatory cytokines were measured by enzyme-linked immunosorbent assay analysis. Cell Counting Kit-8 and flow cytometry were used for cell activity and apoptosis evaluation. The targeting sites between microRNA (miR)-127-5p and cadherin 11 (CDH11) were predicted TargetScan and miRbase database and confirmed by Dual-luciferase reporter assays. CHIP and EMS assay were employed to confirm the binding of LEF1and DNM3OS promoter. Our results showed that DNM3OS was found to upregulated, while miR-127-5p was downregulated in severe FJOA patients and inflammation-induced chondrosarcoma SW1353 cells. DNM3OS reduced cell activity, induced cell apoptosis and extracellular matrix (ECM) degradation by sponging miR-127-5p in vitro. miR-127-5p targeted CDH11 and inhibited wnt3a/ß-catenin pathway to regulate OA in vitro. LEF1 promoted DNM3OS transcription to form a positively feedback in activated wnt3a/ß-catenin pathway. In vivo rat model also confirmed that DNM3OS aggravated FJOA. In summary, DNM3OS/miR-127-5p/CDH11 enhanced Wnt3a/ß-Catenin/LEF-1 pathway to form a positive feedback and aggravate spinal FJOA.

Genetic mutation profiling reveals biomarkers for targeted therapy efficacy and prognosis in non-small cell lung cancer.

Introduction: The genetic heterogeneity of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations may affect clinical responses and outcomes to EGFR tyrosine kinase inhibitors (EGFR-TKIs). This study aims to investigate the genomic factors that influence the efficacy and clinical outcomes of first-line, second-line and third-line treatments in NSCLC and explore the heterogeneity of resistance mechanisms. Materials and methods: This real-world study comprised 65 patients with EGFR mutant NSCLC. Molecular alterations were detected using a customized DNA panel before and after administering targeted therapy. The efficacy and prognosis of each treatment line were evaluated. Results: In first-generation EGFR-TKIs treatment, gefitinib showed favorable efficacy compared to icotinib and erlotinib, particularly in patients with EGFR L858R mutations. The resistance mechanisms to first-generation EGFR-TKIs varied among different EGFR mutation cohorts and different first-generation EGFR-TKIs. In second-line EGFR-TKIs treatment, EPH receptor A3 (EPHA3), IKAROS family zinc finger 1 (IKZF1), p21 (RAC1) activated kinase 5 (PAK5), DNA polymerase epsilon, catalytic subunit (POLE), RAD21 cohesin complex component (RAD21) and RNA binding motif protein 10 (RBM10) mutations were markedly associated with poorer progression-free survival (PFS). Notably, EPHA3, IKZF1 and RBM10 were identified as independent predictors of PFS. The mechanisms of osimertinib resistance exhibited heterogeneity, with a higher proportion of non-EGFR-dependent resistant mutations. In third-line treatments, the combination of osimertinib and anlotinib demonstrated superior efficacy compared to other regimens. Glutamate ionotropic receptor NMDA type subunit 2A (GRIN2A) mutation was an independent risk indicator of shorter OS following third-line treatments. Conclusions: Comprehending the tumor evolution in NSCLC is advantageous for assessing the efficacy and prognosis at each stage of treatment, providing valuable insights to guide personalized treatment decisions for patients.

Discovery of novel EGFR-PROTACs capable of degradation of multiple EGFR-mutated proteins.

Although three generations of Epidermal growth factor receptor (EGFR) - TK inhibitors have been approved for the treatment of Non-small-cell lung cancers (NSCLC), their clinical application is still largely hindered by acquired drug resistance mediated new EGFR mutations and side effects. The Proteolysis targeting chimera (PROTAC) technology has the potential to overcome acquired resistance from mutant EGFR through a novel mechanism of action. In this study, we developed the candidate degrader IV-3 by structural modifications of the lead compound 13, which exhibited limited antiproliferative activity against HCC-827 cells. Compared to compound 13, IV-3 exhibited remarkable anti-proliferative activity against HCC-827 cells, NCI-H1975 cells, and NCI-H1975-TM cells (IC50 = 0.009 µM, 0.49 µM and 3.24 µM, respectively), as well as significantly inducing degradation of EGFR protein in these cell lines (DC50 = 17.93 nM, 0.25 µM and 0.63 µM, respectively). Further investigations confirmed that IV-3 exhibited superior anti-tumor activity in all xenograft tumor models through the degradation of mutant EGFR protein. Moreover, IV-3 showed no inhibitory activity against A431 and A549 cells expressing wild-type EGFR, thereby eliminating potential toxic side effects emerging from wild-type EGFR inhibition. Overall, our study provides promising insights into EGFR-PROTACs as a potential therapeutic strategy against EGFR-acquired mutation.

Targeted deletion of Kif18a protects from colitis-associated colorectal (CAC) tumors in mice through impairing Akt phosphorylation.

Kinesins are a superfamily of molecular motors involved in cell division or intracellular transport. They are becoming important targets for chemotherapeutic intervention of cancer due to their crucial role in mitosis. Here, we demonstrate that the kinesin-8 Kif18a is overexpressed in murine CAC and is a crucial promoter during early CAC carcinogenesis. Kif18a-deficient mice are evidently protected from AOM-DSS-induced colon carcinogenesis. Kif18A is responsible for proliferation of colonic tumor cells, while Kif18a ablation in mice promotes cell apoptosis. Mechanistically, Kif18a is responsible for induction of Akt phosphorylation, which is known to be associated with cell survival regulation. In conclusion, Kif18a is critical for colorectal carcinogenesis in the setting of inflammation by mechanisms of increased PI3K-AKT signaling. Inhibition of Kif18A activity may be useful in the prevention or chemotherapeutic intervention of CAC.

Gandouling alleviates nerve injury through PI3K/Akt/FoxO1 and Sirt1/FoxO1 signaling pathway to inhibit autophagy in the rats model of Wilson's disease.

INTRODUCTION: Previous studies have shown that Gandouling (GDL) may alleviate the nerve damage caused by Wilson's disease (WD) by inhibiting the autophagy of nerve cell mitochondria. However, its mechanisms are still unclear. Revealing the therapeutic mechanism of GDL is beneficial for its clinical application and provides theoretical support for the development of new formulations for treating WD. METHOD: This time we found that the oxidative stress level in the body of the copper-overloaded WD rates increased, neurons in the hippocampus were damaged, and autophagy occurred. GDL reversed these situations and significantly improved the learning, memory, and spatial cognitive abilities of the high-copper-loaded WD rates. After GDL intervention, the expression of phosphatidylinositol-3 kinase (PI3K), phosphorylated serine-threonine protein kinase (AKT), and phosphorylated forkhead box protein O1 (FoxO1) significantly increased, whereas FoxO1 in the nucleus decreased and phosphorylated FoxO1 in the cytoplasm also significantly raised. In addition, the expression of Sirt1 significantly declined, and Ac-FoxO1 in the nucleus also significantly increased. RESULTS: These data indicated that GDL may promote the phosphorylation of FoxO1 and promote its nucleation by activating the PI3K/AKT/FoxO1 signaling pathway and inhibit Ac-FoxO1 hydrolysis in the nucleus through the Sirt1/FoxO1 signaling pathway to suppress the transcriptional activity of FoxO1. CONCLUSION: Furthermore, it inhibited the expression of autophagy genes Atg12 and Gabarapl1. In summary, our work provides new insights into the potential mechanisms of GDL repairing WD neuronal damage through autophagy pathways.

Integrated Metabolomics and Network Pharmacology to Reveal the Mechanisms of Gandouling Tablets Against Copper-Overload-Induced Neuronal Injury in Rats with Wilson's Disease.

Purpose: Gandouling Tablets (GDL), a proprietary Chinese medicine, have shown a preventive effect against Wilson's disease (WD)-induced neuronal damage in previous studies. However, the potential mechanisms need additional investigation. Combining metabonomics and network pharmacology revealed the GDL pathway against WD-induced neuronal damage. Methods: The WD rat model with a high copper load was developed, and nerve damage was assessed. Total metabonomics was used to identify distinct hippocampus metabolites and enriched metabolic pathways in MetaboAnalyst. The GDL's possible targets against WD neuron damage were then determined by network pharmacology. Cytoscape constructed compound metabonomics and pharmacology networks. Moreover, molecular docking and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) validated key targets. Results: GDL reduced WD-induced neuronal injury. Twenty-nine GDL-induced metabolites may protect against WD neuron injury. According to network pharmacology, we identified three essential gene clusters, of which genes in cluster 2 had the most significant impact on the metabolic pathway. A comprehensive investigation identified six crucial targets, including UGT1A1, CYP3A4, CYP2E1, CYP1A2, PIK3CB, and LPL, and their associated core metabolites and processes. Four targets reacted strongly with GDL active components. GDL therapy improved five targets' expression. Conclusion: This collaborative effort revealed the mechanisms of GDL against WD neuron damage and a way to investigate the potential pharmacological mechanisms of other Traditional Chinese Medicine (TCM).

Advancing Concrete Mix Proportion through Hybrid Intelligence: A Multi-Objective Optimization Approach.

Concrete mixture design has been a key focus in concrete research. This study presents a new method for concrete mixture design by combining artificial neural networks (ANN), genetic algorithms (GA), and Scipy libraries for hybrid intelligent modeling. This method enables the prediction of concrete mechanical properties and the optimization of mix proportions with single or multi-objective goals. The GA is used to optimize the structure and weight parameters of ANN to improve prediction accuracy and generalization ability (R2 > 0.95, RMSE and MAE < 10). Then, the Scipy library combined with GA-ANN is used for the multi-objective optimization of concrete mix proportions to balance the compressive strength and costs of concrete. Moreover, an AI-based concrete mix proportion design system is developed, utilizing a user-friendly GUI to meet specific strength requirements and adapt to practical needs. This system enhances optimization design capabilities and sets the stage for future advancements. Overall, this study focuses on optimizing concrete mixture design using hybrid intelligent modeling and multi-objective optimization, which contributes to providing a novel and practical solution for improving the efficiency and accuracy of concrete mixture design in the construction industry.

[Nonsense-mediated mRNA decay and human monogenic disease].

Nonsense-mediated mRNA decay (NMD) is a widespread quality control mechanism in eukaryotic cells. It can recognize and degrade aberrant transcripts harbouring a premature translational termination codon (PTC), and thereby prevent the production of C-terminally truncated proteins which might be deleterious. Approximately, 30% of human genetic diseases are caused by transcripts containing PTCs. These transcripts are potential targets of NMD. As for monogenic diseases, NMD has effects on the phenotype or mode of inheritance. Here, we explain the mechanism of this surveillance pathway, and take several neuromuscular disorders as examples to discuss its influence for human monogenic diseases. The deeper understanding for NMD will shed light on the nosogenesis and therapies of monogenic diseases.