Mutant Type Research Articles

TAF15 inhibits p53 nucleus translocation and promotes HCC cell 5-FU resistance via post-transcriptional regulation of UBE2N.

Chemotherapy resistance is an important factor responsible for the low 5-year survival rate of hepatocellular carcinoma (HCC) patients. Ubiquitin-conjugating enzyme E2N (UBE2N) is a cancer-associated ubiquitin-conjugating enzyme that is expressed in HCC tissues, and its high expression is associated with a poor prognosis. This study explored the role played by UBE2N in development of 5-fluorouracil (5-FU) resistance in HCC cells. Three HCC cell lines (HepG2 [p53 wild type], Huh7 [p53 point mutant type], Hep3B [p53 non-expression type]), and one normal liver cell line (MIHA) were used in our present study. The IC50 value of 5-FU was determined using a cell counting kit-8 (CCK-8) assay. Cell viability was assessed by colony formation assays. TUNEL assays and flow cytometry were used to analyze cell apoptosis. RNA pull-down and RNA immunoprecipitation (RIP) assays were performed to confirm the binding relationship between UBE2N mRNA and TAF15 protein. Our results showed that TAF15 and UBE2N were highly expressed in HCC cells. UBE2N inhibited the translocation of p53 protein into the cell nucleus to increase 5-FU resistance, as reflected by an increased IC50 value, an increase in cell viability, and a reduction in cell apoptosis. Overexpression of p53 reduced 5-FU resistance, but that effect could be reversed by UBE2N overexpression. TAF15 protein bound to and stabilized UBE2N mRNA, thereby inhibiting p53 translocation into the nucleus and promoting 5-FU resistance in HCC cells. Collectively, our present study identified a novel mechanism by which TAF15/UBE2N regulates p53 distribution to increase 5-FU resistance. Our results also suggest potential therapeutic strategies for treating HCC.

OsNAL11 and OsGASR9 Regulate the Low-Temperature Germination of Rice Seeds by Affecting GA Content

Low temperatures cause serious threat to rice seed emergence, which has become one of the main limiting factors in the production of direct seeding rice. It is of great importance to study the genes controlling low-temperature tolerance during seed germination and to mine the possible regulatory mechanism for developing new rice varieties with immense low-temperature germination ability. In the current research study, two types of mutants of nal11 and gasr9, derived from the WT (wild type) ZH11, were used for the analysis of low-temperature germinability. The results showed that the nal11 and gasr9 mutants displayed no significant difference in germination rate with ZH11 at room temperature, but the mutants showed significantly lower germination rates, germination potential and germination index, and slowed seedling growth in the simulated direct seeding experiments at low temperatures compared to ZH11. Additionally, the activity of POD, SOD, CAT, and anti-superoxide anion radial activity were significantly reduced, but the levels of MDA and H2O2 were significantly higher in the nal11 and gasr9 mutant seeds that were germinated at low temperatures compared to ZH11. Further analysis revealed that the levels of total active GA, especially GA4 and GA7, were significantly lower in the nal11 and gasr9 mutants than that in ZH11 during low-temperature germination. Based on qRT-PCR analysis, the expression levels of some GA synthesis-related genes were higher, whereas some were lower in the nal11 and gasr9 mutants than those in ZH11, however, the GA metabolism-related genes OsGA2ox8 and OsGA2ox10 and the GA signaling negative regulator gene SLR1 were significantly up-regulated in both nal11 and gasr9 mutants at several time points during low-temperature germination. This may explain the lower GA levels in the nal11 and gasr9 mutants. Furthermore, the interaction between the OsNAL11 and OsGASR9 proteins was confirmed by Y2H, LUC, and Co-IP assays. This study provides preliminary insights into the regulatory mechanism of the OsNAL11 and OsGASR9 genes, which control the low-temperature germination of rice seeds by affecting the GA pathway. Our study will provide the basis for further mining the molecular mechanisms of low-temperature germination in rice and valuable theoretical reference for breeding varieties with strong low-temperature germinability.

Establishment of potential reference measurement procedure and reference materials for EML4-ALK fusion variants measurement

Echinoderm microtubule-associated protein 4 (EML4) - anaplastic lymphoma kinase (ALK) fusion gene detection is of great significance in personalized tumor treatment. With the development of EML4-ALK fusion variants detection, it is necessary to establish traceability to ensure the consistency and comparability of its detection results in clinical practice. The establishment of traceability relies on SI traceable reference materials (RMs) and potential reference measurement procedures (RMPs). In this study, a potential RMP for the quantitative detection of V1 and V3 fusion mutations and the reference type (ALK-ref, including wild type, V1 and V3 mutant type) based on reverse transcription dPCR (RT-dPCR) and EML4-ALK fusion gene RMs were established. The proposed potential RMP has high specificity, good inter-laboratory reproducibility (CV < 7.3%) and good linear relationship (0.92 < slope < 1.06, R2 ≧ 0.99). The limit of detection (LoD) of V1, V3, and ALK-ref are 2 copies/reaction, 2 copies/reaction, and 1 copy/reaction, respectively. Interlaboratory studies using the EML4-ALK RMs and potential RMP showed that participating laboratories can produce consistent copy concentrations of fusion variant and ALK-ref as well as the ratio of EML4-ALK/ALK-ref. The established potential RMP with high specificity and accuracy can be used to characterize the EML4-ALK RMs, and the potential RMP and RM are useful to establish the traceability of EML4-ALK fusion measurement to improve the comparability and consistency in clinical tests.

Development of a High-Resolution Melting Method for the Detection of Clarithromycin-Resistant Helicobacter pylori in the Gastric Microbiome.

Background: The issue of Helicobacter pylori (H. pylori) resistance to clarithromycin (CLR) has consistently posed challenges for clinical treatment. Hence, a rapid susceptibility testing (AST) method urgently needs to be developed. Methods: In the present study, 35 isolates of H. pylori were isolated from 203 gastritis patients of the Guangzhou cohort, and the antimicrobial resistance phenotypes were associated with their genomes to analyze the relevant mutations. Based on these mutations, a rapid detection system utilizing high-resolution melting (HRM) curve analysis was designed and verified by the Shenzhen cohort, which consisted of 38 H. pylori strains. Results: Genomic analysis identified the mutation of the 2143 allele from A to G (A2143G) of 23S rRNA as the most relevant mutation with CLR resistance (p < 0.01). In the HRM system, the wild-type H. pylori showed a melting temperature (Tm) of 79.28 ± 0.01 °C, while the mutant type exhibited a Tm of 79.96 ± 0.01 °C. These differences enabled a rapid distinction between two types of H. pylori (p < 0.01). Verification examinations showed that this system could detect target DNA as low as 0.005 ng/μL in samples without being affected by other gastric microorganisms. The method also showed a good performance in the Shenzhen validation cohort, with 81.58% accuracy, and 100% specificity. Conclusions: We have developed an HRM system that can accurately and quickly detect CLR resistance in H. pylori. This method can be directly used for the detection of gastric microbiota samples and provides a new benchmark for the simple detection of H. pylori resistance.

Novel Approach for Detection of BRAF and KRAS Mutations Using ARMS (amplification refractory mutation system) Primers for Colon Cancer Therapeutic Response Prediction

Abstract Introduction/Objective KRAS and BRAF V600E mutations are confirmed predictive biomarkers for anti-EGFR monoclonal antibody therapy response and prognosis for colorectal cancer and often considered mutually exclusive with few exceptions (0.05% of mCRC cases). We have developed a multiplex PCR assay utilizing ARMS (amplification refractory mutation system) Primers and Probes for screening and further testing of BRAF mutation status in a 2 step process. Methods/Case Report The Multiplex PCR Assay or BRAF/KRAS mutation was designed as a 5 color assay. Primers and Probes consisted of Wild Type Kras and wild and mutant (V600E/V600K) type BRAF primers and probes. For specimen, Positive and negative control standards made of engineered positive control plasmids for wild type KRAS &amp; BRAF and mutant type V600E &amp; V600K BRAF as well as internal control of bet actin and 2 Patient samples with known BRAF V600E mutant status were run. Mutant type V600E &amp; V600K BRAF were mixed with wild type V600 controls in different ratios as well. Results were interpreted as BRAF wild KRAS wild/ BRAF wild KRAS mutant/ BRAF V600E KRAS wild/BRAF V600K KRAS wild type and NGS was recommended in cases of concurrent BRAF and KRAS mutations. Results (if a Case Study enter NA) Positive/negative/internal control standards for wild type KRAS &amp; BRAF and mutant type V600E &amp; V600K BRAF showed expected results in both CFX 96 and ABI 7500 runs in triplicates. Graphs were able to determine separate Ct values and sigmoidal amplification plots that readily differentiated KRAS wild/BRAF wild/ BRAF V600E types. Mutant type V600E &amp; V600K BRAF that were mixed with wild type V600 controls in different ratios all showed expected results and were able to differentiate between wild and mutant types with accuracy. Also, the 2 patient samples with known (Sanger sequencing-diagnosed) V600E mutation status showed positive Ct values for KRAs wild and V600E probes and negative CT values for V600 wild/V600K probes. No invalid runs or unexpected control results were observed. Conclusion The ARMS BRAF/KRAS multiplex test is a efficient, accurate 2 step assay. In the patients and positive and negative controls prepared, it was able to reliably diagnose KRAS and BRAF mutation status in a matter of 4 hours running time. Given the fact that this is a cost effective assay that can utilize most commonly used Real time PCR machines this test may be clinically implemented if larger clinical trials utilizing tissue samples from a larger number of colon cancer patients are conducted.

Analysis of low-density lipoprotein receptor gene mutations in a family with familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a common monogenic autosomal dominant disorder, primarily mainly caused by pathogenic mutations in the low-density lipoprotein receptor (LDLR) gene. Through phenotypic-genetic linkage analysis, two LDLR pathogenic mutations were identified in FH families: c.G1027A (p.Gly343Ser) and c.G1879A (p.Ala627Thr). Whole exome sequencing was conducted on the proband with familial hypercholesterolemia to identify the target gene and screen for potential pathogenic mutations. The suspicious responsible mutation sites in 14 family members were analyzed using Sanger sequencing to assess genotype-phenotype correlations. Mutant and wild type plasmids were constructed and transfected into HEK293T cells to evaluate LDLR mRNA and protein expression. In parallel, bioinformatics tools were employed to predict structural and functional changes in the mutant LDLR. Immunofluorescence analysis revealed no significant difference in the intracellular localization of the p.Gly343Ser mutation, whereas protein expression of the p.Ala627Thr mutation was decreased and predominantly localized in the cytoplasm. Western blotting has showed that protein expression levels of the mutant variants were markedly declined in both cell lysates and supernatants. Enzyme linked immunosorbent assay has demonstrated that LDLR protein levels in the supernatant of cell culture medium was not significant different from those of the wild-type group. However, LDLR protein levels in the cell lysate of both the Gly343Ser and Ala627Thr variants groups were significantly lower than those in the wild-type group. Bioinformatic predictions further suggested that these mutations may affect post-translational modifications of the protein, providing additional insight into the mechanisms underlying the observed reduction in protein expression. In this study, we identified two heterozygous pathogenic variants in the LDLR gene, c.G1027A (p.Gly343Ser) and c.G1879A (p.Ala627Thr), in a family with familial hypercholesterolemia. We also conducted preliminary investigations into the mechanisms by which these mutations contribute to disease pathology.

SnRNA-seq reveals differential functional transcriptional pathway alterations in three mutant types of dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a leading cause of heart failure, characterized by ventricular dilation, thinning of the ventricular walls, and systolic dysfunction in either the left or both ventricles, often accompanied by fibrosis. Human cardiac tissue is composed of various cell types, including cardiomyocytes (CMs), fibroblasts (FBs), endothelial cells (ECs), macrophages, lymphocytes and so on. In DCM patients, these cells frequently undergo functional and phenotypic changes, contributing to contractile dysfunction, inflammation, fibrosis, and cell death, thereby increasing the risk of heart failure. This study focuses on DCM patients with mutations (LMNA, RBM20, and TTN) and analyzes functional changes in subpopulations of four cardiac cell types. The study involves functional annotation of subpopulations within each cell type and explores the association between gene mutations and specific functions and pathways. Additionally, the SCENIC method is employed of a particular cell subpopulation with significant functional importance, aiming to identify key transcriptional regulators in specific cell states. By analyzing the expression levels of ligand-receptor pairs in vCM4, vFB2, EC5.0, T cells, and NK cells across the DCM mutant genotypes, we predicted their signaling pathways and communications. This research provides insights into the molecular mechanisms of DCM and potential therapeutic targets.

Fractal dimension and lacunarity measures of glioma subcomponents are discriminative of the grade of gliomas and IDH status.

Since the overall glioma mass and its subcomponents-enhancing region (malignant part of the tumor), non-enhancing (less aggressive tumor cells), necrotic core (dead cells), and edema (water deposition)-are complex and irregular structures, non-Euclidean geometric measures such as fractal dimension (FD or "fractality") and lacunarity are needed to quantify their structural complexity. Fractality measures the extent of structural irregularity, while lacunarity measures the spatial distribution or gaps. The complex geometric patterns of the glioma subcomponents may be closely associated with the grade and molecular landscape. Therefore, we measured FD and lacunarity in the glioma subcomponents and developed machine learning models to discriminate between tumor grades and isocitrate dehydrogenase (IDH) gene status. 3D fractal dimension (FD3D) and lacunarity (Lac3D) were measured for the enhancing, non-enhancing plus necrotic core, and edema-subcomponents using preoperative structural-MRI obtained from the The Cancer Genome Atlas (TCGA) and University of California San Francisco Preoperative Diffuse Glioma MRI (UCSF-PDGM) glioma cohorts. The FD3D and Lac3D measures of the tumor-subcomponents were then compared across glioma grades (HGGs: high-grade gliomas vs. LGGs: low-grade gliomas) and IDH status (mutant vs. wild type). Using these measures, machine learning platforms discriminative of glioma grade and IDH status were developed. Kaplan-Meier survival analysis was used to assess the prognostic significance of FD3D and Lac3D measurements. HGG exhibited significantly higher fractality and lower lacunarity in the enhancing subcomponent, along with lower fractality in the non-enhancing subcomponent compared to LGG. This suggests that a highly irregular and complex geometry in the enhancing-subcomponent is a characteristic feature of HGGs. A comparison of FD3D and Lac3D between IDH-wild type and IDH-mutant gliomas revealed that mutant gliomas had ~2.5-fold lower FD3D in the enhancing-subcomponent and higher FD3D with lower Lac3D in the non-enhancing subcomponent, indicating a less complex and smooth enhancing subcomponent, and a more continuous non-enhancing subcomponent as features of IDH-mutant gliomas. Supervised ML models using FD3D from both the enhancing and non-enhancing subcomponents together demonstrated high-sensitivity in discriminating glioma grades (~97.9%) and IDH status (~94.4%). A combined fractal estimation of the enhancing and non-enhancing subcomponents using MR images could serve as a non-invasive, precise, and quantitative measure for discriminating glioma grade and IDH status. The combination of 2-hydroxyglutarate-magnetic resonance spectroscopy (2HG-MRS) with FD3D and Lac3D quantification may be established as a robust imaging signature for glioma subtyping.

A FRAMEWORK TO CONNECT VIRAL QUASISPECIES, MICROBIOME, AND HOST

COVID-19 is a pandemic that was detected in Wuhan, China, in 2019 and spread all around the world. COVID-19 is caused by SARS-CoV-2, which spreads through close contact. Many studies have been conducted on the transmission, virulence, and immune response of SARS-CoV-2. The intracellular mechanism of action of the virus and various host interaction pathways are also known, and there are many studies on the mutant types of the virus. Spike protein, which is one of the most important structural proteins of the virus, allows the virus to enter the cell. In this study, it has been investigated whether such an interaction occurs between Spike protein variants and host microbiota. For this purpose, the association of Sars-CoV-2 variants with different host factors that belong to the urban ecosystems has been analyzed statistically and host factors associated with different microbiota compositions were analyzed via a Bayesian Network. As a result, it is suggested that different variants of Spike protein may exist in the hosts that have dissimilar microbial compositions, and the microbiota may be a carrier that allows external factors -such as environment and human organisation- acting on hosts in the population to influence the selection of viral mutants

Dynamic Analysis of the Fruit Sugar-Acid Profile in a Fresh-Sweet Mutant and Wild Type in 'Shatangju' (Citrus reticulata cv.).

Citrate is a major determinant of fruit flavor quality. Currently, citrus species and/or varieties with significant alterations in citrate level have greatly advanced the molecular basis of citrate accumulation in fruit. However, in-depth dissections of the molecular mechanism specific to citrate accumulation are still limited due to the lack of mutants, especially within one single variety. In this study, a fresh-sweet 'Shatangju' mutant (Citrus reticulata cv.) was obtained during a survey of citrus resources in Guangdong, China, and the phenotype, fruit morphology, and primary flavor profiles were comparatively analyzed. Unlike the wild-type 'Shatangju' (WT), the mutant (MT) material exhibited a dwarfed and multi-branched tree shape, delayed flowering and fruit ripening at maturity, a prolonged fruit tree-retention time, and a decreased single fruit weight at maturity. Dynamic measurement of the metabolite levels further suggested that the contents and fluctuation patterns of vitamin C, malate, quinate, and oxalate showed no obvious difference between MT and MT fruits, while the citrate level in MT fruits significantly decreased over various developmental stages, ranging from 0.356 to 1.91 mg g-1 FW. In addition, the accumulation patterns of the major soluble sugars (sucrose, fructose, and glucose), as well as the sugar/acid ratio, were also altered in MT fruits during development. Taken together, this study provides a novel acid-free 'Shatangju' mutant, which can serve as a powerful tool for the research of fruit flavor quality, especially for the comprehensive understanding of the molecular mechanism of citrate accumulation in fruits.

Improvement of substrate specificity of the direct electron transfer type FAD-dependent glucose dehydrogenase catalytic subunit

The heterotrimeric flavin adenine dinucleotide (FAD) dependent glucose dehydrogenase derived from Burkholderia cepacia (BcGDH) has many exceptional features for its use in glucose sensing—including that this enzyme is capable of direct electron transfer with an electrode in its heterotrimeric configuration. However, this enzyme’s high catalytic activity towards not only glucose but also galactose presents an engineering challenge. To increase the substrate specificity of this enzyme, it must be engineered to reduce its activity towards galactose while maintaining its activity towards glucose. To aid in these mutagenesis studies, the crystal structure composed of BcGDH’s small subunit and catalytic subunit (BcGDHγα), in complex with D-glucono-1,5-lactone was elucidated and used to construct the three-dimensional model for targeted, site-directed mutagenesis. BcGDHγα was then mutated at three different residues, glycine 322, asparagine 474 and asparagine 475. The single mutations that showed the greatest glucose selectivity were combined to create the resulting mutant, α-G322Q-N474S-N475S. The α-G322Q-N474S-N475S mutant and BcGDHγα wild type were then characterized with dye-mediated dehydrogenase activity assays to determine their kinetic parameters. The α-G322Q-N474S-N475S mutant showed more than a 2-fold increase in Vmax towards glucose and this mutant showed a lower activity towards galactose in the physiological range (5 mM) of 4.19 U mg−1, as compared to the wild type, 86.6 U mg−1. This resulting increase in specificity lead to an 81.7 gal/glc % activity for the wild type while the α-G322Q-N474S-N475S mutant had just 10.9 gal/glc % activity at 5 mM. While the BcGDHγα wild type has high specificity towards galactose, our engineering α-G322Q-N474S-N475S mutant showed concentration dependent response to glucose and was not affected by galactose.

Gaining Insight into the Catalytic Mechanism of the R132H IDH1 Mutant: A Synergistic DFT Cluster and Experimental Investigation.

Human isocitrate dehydrogenase 1 (IDH1) is an enzyme that is found in humans that plays a critical role in aerobic metabolism. As a part of the citric acid cycle, IDH1 becomes responsible for catalyzing the oxidative decarboxylation of isocitrate to form α-ketoglutarate (αKG), with nicotinamide adenine dinucleotide phosphate (NADP+) as a cofactor. Strikingly, mutations of the IDH1 enzyme have been discovered in several cancers including glioblastoma multiforme (GBM), a highly aggressive form of brain cancer. It has been experimentally determined that single-residue IDH1 mutations occur at a very high frequency in GBM. Specifically, the IDH1 R132H mutation is known to produce (D)2-hydroxyglutarate (2HG), a recognized oncometabolite. Using the previously determined catalytic mechanism of IDH1, a DFT QM model was developed to study the mechanistic properties of IDH1 R132H compared to wild type enzyme. Validating these insights, biochemical in vitro assays of metabolites produced by mutant vs wild type enzymes were measured and compared. From the results discussed herein, we discuss the mechanistic impact of mutations in IDH1 on its ability to catalyze the formation of αKG and 2HG.

Biological responses of an elite centipedegrass [Eremochloa ophiuroides (Munro) Hack.] cultivar (Ganbei) to carbon ion beam irradiation.

Carbon ion beam irradiation (CIBI) is a highly efficient mutagenesis for generating mutations that can be used to expand germplasm resources and create superior new germplasm. The study investigated the effects of different doses of CIBI (50 Gy, 100 Gy, 150 Gy, 200 Gy and 300 Gy) on seed germination and seedling survival, seedling morphological and physiological traits of an elite centipedegrass cultivar Ganbei. The results showed that irradiation greater than 50 Gy cause inhibition of seed germination, and the semi-lethal dose (LD50) is around 90 Gy for CIBI treated seeds of Ganbei. A carbon ion beam-mutagenized centipedegrass population was generated from Ganbei, with irradiation dosages from 50 Gy to 200 Gy. More than ten types of phenotypic variations and novel mutants with heritable tendencies mainly including putative mutants of stolon number, length and diameter, of internode length, of leaf length and width, of leaf chlorophyll content, of stolon growth rate, of aboveground tissue dry weight, of sward height were identified. While the total sugar content of the plants from irradiated seeds showed no obvious change in all treatments as compared to the control, the crude protein content displayed significant reduction at a high-dose treatment of 200 Gy. Genetic polymorphism was detected in mutagenized centipedegrass population using SSR-PCR analysis, suggesting that CIBI caused alteration of larger fragments of the DNA sequence. As a result, a preliminary batch of mutants was screened in this study. In summary, carbon ion beam mutagenesis is an effective way for developing centipedegrass germplasm with wider variation, and treating seeds with CIBI at a dosage of ~100 Gy could be effective in centipedegrass mutation breeding.

β-glucan combined with Envafolimab and Endostar as immune rechallenge for metastatic non-small cell lung cancer

BackgroundImmune checkpoint inhibitor rechallenge has emerged as a prominent study area in non-small cell lung cancer (NSCLC). β-glucan was reported to reverse resistance to anti-PD-1/PD-L1 inhibitors by regulating the tumor microenvironment. In this self-initiated clinical trial (ChiCTR2100054796), NSCLC participants who have previously failed anti-PD-1 therapy received β-glucan (500 mg, bid, d1-21), Envafolimab (300 mg, d1) and Endostar (210 mg, civ72h) every 3 weeks until disease progression or unacceptable toxicity. The clinical efficacy and adverse events were observed, while serum samples were collected for proteomic analysis.ResultsTwenty Three patients were enrolled from January 2022 to March 2023 (median age, 65 years; male, n = 18 [78.3%]; squamous NSCLC, n = 9 [39.1%]; mutant type, n = 13 [56.5%]). The overall response rate (ORR) was 21.7% and disease control rate (DCR) was 73.9%. Median progression-free survival (mPFS) and median overall survival (mOS) was 4.3 months [95% CI: 2.0–6.6] and 9.8 months [95% CI: 7.2–12.4], respectively. The mPFS between PD-L1 positive and negative subgroup has significant difference (6.3 months vs. 2.3 months, p = 0.002). Treatment-related adverse events (TRAEs) occurred in 52.2% of patients. The most common TRAEs were hypothyroidism (26.1%) and fatigue (26.1%). 2 (8.7%) grade 3 adverse events were reported. No adverse reaction related deaths have been observed. Proteomic analysis revealed that the levels of CASP-8, ARG1, MMP12, CD28 and CXCL5 correlated with resistance to the treatment while the levels of CD40-L and EGF related to the favorable response.Conclusionβ-glucan combined with Envafolimab and Endostar has considerable efficacy and safety for immune rechallenge in metastatic NSCLC patients who failed of anti-PD-1 treatment previously, especially for PD-L1 positive patients.

Marine Compound-Carpatamide D as a Potential Inhibitor Against TOP2A and Its Mutant D1021Y in Colorectal Cancer: Insights from DFT, MEP and Molecular Dynamics Simulation.

Colorectal cancer (CRC) ranks as the third most prevalent cancer globally, hence there is an urgent need for new and effective therapeutic options. DNA topoisomerase 2A (TOP2A) plays a crucial role in the cell cycle and is involved in CRC progression, making it essential to identify structural and functional relevant alterations. Among the 24 mutations, our findings indicated that mutation D1021Y has the most deleterious effect on the TOP2A protein. Based on virtual screening of 31,561 compounds, we identified three lead candidates: 17683 (nigrospoxydon C), 28461 (carpatamide D), and 28853 (6'-O-acetyl-isohomaarbutin), which showed promising inhibitory effect against TOP2A and its mutant form. These compounds were assessed for their stability using density functional theory (DFT) analysis, where carpatamide D possessed the least energy gap of 4.398eV showing its high reactivity among all. Further, molecular docking also shows the carpatamide D as the top candidate, which exhibited favourable docking energy against the TOP2A wild type (- 7.47kcal/mol) and with D1021Y mutant (- 7.62kcal/mol) as compared to reference compound PK1, which showed - 6.11kcal/mol TOP2A wild type and - 6.24kcal/mol against mutant type. The molecular dynamics simulation was performed to analyse the dynamics and stability of complex, which revealed TOP2A_28641 and D1021Y_28641 complexes to be stable with least root-mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF). Molecular mechanics/Poisson-Boltzmann surface area calculations indicated that TOP2A_28641 and D1021Y_28641 complexes exhibited the lowest binding energy of - 23.55kcal/mol and - 25.03kcal/mol, respectively. Our findings suggest carpatamide D as a promising lead compound for the TOP2A_D1021Y targeted cancer therapies, which needs further experimental validation.

Therapeutic effects of focused ultrasound on vulvar squamous intraepithelial lesions in rat

Objective In this study, we established a Sprague-Dawley rat model of vulvar squamous intraepithelial lesions and investigated the impact of focused ultrasound on the expression of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and mutant type p53 (mtp53) in the vulvar skin of rats with low-grade squamous intraepithelial lesions (LSIL). Materials and methods The vulvar skin of 60 rats was treated with dimethylbenzanthracene (DMBA) and mechanical irritation three times a week for 14 weeks. Rats with LSIL were randomly allocated into the experimental group or the control group. The experimental group was treated with focused ultrasound, while the control group received sham treatment. Results After 14 weeks treatment of DMBA combined with mechanical irritation, LSIL were observed in 44 (73.33%) rats, and high-grade squamous intraepithelial lesions (HSIL) were observed in 14 (23.33%) rats. 90.91% (20/22) of rats showed normal pathology and 9.09% (2/22) of rats exhibited LSIL in the experimental group at four weeks after focused ultrasound treatment. 22.73% (5/22) of rats exhibited LSIL, 77.27% (17/22) of rats progressed to HSIL in the control group. Compared with the control-group rats, the levels of HIF-1α, VEGF and mtp53 were significantly decreased in experimental-group rats (p < 0.05). Conclusions These results indicate that DMBA combined with mechanical irritation can induce vulvar squamous intraepithelial lesion in SD rats. Focused ultrasound can treat LSIL safely and effectively, prevent the progression of vulvar lesions, and improve the microenvironment of vulvar tissues by decreasing the localized expression of HIF-1α, VEGF, and mtp53 in rats.

Application of CRISPR/Cas9 system to knock out GluB gene for developing low glutelin rice mutant

The nutritional quality improvement is among the most integral objective for any rice molecular breeding programs. The seed storage proteins (SSPs) have greater role to determine the nutritional quality of any cereal grains. Rice contains relatively balanced amino acid composition and the SSPs are fractioned into albumins (ALB), globulins (GLO), prolamins (PRO) and glutelins (GLU) according to differences in solubility. GLUs are further divided into subfamilies: GluA, GluB, GluC, and GluD depending on resemblance in amino acid. The GLU protein accounts for 60–80% of total protein contents, encoded by 15 genes located on different chromosomes of rice genome. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system was employed to knockout Glu-B (LOC-Os02g15070) gene in non-basmati rice PK386 cultivar. The mutant displayed two base pair and three base pair mutation in the targeted regions. The homozygous mutant plant displayed reduction for both in total protein contents and GLU contents whereas, elevation in GLO, ALB and PRO. Moreover, the mutant plant also displayed reduction in physio-chemical properties e.g., total starch, amylose and gel consistency. The agronomic characteristics of both mutant and wild type displayed non-significant differences along with increase in higher percentage of chalkiness in mutant plants. The results obtained from scanning electron microscopy showed the loosely packed starch granules compared to wild type. The gene expression analysis displayed the lower expression of gene at 5 days after flowering (DAF), 10 DAF, 15 DAF and 20 DAF compared to wild type. GUS sub-cellular localization showed the staining in seed which further validated the results obtained from gene expression. Based on these findings it can be concluded Glu-B gene have significant role in controlling GLU contents and can be utilized in breeding programs to enhance the nutritional quality of rice, and may serve as healthy diet for patient allergic with high GLU contents.

Tumor markers in non-small cell lung cancer spine metastasis: an assessment of prognosis and overall survival

PurposeThe identification of gene mutations in the modern medical workup of metastatic spine tumors has become more common but has not been highly utilized in surgical planning. Potential utility of these genetic markers as surrogates for cancer behavior in current prognosis scoring systems and overall survival (OS) remains underexplored in existing literature. This study seeks to investigate the association of frequently identified tumor markers, EGFR, ALK, and PD-L1, in metastatic non-small cell lung cancer (NSCLC) to the spine with Tokuhashi prognosis scoring and OS.MethodsPatients with NSCLC metastasis to spine were identified through chart review. EGFR, ALK, and PD-L1 wild type vs. mutant type were identified from targeted chemotherapy genetic testing. Multiple linear regression was performed to assess gene profile contributions to Tokuhashi score. Cox Proportional Hazards models were generated for each tumor marker to assess the relationship between each marker and OS.ResultsA total of 119 patients with NSCLC spine metastasis were identified. We employed a multiple linear regression analysis to investigate the influence of EGFR, ALK, and PD-L1 genotypes on the Tokuhashi score, revealing statistically significant relationships overall (p = 0.002). Individual genotype contributions include EGFR as a non-significant contributor (p = 0.269) and ALK and PD-L1 as significant contributors (p = 0.037 and p = 0.001 respectively). Overall survival was not significantly associated with tumor marker profiles through Kaplan-Meier analysis (p = 0.46) or by multivariable analysis (p = 0.108).ConclusionALK and PD-L1 were significantly associated with Tokuhashi score while EGFR was not. Tumor markers alone were not predictive of OS. These findings indicate that genetic markers found in NSCLC metastases to the spine may demonstrate prognostic value. Therefore, employing standard tumor markers could enhance the identification of appropriate surgical candidates, although they demonstrate limited effectiveness in predicting overall survival.

Gene inactivation of lysyl oxidase in smooth muscle cells reduces atherosclerosis burden and plaque calcification in hyperlipidemic mice

Background and aimsLysyl oxidase (LOX) catalyzes the crosslinking of collagen and elastin to maintain tensile strength and structural integrity of the vasculature. Excessive LOX activity increases vascular stiffness and the severity of occlusive diseases. Herein, we investigated the mechanisms by which LOX controls atherogenesis and osteogenic differentiation of vascular smooth muscle cells (SMC) in hyperlipidemic mice. MethodsGene inactivation of Lox in SMC was achieved in conditional knockout mice after tamoxifen injections. Atherosclerosis burden and vascular calcification were assessed in hyperlipidemic conditional [Loxf/fMyh11-CreERT2ApoE−/−] and sibling control mice [Loxwt/wtMyh11-CreERT2ApoE−/−]. Mechanistic studies were performed with primary aortic SMC from Lox mutant and wild type mice. ResultsInactivation of Lox in SMCs decreased > 70 % its RNA expression and protein level in the aortic wall and significantly reduced LOX activity without compromising vascular structure and function. Moreover, LOX deficiency protected mice against atherosclerotic burden (13 ± 2 versus 23 ± 1 %, p < 0.01) and plaque calcification (5 ± 0.4 versus 11.8 ± 3 %, p < 0.05) compared to sibling controls. Interestingly, gene inactivation of Lox in SMCs preserved the contractile phenotype of vascular SMC under hyperlipidemic conditions as demonstrated by single-cell RNA sequencing and immunofluorescence. Mechanistically, the absence of LOX in SMC prevented excessive collagen crosslinking and the subsequent activation of the pro-osteogenic FAK/β-catenin signaling axis. ConclusionsLox inactivation in SMC protects mice against atherosclerosis and plaque calcification by reducing SMC modulation and FAK/β-catenin signaling.

Integrated multi-omics characterization of SMAD4 mutant colorectal cancer

Colorectal cancer is one of the most common cancers around the world, which is a severe threat to people’s health. SMAD4 belongs to the dwarfin/SMAD family, which plays a crucial role in TGF-β and BMP signal pathways. As the molecular characterization of colon cancer patients following SMAD4 mutations remains unclear, we integrated multi-omics data of SMAD4 mutant patients to reveal the profile of molecular characterization of SMAD4 mutation. A missense mutation is the most common mutant type of SMAD4. Patients with SMAD4 mutation had worse survival. Tumor tissues from patients carrying the SMAD4 mutation showed a reduction in various immune cells, such as CD4 + memory T cells and memory B cells. Many differential genes were identified compared to the SMAD4 mutation-free group and could be significantly enriched for tumor- and immune-related signaling pathways. In addition, the mutant group had different drug sensitivities than the non-mutant group.