[Correlation of miR-155 Expression with Drug Sensitivity of FLT3-ITD+ Acute Myeloid Leukemia Cell Line and Its Mechanism].

OBJECTIVE: To investigate the correlation of miR-155 expression with drug sensitivity of FLT3-ITD+ acute myeloid leukemia (AML) cell line and its potential regulatory mechanism. METHODS: By knocking out miR-155 gene in FLT3-ITD+ AML cell line MV411 through CRISPR/Cas9 gene-editing technology, monoclonal cells were screened. The genotype of these monoclonal cells was validated by PCR and Sanger sequencing. The expression of mature miRNA was measured by RT-qPCR. The treatment response of doxorubicin, quizartinib and midostaurin were measured by MTT assay and IC50 of these drugs were calculated to identify the sensitivity. Transcriptome sequencing was used to analyze change of mRNA level in MV411 cells after miR-155 knockout, gene set enrichment analysis to analyze change of signaling pathway, and Western blot to verify expressions of key molecules in signaling pathway. RESULTS: Four heterozygotes with gene knockout and one heterozygote with gene insertion were obtained through PCR screening and Sanger sequencing. RT-qPCR results showed that the expression of mature miR-155 in the monoclonal cells was significantly lower than wild-type clones. MTT results showed that the sensitivity of MV411 cells to various anti FLT3-ITD+ AML drugs increased significantly after miR-155 knockout compared with wild-type clones. RNA sequencing showed that the mTOR signaling pathway and Wnt signaling pathway were inhibited after miR-155 knockout. Western blot showed that the expressions of key molecules p-mTOR, Wnt5α and ß-catenin in signaling pathway were down-regulated. CONCLUSION: Drug sensitivity of MV411 cells to doxorubicin, quizartinib and midostaurin can be enhanced significantly after miR-155 knockout, which is related to the inhibition of multiple signaling pathways including mTOR and Wnt signaling pathways.

Observation of Nonlinear Exceptional Points with a Complete Basis in Dynamics.

The exotic physics associated with exceptional points (EPs) is always under the scrutiny of theoretical and experimental science. Recently, considerable effort has been invested in the combination of nonlinearity and non-Hermiticity. The concept of nonlinear EPs (NEPs) has been introduced, which can avoid the loss of completeness of the eigenbasis in dynamics while retaining the key features of linear EPs. Here, we present the first direct experimental demonstration of a NEP based on two non-Hermition coupled circuit resonators combined with a nonlinear saturable gain. At the NEP, the response of the eigenfrequency to perturbations demonstrates a third-order root law and the eigenbasis of the Hamiltonian governing the system dynamics is still complete. Our results bring this counterintuitive aspect of the NEP to light and possibly open new avenues for applications.

Nonlinear Exceptional Points with a Complete Basis in Dynamics.

Exceptional points (EPs) are special spectral singularities at which two or more eigenvalues, and their corresponding eigenvectors, coalesce and become identical. In conventional wisdom, the coalescence of eigenvectors inevitably leads to the loss of completeness of the eigenbasis. Here, we show that this scenario breaks down in general at nonlinear EPs (NEPs). As an example, we realize a fifth-order NEP (NEP_{5}) within only three coupled resonators with both a theoretical model and simulations in circuits. One stable and another four auxiliary steady eigenstates of the nonlinear Hamiltonian coalesce at the NEP_{5}, and the response of eigenfrequency to perturbations demonstrates a fifth-order root law. Intriguingly, the biorthogonal eigenbasis of the Hamiltonian governing the system dynamics is still complete, and this fact is corroborated by a finite Petermann factor instead of a divergent one at conventional EPs. Consequently, the amplification of noise, which diverges at other EPs, converges at our NEP_{5}. Our finding transforms the understanding of EPs and shows potential for miniaturizing various key applications operating near EPs.

Nonlinearity-enabled higher-order exceptional singularities with ultra-enhanced signal-to-noise ratio.

Higher-order exceptional points (HOEPs) with extraordinary responsivity are expected to exhibit a vastly improved performance in detection-related applications. However, over the past few years, such an approach has been questioned due to several potential drawbacks, including the stringent parameter requirements, fundamental resolution limits and noise. Here, exploring the consequence of nonlinear gain saturation in exceptional singularities of non-Hermitian systems, we offer a feasible scheme to overcome all the above difficulties. We provide a simple and intuitive example by demonstrating with both theory and circuit experiments an 'exceptional nexus' ('EX'), a HOEP with an ultra-enhanced signal-to-noise ratio (SNR), in only two coupled resonators with the aid of nonlinear gain. The tedious parameter tuning in a six-dimensional hyper-dimensional space is reduced to two dimensions. The feedback mechanism of nonlinear saturable gain can give a solution to the ongoing debate on the SNR of EPs in other linear systems. Our findings advance the fundamental understanding of the peculiar topology of nonlinear non-Hermitian systems, significantly reduce the practical difficulty in EP sensing and possibly open new avenues for applications.

Elastic fiber degradation in the development of pediatric granuloma annulare: Report of 39 cases.

BACKGROUND: Granuloma annulare (GA) has diverse clinical manifestations, multiple subtypes, and unknown etiology and pathogenesis. Existing studies regarding GA in children are scarce. AIM: To examine the correlation between clinical manifestation and histopathology of pediatric GA. METHODS: A total of 39 patients under 18 years of age with both a clinical and pathological diagnosis of GA at Kunming Children's Hospital from 2017 to 2022 were retrieved. Their medical records were consulted, and clinical data of the children were recorded and summarized, including gender, age, disease site, etc. Existing wax blocks of skin lesion specimens of children and pathological films were retrieved for further study and relevant histology, including hematoxylin-eosin, Alcian blue, elastic fiber (Victoria blue-Lichon red method), and antacid staining. Finally, the children's clinical manifestations, histopathological results, and special staining characteristics were analyzed. RESULTS: The clinical manifestations of granuloma annulare in children were diverse: 11 cases presented with a single lesion, 25 with multiple lesions, and 3 with generalized lesions. The pathological typing comprised histiocytic infiltration, palisading granuloma, epithelioid nodular, and mixed types in 4, 11, 9, and 15 cases, respectively. Thirty-nine cases were negative for antacid staining. The positive rate of Alcian blue staining was 92.3%, and that of elastic fiber staining was 100%. The degree of elastic fiber dissolution and granuloma annulare histopathological typing were positively correlated (r = 0.432, P < 0.05). No correlation was found between clinical presentation and histopathological typing of the granuloma annulare in children. In the pathological diagnosis of granuloma annulare, the positive elastic fiber staining rate was higher than that of Alcian blue staining. A correlation was found between elastic fiber dissolution degree and histopathological staging. However, the differences in pathological staging may have been related to the pathological manifestation of granuloma annulare at different periods. CONCLUSION: Elastic fiber degradation may be a critical step in the pathogenesis of pediatric granuloma annulare. This is also one of the first studies focused on granuloma annulare in children.

[Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD+AML].

OBJECTIVE: Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes. METHODS: The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib. RESULTS: In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate. CONCLUSION: The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.

[Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD+AML].

OBJECTIVE: Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes. METHODS: The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib. RESULTS: In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate. CONCLUSION: The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.

Radiomics in Antineoplastic Agents Development: Application and Challenge in Response Evaluation.

The recent spring up of the antineoplastic agents and the prolonged survival bring both challenge and chance to radiological practice. Radiological methods including CT, MRI and PET play an increasingly important role in evaluating the efficacy of these antineoplastic drugs. However, different antineoplastic agents potentially induce different radiological signs, making it a challenge for radiological response evaluation, which depends mainly on one-sided morphological response evaluation criteria in solid tumors (RECIST) in the status quo of clinical practice. This brings opportunities for the development of radiomics, which is promising to serve as a surrogate for response evaluations of anti-tumor treatments. In this article, we introduce the basic concepts of radiomics, review the state-of-art radiomics researches with highlights of radiomics application in predictions of molecular biomarkers, treatment response, and prognosis. We also provide in-depth analyses on major obstacles and future direction of this new technique in clinical investigations on new antineoplastic agents.

[Identify Myeloid Differentiation-Related MiRNAs Response to ATRA Induction by RNA Sequencing and CRISPR/Cas9 Gene Editing].

OBJECTIVE: To identify differentiation related miRNA and evaluate roles of miRNA during ATRA induced myeloid differentiation. METHODS: The small RNA sequencing was used to analyze differential expressed miRNAs in ATRA induced NB4 cells. Then the several up or down-regulated miRNA were selected as the research candidates. SgRNAs targeting the genome of each miRNA were designed and NB4 cells with inducible expression of Cas9 protein were generated. After transduced sgRNA into NB4/Cas9 cells, the mutation level by PCR and surveyor assay were evaluated. The cell differentiation level was investigated by surface CD11b expression via flow cytometry. RESULTS: A total of 410 mature miRNAs which expressed in NB4 cells were detected out after treated by ATRA, 74 miRNAs were up-regulated and 55 were down-regulated miRNAs with DNA cleavage generated by CRISPR/Cas9 was assayed directly by PCR or surveyor assay, quantitative PCR showed that the expression of miRNA was downregulated, which evaluated that gene edition successfully inhibitied the expression of mature miRNA. MiR-223 knockout showed the myeloid differentation of NB4 significantly inhibitied, while miRNA-155 knockout showed the myeloid differentation of NB4 cells significantly increased. CONCLUSION: CRISPR/Cas9 is a powerful tool for gene editing and can lead to miRNA knockout. Knockouts of miR-223 and miR-155 have shown a differentiation-related phenotype, and the potential mechanism is the integrative regulation of target genes.

In vivo assessment of Lauren classification for gastric adenocarcinoma using diffusion MRI with a fractional order calculus model.

OBJECTIVES: To evaluate the performance of a fractional order calculus (FROC) diffusion model for imaging-based assessment of Lauren classification in gastric adenocarcinoma. METHODS: In this study, 43 patients (15 females, 28 males) with gastric adenocarcinoma underwent MRI at 1.5 T. According to pathology-based Lauren classification, 10 patients had diffuse-type, 20 had intestinal-type, and 13 had mixed-type lesions. The diffuse and mixed types were combined as diffuse-and-mixed type to be differentiated from the intestinal type using diffusion MRI. Diffusion-weighted images were acquired by using eleven b-values (0-2000 s/mm2). Three FROC model parameters comprising diffusion coefficient D, intravoxel diffusion heterogeneity ß, and a microstructural quantity µ, together with a conventional apparent diffusion coefficient (ADC), were estimated. The mean parameter values in the tumour were computed by using a percentile histogram analysis. Individual or linear combinations of the mean parameters in the tumour were used to differentiate the diffuse-and-mixed type from the intestinal type using descriptive statistics and receiver operating characteristic (ROC) analyses. RESULTS: Significant differences were observed between diffuse-and-mixed-type and intestinal-type lesions in D (0.99 ± 0.20 µm2/ms vs. 1.11 ± 0.23 µm2/ms; p = 0.036), ß (0.37 ± 0.08 vs. 0.43 ± 0.11; p = 0.043), µ (7.92 ± 2.79 µm vs. 9.87 ± 1.52 µm; p = 0.038), and ADC (0.81 ± 0.34 µm2/ms vs. 0.96 ± 0.19 µm2/ms; p = 0.033). Among the individual parameters, µ produced the largest area under the ROC curve (0.739). The combinations of (D, ß, µ) and (ß and µ) produced the best overall performance with a sensitivity of 0.739, specificity of 0.750, accuracy of 0.744, and area under the curve of 0.793 (95% confidence interval: 0.657-0.929). CONCLUSION: Diffusion MRI with the FROC model holds promise for non-invasive assessment of Lauren classification for gastric adenocarcinoma. KEY POINTS: • High b-value diffusion MRI with a FROC model that is sensitive to tissue microstructures can differentiate the diffuse-and-mixed type from intestinal type of gastric adenocarcinoma. • The combination of FROC parameters produced the best result for distinguishing the diffuse-and-mixed type from the intestinal type with an area under the receiver operating characteristic curve of 0.793. • The FROC model parameters, individually or conjointly, hold promise for repeated, non-invasive evaluations of gastric adenocarcinoma at various time points throughout disease progression or regression to complement conventional Lauren classification.