A novel integrated lateral flow immunoassay platform for the detection of cardiac troponin I using hierarchical dendritic copper-nickel nanostructures.

Cardiac troponin I (cTnI) is a critical biomarker for the diagnosis of acute myocardial infarction (AMI). Herein, we report a novel integrated lateral flow immunoassay (LFIA) platform for highly sensitive point-of-care testing (POCT) of cTnI using hierarchical dendritic copper-nickel (HD-nanoCu-Ni) nanostructures. The electrodeposited HD-nanoCu-Ni film (∼22 µm thick) on an ITO-coated glass substrate exhibits superior capillary action and structural integrity. These properties enable efficient sample transport and antibody immobilization, making it a compelling alternative to conventional multi-component paper-based LFIA test strips, which are often plagued by structural fragility and susceptibility to moisture damage. The biofunctionalized HD-nanoCu-Ni substrates were laser-etched with lateral flow channels, including a sample loading/conjugate release zone, a test zone, and a control zone. Numerical simulations were used to further optimize the design of these channels to achieve optimal fluid flow and target capture. The HD-nanoCu-Ni LFIA device utilizes a fluorescence quenching based sandwich immunoassay format using antibody-labeled gold nanoparticles (AuNPs) as quenchers. Two different fluorescent materials, fluorescein isothiocyanate (FITC) and CdSe@ZnS quantum dots (QDs), were used as background fluorophores in the device. Upon the formation of a sandwich immunocomplex with cTnI on the HD-nanoCu-Ni device, introduced AuNPs led to the fluorescence quenching of the background fluorophores. The total assay time was approximately 15 min, demonstrating the rapid and efficient nature of the HD-nanoCu-Ni LFIA platform. For FITC, both inner filter effect (IFE) and fluorescence resonance energy transfer (FRET) contributed to the AuNP-mediated quenching. In the case of CdSe@ZnS QDs, IFE dominated the AuNP-induced quenching. Calibration curves were established based on the relationship between the fluorescence quenching intensity and cTnI concentration in human serum samples, ranging from 0.5 to 128 ng/mL. The limits of detection (LODs) were determined to be 0.27 ng/mL and 0.40 ng/mL for FITC and CdSe@ZnS QDs, respectively. A method comparison study using Passing-Bablok regression analysis on varying cTnI concentrations in human serum samples confirmed the equivalence of the HD-nanoCu-Ni LFIA platform to a commercial fluorescence cTnI LFIA assay kit, with no significant systematic or proportional bias observed.

FLT3-selective PROTAC: Enhanced safety and increased synergy with Venetoclax in FLT3-ITD mutated acute myeloid leukemia.

Acute myeloid leukemia (AML) patients carrying Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations often face a poor prognosis. While some FLT3 inhibitors have been used clinically, challenges such as short efficacy and poor specificity persist. Proteolytic targeting chimera (PROTAC), with its lower ligand affinity requirement for target proteins, offers higher and rapid targeting capability. Gilteritinib, used as the ligand for the target protein, was connected with different E3 ligase ligands to synthesize several series of PROTAC targeting FLT3-ITD. Through screening and structural optimization, the optimal lead compound PROTAC Z29 showed better specificity than Gilteritinib. Z29 induced FLT3 degradation through the proteasome pathway and inhibited tumor growth in subcutaneous xenograft mice. We verified Z29's minimal impact on platelets in a patient-derived xenografts (PDX) model compared to Gilteritinib. The combination of Z29 and Venetoclax showed better anti-tumor effects, lower platelet toxicity, and lower hepatic toxicity in FLT3-ITD+ models. The FLT3-selective PROTAC can mitigate the platelet toxicity of small molecule inhibitors, ensuring safety and efficacy in monotherapy and combination therapy with Venetoclax. It is a promising strategy for FLT3-ITD+ patients, especially those with platelet deficiency or liver damage.

Development of a risk assessment model for cardiac injury in patients newly diagnosed with acute myeloid leukemia based on a multicenter, real-world analysis in China.

BACKGROUND: Studies have revealed that acute myeloid leukemia (AML) patients are prone to combined cardiac injury. We aimed to identify hematological risk factors associated with cardiac injury in newly diagnosed AML patients before chemotherapy and develop a personalized predictive model. METHODS: The population baseline, blood test, electrocardiogram, echocardiograph, and genetic and cytogenetic data were collected from newly diagnosed AML patients. The data were subdivided into training and validation cohorts. The independent risk factors were explored by univariate and multivariate logistic regression analysis respectively, and data dimension reduction and variable selection were performed using the least absolute shrinkage and selection operator (LASSO) regression models. The nomogram was generated and the reliability and generalizability were verified by receiver operating characteristic (ROC) curves, the area under the curve (AUC) and calibration curves in an external validation cohort. RESULTS: Finally, 499 AML patients were included. After univariate logistic regression, LASSO regression and multivariate logistic regression analysis, abnormal NT-proBNP, NPM1 mutation, WBC, and RBC were independent risk factors for cardiac injury in AML patients (all P < 0.05). The nomogram was constructed based on the above four variables with high accuracy. The area under the curve was 0.742, 0.750, and 0.706 in the training, internal validation, and external validation cohort, respectively. The calibration curve indicated that the model has good testing capability. The Kaplan-Meier curve showed that the higher the risk of combined cardiac injury in AML patients, the lower their probability of survival. CONCLUSIONS: This prediction nomogram identifies hematological risk factors associated with cardiac injury in newly diagnosed AML patients and can help hematologists identify the risk and provide precise treatment options.

Comprehensive analysis of clinical prognostic features and tumor microenvironment landscape of CD11b+CD64+ patients with acute myeloid leukemia.

BACKGROUND: Immunophenotyping surface molecules detected in the clinic are mainly applied in diagnostic confirmation and subtyping. However, the immunomodulatory molecules CD11b and CD64, are highly associated with leukemogenesis. Hence, the prognostic value of them and their potential biological functions merit further investigation. METHODS: Flow cytometry was operated to detect immunophenotypic molecules from AML bone marrow samples. Multivariate cox regression, Kaplan-Meier analyses, and nomogram were conducted to predict survival. Transcriptomic data, lymphocyte subsets, and immunohistochemical staining were incorporated to identify potential biological functions of prognostic immunophenotype in acute myeloid leukemia (AML). RESULTS: We classified 315 newly diagnosed AML patients of our center based on the expression of CD11b and CD64. The CD11b+CD64+ populations were identified as independent risk factors for overall survival and event-free survival of AML, exhibiting specific clinicopathological features. The predictive models based on CD11b+CD64+ showed high classification performance. In addition, the CD11b+CD64+ subset, characterized by high inhibitory immune checkpoints, M2-macrophage infiltration, low anti-tumor effector cells infiltration, as well as abnormal somatic mutation landscape, presented a distinctive tumor microenvironmental landscape. The CD11b+CD64+ population showd a higher expression of BCL2, and the drug sensitivity indicated that they presented a lower half-maximal inhibitory concentration value for BCL2 inhibitor, and could benefit more from the above medicine. CONCLUSIONS: This work might be of benefit to enhanced understanding of CD11b+CD64+ in the prognosis and leukemogenesis, and yielded novel biomarkers to guide immunotherapy and targeted therapy for AML.

Identification and validation of immunohistochemical marker panels to predict the prognosis of muscle invasive bladder cancer.

Background: Currently, the treatment regimen of bladder cancer depends on the stage and grade. Yet, patients with similar histopathological characteristics may have distinct prognosis. Luminal/basal subtyping had proved to be a satisfactory subtyping method. Here we intended to evaluate immunohistochemistry, a more clinically-practical method, in luminal/basal classification and further risk-stratification. Methods: Patients diagnosed with urothelial carcinoma of the bladder in Changhai Hospital were retrospectively recruited and corresponding formalin-fixed paraffin embedded blocks were acquired. Tissue microarrays (TMAs) of these patients were established followed by immunohistochemical (IHC) staining of 14 markers. Patients were classified into luminal or basal subtype according to CK5/6, CK14, CK20 and GATA3 expression. Further subtyping of luminal and basal tumors was performed according to the expression of other markers. Results: A total of 236 patients were included: 163 and 73 patients were assigned to training and validation cohorts, respectively. Patients with basal tumor were related with poorer prognosis compared to those with luminal tumor (P=0.025 and 0.008 in training and validation cohorts, respectively). We further revealed luminal muscle invasive bladder cancer (MIBC) patients could be further categorized into subgroups with different risks. Cytoplasmic YAP1 and CCNB1 were selected as classifier, patients with low expression of cytoplasmic YAP1 or CCNB1 were independent risk factor for poorer prognosis (hazard ratio =2.19, P=0.04). Conclusions: Molecular subtyping into luminal/basal subtype and risk stratification method using a 2-marker method by immunohistochemistry can be an economical, clinically practical method to predict patient prognosis and could help to develop treatment strategy and follow-up schedule in clinical practice.

dSTORM-Based Single-Cell Protein Quantitative Analysis Can Effectively Evaluate the Degradation Ability of PROTACs.

As an emerging therapeutic strategy, proteolysis-targeting chimeras (PROTACs) have been proven to be superior to traditional drugs in many aspects. However, due to their unique mechanism of action, existing methods for evaluating the degradation still have many limitations, which seriously restricts the development of PROTACs. In this methodological study, using direct stochastic optical reconstruction microscopy (dSTORM)-based single-cell protein quantitative analysis, we systematically investigated the dynamic degradation characteristics of FLT3 protein during PROTACs treatment. We found that the distribution of FLT3 varies between FLT3-ITD mutation and FLT3-WT cells. PROTACs had an obvious time-course effect on protein degradation and present two distinct phases; this provided a basis for deciding when to evaluate protein degradation. High concentrations of PROTACs were more effective than long-time administration because a higher Dmax was achieved. Two-color dSTORM-based colocalization analysis efficiently detected the proportion of ternary complexes, making it very useful in screening PROTACs. Taken together, our findings show that the dSTORM method is an ideal tool for evaluating PROTACs and will accelerate the development of new PROTACs.

Cardiac-Related Lesions in Newly Diagnosed Patients With Acute Leukemia: A Chinese Population-Based Real-World Study.

The relationship between newly diagnosed acute leukemia (AL) and heart-related lesions remains unclear. This study aimed to investigate baseline cardiac function and risk of cardiovascular diseases (CVDs) in patients with new-onset AL, and provide data on cardiac management strategies for patients with AL. We retrospectively collected data on baseline characteristics, echocardiography, and biochemical blood indicators (e.g., myocardial enzymes) from 408 patients, 200 with newly diagnosed AL, 103 with coronary artery disease (CAD), and 105 controls from January 1, 2015 to August 31, 2019. The creatine kinase isoenzyme myocardial band, lactate dehydrogenase, highly sensitive troponin-I, and B-type natriuretic peptide levels and left ventricular internal diameter (LVID) were significantly higher in patients with newly diagnosed AL than in the control group. The degree of cardiac damage was lower in newly diagnosed AL patients than in CAD patients. The best predictor of heart damage was LVID (AUC [area under the curve] = 0.709; 95% CI [confidence interval]: 0.637-0.781; p < 0.001), and independent prognostic risk factors were age and ejection fraction (HR [hazard ratio] = 1.636; 95% CI: 1.039-2.575; p = 0.033). The ratio of leukemia blasts among patients with AL was positively correlated with cardiac damage. Our data indicated that newly diagnosed AL patients had certain myocardial damage before treatment. Clinicians need to pay attention to these manifestations, which may be related to the prognosis.

Shengxuening Extracted from Silkworm Excrement Mitigates Myelosuppression via SCF-Mediated JAK2/STAT3 Signaling.

Shengxuening (SXN) is a Chinese patent medicine with main ingredients (including chlorophyll derivatives and sodium iron chlorophyllin) extracted from silkworm excrement. SXN exhibited efficacy in clinical trials of renal anemia and iron deficiency anemia; however, the specific mechanisms remain unclear. This study found that SXN increased the number of peripheral blood cells and improved the bone marrow morphology in myelosuppressed mouse model, reversed the reduction in body weight and spleen indices, and increased the serum levels of erythropoietin and granulocyte-macrophage colony-stimulating factor. Quantitative real-time PCR array and Western blot analysis showed the enhanced expression of stem cell factor (SCF), JAK2, and STAT3 in the liver. These results suggested that SXN promoted the recovery of hemopoietic function in myelosuppressed models by increasing the secretion of hematopoietic factors and activating the JAK2/STAT3 pathway. Therefore, this medicine may be applied as therapeutic pharmaceutical drug to mitigate myelosuppression.

Targeting acute myeloid leukemia stem cells: Current therapies in development and potential strategies with new dimensions.

High relapse rate of acute myeloid leukemia (AML) is still a crucial problem despite considerable advances in anti-cancer therapies. One crucial cause of relapse is the existence of leukemia stem cells (LSCs) with self-renewal ability, which contribute to repeated treatment resistance and recurrence. Treatments targeting LSCs, especially in combination with existing chemotherapy regimens or hematopoietic stem cell transplantation might help achieve a higher complete remission rate and improve overall survival. Many novel agents of different therapeutic strategies that aim to modulate LSCs self-renewal, proliferation, apoptosis, and differentiation are under investigation. In this review, we summarize the latest advances of different therapies in development based on the biological characteristics of LSCs, with particular attention on natural products, synthetic compounds, antibody therapies, and adoptive cell therapies that promote the LSC eradication. We also explore the causes of AML recurrence and proposed potential strategies with new dimensions for targeting LSCs in the future.

Novel ADAM-17 inhibitor ZLDI-8 inhibits the metastasis of hepatocellular carcinoma by reversing epithelial-mesenchymal transition in vitro and in vivo.

AIMS: Epithelial-mesenchymal transition (EMT) is one of the important regulators of metastasis in advanced hepatocellular carcinoma (HCC). Blocking the Notch signaling pathway and then reversing the EMT process is a hot spot in clinical tumor research. Here, we aimed to investigate the effect and underlying mechanisms of ADAM-17 (a key cleavage enzyme of Notch pathway) inhibitor ZLDI-8 we found before on the metastasis of hepatocellular carcinoma in vitro and in vivo. MAIN METHODS: The cell viability of HCC cells was evaluated by MTT and colony formation assays. Migration and invasion were assessed respectively with wound healing and transwell assays. The expression and location of proteins were detected by western blot and immunofluorescence, respectively. The effects of ZLDI-8 on metastasis of liver cancer in vivo were investigated in a tail vein injection model. KEY FINDINGS: In the present work, ZLDI-8 significantly inhibited proliferation, migration, invasion and EMT phenotype of highly aggressive MHCC97-H and LM3 cells. Moreover, ZLDI-8 could inhibit the migration and invasion of HepG2 and Bel7402 cells induced by TGF-ß1. ZLDI-8 suppressed the protein expression of interstitial markers and increased that of epithelial markers. Meanwhile, ZLDI-8 decreased the expression of proteins in the Notch signaling pathway. Finally, ZLDI-8 blocks metastasis in the lung metastasis model in vivo. SIGNIFICANCE: ZLDI-8 suppressed the metastasis of hepatocellular carcinoma, which was associated with reversing the EMT process and regulating Notch signaling pathway. The study laid the foundation for the discovery of drugs that reverse EMT to inhibit advanced HCC metastasis.

Development of a cross-priming isothermal amplification assay based on the glycoprotein B gene for instant and rapid detection of feline herpesvirus type 1.

A cross-priming isothermal amplification (CPA) assay was developed for detection of feline herpesvirus type 1 (FHV-1). In this assay, the target fragment of the FHV-1 glycoprotein B gene is amplified rapidly by Bst DNA polymerase at a constant temperature (63 °C, 45 min), using a simple thermostat. The assay had no cross-reactions with four types of feline viruses, and the detection limit was 100 copies/µl. The positive rate of clinical samples from CPA was 100% consistent with qPCR but higher than ordinary PCR, indicating its superiority to ordinary PCR. Visualization was achieved using SYBR Green I dye.

Antiproliferative and Immunoregulatory Effects of Azelaic Acid Against Acute Myeloid Leukemia via the Activation of Notch Signaling Pathway.

Acute myeloid leukemia (AML) is a common type of hematological malignancy that can progress rapidly. AML has a poor prognosis and a high incidence of relapse due to therapeutic resistance. Azelaic acid (AZA), a small molecular compound is known to exhibit antitumor effect on various tumor cells. This study aimed to evaluate the antiproliferative and immunoregulatory effects of AZA against AMLviathe activation of the notch signaling pathway. We found that AZA can inhibit the proliferation of AML cells. In addition, laser confocal microscopy showed AZA-treated AML cells began to swelling and undergo cytoplasmic vacuolization. Importantly, AZA promoted the proliferation of NK and T cells and increased the secretion of TNF-αand IFN-γ. AZA also increased the expression levels of CD107a and TRAIL in NK cells, and CD25 and CD69 in T cells to influence their activation and cytotoxic ability. AZA-treated NK cells can kill AML cells more efficiently at the single-cell level as observed under the microfluidic chips. Further mechanistic analysis using protein mass spectrometry analysis and Notch signaling reporter assay demonstrated that Notch1and Notch2 were up-regulated and the Notch signaling pathway was activated. Moreover, combining AZA with the Notch inhibitor, RO4929097, decreased the expression of Notch1and Notch2, and downstream HES1 and HEY1, which rendered AML cells insensitive to AZA-induced apoptosis and alleviated AZA-mediated cytotoxicity in AML. In vivo, AZA relieved the leukemic spleen infiltration and extended the survival. The percentage of CD3-CD56+NK cells and CD4+CD8+T cells as well as the secretion of cytotoxic cytokines was increased after the treatment of AZA. The overall findings reveal that AZA is a potential Notch agonist against AML in activating the Notch signaling pathway.

Efficacy of SXN in the Treatment of Iron Deficiency Anemia: A Phase IV Clinical Trial.

Shengxuening (SXN) tablet is extracted from the excrement of the silkworm and has effects on hematopoiesis. The main components of SXN are chlorophyll derivatives and sodium iron chlorophyllin (SIC). The present study aims to investigate the efficiency and safety of SXN on iron deficiency anemia. This phase IV, multicenter, open-label, randomized clinical trial was conducted in 31 hospitals in China from June 2001 to April 2002. Adults and children were randomly divided into low-dose (L-SXN), medium-dose (M-SXN), and high-dose (H-SXN) groups, respectively. The course of treatment was 1 month. Peripheral hemogram levels and iron status were examined before and after treatment. Adults in all three dose groups demonstrated a significant increase in hemoglobin (HGB) concentration. Children who received SXN treatment in medium and high doses also demonstrated increased HGB concentration. Reticulocyte counts increased at the end of treatment in the M-SXN and H-SXN adult groups and in the M-SXN child group. For both children and adults, SXN in the three dose groups was found to significantly elevate red blood cell level, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. The total effective rate in the SXN-treated group reached 84.8%. The incidence of adverse events was 4.07%. The most common side effects were nausea (2.83%), diarrhea (0.74%), and rash (0.25%). SXN was proved to be efficient and safe for adults and children with iron deficiency anemia.