Rapid Separation and Display of Active Fibrinogenolytic Agents in Sipunculus nudus through Fibrinogen-Polyacrylamide Gel Electrophoresis.

Fibrinogenolytic agents that can dissolve fibrinogen directly have been widely used in anti-coagulation treatment. Generally, identifying new fibrinogenolytic agents requires the separation of each component first and then checking their fibrinogenolytic activities. Currently, polyacrylamide gel electrophoresis (PAGE) and chromatography are mostly used in the separating stage. Meanwhile, the fibrinogen plate assay and reaction products based PAGE are usually adopted to display their fibrinogenolytic activities. However, because of the spatiotemporal separation of those two stages, it is impossible to separate and display the active fibrinogenolytic agents with the same gel. To simplify the separating and displaying processes of fibrinogenolytic agent identification, we constructed a new fibrinogen-PAGE method to rapidly separate and display the fibrinogenolytic agents of peanut worms (Sipunculus nudus) in this study. This method includes fibrinogen-PAGE preparation, electrophoresis, renaturation, incubation, staining, and decolorization. The fibrinogenolytic activity and molecular weight of the protein can be detected simultaneously. According to this method, we successfully detected more than one active fibrinogenolytic agent of peanut wormhomogenate within 6 h. Moreover, this fibrinogen-PAGE method is time and cost-friendly. Furthermore, this method could be used to study the fibrinogenolytic agents of the other organisms.

A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots.

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of cerebral blood clots is important for diagnosis, treatment, and prognosis. However, the current approaches to studying the cell components of the clots are mainly based on in situ staining, which is unsuitable for the comprehensive study of the cell components because cells are tightly wrapped in the clots. Previous studies have successfully isolated a fibrinolytic enzyme (sFE) from Sipunculus nudus, which can degrade the cross-linked fibrin directly, releasing the cell components. This study established a comprehensive method based on the sFE to study the cell components of cerebral thrombus. This protocol includes clot dissolving, cell releasing, cell staining, and routine blood examination. According to this method, the cell components could be studied quantitatively and qualitatively. The representative results of experiments using this method are shown.

Fibrinolytic drugs induced hemorrhage: mechanisms and solutions.

Thrombosis has been emerging as a major global life-threatening issue with high mortality and serious complications, especially in the post-COVID-19 era. Compared with the commonly used plasminogen activators thrombolytic drugs, fibrinolytic drugs are no longer heavily dependent on the patients' own plasminogen, which are poorly expressed in most patients. As a novel "direct acting" thrombolytic agent, fibrinolytic drugs are considered to have stronger thrombolytic efficacy and safety than the widely used plasminogen activators. However, the risk of their hemorrhage remains a major concern. Herein, the molecular mechanisms and solutions were summarized for the first time based on the systematic review of the latest developments, which could shed new light on the development of novel safety fibrinolytic drugs in the future.

Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus.

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin directly, showing great advantages over traditional thrombolytic agents. However, due to the lack of structural information, all the purification programs for sFE are based on multistep chromatography purifications, which are too complicated and costly. Here, an affinity purification protocol of sFE is developed for the first time based on a crystal structure of sFE; it includes preparation of the crude sample and the lysine/arginine-agarose matrix affinity chromatography column, affinity purification, and characterization of the purified sFE. Following this protocol, a batch of sFE can be purified within 1 day. Moreover, the purity and activity of the purified sFE increases to 92% and 19,200 U/mL, respectively. Thus, this is a simple, inexpensive, and efficient approach for sFE purification. The development of this protocol is of great significance for the further utilization of sFE and other similar agents.

Advances in aptamer-based sensing assays for C-reactive protein.

C-reactive protein (CRP), a non-specific acute-phase indicator of inflammation, has been widely recognized for its value in clinical diagnostic applications. With the advancement of testing technologies, there have been many reports on fast, simple, and reliable methods for CRP testing. Among these, the aptamer-based biosensors are the focus and hotspot of research for achieving high-sensitivity analysis of CRP. This review summarizes the progress of in vitro aptamer screening for CRP and the recent advances in aptamer-based CRP sensor applications, thus developing insight for the new CRP aptasensor design strategy.

Novel Ferrocene Derivatives Induce Apoptosis through Mitochondria-Dependent and Cell Cycle Arrest via PI3K/Akt/mTOR Signaling Pathway in T Cell Acute Lymphoblastic Leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is one of the most common causes of death in pediatric malignancies. However, the clinical chemotherapy for T-ALL has been limited by numerous side effects, emphasizing that novel anti-T-ALL drugs are urgently needed. Herein, a series of 2-acyl-1-dimethylaminomethyl-ferrocenes for cancer therapy have been evaluated. Among them, F1 and F3 exhibited potent cytotoxicity against T-ALL cell lines, especially Jurkat cells, with low cytotoxicity for normal cells. Further mechanistic studies revealed that F1 and F3 could induce apoptosis in Jurkat cells by destructing mitochondrial membrane, enhancing reactive oxygen species (ROS) generation, decreasing the Bcl-2/Bax ratio, releasing Cytochrome c, and increasing the expression of Cleaved Caspase-9/-3 and Cleaved PARP. Additionally, F1 and F3 could suppress cell proliferation and arrest the cell cycle at G0/G1 phase through the PI3K/Akt/mTOR signaling pathway by down-regulating the expression of CDK6, Cyclin D1, p-Akt, p-GSK-3ß, p-mTOR, p-p70 S6K, and up-regulating the expression of P21 and P27, which would also be a possible mechanism. Consequently, ferrocene derivatives F1 and F3 could induce apoptosis through a mitochondria-dependent pathway mediated by ROS, and cell cycle arrest at G0/G1 phase via the PI3K/Akt/mTOR signaling pathway in Jurkat cells. The present study provided fundamental insights into the clinical application of F1 and F3 for the treatment of T-ALL.

Novel Ferrocene Derivatives Induce G0/G1 Cell Cycle Arrest and Apoptosis through the Mitochondrial Pathway in Human Hepatocellular Carcinoma.

In this study, detailed information on hepatocellular carcinoma (HCC) cells (HepG-2, SMMC-7721, and HuH-7) and normal human liver cell L02 treated by ferrocene derivatives (compounds 1, 2 and 3) is provided. The cell viability assay showed that compound 1 presented the most potent and selective anti-HCC activity. Further mechanism study indicated that the proliferation inhibition effect of compound 1 was associated with the cycle arrest at the G0/G1 phase and downregulation of cyclin D1/CDK4. Moreover, compound 1 could induce apoptosis in HCC cells by loss of mitochondrial membrane potential (ΔΨm), accumulation of reactive oxygen species (ROS), decrease in Bcl-2, increase in BAX and Bad, translocation of Cytochrome c, activation of Caspase-9, -3, and cleavage of PARP. These results indicated that compound 1 would be a promising candidate against HCC through G0/G1 cell cycle arrest-related proliferation inhibition and mitochondrial pathway-dependent apoptosis.

Epstein-Barr virus is a promoter of lymphoma cell metastasis.

It is well-known that Epstein-Barr virus (EBV) is the promoter of cell tumourigenesis. We found that EBV is also a promoter of lymphoma cell dissemination, because we found the typical morphopathological phenomenon of cell adhesion, which confirmed that the adhesion of tumour cells was higher than that of normal cells. We also observed that tumour cells disrupted the dynamic pathological changes of vascular endothelial cells, and this made it clear that the rate of tumour cell metastasis was directly proportional to the degree of EBV infection. Furthermore, when we discovered exosomes, it was considered that this was associated with cancer stem cells, suggesting the formation of a microenvironment before tumour cell metastasis. In addition, competitive inhibition was found in cell adhesion, indicating the breakthrough point of preventing tumour cell metastasis, which has clinical reference value for tumour immunotherapy.

A sandwich ELISA-like detection of C-reactive protein in blood by citicoline-bovine serum albumin conjugate and aptamer-functionalized gold nanoparticles nanozyme.

C-reactive protein (CRP) level in blood is associated with the risk of developing cardiovascular events in higher-risk populations. We present a sandwich ELISA-like assay for the determination of CRP in blood by citicoline-bovine serum albumin (citicoline-BSA) conjugate and aptamer-functionalized gold nanoparticles (aptamer-AuNPs) nanozyme. The CRP in the blood sample was selectively adsorbed to the ELISA plate coated by citicoline-BSA, and then incubated with added aptamer-AuNPs. AuNPs exhibited peroxidase activity and oxidized 3,3'5,5'-tetramethylbenzidine from colorless to blue, achieving the measurement at 652 nm. The amplified signal increased linearly in a wide range from 0.1 to 200 ng mL-1 and with a detection limit of 8 pg mL-1. Finally, the method was further tested using rat blood from an isoproterenol-induced myocardial infarction experimental model to confirm its applicability. The developed method could directly determine CRP in blood sample after dilution with high accuracy and sensitivity. This method has many advantages, such as easiness to prepare materials, good stability between batches, high specificity, low detection limit, low-cost, easiness to operate with simple instruments, the most remarkable of which is its excellent lot-to-lot stability over the classical ELISA.

Association of N6-methyladenosine with viruses and virally induced diseases.

N6-methyladenosine (m6A) modification, which alters gene expression, is the most prevalent internal modification of eukaryotic mRNA. m6A modification is dynamic and reversible that is regulated by three associated protein groups: methyltransferases or writers, demethylases or erasers, and m6A-binding proteins or readers. m6A modification is involved in all phases of RNA life, from RNA folding and structure, stability, splicing, nuclear export, translational modulation to RNA degradation. Recent findings show that the abnormal level of m6A modification causes aberrant expression of important viral genes. Here, we reviewe m6A role in gene expression and its contribution to the development  of human viral diseases. Particularly, we would focus on viruses associated with human diseases including HIV-1, IAV, HBV, HCV, EBV and so on to find a novel approach and provide a new sight for the innovative treatment of human viral diseases.

Association of N6-methyladenosine with viruses and related diseases.

BACKGROUND: N6-methyladenosine (m6A) modification is the most prevalent internal modification of eukaryotic mRNA modulating gene expression. m6A modification is a dynamic reversible process regulated by three protein groups: methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers). m6A modification is involved in all phases of RNA metabolism, including RNA folding, stability, splicing, nuclear exporting, translational modulation and degradation. MAIN BODY: In recent years, numerous studies have reported that abnormal m6A modification causes aberrant expression of important viral genes. Herein, we review the role of m6A in viral lifecycle and its contribution to the pathogenesis of human diseases. Particularly, we focus on the viruses associated with human diseases such as HIV-1, IAV, HBV, HCV, EBV and many others. CONCLUSIONS: A better understanding of m6A-virus relationship would provide new insights into the viral replication process and pathogenesis of human diseases caused by viruses. In addition, exploration of the role of m6A in disease-causing viruses will reveal novel approaches for the treatment of such diseases.

Determination of Flavonoid Glycosides by UPLC-MS to Authenticate Commercial Lemonade.

So far, there is no report on the quality evaluation of lemonade available in the market. In this study, a sample preparation method was developed for the determination of flavonoid glycosides by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) based on vortex-assisted dispersive liquid-liquid microextraction. First, potential flavonoids in lemonade were scanned and identified by ultra-performance liquid chromatography-time of flight mass spectrometry (UPLC-TOF/MS). Five flavonoid glycosides were identified as eriocitrin, narirutin, hesperidin, rutin, and diosmin according to the molecular formula provided by TOF/MS and subsequent confirmation of the authentic standard. Then, an ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QqQ/MS) method was developed to determine these five flavonoid glycosides in lemonade. The results showed that the content of rutin in some lemonade was unreasonably high. We suspected that many illegal manufacturers achieved the goal of low-cost counterfeiting lemonade by adding rutin. This suggested that it was necessary for relevant departments of the state to make stricter regulations on the quality standards of lemonade beverages.

IL-8 Secreted from M2 Macrophages Promoted Prostate Tumorigenesis via STAT3/MALAT1 Pathway.

Prostate cancer (PCa) is a major health problem in males. Metastasis-associated with lung adenocarcinoma transcript-1 (MALAT1), which is overexpressed in PCa tissue, is associated with physiological and pathological conditions of PCa. M2 macrophages are major immune cells abundant in the tumor microenvironment. However, it remains unknown whether M2 macrophages are involved in the effects or not, and molecular mechanisms of MALAT1 on PCa progression have not yet been comprehensively explored. Here we reported that, M2 macrophages (PMA/IL-4 treated THP1) induced MALAT1 expression in PCa cell lines. Knockdown MALAT1 expression level in PCa cell lines inhibited cellular proliferation, invasion, and tumor formation. Further mechanistic dissection revealed that M2 macrophages secreted IL-8 was sufficient to drive up MALAT1 expression level via activating STAT3 signaling pathway. Additional chromatin immunoprecipitation (ChIP) and luciferase reporter assays displayed that STAT3 could bind to the MALAT1 promoter region and transcriptionally stimulate the MALAT1 expression. In summary, our present study identified the IL-8/STAT3/MALAT1 axis as key regulators during prostate tumorigenesis and therefore demonstrated a new mechanism for the MALAT1 transcriptional regulation.

LncRNAs and miRNAs: potential biomarkers and therapeutic targets for prostate cancer.

Prostate cancer (PCa) is the second lethal disease for men in western countries. Although androgen receptor (AR) signaling has been widely investigated, noncoding RNAs (ncRNAs), deficient of open reading frame, have also received considerable attention. Growing studies showed that the aberrant ncRNAs expression contributed to cell proliferation, metastasis and drug resistance in PCa. Therefore, therapeutically targeting ncRNAs may synergize androgen deprivation therapy (ADT) to have a better effect to fight against PCa, especially castration-resistant prostate cancer (CRPC). This review would systematically summarize the multicellular events controlled by ncRNAs and give a snapshot of future scientific activities and clinical applications.

[Adeno-associated vector mediated intracellular biological activity of human Kallistatin].

Human tissue kallikrein-binding protein (Kallistatin, KAL), a secretory protein that participates in the regulation of multiple signaling pathways by binding to the extracellular receptor, however, at present has not been reported about the intracellular activity, and whether it has the similar biological activity with extracellular activity. Here we constructed no signal peptide KAL (NSK) into the adeno-associated virus vector to explore the intracellular activity of KAL. Both the endothelial cell and lung cancer cells could express KAL, but not secreted after rAAV2-NSK transfection. The proliferation and migration of human umbilical vein endothelial cells (HUVECs) were inhibited, but the apoptosis rate was not affected. The proliferation rates, mobility and tubule formation of all the three tested lung cancer cells, such as NCI-H446, NCI-H460 and A549, were inhibited to different extents. This cellular study not only confirmed the intracellular activity, but also suggested it may serve as a kind of "balance factor" in multi-targeted controlling, which may provide a new train of thoughts to explain the regulatory contradiction in PI3K-Akt signaling pathways by KAL.

Promising coagulation factor VIII bypassing strategies for patients with haemophilia A.

Haemophilia A is an X-linked recessive monogenic hereditary bleeding disorder caused by a deficiency or functional defect in coagulation factor VIII (FVIII). Typically, only 30% haemophilia A patients are treated with FVIII-specific products successfully. Therefore, other promising clotting factors and FVIII-bypassing factors exhibiting sufficient FVIII-independent activity, low immunogenicity and prolonged half-life are needed to conquer this malady. Here, we will systematically review the current status of the diverse FVIII-bypassing factors for the treatment of FVIII-insensitive haemophilia A patients.

Potential of DNMT and its Epigenetic Regulation for Lung Cancer Therapy.

Lung cancer, the leading cause of mortality in both men and women in the United States, is largely diagnosed at its advanced stages that there are no effective therapeutic alternatives. Although tobacco smoking is the well established cause of lung cancer, the underlying mechanism for lung tumorigenesis remains poorly understood. An important event in tumor development appears to be the epigenetic alterations, especially the change of DNA methylation patterns, which induce the most tumor suppressor gene silence. In one scenario, DNA methyltransferase (DNMT) that is responsible for DNA methylation accounts for the major epigenetic maintenance and alternation. In another scenario, DNMT itself is regulated by the environment carcinogens (smoke), epigenetic and genetic information. DNMT not only plays a pivotal role in lung tumorigenesis, but also is a promising molecular bio-marker for early lung cancer diagnosis and therapy. Therefore the elucidation of the DNMT and its related epigenetic regulation in lung cancer is of great importance, which may expedite the overcome of lung cancer.