TOR-mediated Ypt1 phosphorylation regulates autophagy initiation complex assembly.

The regulation of autophagy initiation is a key step in autophagosome biogenesis. However, our understanding of the molecular mechanisms underlying the stepwise assembly of ATG proteins during this process remains incomplete. The Rab GTPase Ypt1/Rab1 is recognized as an essential autophagy regulator. Here, we identify Atg23 and Atg17 as binding partners of Ypt1, with their direct interaction proving crucial for the stepwise assembly of autophagy initiation complexes. Disruption of Ypt1-Atg23 binding results in significantly reduced Atg9 interactions with Atg11, Atg13, and Atg17, thus preventing the recruitment of Atg9 vesicles to the phagophore assembly site (PAS). Likewise, Ypt1-Atg17 binding contributes to the PAS recruitment of Ypt1 and Atg1. Importantly, we found that Ypt1 is phosphorylated by TOR at the Ser174 residue. Converting this residue to alanine blocks Ypt1 phosphorylation by TOR and enhances autophagy. Conversely, the Ypt1S174D phosphorylation mimic impairs both PAS recruitment and activation of Atg1, thus inhibiting subsequent autophagy. Thus, we propose TOR-mediated Ypt1 as a multifunctional assembly factor that controls autophagy initiation via its regulation of the stepwise assembly of ATG proteins.

BRD9-SMAD2/3 Orchestrates Stemness and Tumorigenesis in Pancreatic Ductal Adenocarcinoma.

BACKGROUND & AIMS: The dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is linked to the presence of pancreatic cancer stem-like cells (CSCs) that respond poorly to current chemotherapy regimens. The epigenetic mechanisms regulating CSCs are currently insufficiently understood, which hampers the development of novel strategies for eliminating CSCs. METHODS: By small molecule compound screening targeting 142 epigenetic enzymes, we identified that bromodomain-containing protein BRD9, a component of the BAF histone remodeling complex, is a key chromatin regulator to orchestrate the stemness of pancreatic CSCs via cooperating with the TGFß/Activin-SMAD2/3 signaling pathway. RESULTS: Inhibition and genetic ablation of BRD9 block the self-renewal, cell cycle entry into G0 phase and invasiveness of CSCs, and improve the sensitivity of CSCs to gemcitabine treatment. In addition, pharmacological inhibition of BRD9 significantly reduced the tumorigenesis in patient-derived xenografts mouse models and eliminated CSCs in tumors from pancreatic cancer patients. Mechanistically, inhibition of BRD9 disrupts enhancer-promoter looping and transcription of stemness genes in CSCs. CONCLUSIONS: Collectively, the data suggest BRD9 as a novel therapeutic target for PDAC treatment via modulation of CSC stemness.

Direct cellular reprogramming techniques for cardiovascular regenerative therapeutics.

Cardiovascular diseases remain a leading cause of hospitalization affecting approximately 38 million people worldwide. While pharmacological and revascularization techniques can improve the patient's survival and quality of life, they cannot help reversing myocardial infarction injury and heart failure. Direct reprogramming of somatic cells to cardiomyocyte and cardiac progenitor cells offers a new approach to cellular reprogramming and paves the way for translational regenerative medicine. Direct reprogramming can bypass the pluripotent stage with the potential advantage of non-immunogenic cell products, reduced carcinogenic risk, and no requirement for embryonic tissue. The process of directly reprogramming cardiac cells was first achieved through the overexpression of transcription factors such as GATA4, MEF2C, and TBX5. However, over the past decade, significant work has been focused on enhancing direct reprogramming using a mixture of transcription factors, microRNAs, and small molecules to achieve cardiac cell fate. This review discusses the evolution of direct reprogramming, recent progress in achieving efficient cardiac cell fate conversion, and describes the reprogramming mechanisms at a molecular level. We also explore various viral and non-viral delivery methods currently being used to aid in the delivery of reprogramming factors to improve efficiency. However, further studies will be needed to overcome molecular and epigenetic barriers to successfully achieve translational cardiac regenerative therapeutics.

Rewiring of 3D Chromatin Topology Orchestrates Transcriptional Reprogramming and the Development of Human Dilated Cardiomyopathy.

BACKGROUND: Transcriptional reconfiguration is central to heart failure, the most common cause of which is dilated cardiomyopathy (DCM). The effect of 3-dimensional chromatin topology on transcriptional dysregulation and pathogenesis in human DCM remains elusive. METHODS: We generated a compendium of 3-dimensional epigenome and transcriptome maps from 101 biobanked human DCM and nonfailing heart tissues through highly integrative chromatin immunoprecipitation (H3K27ac [acetylation of lysine 27 on histone H3]), in situ high-throughput chromosome conformation capture, chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin using sequencing, and RNA sequencing. We used human induced pluripotent stem cell-derived cardiomyocytes and mouse models to interrogate the key transcription factor implicated in 3-dimensional chromatin organization and transcriptional regulation in DCM pathogenesis. RESULTS: We discovered that the active regulatory elements (H3K27ac peaks) and their connectome (H3K27ac loops) were extensively reprogrammed in DCM hearts and contributed to transcriptional dysregulation implicated in DCM development. For example, we identified that nontranscribing NPPA-AS1 (natriuretic peptide A antisense RNA 1) promoter functions as an enhancer and physically interacts with the NPPA (natriuretic peptide A) and NPPB (natriuretic peptide B) promoters, leading to the cotranscription of NPPA and NPPB in DCM hearts. We revealed that DCM-enriched H3K27ac loops largely resided in conserved high-order chromatin architectures (compartments, topologically associating domains) and their anchors unexpectedly had equivalent chromatin accessibility. We discovered that the DCM-enriched H3K27ac loop anchors exhibited a strong enrichment for HAND1 (heart and neural crest derivatives expressed 1), a key transcription factor involved in early cardiogenesis. In line with this, its protein expression was upregulated in human DCM and mouse failing hearts. To further validate whether HAND1 is a causal driver for the reprogramming of enhancer-promoter connectome in DCM hearts, we performed comprehensive 3-dimensional epigenome mappings in human induced pluripotent stem cell-derived cardiomyocytes. We found that forced overexpression of HAND1 in human induced pluripotent stem cell-derived cardiomyocytes induced a distinct gain of enhancer-promoter connectivity and correspondingly increased the expression of their connected genes implicated in DCM pathogenesis, thus recapitulating the transcriptional signature in human DCM hearts. Electrophysiology analysis demonstrated that forced overexpression of HAND1 in human induced pluripotent stem cell-derived cardiomyocytes induced abnormal calcium handling. Furthermore, cardiomyocyte-specific overexpression of Hand1 in the mouse hearts resulted in dilated cardiac remodeling with impaired contractility/Ca2+ handling in cardiomyocytes, increased ratio of heart weight/body weight, and compromised cardiac function, which were ascribed to recapitulation of transcriptional reprogramming in DCM. CONCLUSIONS: This study provided novel chromatin topology insights into DCM pathogenesis and illustrated a model whereby a single transcription factor (HAND1) reprograms the genome-wide enhancer-promoter connectome to drive DCM pathogenesis.

STING controls opioid-induced itch and chronic itch via spinal tank-binding kinase 1-dependent type I interferon response in mice.

BACKGROUND: Patients receiving epidural or intrathecal opioids administration for neuraxial analgesia frequently suffer from an irritating itch. STING (stimulator of interferon genes), an innate immune modulator, is strongly implicated in pain pathogenesis via neuron-immune modulation. Given that pain and itch share some common neurocircuits, we evaluate the therapeutic potential of STING agonists in opioid-induced itch and chronic itch. METHODS: Opioids (morphine, fentanyl and sufentanil) were intrathecally injected to induce acute itch. Chronic itch was induced by dry skin and contact dermatitis. Opioids analgesic effect, itch-induced scratching behavior, spinal expression of STING, phosphorylation of TBK1 (tank-binding kinase 1), IRF3 (interferon regulatory factor-3) and ERK (extracellular signal-regulated kinase), as well as production of IFN-α and IFN-ß were examined. STING agonists (DMXAA and ADU-S100), TBK1 inhibitor, recombinant IFN-α and IFN-ß elucidated the mechanism and treatment of itch. Whole-brain functional connectivity was evaluated using resting-state fMRI. RESULTS: We report the primary expression of STING protein by the spinal dorsal horn neurons. Intraperitoneal injection of DMXAA dose-dependently reduces morphine-induced scratch bouts, without impairing morphine antinociception. Simultaneously, DMXAA alleviates fentanyl- and sufentanil-induced itching-like behavior, and chronic scratching behavior caused by dry skin and contact dermatitis. Furthermore, DMXAA drastically increases spinal phosphorylation of TBK1 and IRF3 following morphine exposure, dry skin and contact dermatitis. DMXAA-induced anti-pruritus effects and spinal productions of IFN-α and IFN-ß are compensated by intrathecal delivery of the TBK1 inhibitor. Also, ADU-S100, recombinant IFN-α and IFN-ß exhibits remarkable attenuation in scratching behaviors after morphine injection and dermatitis. Recombinant IFN-α inhibits morphine-induced spinal phosphorylation of ERK. Finally, DMXAA prevents dermatitis-induced the increase of cerebral functional connectivity between regions of interests such as primary somatosensory cortex, piriform cortex, retrosplenial cortex, colliculus and ventral thalamus. CONCLUSIONS: STING activation confers protection against opioid-induced itch and chronic itch through spinal up-regulation of TBK1-IRF3-type I interferon cascades in mice, suggesting that STING agonists are promising candidates in translational development for pruritus relief.

Establishment of matrix metalloproteinase 3 time-resolved immunoassay and some potential clinical applications.

AIM: To develop a highly sensitive time-resolved fluorescence immunoassay (TRFIA) for the detection of serum matrix metalloproteinase-3 (MMP-3) and to assess MMP-3's clinical value in patients with colorectal cancer (CRC).st. METHODS: MMP-3 levels were established using the double antibody sandwich technique. The MMP-3 TRFIA technique was developed and optimized, and its linearity, sensitivity, accuracy, specificity, and recovery were assessed. Then, serum concentrations in healthy individuals and patients with CRC were determined by MMP-3 TRFIA. RESULTS: The linear range of MMP-3 TRFIA was 0.73-500 ng/mL. MMP-3 TRFIA had an intra-batch precision range of 2.16%-7.10% percent and an inter-batch precision range of 3.99%-11.21%. MMP-3, tumor-associated trypsinogen 2, and AFP had no cross reaction.The recovery is between 90% and 110%, and had no serum interference. Patients with CRC had serum MMP-3 levels (73.95 ± 78.43 ng/mL) that were considerably higher than those of healthy individuals (21.45 ± 11.12 ng/mL), and those with metastasis had serum MMP-3 levels (95.89 ± 76.21 ng/mL) that were considerably higher than those of patients without metastasis (52.74 ± 47.25 ng/mL). CONCLUSIONS: A highly sensitive MMP-3 TRFIA assay was successfully developed, and serum MMP-3 may be associated with CRC invasion and metastasis. Therefore, MMP-3 can be used in the auxiliary diagnosis of CRC.

Development of amplified luminescent proximity homogeneous assay for quantitation of gastrin-17.

A highly sensitive and convenient amplified luminescent proximity homogeneous assay (AlphaLISA) method with high throughput and automation potential was developed for quantitation of serum Gastrin-17 (G-17) levels, which can facilitate the early diagnosis of atrophic gastritis in people at high risk of gastric cancer using a non-invasive approach. In this study, donor and acceptor beads with modified carboxyl groups on the surface were directly coupled to anti-G-17 antibodies through activation was proposed for application in the development of the new AlphaLISA, which can effectively simplify the steps and shorten the reaction time to achieve faster detection. Therefore, the G-17-AlphaLISA only needs to react for 15 min to obtain good analysis results. The proposed method has a wider detection range than commercial enzyme-linked immunosorbent assay (ELISA) kits (0.12-112.8 pmol/L > 0.5-40 pmol/L). In addition, results of G-17-AlphaLISA and ELISA had good correlation and agreement (ρ = 0.936). Importantly, the developed method may be more suitable for the large-scale screening of people at high risk for gastric cancer than traditional ELISA and provides a novel solution for other biomarkers that require accurate, highly sensitive, and high throughput detection.

The expression of SERPINE1 in colon cancer and its regulatory network and prognostic value.

BACKGROUND: Serpin Peptidase Inhibitor 1 (SERPINE1) promotes cancer progression by making it easier for cancer cells to spread to surrounding normal tissue. We expect to understand the prognostic value and regulatory network of SERPINE1 in colon cancer using bioinformatics methods. METHODS: The expression of target gene SERPINE1 in varying cancers was analyzed by the Tumor Immune Estimation Resource (TIMER) database. SERPINE1 expression in Colon Adenocarcinoma and normal tissue samples was assessed by starBase and UALCAN databases. SERPINE1 expression in clinical tissues was assayed using quantitative reverse transcription Polymerase Chain Reaction (qRT-PCR). SERPINE1 expression was detected in colon cancer patients with various clinical features (age, gender, nodal metastasis status, race, stages, and subtype) using analysis of variance. Survival curve was used to analyze the effect of high and low expression of SERPINE1 on the survival time of patients with different clinical phenotypes. Gene Set Enrichment Analysis (GSEA) was conducted on the results of LinkFinder calculation using LinkInterpreter module, which was combined with Pearson correlation analysis to obtain the kinase targets and miRNA targets, transcription factor targets, and corresponding signaling pathways associated with SERPINE1. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed on GSEA result. Finally, Gene Multiple Association Network Integration Algorithm (GeneMANIA) was utilized to establish a network of genes related to the kinases MAPK1, miR-18a, and SRF_Q, and biological functions were analyzed. RESULTS: Based on TIMER, starBase, and UALCAN databases, SERPINE1 was found to be remarkably highly expressed in colon cancer patients, which was further verified by clinical tissue. It was also associated with different clinical features (nodal metastasis status, stages, subtypes). Additionally, survival analysis showed that patients with low expression of SERPINE1 had a longer survival time, suggesting that SERPINE1 was a prognostic risk factor for colon cancer. Pearson correlation analysis revealed that the expression of Integrin Alpha 5 (ITGA5), Matrix Metallopeptidase 19 (MMP19), and ADAM Metallopeptidase with Thrombospondin Type 1 Motif, 4 (ADAMTS4) had the highest correlation with that of SERPINE1. The GSEA results indicated that these genes were mainly enriched in the pathways of RNA expression and kinases. Finally, GeneMANIA analysis was introduced to construct the molecular network of SERPINE1. CONCLUSION: Overall, our bioinformatics analyses comprehensively described the networks involved SERPINE1 in colon cancer and the potentially associated molecular mechanisms.

CRISPR activation of endogenous genes reprograms fibroblasts into cardiovascular progenitor cells for myocardial infarction therapy.

Fibroblasts can be reprogrammed into cardiovascular progenitor cells (CPCs) using transgenic approaches, although the underlying mechanism remains unclear. We determined whether activation of endogenous genes such as Gata4, Nkx2.5, and Tbx5 can rapidly establish autoregulatory loops and initiate CPC generation in adult extracardiac fibroblasts using a CRISPR activation system. The induced fibroblasts (>80%) showed phenotypic changes as indicated by an Nkx2.5 cardiac enhancer reporter. The progenitor characteristics were confirmed by colony formation and expression of cardiovascular genes. Cardiac sphere induction segregated the early and late reprogrammed cells that can generate functional cardiomyocytes and vascular cells in vitro. Therefore, they were termed CRISPR-induced CPCs (ciCPCs). Transcriptomic analysis showed that cell cycle and heart development pathways were important to accelerate CPC formation during the early reprogramming stage. The CRISPR system opened the silenced chromatin locus, thereby allowing transcriptional factors to access their own promoters and eventually forming a positive feedback loop. The regenerative potential of ciCPCs was assessed after implantation in mouse myocardial infarction models. The engrafted ciCPCs differentiated into cardiovascular cells in vivo but also significantly improved contractile function and scar formation. In conclusion, multiplex gene activation was sufficient to drive CPC reprogramming, providing a new cell source for regenerative therapeutics.

Establishment and clinical application of time-resolved immunofluorescence assay of lipoprotein-associated phospholipase A2.

AIM: This study aimed to establish a highly sensitive time-resolved fluorescence immunoassay (TRFIA) for the detection of serum lipoprotein-associated phospholipase A2 (Lp-PLA2) and evaluate the clinical application value of Lp-PLA2 in patients with breast cancer. METHODS: The level of Lp-PLA2 was detected using the double-antibody sandwich method. First, the Lp-PLA2-TRFIA method was established, and the method was evaluated on the basis of linearity, sensitivity, precision, specificity, and recovery rate. Then, the fluorescence counts in serum of healthy subjects and patients with breast cancer were detected by Lp-PLA2-TRFIA, and the levels of Lp-PLA2 were calculated using a standard curve. RESULTS: Lp-PLA2-TRFIA had a wide linear range (43.48-2000 ng/mL). The intra-assay precisions of Lp-PLA2-TRFIA ranged from 2.66% to 4.84% (<10%), and the inter-assay precisions were between 5.39% and 6.95% (<15%). No cross-reaction was observed among Lp-PLA2, Tumor-associated trypsinogen-2, and T-cell immunoglobulin mucin 3. In addition, the recovery rates were between 90% and 100%. The serum Lp-PLA2 levels of patients with breast cancer were significantly higher than those of healthy subjects. CONCLUSIONS: We successfully established a highly sensitive Lp-PLA2-TRFIA method, and found serum Lp-PLA2 may be associated with dyslipidemia in breast cancer and could be used for auxiliary diagnose.

Development and application of amplified luminescent proximity homogeneous assay for quantitation of heparin-binding protein.

A fast and highly sensitive amplified luminescent proximity homogeneous assay (AlphaLISA) method was developed for quantitation of plasma heparin-binding protein levels. In this study, a method directly coupling donor and acceptor beads modified with aldehyde groups to anti-HBP antibodies was proposed, which can effectively simplify the steps and shorten the reaction time to achieve faster detection. Therefore, the developed method required only 15 min of reaction time to generate results. Compared with the approved commercial kit, the developed method had a wider linear range (2.78-500 ng/mL). The excellent linear range means that the method can better exploit the value of HBP in clinical applications. Meanwhile, results of amplified luminescent proximity homogeneous assay and fluorescence dry quantitative immunoassay had good correlation and consistency (ρ = 0.9181). Moreover, the plasma HBP concentrations of patients with bacterial infection were significantly higher than those of healthy individuals (P < 0.0001), indicating the potential applicability of the proposed method for predicting the incidence of bacterial infections. Importantly, the newly developed method is expected to serve as an alternative to the traditional assay method and provides a completely new platform for other biomarkers that require rapid detection.

Establishment and Clinical Application of a Highly Sensitive Time-Resolved Fluorescence Immunoassay for Tumor-Associated Trypsinogen-2.

To establish a rapid and highly sensitive assay for tumor-associated trypsinogen-2 (TAT-2) based on the time-resolved fluorescence immunoassay (TRFIA) and evaluate its potential clinical value in patients with lung cancer. The double-antibody sandwich method was used in detecting TAT-2 antigen concentrations, and two types of TAT-2 antibodies (coating antibodies and Eu3+ labeled antibodies) were used. A TAT-2-TRFIA method was then established, evaluated, and used in detecting the serum TAT-2 levels of healthy subjects and patients with lung cancer. The linear range of the TAT-2-TRFIA method was 1.53-300 ng/mL, the intra-assay coefficient of variation (CV) were between 1.67% and 8.42%, and the inter-assay CV were between 4.29% and 11.44%. The recovery rates of TAT-2-TRFIA were between 99.17% and 107.06%. The cross-reactivities of trypsin and T-cell immunoglobulin mucin 3 were 0.02% and 0.82%, respectively. The serum TAT-2 levels of patients with lung cancer were higher than those of healthy subjects (P < 0.001). Combined with TAT-2, the sensitivity and specificity of CEA and CA-125 for lung cancer improved significantly. Conclusion: We successfully established a highly sensitive TAT-2-TRFIA method, which was able to facilitate the timely diagnosis of lung cancer.

Establishment of Galectin-3 Time-resolved Fluoroimmunoassay and its Application in Idiopathic Membranous Nephropathy.

The aim of this study was to establish a time-resolved fluorescent immunoassay (TRFIA) for the detection of serum Galectin-3 (Gal-3) and apply this method to evaluate the clinical significance of serum Gal-3 in predicting Idiopathic Membranous Nephropathy (IMN) progression. The Gal-3-TRFIA was established using the double antibody sandwich method, with the capture antibodies coated on a 96-well microplate and the detection antibodies chelated with Europium (III) (Eu3+). Serum Gal-3 was detected in 81 patients with IMN and 123 healthy controls to further evaluate the value of the Gal-3 in staging of IMN. The sensitivity of the Gal-3-TRFIA assay was 0.85 ng/mL, and the detection range was 0.85-1000 ng/mL. The Gal-3 intra-batch and inter-batch coefficients of variation were 3.45% and 5.12%, respectively. The correlation coefficient (R) between the Gal-3-TRFIA assay and commercially available enzyme-linked immunosorbent assay kits was 0.83. The serum Gal-3 concentration was higher in patients with IMN (65.57 ± 55.90 ng/mL) compared to healthy controls (16.29 ± 9.91 ng/mL, P < 0.0001). In this study, a wide detection range Gal-3-TRFIA assay was developed using lanthanide (Eu3+) chelates for the detection of Gal-3 concentrations in serum. Gal-3 concentration is elevated in patients with IMN.

Establishment of a time-resolved immunoassay for acute kidney injury based on the detection of Kim-1.

AIM: To establish a highly sensitive time-resolved fluorescence immunoassay (TRFIA) of kidney injury molecule-1 (Kim-1) and evaluate its clinical value in acute kidney injury (AKI). METHODS: The Kim-1-TRFIA was established by the double-antibody sandwich method, and the method was evaluated. The established Kim-1-TRFIA was used to detect the concentration of Kim-1 in the serum of healthy controls and patients with AKI. RESULTS: The optimal coating antibody concentration and optimal Eu3+ -labeled antibody dilution ratio for Kim-1-TRFIA are 1 µg/ml and 1:140, respectively. The linear range is 42.71-4666.69 pg/ml. The intra- and inter-assay coefficients of variation are <10%. The specificity of our Kim-1-TRFIA is acceptable. The recovery is between 95.14% and 102.84%. The concentration of Kim-1 in the serum of patients with AKI is 126.50 ± 67.99 pg/ml, which is significantly higher than that in the serum of healthy controls (49.72 ± 16.40 pg/ml, p < 0.001). Staging patients with AKI by glomerular filtration rate shows that the serum concentration of Kim-1 increases significantly with increasing disease severity (p < 0.05). CONCLUSION: A highly sensitive Kim-1-TRFIA was established. With this immunoassay, a good differential diagnosis can be made, and healthy people and AKI patients can be differentiated by detecting the concentration of Kim-1 in the serum. Moreover, the severity of AKI patients can be determined.

Mitochondrial nucleoid in cardiac homeostasis: bidirectional signaling of mitochondria and nucleus in cardiac diseases.

Metabolic function and energy production in eukaryotic cells are regulated by mitochondria, which have been recognized as the intracellular 'powerhouses' of eukaryotic cells for their regulation of cellular homeostasis. Mitochondrial function is important not only in normal developmental and physiological processes, but also in a variety of human pathologies, including cardiac diseases. An emerging topic in the field of cardiovascular medicine is the implication of mitochondrial nucleoid for metabolic reprogramming. This review describes the linear/3D architecture of the mitochondrial nucleoid (e.g., highly organized protein-DNA structure of nucleoid) and how it is regulated by a variety of factors, such as noncoding RNA and its associated R-loop, for metabolic reprogramming in cardiac diseases. In addition, we highlight many of the presently unsolved questions regarding cardiac metabolism in terms of bidirectional signaling of mitochondrial nucleoid and 3D chromatin structure in the nucleus. In particular, we explore novel techniques to dissect the 3D structure of mitochondrial nucleoid and propose new insights into the mitochondrial retrograde signaling, and how it regulates the nuclear (3D) chromatin structures in mitochondrial diseases.

Establishment of heparin-binding protein time-resolved immunoassay and some potential clinical applications.

AIM: To establish a highly sensitive time-resolved fluorescence immunoassay of heparin-binding protein (HBP-TRFIA) and evaluate its application value for bacterial or fungal infections in tumor patients. METHODS: Two types of HBP monoclonal specific antibodies against different epitopes of the antigen molecule were used as coating antibodies and Eu3+-labeled antibodies, respectively. The double-antibody sandwich method was used in establishing HBP-TRFIA, and the methodology was evaluated. The established HBP-TRFIA was used in detecting HBP concentration in the plasma samples of healthy individuals, patients with bacterial or fungal infections, and infected or uninfected patients with various types of tumors. RESULTS: The linear range of HBP-TRFIA was (0.11-530 ng/mL). Plasma HBP concentrations detected through HBP-TRFIA were consistent with the results of fluorescence quantitative immunochromatography (ρ = 0.964). The plasma HBP concentrations of infected tumor patients were significantly higher than those of uninfected tumor patients (P < 0.01). CONCLUSION: This study successfully established a highly sensitive HBP-TRFIA, which was highly comparable to commercially available fluorescent quantitative immunochromatographic kits and was able to facilitate the timely diagnosis of bacterial or fungal infections in patients with tumor.

Luteolin enhances the antitumor efficacy of oncolytic vaccinia virus that harbors IL-24 gene in liver cancer cells.

BACKGROUND: Interleukin 24 (IL-24) is an IL-10 family member and a secreted cytokine characterized by cancer-targeted toxicity and can activate apoptosis by sensitizing cancer cells to chemotherapy. Cytotoxic effects of luteolin on different types of cancer cells suppress their growth by acting on the components of the apoptosis signaling cascade. Therefore, our study aimed to prove whether oncolytic vaccinia virus (VV) that harbors IL-24 (VV-IL-24) combine with luteolin exerts a synergistic inhibitory effect in liver cancer cells. METHODS: Impacts on cell viability of VV-IL-24 and luteolin were assessed by MTT in various liver cancer cell lines. Then, liver cancer cell apoptosis was analyzed via flow cytometry and Western blotting. Besides, the MHCC97-H xenograft mouse model was employed as a means of assessing in vivo antitumor efficacy. RESULTS: MTT assay confirmed that the combination treatment decreased liver cancer cells viability to a greater degree than treatment with VV-IL-24 or luteolin alone. Flow cytometry and Western blot assay proved that VV-IL-24 plus luteolin induced more liver cancer cells apoptosis than single treatment. Furthermore, in the MHCC97-H xenograft model, 15 days of treatment with VV-IL-24 plus luteolin inhibited tumor growth significantly more than single treatment. CONCLUSION: These data confirm that the synergistic mechanism of VV-IL-24 and luteolin elicits a stronger tumor growth inhibition than any single therapy. Thus, the combination of VV-IL-24 and luteolin could provide the basis for preclinical research in the treatment of liver cancer.

Establishment of time-resolved fluoroimmunoassay of IgG4 based on magnetic microspheres.

BACKGROUND: The abnormal increase in serum IgG4 level is an important clinical symptom of IgG4-related disease (IgG4-RD), and the detection of serum IgG4 level is a powerful tool for the diagnosis of IgG4-RD. This study was conducted to establish a simple and rapid immunoassay for the determination of human serum IgG4 levels. METHODS: Based on the competition method, a novel immunoassay was established for the determination of human serum IgG4 using a combination of time-resolved fluoroimmunoassay (TRFIA) and magnetic microspheres. IgG4 was coupled with magnetic microspheres and competed with IgG4 in the samples to bind the Eu3+ -labeled anti-IgG4 antibody. The immunocomplex was separated and washed in a magnetic field, and the fluorescence counts were measured according to the number of dissociated europium ions. RESULTS: The analytical sensitivity of IgG4-TRFIA based on magnetic microspheres was 0.006 g/L, and the detection range was 0.006-20 g/L under optimal conditions. The precision, recovery, and specificity of this immunoassay were demonstrated to be acceptable. The clinical application of IgG4-TRFIA based on magnetic microspheres was evaluated and compared with that of immunonephelometry. The results showed that the two detection methods had a good correlation, with a correlation coefficient of .9871. CONCLUSION: IgG4-TRFIA based on magnetic microspheres has the advantages of high sensitivity, wide detection range, and short analysis time and has the potential to become a useful tool for the diagnosis of IgG4-RD.

Establishment of interleukin-18 time-resolved fluorescence immunoassay and its preliminary application in liver disease.

BACKGROUND: To establish a time-resolved fluorescence immunoassay of interleukin (IL)-18 (IL-18-TRFIA) and detect its concentration in different liver disease serum samples. METHODS: The IL-18 coating antibody and the Eu3+ -labeled detection antibody were used for the IL-18-TRFIA to detect serum IL-18 concentration in patients with liver cancer, hepatitis B, hepatitis C, autoimmune hepatitis, fatty liver disease, and healthy controls. The double-antibody sandwich method was used and methodological evaluation was performed. RESULTS: The average intra- and inter-assay coefficient of variation for IL-18-TRFIA was 4.80% and 5.90%, respectively. The average recovery rate was 106.19 ± 3.44%. The sensitivity (10.96 pg/mL) was higher than that obtained using the ELISA method (62.5 pg/mL). The detection range was 10.96-1000 pg/mL. IL-6 and galectin-3 did not cross-react with IL-18-TRFIA. The serum concentration of IL-18 was (776.99; 653.48-952.39 pg/mL) in hepatitis C, (911; 775.55-1130.03 pg/mL) in fatty liver, (1048.88; 730.04-1185.10 pg/mL) in liver cancer, and (949.12; 723.70-1160.28 pg/mL) in hepatitis B. Moreover, IL-18 serum levels were significantly higher in patients than the healthy controls (483.09; 402.52-599.70/mL) (p < 0.0001). Autoimmune hepatitis with a serum IL-18 concentration of 571.62; 502.47-730.31 pg/mL was not significantly different from the healthy controls (p > 0.05). CONCLUSION: We established a highly sensitive IL-18-TRFIA method that successfully detected serum IL-18 concentrations in different liver diseases. Furthermore, IL-18 serum concentration was higher in patients with liver cancer, hepatitis C, hepatitis B, and fatty liver disease compared to healthy controls.

An oncolytic adenovirus delivering TSLC1 inhibits Wnt signaling pathway and tumor growth in SMMC-7721 xenograft mice model.

Tumor suppressor in lung cancer-1 (TSLC1) was first identified as a tumor suppressor for lung cancer, and frequently downregulated in various types of cancers including hepatocellular carcinoma (HCC). The Wnt pathway plays a critical role in tumorigenesis, migration, and invasion in HCC. However, the function of TSLC1 in modulating Wnt signaling in HCC is unclear. In this study, we evaluated the effect of TSLC1-armed oncolytic adenovirus (S24-TSLC1) on the Wnt/ß-catenin pathway, cell viability, invasion and migration abilities of HCC in vitro and the growth of SMMC-7721-xenografted tumor in mice model. We detected the expression of TSLC1 in tumor samples and HCC cell lines. The results showed that TSLC1 expression was low in HCC, but high in pericarcinomatous tissue and normal cells, which implied that TSLC1 is a tumor suppressor of liver cancer. S24-TSLC1 exhibited an antitumor effect on HCC cell growth in vitro, but did little damage to normal liver cells. Overexpression of TSLC1 downregulated the transcriptional activity of TCF4/ß-catenin and inhibited the mRNA or protein expression of Wnt target genes cyclinD1 and c-myc. S24-TSLC1 also inhibited the invasion and migration of HCC cells. Animal experiments further confirmed that S24-TSLC1 significantly inhibited tumor growth of the SMMC-7721-xenografted tumor. In conclusion, TSLC1 could downregulate the Wnt signal pathway and suppress HCC cell growth, migration and invasion, suggesting that S24-TSLC1 may be a potent antitumor agent for future clinical trials in liver cancer treatment.