Study on the mechanism of S100A4-mediated cancer oncogenesis in uveal melanoma cells through the integration of bioinformatics and in vitro experiments.

BACKGROUND: The elevated metastasis rate of uveal melanoma (UM) is intricately correlated with patient prognosis, significantly affecting the quality of life. S100 calcium-binding protein A4 (S100A4) has tumorigenic properties; therefore, the present study investigated the impact of S100A4 on UM cell proliferation, apoptosis, migration, and invasion using bioinformatics and in vitro experiments. METHODS: Bioinformatic analysis was used to screen S100A4 as a hub gene and predict its possible mechanism in UM cells, and the S100A4 silencing cell line was constructed. The impact of S100A4 silencing on the proliferative ability of UM cells was detected using the Cell Counting Kit-8 and colony formation assays. Annexin V-FITC/PI double fluorescence and Hoechst 33342 staining were used to observe the effects of apoptosis on UM cells. The effect of S100A4 silencing on the migratory and invasive capabilities of UM cells was assessed using wound healing and Transwell assays. Western blotting was used to detect the expression of related proteins. RESULTS: The present study found that S100A4 is a biomarker of UM, and its high expression is related to poor prognosis. After constructing the S100A4 silencing cell line, cell viability, clone number, proliferating cell nuclear antigen, X-linked inhibitor of apoptosis protein, and survivin expression were decreased in UM cells. The cell apoptosis rate and relative fluorescence intensity increased, accompanied by increased levels of Bax and caspase-3 and decreased levels of Bcl-2. Additionally, a decrease in the cell migration index and relative invasion rate was observed with increased E-cadherin expression and decreased N-cadherin and vimentin protein expression. CONCLUSION: S100A4 silencing can inhibit the proliferation, migration, and invasion and synchronously induces apoptosis in UM cells.

A novel Fas ligand plays an important role in cell apoptosis of Crassostrea hongkongensis: molecular cloning, expression profiles and functional identification of ChFasL.

Background: Apoptosis regulates normal development, homeostasis, immune tolerance and response to environmental stress by eliminating unwanted or diseased cells, and plays a key role in non-specific immunity of invertebrates. The exogenous pathway mediated by death receptors and death ligands is a very important pathway for cell apoptosis. Death ligands are mainly members of the tumour necrosis factor (TNF) family, of which FasL is an important member. The deep involvement of FasL in vertebrates cell apoptosis and immunity has been reported many times, but there is limited research on the FasL gene in shellfish, and its functional importance in oyster cell apoptosis and immunity remains unclear. Methods: The full length of ChFasL was identified and cloned based on the genome of Crassostrea hongkongensis. Quantitative PCR was used to detect the relative expression of ChFasL in different developmental stages and tissues, as well as the changes of relative expression in hemocytes after bacterial infection. The expression position of ChFasL in HEK293T cells was also located by subcellular localization, and the effect of increased recombinant protein content on the activity of reporter genes p53 and p21 was studied by dual-fluorescence reporter gene. Finally, the changes of apoptosis rate in hemocytes after ChFasL silencing was identified by RNA interference technology. Results: We identified a novel FasL gene from C. hongkongensis and named it ChFasL. We found that ChFasL has potential N-linked glycosylation site, a transmembrane domain and a TNF region, which was a typical characteristics of TNF family. ChFasL was expressed in all developmental stages of larvae and in all tissues of oysters. After stimulation by V. alginolyticus or S. haemolyticus, its relative expression in hemocytes increased significantly, suggesting that ChFasL was deeply engaged in the immune response process of C. hongkongensis to external microbial stimulation. The results of subcellular localization showed that ChFasL was mainly distributed in the cytoplasm of HEK293T cells. With the overexpression of the recombinant protein pcDNA3 1- ChFasL, the activity of p53 and p21 significantly increased, showing a positive regulatory effect. Moreover, after dsRNA successfully reduced the relative expression of ChFasL, the apoptosis rate of hemocytes was significantly lower than that the dsGFP group. Conclusion: These results comprehensively confirmed the important role of ChFasL in the apoptosis process of C. hongkongensis, which provided the basis and premise for the in-depth understanding of the immune function of apoptosis in molluscs, and also contributed to the research on the pathogenic death mechanism and disease resistance breeding of marine bivalves.

Generation of a CD70-Specific Fusion Nanobody with IgG Recruiting Capacity for Tumor Killing.

Purpose: Due to its competitive advantages such as small size, high stability, easy production, and good tissue penetration compared with monoclonal antibodies (mAb), nanobodies (Nbs) were considered the next generation of therapeutics. However, the absence of Fc fragments and Fc-triggered immune effectors limits their clinical applications. In order to overcome these limitations, we develop a novel approach by attaching an IgG binding domain (IgBD) to Nbs for recruiting endogenous IgG and recovering the immune effectors for tumor killing. Material and Methods: We linked a Streptococcal Protein G-derived IgBD, termed C3Fab, at the C-terminus of a CD70-specific Nb 3B6 to construct an endogenous IgG recruitment antibody (termed EIR). The recombinant Nb3B6-C3Fab was expressed in E. coli BL21 (DE3) and purified by nickel affinity chromatography. We further evaluated the binding, recruitment of IgG, and the serum half-life of Nb3B6-C3Fab. The tumor-killing effects on CD70 positive cells mediated by antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity were also detected. Results: We successfully constructed a IgBD fused Nb3B6-C3Fab with high affinity for CD70 and mouse IgG (mIgG). Nb3B6-C3Fab can specifically bind to CD70 positive tumor cells and recruit mIgG on the cell surface. Ligating of Nb3B6 with C3Fab increased its serum half-life in mice almost 39-fold from 0.96 h to 37.67 h. Moreover, we demonstrated remarkable cytotoxicity of Nb3B6-C3Fab to CD70 positive tumor cells via C3Fab by immune effector cells. Conclusion: Our study demonstrates that IgBD fusion endows Nbs with the ability for endogenous IgG recruitment and half-life promotion. Linking IgBD to Nbs is an effective strategy to recovering immune effectors for tumor killing.

Nox4-and Tf/TfR-mediated peroxidation and iron overload exacerbate neuronal ferroptosis after intracerebral hemorrhage: Involvement of EAAT3 dysfunction.

Intracerebral hemorrhage (ICH) induces high mortality and disability. Neuronal death is the principal factor to unfavourable prognosis in ICH. However, the mechanisms underlying this association remain unclear. In this study, we investigated the molecular mechanisms by which neuronal ferroptosis occurs after ICH and whether the use of corresponding modulators can inhibit neuronal death and improve early outcomes in a rat ICH model. Our findings indicated that Nox4 and TF/TfR were upregulated in the perihematomal tissues of ICH rats. Oxidative stress and iron overload induced by Nox4 and TF/TfR promoted neuronal ferroptosis post-ICH. In contrast, application of Nox4-siRNA and the deferoxamine (DFO) attenuated peroxidation and iron deposition in the hemorrhagic brain, alleviated neuronal ferroptosis, and improved sensorimotor function in ICH rats. Additionally, our findings indicated that the post-ICH neuronal reduced glutathione (GSH) depletion were not related to dysfunctional glutamine delivery in astrocytes but rather to downregulation of EAAT3 due to lipid peroxidation-induced dysfunction in the neuronal membrane. These findings indicate that ferroptosis is involved in neuronal death in model rats with collagenase-induced ICH. Oxidative stress and iron overload induced by Nox4 and TF/TfR exacerbate ferroptosis after ICH, while Nox4 downregulation and iron chelation exert neuroprotective effects. The present results highlight the cysteine importer EAAT3 as a potential biomarker of ferroptosis and provide insight into the neuronal death process that occurs following ICH, which may aid in the development of translational treatment strategies for ICH.

Circ_0000284 upregulates RHPN2 to facilitate pancreatic cancer proliferation, metastasis, and angiogenesis through sponging miR-1179.

BACKGROUND: Circular RNA (circRNA) has been confirmed to be a key regulator for pancreatic cancer (PC) progression, but the role of circ_0000284 in PC development remains unclear. METHODS: Quantitative real-time PCR was used to measure the expression of circ_0000284, microRNA (miR)-1179, and rhophilin 2 (RHPN2). PC cell proliferation, metastasis, angiogenesis, and apoptosis were assessed by EdU assay, transwell assay, tube formation assay, and flow cytometry. Relative protein expression was determined by western blot analysis. The interaction between miR-1179 and circ_0000284 or RHPN2 was confirmed by dual-luciferase reporter assay and RNA pull-down assay. RESULTS: Circ_0000284 was significantly upregulated in PC tissues and cells, and its knockdown inhibited PC cell proliferation, migration, invasion, and angiogenesis while promoting apoptosis. MiR-1179 was downregulated in PC tissues and cells, and it could be sponged by circ_0000284. Moreover, the miR-1179 inhibitor reversed the regulation of circ_0000284 knockdown on PC cell progression. The highly expressed RHPN2 was found in PC tissues and cells, and it could be targeted by miR-1179. Also, circ_0000284 sponged miR-1179 to regulate RHPN2 expression. Overexpressed RHPN2 could reverse the regulation of circ_0000284 knockdown on PC cell progression. In addition, interference of circ_0000284 was discovered to repress PC tumor growth by regulating miR-1179/RHPN2.RHPN2. CONCLUSION: To sum up, our data confirmed that circ_0000284 facilitated PC malignant progression depending on the regulation of miR-1179/RHPN2 axis, suggesting that circ_0000284 might be a potential target for PC treatment.

Identification and characterization of blocking nanobodies against human CD70.

CD70 is overexpressed in a variety of solid and hematological tumors and plays a role in tumor proliferation and evasion of immune surveillance. Targeting and blocking its binding to the receptor CD27 have the potential to treat CD70-dependent tumors. To generate novel CD70 blocking agents, we screen a human CD70-immunized camel VHH phage display library and isolate two blocking nanobodies against human CD70 targeting different epitopes. Upon enrichment by three rounds of biopanning, two strategies are employed to identify CD70 blockers. One named affinity selection is used for detecting clones with CD70 binding by conventional PE-ELISA. However, no clone with a blocking effect is obtained from 188 enriched clones by this method. The alternative strategy named competitive selection is based on the inhibiting capacity of CD70-CD27 binding by enriched VHHs. By this method, two clones, Nb-2B3 and Nb-3B6, with strong blocking capacity are obtained from 20 enriched VHHs, suggesting the efficiency of this strategy. Furthermore, Nb-2B3 and Nb-3B6 specifically bind to CD70-positive SKOV3 and Raji cells at low concentrations. Meanwhile, Nb-2B3 has no competitive effect on the binding of Nb-3B6 to CD70, and vice versa, indicating that they target two different epitopes on CD70. Our data show that nanobodies Nb-2B3 and Nb-3B6 are potential attractive theranostic agents for CD70-expressing cancers.

Effect of Gene 33/Mig6/ERRFI1 on hexavalent chromium-induced transformation of human bronchial epithelial cells depends on the length of exposure.

Hexavalent chromium (Cr(VI)) compounds are environmental and occupational lung carcinogens. The present study followed the chronic effect of Cr(VI) on the neoplastic transformation of BEAS-2B lung bronchial epithelial cells with or without deletion of Gene 33 (Mig6, EFFRI1), a multifunctional adaptor protein. We find that Gene 33-deleted cells exhibit increased anchorage-independent growth compared to control cells after transformed by 8-week but not 24-week Cr(VI) exposure. Gene 33-deleted cells show a higher level of cell proliferation and are more resistant to acute Cr(VI) toxicity compared to control cells after transformed by 8-week but not 24-week Cr(VI) exposure, despite that 24-week-transformed cells have increased resistance to acute Cr(VI) toxicity. However, Gene 33-deleted cells show increased migration after transformed by both 8-week and 24-week Cr(VI) exposures. Furthermore, only cells transformed by 24 weeks of Cr(VI) exposure can form subcutaneous tumors in nude mice. Although no significant difference in the size of tumors formed by the two cell types, there is a marked difference in the histological manifestation and more MMP3 expression in tumors from Gene 33-deleted cells. Our results demonstrate progressive neoplastic transformation of BEAS-2B cells and the adaptation of these cells to Gene 33 deletion during chronic exposure to Cr(VI).

lncRNA IUR upregulates miR-34a to inhibit pancreatic adenocarcinoma cell migratory and invasive abilities.

The long non-coding RNA (lncRNA) imatinib-upregulated (IUR) has been recently reported as a tumor suppressor in leukemia. Preliminary microarray data revealed a downregulation of IUR in pancreatic adenocarcinoma (PAAD) and a positive correlation with microRNA-34a (miR-34a) expression. The present study aimed to investigate the role of IUR in PAAD. This study included samples from 58 patients with PAAD and the PAAD cell lines Capan-2 and HPAC. Reverse transcription quantitative PCR was performed to determine gene expression levels. Cell transfections were carried out to assess gene interactions between IUR, miR-34a and CD44. Transwell assays were performed to explore the effects of transfections on cell invasive and migratory abilities. The results demonstrated that IUR was downregulated in PAAD tissue compared with adjacent non-tumor tissue samples and that low expression levels of IUR correlated with poor survival in patients with PAAD. In PAAD tissue samples, the expression of IUR positively correlated with miR-34a expression but negatively correlated with CD44 expression, which is a target of miR-34a. In PAAD cells, overexpression of IUR resulted in miR-34a upregulation and CD44 downregulation. miR-34a overexpression did not affect the expression of IUR but downregulated CD44. In PAAD cells, overexpression of IUR and miR-34a led to decreased invasive and migratory abilities. However, CD44 overexpression played an opposite role and attenuated the effects of IUR and miR-34a overexpression. In conclusion, the results from this study demonstrated that IUR may upregulate miR-34a expression in order to inhibit PAAD cell migration and invasion by downregulating CD44.

High affinity monoclonal antibody targeting Siglec-15 for cancer immunotherapy.

BACKGROUND AND AIM: Recently, Siglec-15 has been proved as a novel immune suppressor and a potential target for normalization cancer immunotherapy, which is non-redundant to the well-known PD-L1/PD-1 pathway. Herein, anti-Siglec-15 mAb, a monoclonal antibody (mAb) with a high affinity against Siglec-15, was prepared. METHODS: The engineered CHO-K1 Siglec-15 cell line was constructed to heterologously expressed Siglec-15 for the affinity test with the mAb. Antigens Siglec-15-mIgG and Siglec-15-his were recombinantly expressed by 293F cells and purified by high-performance liquid chromatography (HPLC). Hybridoma cell line against Siglec-15 was prepared and validated by enzyme-linked immunoabsorbant assay (ELISA) and fluorescent-activated cell sorting (FACS). Finally, the anti-Siglec-15 mAb was produced, purified, and confirmed by SDS-PAGE, ELISA, and FACS. RESULTS: The EC50 of the anti-Siglec-15 mAb with Siglec-15 is 76.65 ng/mL, lower than that of the positive control 5G12 (90.7 ng/mL), indicating a high affinity of the anti-Siglec-15 mAb. In vitro and in vivo studies verified that the anti-Siglec-15 mAb blocks the Siglec-15-mediated suppression of T cell and moderately prevents the tumor growth. CONCLUSIONS: The anti-Siglec-15 mAb can be considered as an effective immunotherapy for tumor suppression. RELEVANCE FOR PATIENTS: The anti-Siglec-15 mAb prepared in this study is useful as an immune checkpoint inhibitor against Siglec-15 for normalization cancer immunotherapy. This immunotherapy provides an alternative treatment for cancer patients who are refractory to the well-known PD-L1/PD-1-targeting therapies.

Investigation of Interface Thermal Resistance between Polymer and Mold Insert in Micro-Injection Molding by Non-Equilibrium Molecular Dynamics.

Micro-injection molding has attracted a wide range of research interests to fabricate polymer products with nanostructures for its advantages of cheap and fast production. The heat transfer between the polymer and the mold insert is important to the performance of products. In this study, the interface thermal resistance (ITR) between the polypropylene (PP) layer and the nickel (Ni) mold insert layer in micro-injection molding was studied by using the method of non-equilibrium molecular dynamics (NEMD) simulation. The relationships among the ITR, the temperature, the packing pressure, the interface morphology, and the interface interaction were investigated. The simulation results showed that the ITR decreased obviously with the increase of the temperature, the packing pressure and the interface interaction. Both rectangle and triangle interface morphologies could enhance the heat transfer compared with the smooth interface. Moreover, the ITR of triangle interface was higher than that of rectangle interface. Based on the analysis of phonon density of states (DOS) for PP-Ni system, it was found that the mismatch between the phonon DOS of the PP atoms and Ni atoms was the main cause of the interface resistance. The frequency distribution of phonon DOS also affected the interface resistance.

Spleen contributes to restraint stress induced hepatocellular carcinoma progression.

The spleen is the largest secondary immune organ and plays a critical role in the progression of tumor. Psychological stress promotes tumor progression through inhibiting antitumor immune. However, the role of spleen in tumor progression induced by stress is unclear. Here, we showed that restraint stress promoted tumor growth, increased the percentage of CD11b+Gr-1+ MDSC while decreased the percentages of CD3-NK1.1+ NK and CD3+NK1.1+ NKT in the tumor tissues. Restraint stress decreased the percentages of CD3+CD4+ T lymphocytes and CD3+CD8+ T lymphocytes while increased the percentage of CD11b+Gr-1+ MDSC in the blood of tumor-bearing mice. Restraint stress increased the percentages of CD3+CD4+ T lymphocytes, CD3+CD8+ T lymphocytes, CD4+PD1+ T lymphocytes and CD8+PD1+ T lymphocytes while decreased the percentage of CD11b+Gr-1+ MDSC in the spleen of tumor-bearing mice. Interestingly, splenectomy inhibited tumor growth and attenuated the changes of CD3+CD4+ T lymphocytes, CD3+CD8+ T lymphocytes, and CD11b+Gr-1+ MDSC in blood induced by chronic restraint stress. Finally, splenectomy blocked the increases of CD11b+Gr-1+ MDSC but did not attenuate the decreases of CD3-NK1.1+ NK and CD3+NK1.1+ NKT in tumor tissue induced by chronic stress. Together, these data indicate that chronic restraint stress promotes hepatocellular carcinoma growth and suppresses the antitumor immunity of tumor-bearing mice. Splenectomy could inhibit tumor growth and partly block the decrease of antitumor immune activity induced by stress.

Splenic serum from portal hypertensive patients enhances liver stem cell proliferation and self-renewal via the IGF-II/ERK signaling pathway.

BACKGROUND: Hypersplenism is a serious complication of portal hypertension (PH) and can affect the prognosis of liver disease. Liver stem cells (LSCs) are involved in liver regeneration and hepatocarcinogenesis after liver cirrhosis. AIM: To explore the effects and mechanism of the spleen on the proliferation and differentiation of LSCs in PH due to liver cirrhosis. METHODS: Fetal liver stem cells (FLSCs) were treated with splenic serum from liver cirrhosis patients with hypersplenism and control serum from healthy volunteers, and the proliferation, self-renewal, and IGF-II/ERK signaling pathway of FLSCs were then evaluated. RESULTS: We found that splenic serum from PH patients promoted FLSC proliferation, colony formation, and Ki-67 expression in vitro. Splenic serum from PH also enhanced FLSC spheroid formation in vitro. Mechanistically, we determined that insulin-like growth factor (IGF)-II concentration was elevated in splenic serum from PH patients and could promote FLSC proliferation and self-renewal. Furthermore, both IGF-II and splenic serum from PH patients enhanced ERK signaling activation through IGF-I receptor (IGF-I R) in FLSCs. Consistently, blocking IGF-I R or ERK signaling could attenuate the effects of splenic serum from PH patients on FLSCs. CONCLUSIONS: The spleen in PH patients promotes FLSC proliferation and self-renewal through the IGF-II/ERK signaling pathway.

Ring1 promotes the transformation of hepatic progenitor cells into cancer stem cells through the Wnt/ß-catenin signaling pathway.

The proliferation of hepatic progenitor cells (HPCs) is observed in reactive conditions of the liver and primary liver cancers. Ring1 as a member of polycomb-group proteins which play vital roles in carcinogenesis and stem cell self-renewal was increased in HCC patients and promoted proliferation and survival of cancer cell by degrading p53. However, the mechanisms of Ring1 driving the progression of hepatocarcinogenesis have not been elucidated. In this study, forced expression Ring1 and Ring1 siRNA lentiviral vectors were utilized to stably overexpression and silence Ring1 in HPC cell line (WB-F344), respectively. Our finding indicated that overexpression of Ring1 in HPCs promoted colony formation, cell multiplication, and invasion in vitro, conversely depletion of Ring1 repressed the biological functions of HPCs relative to controls. The expression of ß-catenin was upregulated in the HPCs with overexpression of Ring1, and the correlation analysis also showed that ß-catenin and Ring1 had a significant correlation in the liver cancer tissues and adjacent tissues. The activation of the Wnt/ß-catenin signaling pathway significantly increased the expression of liver cancer stem cells related (LCSCs)-related molecular markers CD90 and EpCAM, which led to the transformation of HPCs into LCSCs. Most importantly, the injection of HPCs with overexpressed Ring1 into the subcutaneous of nude mice leads to the formation of poorly differentiated HCC neoplasm. Our findings elucidate that overexpression of Ring1 the activated Wnt/ß-catenin signaling pathway and drove the transformation of HPCs into cancer stem cell-like cells, suggesting Ring1 has extraordinary potential in early diagnosis of HCC.

Chronic restraint stress promotes hepatocellular carcinoma growth by mobilizing splenic myeloid cells through activating ß-adrenergic signaling.

Psychological stress promotes tumor progression and has a large impact on the immune system, particularly the spleen. The spleen plays an important role in tumor behavior. However, the role and mechanism of the spleen in hepatocellular carcinoma progression induced by stress is unclear. Here, we showed that the spleen plays a critical role in hepatocellular carcinoma growth induced by restraint stress. Our results demonstrated that restraint stress promoted hepatocellular carcinoma growth, changed the spleen structure, and redistributed splenic myeloid cells to tumor tissues. Interestingly, we found that splenectomy could inhibit hepatocellular carcinoma growth and prevent increases in myeloid cells and macrophages in tumor tissues in stressed mice. Restraint stress significantly elevated the concentration of norepinephrine in the spleen, serum and tumor tissues. Meanwhile, propranolol, an inhibitor of ß-adrenergic signaling, could inhibit hepatocellular carcinoma growth and prevent the redistribution of splenic myeloid cells induced by restraint stress, suggesting that restraint stress promotes hepatocellular carcinoma growth and redistributes splenic myeloid cells through ß-adrenergic signaling. Mechanistic studies revealed that restraint stress upregulated the expressions of CXCL2/CXCL3 in tumor tissues and changed the expression of CXCR2 in myeloid cells. SB225002, an inhibitor of CXCR2, could prevent the recruitment of myeloid cells in tumor tissues and inhibit tumor growth in stressed mice. Together, these data indicate that chronic restraint stress promotes hepatocellular carcinoma growth by mobilizing splenic myeloid cells to tumor tissues via activating ß-adrenergic signaling. The CXCR2-CXCL2/CXCL3 axis contributed to the recruitment of myeloid cells in tumor tissues induced by restraint stress.

Concurrent detection of lysosome and tissue transglutaminase activation in relation to cell cycle position during apoptosis induced by different anticancer drugs.

Described is the new cytometric approach do detect either stimulation or a collapse of lysosomal proton pump (lysosomes rupture) combined with activation of transglutaminase 2 (TG2) during induction of apoptosis. Apoptosis of human lymphoblastoid TK6 cells was induced by combination of 2-deoxyglucose with the isoquinoline alkaloid berberine, by DNA topoisomerase I inhibitor camptothecin, its analog topotecan, topoisomerase II inhibitors etoposide or mitoxantrone, as well as by the cytotoxic anticancer ribonuclease ranpirnase (onconase). Activity of the proton pump of lysosomes was assessed by measuring entrapment and accumulation of the basic fluorochrome acridine orange (AO) resulting in its metachromatic red luminescence (F>640 ) within these organelles. Activation of TG2 was detected in the same cell subpopulation by the evidence of crosslinking of cytoplasmic proteins revealed by the increased intensity of the side light scatter (SSC) as well as following cell lysis by detergent, by its red fluorescence after staining by sulforhodamine 101. Because at low AO concentration nuclear DNA of the lysed cells was stoichiometrically stained green (F530 ) its quantity provided information on effects of the drug treatments on cell cycle in relation to activation of TG2. The data reveal that activation of lysosomal proton pump was evident in subpopulations of cells treated with 2-deoxyglucose plus berberine, topotecan, etoposide and mitoxantrone but not with ranpirnase. The collapse of lysosomal proton pump possibly reporting rupture of these organelles was observed in definite cell subpopulations after treatment with each of the studied drugs. Because regardless of the inducer of apoptosis TG2 activation invariably was correlated with lysosomes rupture it is likely that it was triggered by calcium ions or protons released from the ruptured lysosomes. This new methodological approach offers the means to investigate mechanisms and factors affecting autophagic lysosomes proton pump activity vis-à-vis TG2 activation that are common in several pathological states. © 2019 International Society for Advancement of Cytometry.

Spleen Regulates Hematopoietic Stem/Progenitor Cell Functions Through Regulation of EGF in Cirrhotic Hypersplenism.

BACKGROUND: Hematopoietic abnormality is a common cause of cirrhotic hypersplenism (CH) complications and death; it causes serious adverse effects and is associated with bleeding, anemia, infection in CH patients. However, the underlying mechanism is unclear. AIMS: We aimed to investigate the effects of the spleen on hematopoiesis and hematopoietic stem/progenitor cells (HSPCs) in CH patients. METHODS: Eleven CH patients were enrolled to assess the effects of the spleen on HSPC functions. Hematopoietic changes were examined by flow cytometry analysis. HSPC functions were detected with colony-forming assays and in vitro cell cultures. Enzyme-linked immunosorbent assay (ELISA) was used to test the concentration of epithelial growth factor (EGF). RESULTS: The number of HSPCs was decreased in CH patients and was rescued after splenectomy. Serum from CH patients dysregulated HSPCs function, and serum from splenectomy patients restored the dysregulated HSPC function in vitro. The concentration of EGF was decreased in CH patients and was restored to normal level after splenectomy. EGF rescued the dysregulated HSPCs function in vitro. CONCLUSIONS: The spleen can regulate the functions of HSPCs in CH patients by regulating EGF signaling. EGF may be a therapeutic target for CH treatment.

Single-cell RNA sequencing reveals an altered gene expression pattern as a result of CRISPR/cas9-mediated deletion of Gene 33/Mig6 and chronic exposure to hexavalent chromium in human lung epithelial cells.

Gene 33 (Mig6, ERRFI1) is an adaptor protein with multiple cellular functions. We recently reported that depletion of this protein promotes lung epithelial cell transformation induced by hexavalent chromium [Cr(VI)]. However, the early molecular events that mediate this process are not clear. In the present study, we used single-cell RNA sequencing to compare gene expression profiles between BEAS-2B lung epithelial cells chronically exposed to a sublethal dose of Cr(VI) with or without CRISPR/cas9-mediated deletion of Gene 33. Our data reveal 83 differentially expressed genes. The most notable changes are genes associated with cell adhesion, oxidative stresses, protein ubiquitination, epithelial-mesenchymal transition/metastasis, and WNT signaling. Up-regulation of some neuro-specific genes is also evident, particularly ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a deubiquitinase and potential biomarker for lung cancer. Gene 33 deletion and/or Cr(VI) exposure did not cause discernable changes in cell morphology. However, Gene 33 deletion led to a modest but significant reduction of cells in the G2/M phase of the cell cycle regardless of Cr(VI) exposure. Gene 33 deletion also significantly reduced cell proliferation. Interestingly, Cr(VI) exposure eliminated the difference in cell proliferation between the two genotypes. Gene 33 deletion also significantly elevated cell migration. Our data indicate that combined Gene 33 deletion and chronic Cr(VI) exposure produces a gene expression pattern and a phenotype resemble those of the transformed lung epithelial cells. Given the known association of UCHL1 with lung cancer, we propose that UCHL1 is an important player in the early stage of lung epithelial cell transformation and tumorigenesis.

ATM Activation and H2AX Phosphorylation Induced by Genotoxic Agents Assessed by Flow- and Laser Scanning Cytometry.

Activation of Ataxia Telangiectasia Mediated protein kinase (ATM) by its phosphorylation on serine 1981 and phosphorylation of histone H2AX on serine 139 (γH2AX) are the key events reporting DNA damage, primarily formation of DNA double strand breaks. These events are detected immunocytochemically in individual cells using phospho-specific Abs. The protocols are presented that describe the methodology of immunofluorescent labeling of cells in conjunction with specific staining of cellular DNA. Flow- and imaging-cytometry, the latter exemplified as laser scanning cytometry, is used to quantify intensity of cellular fluorescence reporting activation of ATM and induction of γH2AX with respect to cellular DNA content, which in turn reports the cell cycle phase. Different protocols are presented for analysis of cells either grown in suspension or attached to surface of culture vessels. Examples of ATM activation and H2AX phosphorylation in response to DNA damage in leukemic HL-60 cells by DNA topoisomerase I inhibitor topotecan, and in lung carcinoma A549 cells by hydrogen peroxide, are presented.

A camel anti-lysozyme CDR3 only domain antibody selected from phage display VHH library acts as potent lysozyme inhibitor.

Mimetics of antibody-binding sites represent particularly interesting targets, however they are difficult to identify. In most cases, naturally derived CDR3 peptides show a much lower activity and affinity. In this study, we identified a CDR3 domain antibody with framework 3 (FR3) and FR4 in the flank by screening a lysozyme-immunized phage display VHH library. This antibody has a potent enzyme inhibiting activity and high thermal stability. With sequence alignment and site-directed mutagenic analysis, we found that the cysteine residue at amino acid position 88 in FR3 might play a key role in maintaining the stability of the CDR3 antibody. The small-sized CDR3 domain antibody might act as a new scaffold for affinity transfer, hence making a useful contribution to the understanding of antigen-antibody interactions.

Synergy of 2-deoxy-D-glucose combined with berberine in inducing the lysosome/autophagy and transglutaminase activation-facilitated apoptosis.

Utilizing a variety of flow cytometric methods evidence was obtained indicating that a combination of the glucose analog 2-deoxy-D-glucose (2-dG) and the plant alkaloid berberine (BRB) produces synergistic effect in the induction of apoptosis in human lymphoblastoid TK6 cells. The synergistic effect is seen at concentrations of the drugs at which each of them alone shows no cytotoxicity at all. The data suggest that the combination of these drugs, which are known in terms of their overall toxicity, side effects and pharmacokinetics may be considered for further studies as chemopreventive and cancer treatment modalities. Of interest are results indicating that rapamycin, which similarly to BRB, suppresses mTOR signaling, when combined with 2-dG shows no synergistic properties. Metformin, on other hand, requires much higher concentration to show the synergy with 2-dG. Also of interest are the findings pertaining to the methodology of the present study. Specifically, dynamic assessment of cellular viability was performed by using the DRAQ7 cell exclusion fluorochrome present in cultures from 0 to 72 h. Concurrent measurement of lysosomal proton pump using acridine orange as the probe shows activation of lysosomes in the cells treated with 2-dG or BRB alone as well as with the drugs combined. Apoptosis was assessed by measuring DNA fragmentation, cell cycle, activation of caspase-3 and tissue transglutaminase (Tgase). A novel cytometric method was developed based on analysis of lysosomal (acidic vesicles) proton pump in live cells followed by cell lysis with detergent and fluorochrome labeling of proteins and DNA to analyze Tgase activation concurrently with cell cycle, in same population of cells. The data show that the cell subpopulation undergoing apoptosis has increased side (right-angle) light scatter likely due to the presence of the crosslinked (solid state) proteins, the consequence Tgase activation.