Oral intake of hydrogen-rich water ameliorated chlorpyrifos-induced neurotoxicity in rats.

Chronic exposure to low-levels of organophosphate (OP) compounds, such as chlorpyrifos (CPF), induces oxidative stress and could be related to neurological disorders. Hydrogen has been identified as a novel antioxidant which could selectively scavenge hydroxyl radicals. We explore whether intake of hydrogen-rich water (HRW) can protect Wistar rats from CPF-induced neurotoxicity. Rats were gavaged daily with 6.75mg/kg body weight (1/20 LD50) of CPF and given HRW by oral intake. Nissl staining and electron microscopy results indicated that HRW intake had protective effects on the CPF-induced damage of hippocampal neurons and neuronal mitochondria. Immunostaining results showed that the increased glial fibrillary acidic protein (GFAP) expression in astrocytes induced by CPF exposure can be ameliorated by HRW intake. Moreover, HRW intake also attenuated CPF-induced oxidative stress as evidenced by enhanced level of MDA, accompanied by an increase in GSH level and SOD and CAT activity. Acetylcholinesterase (AChE) activity tests showed significant decrease in brain AChE activity after CPF exposure, and this effect can be ameliorated by HRW intake. An in vitro study demonstrated that AChE activity was more intense in HRW than in normal water with or without chlorpyrifos-oxon (CPO), the metabolically-activated form of CPF. These observations suggest that HRW intake can protect rats from CPF-induced neurotoxicity, and the protective effects of hydrogen may be mediated by regulating the oxidant and antioxidant status of rats. Furthermore, this work defines a novel mechanism of biological activity of hydrogen by directly increasing the AChE activity.

The Screening and Mechanism of Influenza-Virus Sensitive MDCK Cell Lines for Influenza Vaccine Production.

Influenza is a potentially fatal acute respiratory viral disease caused by the influenza virus. Influenza viruses vary in antigenicity and spread rapidly, resulting in seasonal epidemics. Vaccination is the most effective strategy for lowering the incidence and fatality rates of influenza-related disorders, and it is also an important method for reducing seasonal influenza infections. Mammalian Madin-Darby canine kidney (MDCK) cell lines are recommended for influenza virus growth, and such cell lines have been utilized in several commercial influenza vaccine productions. The limit dilution approach was used to screen ATCC-MDCK cell line subcellular strains that are especially sensitive to H1N1, H3N2, BV, and BY influenza viruses to increase virus production, and research on influenza virus culture media was performed to support influenza virus vaccine development. We also used RNA sequencing to identify differentially expressed genes and a GSEA analysis to determine the biological mechanisms underlying the various levels of susceptibility of cells to influenza viruses. MDCK cell subline 2B6 can be cultured to increase titer and the production of the H1N1, H3N2, BV, and BY influenza viruses. MDCK-2B6 has a significantly enriched and activated in ECM receptor interaction, JAK-STAT signaling, and cytokine receptor interaction signaling pathways, which may result in increased cellular susceptibility and cell proliferation activity to influenza viruses, promote viral adsorption and replication, and elevate viral production, ultimately. The study revealed that MDCK-2B6 can increase the influenza virus titer and yield in vaccine production by increasing cell sensitivity and enhancing proliferative activity.

Targeting IL-17A enhances imatinib efficacy in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia.

Dysregulated hematopoietic niches remodeled by leukemia cells lead to imbalances in immunological mediators that support leukemogenesis and drug resistance. Targeting immune niches may ameliorate disease progression and tyrosine kinase inhibitor (TKI) resistance in Philadelphia chromosome-positive B-ALL (Ph+ B-ALL). Here, we show that T helper type 17 (Th17) cells and IL-17A expression are distinctively elevated in Ph+ B-ALL patients. IL-17A promotes the progression of Ph+ B-ALL. Mechanistically, IL-17A activates BCR-ABL, IL6/JAK/STAT3, and NF-kB signalling pathways in Ph+ B-ALL cells, resulting in robust cell proliferation and survival. In addition, IL-17A-activated Ph+ B-ALL cells secrete the chemokine CXCL16, which in turn promotes Th17 differentiation, attracts Th17 cells and forms a positive feedback loop supporting leukemia progression. These data demonstrate an involvement of Th17 cells in Ph+ B-ALL progression and suggest potential therapeutic options for Ph+ B-ALL with Th17-enriched niches.

Metabolic Responses and Pathway Changes of Vero Cells under High-Vitamin B Medium.

The production efficiency of a cell substrate directly affects the yield of target products such as viruses, while its density is mainly regulated by the type of culture medium and culture conditions. In this study, Vero cells were used as model cells for systematic medium screening, and a high-efficiency medium for biological drug production was identified. Through the results of cell proliferation by a cell counting kit (CCK)-8 assay, 5-Ethynyl-2'-deoxyuridine(EdU) assay, real-time quantitative PCR (RT-qPCR) and Western blotting, we found that adding an appropriate amount of vitamin B to the conventional basic medium can significantly improve and maintain the high-density growth of Vero cells. In addition, the molecular mechanism of the high-density culture of Vero cells promoted by B vitamins is explained for the first time by using the systems multi-omics analysis methods. Here, we determined that B vitamins regulate cell proliferation through the synthesis and metabolism of unsaturated fatty acids, affecting the productivity of cell substrate in industrial production. This study provides an important tool for the screening of key components of cell-based high-efficiency medium.

Alum/CpG Adjuvanted Inactivated COVID-19 Vaccine with Protective Efficacy against SARS-CoV-2 and Variants.

Since the beginning of the COVID-19 pandemic, numerous variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged, including five variants of concern (VOC) strains listed by the WHO: Alpha, Beta, Gamma, Delta and Omicron. Extensive studies have shown that most of these VOC strains, especially the currently dominant variant Omicron, can escape the host immune response induced by existing COVID-19 vaccines to different extents, which poses considerable risk to the health of human beings around the world. In the present study, we developed a vaccine based on inactivated SARS-CoV-2 and an adjuvant consisting of aluminum hydroxide (alum) and CpG. The immunogenicity and safety of the vaccine were investigated in rats. The candidate vaccine elicited high titers of SARS-CoV-2-spike-specific IgG antibody and neutralizing antibody in immunized rats, which not only neutralize the original SARS-CoV-2, but also showed great cross-neutralization activity against the Beta, Delta and Omicron variants.

Vaccination with Omicron Inactivated Vaccine in Pre-vaccinated Mice Protects against SARS-CoV-2 Prototype and Omicron Variants.

In response to the fast-waning immune response and the great threat of the Omicron variant of concern (VOC) to the public, we report the pilot-scale production of an inactivated Omicron vaccine candidate that induces high levels of neutralizing antibody titers to protect against the Omicron virus. Here, we demonstrate that the inactivated Omicron vaccine is safe and effective in recalling immune responses to the HB02, Omicron, and Delta viruses after one or two doses of BBIBP-CorV. In addition, the efficient productivity and good genetic stability of the manufactured inactivated vaccine is proved. These results support the further evaluation of the Omicron vaccine in a clinical trial.

Faciogenital Dysplasia 5 supports cancer stem cell traits in basal-like breast cancer by enhancing EGFR stability.

Most basal-like breast cancers (BLBCs) are triple-negative breast cancers (TNBCs), which have the worst prognosis and distant metastasis-free survival among breast cancer subtypes. Now, no targeted therapies are available for patients with BLBC due to the lack of reliable and effective molecular targets. Here, we performed the BLBC tissue microarray-based immunohistochemical analysis and showed that Faciogenital Dysplasia 5 (FGD5) abundance is associated with poor prognosis in BLBCs. FGD5 deletion decreased the proliferation, invasion, and tumorsphere formation capacity of BLBC cells. Furthermore, genetic inhibition of Fgd5 in mouse mammary epithelial cells attenuated BLBC initiation and progression by reducing the self-renewal ability of tumor-initiating cells. In addition, FGD5 abundance was positively correlated with the abundance of epidermal growth factor receptor (EGFR) in BLBCs. FGD5 ablation decreased EGFR abundance by reducing EGFR stability in TNBC cells in 2D and 3D culture conditions. Mechanistically, FGD5 binds to EGFR and interferes with basal EGFR ubiquitination and degradation induced by the E3 ligase ITCH. Impaired EGFR degradation caused BLBC cell proliferation and promoted invasive properties and self-renewal. To verify the role of the FGD5-EGFR interaction in the regulation of EGFR stability, we screened a cell-penetrating α-helical peptide PER3 binding with FGD5 to disrupt the interaction. Treatment of BLBC patient-derived xenograft-bearing mice with the peptide PER3 disrupting the FGD5-EGFR interaction either with or without chemotherapy reduced BLBC progression. Our study identified FGD5 as a positive modulator of tumor-initiating cells and suggests a potential therapeutic option for the BLBC subtype of breast cancer.

PML-RARα interaction with TRIB3 impedes PPARγ/RXR function and triggers dyslipidemia in acute promyelocytic leukemia.

Although dyslipidemia commonly occurs in patients with acute promyelocytic leukemia (APL) in response to anti-APL therapy, the underlying mechanism and the lipid statuses of patients with newly diagnosed APL remain to be addressed. Methods: We conducted a retrospective study to investigate the lipid profiles of APL patients. PML-RARα transgenic mice and APL cells-transplanted mice were used to assess the effects of APL cells on the blood/liver lipid levels. Subsequently, gene set enrichment analysis, western blot and dual luciferase reporter assay were performed to examine the role and mechanism of PML-RARα and TRIB3 in lipid metabolism regulation in APL patients at pretreatment and after induction therapy. Results: APL patients exhibited a higher prevalence of dyslipidemia before anti-APL therapy based on a retrospective study. Furthermore, APL cells caused secretion of triglycerides, cholesterol, and PCSK9 from hepatocytes and degradation of low-density lipoprotein receptors in hepatocytes, which elevated the lipid levels in APL cell-transplanted mice and Pml-Rarα transgenic mice. Mechanistically, pseudokinase TRIB3 interacted with PML-RARα to inhibit PPARγ activity by interfering with the interaction of PPARγ and RXR and promoting PPARγ degradation. Thus, reduced PPARγ activity in APL cells decreased leptin but increased resistin expression, causing lipid metabolism disorder in hepatocytes and subsequent dyslipidemia in mice. Although arsenic/ATRA therapy degraded PML-RARα and restored PPARγ expression, it exacerbated dyslipidemia in APL patients. The elevated TRIB3 expression in response to arsenic/ATRA therapy suppressed PPARγ activity by disrupting the PPARγ/RXR dimer, which resulted in dyslipidemia in APL patients undergoing therapy. Indeed, the PPAR activator not only enhanced the anti-APL effects of arsenic/ATRA by suppressing TRIB3 expression but also reduced therapy-induced dyslipidemia in APL patients. Conclusion: Our work reveals the critical role of the PML-RARα/PPARγ/TRIB3 axis in the development of dyslipidemia in APL patients, potentially conferring a rationale for combining ATRA/arsenic with the PPAR activator for APL treatment.

TRIB3 promotes MYC-associated lymphoma development through suppression of UBE3B-mediated MYC degradation.

The transcription factor MYC is deregulated in almost all human cancers, especially in aggressive lymphomas, through chromosomal translocation, amplification, and transcription hyperactivation. Here, we report that high expression of tribbles homologue 3 (TRIB3) positively correlates with elevated MYC expression in lymphoma specimens; TRIB3 deletion attenuates the initiation and progression of MYC-driven lymphoma by reducing MYC expression. Mechanistically, TRIB3 interacts with MYC to suppress E3 ubiquitin ligase UBE3B-mediated MYC ubiquitination and degradation, which enhances MYC transcriptional activity, causing high proliferation and self-renewal of lymphoma cells. Use of a peptide to disturb the TRIB3-MYC interaction together with doxorubicin reduces the tumor burden in MycEµ mice and patient-derived xenografts. The pathophysiological relevance of UBE3B, TRIB3 and MYC is further demonstrated in human lymphoma. Our study highlights a key mechanism for controlling MYC expression and a potential therapeutic option for treating lymphomas with high TRIB3-MYC expression.

Disrupting the TRIB3-SQSTM1 interaction reduces liver fibrosis by restoring autophagy and suppressing exosome-mediated HSC activation.

Impaired macroautophagy/autophagy is involved in the pathogenesis of hepatic fibrosis. However, how aberrant autophagy promotes fibrosis is far from understood. Here, we aimed to define a previously unrevealed pro-fibrotic mechanism for the stress protein TRIB3 (tribbles pseudokinase 3)-mediated autophagy dysfunction. Human fibrotic liver tissues were obtained from patients with cirrhosis who underwent an open surgical repair process. The functional implications of TRIB3 were evaluated in mouse models of hepatic fibrosis induced by bile duct ligation (BDL) or thioacetamide (TAA) injection. Human fibrotic liver tissues expressed higher levels of TRIB3 and selective autophagic receptor SQSTM1/p62 (sequestosome 1) than nonfibrotic tissues and the elevated expression of TRIB3 and SQSTM1 was positively correlated in the fibrotic tissues. Silencing Trib3 protected against experimentally induced hepatic fibrosis, accompanied by restored autophagy activity in fibrotic liver tissues. Furthermore, TRIB3 interacted with SQSTM1 and hindered its binding to MAP1LC3/LC3, which caused the accumulation of SQSTM1 aggregates and obstructed autophagic flux. The TRIB3-mediated autophagy impairment not only suppressed autophagic degradation of late endosomes but also promoted hepatocellular secretion of INHBA/Activin A-enriched exosomes which caused migration, proliferation and activation of hepatic stellate cells (HSCs), the effector cells of liver fibrosis. Disrupting the TRIB3-SQSTM1 interaction with a specific helical peptide exerted potent protective effects against hepatic fibrosis by restoring autophagic flux in hepatocytes and HSCs. Together, stress-elevated TRIB3 expression promotes hepatic fibrosis by interacting with SQSTM1 and interfering with its functions in liver-parenchymal cells and activating HSCs. Targeting this interaction is a promising strategy for treating fibroproliferative liver diseases.Abbreviations: 3-MA: 3-methyladenine; AAV: adeno-associated virus; ACTA2/α-SMA: actin, alpha 2, smooth muscle, aorta; BDL: bile duct ligation; BECN1/Beclin 1: beclin 1, autophagy related; CHX: cycloheximide; CQ: chloroquine; Edu: 5-ethynyl-2-deoxyuridine; ESCRT: endosomal sorting complexes required for transport; HSC: hepatic stellate cell; ILV: intralumenal vesicle; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MVB: multivesicular body; PIK3C3: phosphatidylinositol 3-kinase, catalytic subunit type 3; PPI: protein-protein interaction; SQSTM1/p62: sequestosome 1; TAA: thioacetamide; TEM: transmission electron microscopy; TGFB1/TGFß1: transforming growth factor, beta 1; TLR2: toll-like receptor 2; TRIB3: tribbles pseudokinase 3.

Improvement in affinity and thermostability of a fully human antibody against interleukin-17A by yeast-display technology and CDR grafting.

Monoclonal antibodies (mAbs) are widely used in many fields due to their high specificity and ability to recognize a broad range of antigens. IL-17A can induce a rapid inflammatory response both alone and synergistically with other proinflammatory cytokines. Accumulating evidence suggests that therapeutic intervention of IL-17A signaling offers an attractive treatment option for autoimmune diseases and cancer. Here, we present a combinatorial approach for optimizing the affinity and thermostability of a novel anti-hIL-17A antibody. From a large naïve phage-displayed library, we isolated the anti-IL-17A mAb 7H9 that can neutralize the effects of recombinant human IL-17A. However, the modest neutralization potency and poor thermostability limit its therapeutic applications. In vitro affinity optimization was then used to generate 8D3 by using yeast-displayed random mutagenesis libraries. This resulted in four key amino acid changes and provided an approximately 15-fold potency increase in a cell-based neutralization assay. Complementarity-determining regions (CDRs) of 8D3 were further grafted onto the stable framework of the huFv 4D5 to improve thermostability. The resulting hybrid antibody 9NT/S has superior stabilization and affinities beyond its original antibody. Human fibrosarcoma cell-based assays and in vivo analyses in mice indicated that the anti-IL-17A antibody 9NT/S efficiently inhibited the secretion of IL-17A-induced proinflammatory cytokines. Therefore, this lead anti-IL-17A mAb might be used as a potential best-in-class candidate for treating IL-17A related diseases.

TRIB3 supports breast cancer stemness by suppressing FOXO1 degradation and enhancing SOX2 transcription.

The existence of breast cancer stem cells (BCSCs) is a major reason underlying cancer metastasis and recurrence after chemotherapy and radiotherapy. Targeting BCSCs may ameliorate breast cancer relapse and therapy resistance. Here we report that expression of the pseudokinase Tribble 3 (TRIB3) positively associates with breast cancer stemness and progression. Elevated TRIB3 expression supports BCSCs by interacting with AKT to interfere with the FOXO1-AKT interaction and suppress FOXO1 phosphorylation, ubiquitination, and degradation by E3 ligases SKP2 and NEDD4L. The accumulated FOXO1 promotes transcriptional expression of SOX2, a transcriptional factor for cancer stemness, which in turn, activates FOXO1 transcription and forms a positive regulatory loop. Disturbing the TRIB3-AKT interaction suppresses BCSCs by accelerating FOXO1 degradation and reducing SOX2 expression in mouse models of breast cancer. Our study provides insights into breast cancer development and confers a potential therapeutic strategy against TRIB3-overexpressed breast cancer.

Metformin suppresses melanoma progression by inhibiting KAT5-mediated SMAD3 acetylation, transcriptional activity and TRIB3 expression.

Metformin has beneficial effects of preventing and treating cancers on type 2 diabetic patients. However, the role of metformin in non-diabetic cancer patients and the precise molecular mechanisms against cancer have not yet been sufficiently elucidated. We recently reported that the pseudokinase protein TRIB3 acts as a stress sensor linking metabolic stressors to cancer promotion by inhibiting autophagy and ubiquitin-proteasomal degradation systems; genetically abrogating of TRIB3 expression reduces tumourigenesis and cancer progression. Thus, TRIB3 is a potential therapeutic target for diverse cancers. In this study, we found that metformin attenuates melanoma growth and metastasis by reducing TRIB3 expression in non-diabetic C57BL/6 mice and diabetic KK-Ay mice; overexpression of TRIB3 protects metformin from the activation of autophagic flux, the clearance of accumulated tumour-promoting factors and the attenuation of tumour progression. We further elucidated that TRIB3 acts as an adaptor to recruit lysine acetyltransferase 5 (KAT5) to SMAD3 and induce a phosphorylation-dependent K333 acetylation of SMAD3, which sustains transcriptional activity of SMAD3 and subsequently enhances TRIB3 transcription. Metformin suppresses SMAD3 phosphorylation and decreases the KAT5/SMAD3 interaction, to attenuate the KAT5-mediated K333 acetylation of SMAD3, reduce the SMAD3 transcriptional activity and subsequent TRIB3 expression, thereby antagonizes melanoma progression. Together, our study not only defines a molecular mechanism by which metformin protects against melanoma progression by disturbing the KAT5/TRIB3/SMAD3 positive feedback loop in diabetes and non-diabetes mice, but also suggests a candidate diverse utility of metformin in tumour prevention and therapy because of suppressing stress protein TRIB3 expression.