Hemin regulates platelet clearance in hemolytic disease by binding to GPIbα.

Hemolysis is associated with thrombosis and vascular dysfunction, which are the pathological components of many diseases. Hemolytic products, including hemoglobin and hemin, activate platelets (PLT). Despite its activation, the effect of hemolysis on platelet clearance remains unclear, It is critical to maintain a normal platelet count and ensure that circulating platelets are functionally viable. In this study, we used hemin, a degradation product of hemoglobin, as a potent agonist to treat platelets and simulate changes in vivo in mice. Hemin treatment induced activation and morphological changes in platelets, including an increase in intracellular Ca2+ levels, phosphatidylserine (PS) exposure, and cytoskeletal rearrangement. Fewer hemin-treated platelets were cleared by macrophages in the liver after transfusion than untreated platelets. Hemin bound to glycoprotein Ibα (GPIbα), the surface receptor in hemin-induced platelet activation and aggregation. Furthermore, hemin decreased GPIbα desialylation, as evidenced by reduced Ricinus communis agglutinin I (RCA- I) binding, which likely extended the lifetime of such platelets in vivo. These data provided new insight into the mechanisms of GPIbα-mediated platelet activation and clearance in hemolytic disease.

What is the context? Hemolysis is a primary hematological disease. Hemolysis is a pathological complication of several diseases.Hemin, a degradation product of cell-free hemoglobin, has been proven to be a more potent agonist than hemoglobin for directly activating platelets.Platelet membrane glycoproteins (GP), including GPIb-IX and GPIIb/IIIa complexes, play crucial roles in platelet hemostasis.Desialylation (loss of sialic acid residues) of GPIbα, is believed to regulate physiological platelet clearance through liver macrophages and hepatocytes.What is new? In this study, we evaluated the effects of hemolysis on platelet clearance. We first analyzed the influence of hemin at 0-50 µM on platelets in vitro before exploring the mechanism underlying hemin-induced platelet activation and its role in platelet clearance in vitro and in vivo.Our analyses suggest that: Hemin bound to GPIbα on the platelet surface with high affinity.Platelet clearance occurred slowly in the liver and spleen after hemin treatment.Platelets exhibited significant significantly reduced GPIbα surface expression and desialylation after hemin treatment.Platelets exhibited significant significantly reduced GPIbα surface expression and desialylation after hemin treatment.What is the impact? This study provides new insights into the role of hemin in the mechanisms of GPIbα-mediated platelets activation and clearance in diseases associated with hemolysis.

CTRP4/interleukin-6 receptor signaling ameliorates autoimmune encephalomyelitis by suppressing Th17 cell differentiation.

C1q/TNF-related protein 4 (CTRP4) is generally thought to be released extracellularly and plays a critical role in energy metabolism and protecting against sepsis. However, its physiological functions in autoimmune diseases have not been thoroughly explored. In this study, we demonstrate that Th17 cell-associated experimental autoimmune encephalomyelitis was greatly exacerbated in Ctrp4-/- mice compared with WT mice due to increased Th17 cell infiltration. The absence of Ctrp4 promoted the differentiation of naive CD4+ T cells into Th17 cells in vitro. Mechanistically, CTRP4 interfered with the interaction between IL-6 and the IL-6 receptor (IL-6R) by directly competing to bind with IL-6R, leading to suppression of IL-6-induced activation of the STAT3 pathway. Furthermore, the administration of recombinant CTRP4 protein ameliorated disease symptoms. In conclusion, our results indicate that CTRP4, as an endogenous regulator of the IL-6 receptor-signaling pathway, may be a potential therapeutic intervention for Th17-driven autoimmune diseases.

Laponite Lights Calcium Flickers by Reprogramming Lysosomes to Steer DC Migration for An Effective Antiviral CD8+ T-Cell Response.

Immunotherapy using dendritic cell (DC)-based vaccination is an established approach for treating cancer and infectious diseases; however, its efficacy is limited. Therefore, targeting the restricted migratory capacity of the DCs may enhance their therapeutic efficacy. In this study, the effect of laponite (Lap) on DCs, which can be internalized into lysosomes and induce cytoskeletal reorganization via the lysosomal reprogramming-calcium flicker axis, is evaluated, and it is found that Lap dramatically improves the in vivo homing ability of these DCs to lymphoid tissues. In addition, Lap improves antigen cross-presentation by DCs and increases DC-T-cell synapse formation, resulting in enhanced antigen-specific CD8+ T-cell activation. Furthermore, a Lap-modified cocktail (Lap@cytokine cocktail [C-C]) is constructed based on the gold standard, C-C, as an adjuvant for DC vaccines. Lap@C-C-adjuvanted DCs initiated a robust cytotoxic T-cell immune response against hepatitis B infection, resulting in > 99.6% clearance of viral DNA and successful hepatitis B surface antigen seroconversion. These findings highlight the potential value of Lap as a DC vaccine adjuvant that can regulate DC homing, and provide a basis for the development of effective DC vaccines.

A possible strategy for generating polymer chains with an entanglement-free structure.

We propose a possible strategy that may experimentally generate long polymeric chains with an entanglement-free structure. The basic idea is designing the conditions to restrict polymer chains from growing along the surface with an obviously concave curvature. This strategy is proved to effectively reduce the chance of forming both inter- and intra-molecular entanglements, which is quite similar to the self-avoiding random walking of chains on a two dimensional plane. We believe that this kind of chain growth strategy may supply a kind of possible explanation on the formation of the entanglement-free structure of chromosomes, which also have tremendously large molecular weight. Besides, this study also guides experimentalists on synthesizing specific entanglement-free functional polymeric or biological materials.

FAM96A suppresses epithelial-mesenchymal transition and tumor metastasis by inhibiting TGFß1 signals.

Metastasis is the main cause of death in 90% of patients with tumors, and epithelial-mesenchymal transition (EMT) plays a key role in this process. Family with sequence similarity 96 member A (FAM96A) is an evolutionarily conserved protein related to cytosolic iron assembly. However, no research has been conducted on its role in tumor metastasis. Bioinformatics analysis with Kaplan-Meier analysis showed that patients with lower FAM96A expression in different tumors had shorter survival times and poorer prognoses. Here, we identified FAM96A as a crucial regulator of the TGFß signaling pathway because it inhibits TGFß-mediated tumor metastasis and EMT. FAM96A did not affect the proliferation or apoptosis of tumor cells but significantly reduced their migration and invasion abilities in vitro. The colonization and metastatic abilities of FAM96A-knockout tumor cells were significantly enhanced in vivo. Furthermore, the overexpression of exogenous FAM96A inhibited TGFß-induced EMT through the SMAD-mediated pathway and downregulated the expression of endogenous transforming growth factor-ß1 (TGFß1). These findings demonstrated a correlation between EMT and FAM96A gene expression for the first time, which is highly important for revealing the mechanism underlying tumor metastasis.

PTPIP51 inhibits non-small-cell lung cancer by promoting PTEN-mediated EGFR degradation.

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) interacts with two non-receptor tyrosine phosphatases and induces apoptosis. In the present study, we showed that PTPIP51 is downregulated in non-small cell lung cancer (NSCLC), and its elevated expression correlates with improved outcomes. PTPIP51 overexpression in NSCLC cells significantly inhibits downstream epidermal growth factor receptor (EGFR) signaling in PI3K/Akt, RAS/RAF/ERK, and JAK/STAT3 pathways. The efficacy of the EGFR inhibitor gefitinib improves in combination with PTPIP51 to accelerate apoptosis and inhibit NSCLC growth in vivo and in vitro. Here, we demonstrated that PTPIP51 interacts with phosphatase and tensin homolog (PTEN) to form a PTPIP51-PTEN-CK2 complex, which induces phosphorylation of the C-tail region of PTEN (p-PTEN Thr382 and Thr383). This subsequently induces ubiquitylation of EGFR and its degradation via lysosomes. Therefore, PTPIP51 acts as a tumor suppressor in NSCLC by inducing PTEN phosphorylation and by promoting EGFR degradation.

Establishment of a Novel Mouse Hepatocellular Carcinoma Model for Dynamic Monitoring of Tumor Development by Bioluminescence Imaging.

In this study, a novel mouse model of hepatocellular carcinoma (HCC) was established by simultaneously knocking out Pten and p53 suppressor genes and overexpressing c-Met and △90-ß-catenin proto-oncogenes in the livers of mice via hydrodynamic injection (HDI). The mutations were introduced using the CRISPR/Cas9 and Sleeping Beauty transposon systems. In this way, a primary liver cancer model was established within six weeks. In addition, macrophages expressing arginase-1(Arg1) promoter coupled with firefly luciferase were engineered for bioluminescence imaging (BLI) of the tumor microenvironment. This novel, rapidly-generated model of primary hepatocellular carcinoma can be monitored noninvasively, which can facilitate not only applications of the model, but also the development of new drugs and treatment strategies of HCC.

CTRP4 acts as an anti-inflammatory factor in macrophages and protects against endotoxic shock.

Despite the availability of antibiotics, current therapies to treat sepsis are still ineffective and many clinical trials aimed at neutralizing specific inflammatory cytokines have failed, suggesting the urgent need for new treatments. Using two models of LPS-induced endotoxemia and cecal ligation and puncture (CLP)-induced sepsis, we investigated the effects of C1q/TNF-related protein 4(CTRP4) on septic lethality and sepsis-induced inflammation. The effects of CTRP4 on survival, inflammation, organ damage, and bacterial clearance were assessed. Here, we found that CTRP4 decreased the mortalities of mice and alleviated pathological lung injury in mice model. In vivo depletion and adoptive transfer studies showed CTRP4-expressing macrophages as the key cell type inhibiting LPS-induced septic shock. The mechanism associated with the CTRP4 deficiency involved promoting of TLR4 internalization and activation of downstream pathways that resulted in a lethal, prolonged proinflammatory cytokine storm. Treatment of macrophages with exogenous CTRP4 abrogated proinflammatory cytokine production. Our results showed CTRP4 regulates inflammatory response and could be a promising strategy to treat septic shock.

FAM96A Protects Mice From Dextran Sulfate Sodium (DSS)-Induced Colitis by Preventing Microbial Dysbiosis.

Family with sequence similarity 96 member A (FAM96A) is an evolutionarily conserved intracellular protein that is involved in the maturation of the Fe/S protein, iron regulatory protein 1 (IRP1), and the mitochondria-related apoptosis of gastrointestinal stromal tumor cells. In this study, we used a mouse model of chemically induced colitis to investigate the physiological role of FAM96A in intestinal homeostasis and inflammation. At baseline, colons from Fam96a-/- mice exhibited microbial dysbiosis, dysregulated epithelial cell turnover, an increased number of goblet cells, and disordered tight junctions with functional deficits affecting intestinal permeability. After cohousing, the differences between wild-type and Fam96a-/- colons were abrogated, suggesting that FAM96A affects colonic epithelial cells in a microbiota-dependent manner. Fam96a deficiency in mice resulted in increased susceptibility to dextran sulfate sodium (DSS)-induced colitis. Importantly, the colitogenic activity of Fam96a-/- intestinal microbiota was transferable to wild-type littermate mice via fecal microbial transplantation (FMT), leading to exacerbation of DSS-induced colitis. Taken together, our data indicate that FAM96A helps to maintain colonic homeostasis and protect against DSS-induced colitis by preventing gut microbial dysbiosis. This study used gene knockout animals to help to understand the in vivo effects of the Fam96a gene for the first time and provides new evidence regarding host-microbiota interactions.

In vitro study of FUZ as a novel potential therapeutic target in non-small-cell lung cancer.

FUZ is regarded as a planar cell polarity effector that controls multiple cellular processes during vertebrate development. However, the role of FUZ in tumor biology remains poorly studied. Our purpose of this study is to discover the physiological effects and mechanism of FUZ in non-small-cell lung cancer (NSCLC) in vitro. With the help of bioinformatics analysis, we noticed that the expression level of FUZ negatively correlates with prognosis of NSCLC patients. Exogenous FUZ expression markedly promoted cell proliferation of NSCLC cells. The phosphorylation of Erk1/2, STAT3 and related signaling molecules were induced activated after FUZ over-expression. FUZ also plays an important role in cell motility by regulating cell signaling pathways and inducing epithelial to mesenchymal transition (EMT). FUZ promotes EMT along with the up-regulation of N-cadherin, vimentin, Zeb1, Twist1 and decreased level of E-cadherin. Furthermore, we also carried out FUZ directed siRNA treatments to prove the above observations. Knockdown of FUZ resulted in delayed cell growth as well as impaired cell migration and reversed EMT phonotype. Importantly, we reported for the first time that FUZ is a BNIP3-interacting protein. Loss of FUZ resulted in decreased BNIP3 protein level, but no influence on BNIP3 mRNA level, suggesting weakened stability of BNIP3 protein. Overall, our results in vitro show that FUZ is responsible for NSCLC progression and metastasis, suggesting that FUZ can be a potential therapeutic target for NSCLC.

TMEM74 promotes tumor cell survival by inducing autophagy via interactions with ATG16L1 and ATG9A.

Autophagy is a highly inducible system of intracellular degradation that occurs in lysosomes or vacuoles. Transmembrane 74 (TMEM74) has been shown to induce autophagy. However, the mechanism by which TMEM74 stimulates autophagy and the impacts of TMEM74-induced autophagy on tumor cell survival remain unclear. In this study, TMEM74 was shown to increase the autophagic flux process in different tumor cell lines. Further investigations revealed that TMEM74 interacts with ATG16L1 and ATG9A. Moreover, distinctive from the common autophagy models, it is found that TMEM74-related autophagy is independent of BECN1/PI3KC3 complex and ULK1, and TMEM74 may initiate and promote autophagy directly via interactions with ATG16L1 and ATG9A responsible for the nucleation and elongation respectively. Considering the ultimate outcome of TMEM74-induced autophagy in tumor cells, TMEM74-triggered autophagy induces a pro-survival effect on tumor cells, particularly cells under metabolic stress, consistent with alteration of a series of signal pathways. Intriguingly, TMEM74 itself can be downregulated through the autophagic process, which indicates that a potential self-regulatory loop exists so as to maintain an appropriate level of autophagy, avoiding excessive autophagy to commit tumor cells to death. According to the clinical database analysis, the high expression of TMEM74 significantly shortens the surviving periods of patients in several specific cancers indicating that TMEM74 itself can be treated as an effective potential target with clinical values to prolong surviving periods of cancer patients in the future. In conclusion, our study reveals a new mechanism by which autophagy is stimulated by a novel positive modulator through a unique pathway and demonstrates a novel connection between autophagy and cell survival, which undoubtedly serves to broaden our understanding of autophagy.

SZRD1 is a Novel Protein that Functions as a Potential Tumor Suppressor in Cervical Cancer.

SZRD1 is a novel gene screened out by high-throughput platform, and so far there exists no systematic function reports. The purpose of our study is to discover the function and mechanism of this novel human gene. Bioinformatics analysis indicates that SZRD1 is a highly conserved intracellular protein. After overexpression of SZRD1, we found that SZRD1 could arrest the cell cycle in G2 phase and play a role in inhibiting cell proliferation and inducing apoptosis. In contrast, after knockdown of endogenous SZRD1, we concluded that it could promote cell proliferation. The mechanism investigations showed that overexpression of SZRD1 could downregulate the phosphorylation of ERK1/2, AKT, STAT3 and downstream signaling molecules, and then arrest the cells in G2 phase by upregulating P21. Tissue microarray analysis showed that the expression of SZRD1 was downregulated in cervical squamous cell carcinomas compared with normal squamous epithelium, and the ratio of downregulation correlated with the stage of the cancer. Overall, we clarified the function of this novel protein SZRD1, which indicated it may be a potential novel tumor suppressor in cervical cancer.

Autophagy regulatory molecule, TMEM74, interacts with BIK and inhibits BIK-induced apoptosis.

TMEM74 (Transmembrane protein 74), a lysosome transmembrane protein, induces cell autophagy. Knockdown of TMEM74 abolished EBSS-induced autophagy. BIK, belonging to BOP (BH3-only protein) protein family, has been reported to induce cell apoptosis. Autophagy and apoptosis, as different pathways regulated by extra- or intra-cellular signals precisely, both play a crucial role in processes of intra-cellular substrates degradation, energy metabolism and cell survival. However, the relationship between autophagy and apoptosis still remains elusive. To elucidate the putative new relationship and further identify the function of TMEM74, we performed the study mainly using co-immunoprecipitation, immunoblotting, fluorescent location and basic cell biologic experimental techniques. In the present study, for the first time, it is demonstrated that autophagy-related protein TMEM74 co-localizes with apoptosis-related protein BIK in subcellular organelles. The data indicated that TMEM74 associates with BIK via TM domains of TMEM74 and BH3 domain of BIK. Further investigations revealed that TMEM74 inhibits BIK-induced apoptosis by interacting with BIK, as evidenced by the results that autophagosome formation inhibitor could not block the inhibition effect completely. On the contrary, knockdown of TMEM74 and the TM domain-deficient mutant led to deprivation of the function. Overall, the results revealed the autophagy modulator TMEM74 interrelates with apoptosis inducer BIK and inhibits its function, which provides a novel crosstalk point between autophagy and apoptosis to enlarge our understanding of the programmed cell death.