Hyperosmotic Stress Induces Inflammation and Excessive Th17 Response to Blunt T-Cell Immunity in Tilapia.

Despite the advances in study on osmotic physiology in bony fish, the mechanism by which the immune system, especially T-cell immunity, adapts and responds to osmotic stress remains unknown. In the current study, we investigated the response of T cells to hyperosmotic stress in the bony fish Nile tilapia (Oreochromis niloticus). As a euryhaline fish, tilapia was able to adapt to a wide range of salinities; however, hypertonic stress caused inflammation and excessive T-cell activation. Furthermore, hypertonic stress increased the expression of IL-17A in T cells, upregulated the transcription factor RORα, and activated STAT3 signaling, along with IL-6- and TGF-ß1-mediated pathways, revealing an enhanced Th17 response in this early vertebrate. These hypertonic stress-induced events collectively resulted in an impaired antibacterial immune response in tilapia. Hypertonic stress elevated the intracellular ROS level, which in turn activated the p38-MK2 signaling pathway to promote IL-17A production by T cells. Both ROS elimination and the p38-MK2 axis blockade diminished the increased IL-17A production in T cells under hypertonic conditions. Moreover, the produced proinflammatory cytokines further amplified the hypertonic stress signaling via the MKK6-p38-MK2 axis-mediated positive feedback loop. To our knowledge, these findings represent the first description of the mechanism by which T-cell immunity responds to hypertonic stress in early vertebrates, thus providing a novel perspective for understanding the adaptive evolution of T cells under environmental stress.

Fish Uses CTLA-4 Immune Checkpoint to Suppress mTORC1-Controlled T-Cell Glycolysis and Immunity.

As an immune checkpoint, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) suppresses the activation, proliferation, and effector function of T cells, thus preventing an overexuberant response and maintaining immune homeostasis. However, whether and how this immune checkpoint functions in early vertebrates remains unknown. In the current study, using a Nile tilapia (Oreochromis niloticus) model, we investigated the suppression of T cell response by CTLA-4 in bony fish. Tilapia CTLA-4 is constitutively expressed in lymphoid tissues, and its mRNA and protein expression in lymphocytes are upregulated following PHA stimulation or Edwardsiella piscicida infection. Blockade of CTLA-4 signaling enhanced T cell activation and proliferation but inhibited activation-induced T cell apoptosis, indicating that CTLA-4 negatively regulated T cell activation. In addition, blocking CTLA-4 signaling in vivo increased the differentiation potential and cytotoxicity of T cells, resulting in an enhanced T cell response during E. piscicida infection. Tilapia CTLA-4 competitively bound the B7.2/CD86 molecule with CD28, thus antagonizing the CD28-mediated costimulatory signal of T cell activation. Furthermore, inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling, c-Myc, or glycolysis markedly impaired the CTLA-4 blockade-enhanced T cell response, suggesting that CTLA-4 suppressed the T cell response of tilapia by inhibiting mTORC1/c-Myc axis-controlled glycolysis. Overall, the findings indicate a detailed mechanism by which CTLA-4 suppresses T cell immunity in tilapia; therefore, we propose that early vertebrates have evolved sophisticated mechanisms coupling immune checkpoints and metabolic reprogramming to avoid an overexuberant T cell response.

IL-10 Negatively Controls the Primary T Cell Response of Tilapia by Triggering the JAK1/STAT3/SOCS3 Axis That Suppresses NF-κB and MAPK/ERK Signaling.

The braking mechanisms to protect the host from tissue damage and inflammatory disease caused by an overexuberant immune response are common in many T cell subsets. However, the negative regulation of T cell responses and detailed mechanisms are not well understood in early vertebrates. In the current study, using a Nile tilapia (Oreochromis niloticus) model, we investigated the suppression of T cell immunity by IL-10. Tilapia encodes an evolutionarily conserved IL-10, whose expression in lymphocytes is markedly induced during the primary adaptive immune response against Aeromonas hydrophila infection. Activated T cells of tilapia produce IL-10, which in turn inhibits proinflammatory cytokine expression and suppresses PHA-induced T cell activation. Moreover, administration of IL-10 impairs the proliferation of tilapia T cells, reduces their potential to differentiate into Th subsets, and cripples the cytotoxic function, rendering the animals more vulnerable to pathogen attack. After binding to its receptor IL-10Ra, IL-10 activates the JAK1/STAT3 axis by phosphorylation and enhances the expression of the suppressor of cytokine signaling 3 (SOCS3), which in turn attenuates the activation of the NF-κB and MAPK/ERK signaling pathways, thus suppressing the T cell response of tilapia. Our findings elucidate a negative regulatory mechanism of T cell immunity in a fish species and support the notion that the braking mechanism of T cells executed through IL-10 existed prior to the divergence of the tetrapod lineage from teleosts. Therefore, this study, to our knowledge, provides a novel perspective on the evolution of the adaptive immune system.

Nonreciprocal magnon blockade based on nonlinear effects.

We present an alternative scheme to achieve nonreciprocal unconventional magnon blockade (NUMB) in a hybrid system formed by two microwave cavities and one yttrium iron garnet (YIG) sphere, where the pump and signal cavities interact nonlinearly with each other and the signal cavity is coupled to the YIG sphere. It is found that the nonlinear coupling occurs between the pump cavity and magnon modes due to the dispersive interactions among three bosonic modes. Meanwhile, the Kerr nonlinearity is present in the pump cavity. Based on these nonlinear effects, a nonreciprocal magnon blockade could be achieved with the help of the weak parametric driving of the pump cavity. The present work provides an alternative method to prepare single magnon resource, which may be helpful for quantum information processing.

The TAK1/JNK axis participates in adaptive immunity by promoting lymphocyte activation in Nile tilapia.

The transforming growth factor beta-activated kinase 1 (TAK1) / c-Jun N-terminal kinase (JNK) axis is an essential MAPK upstream mediator and regulates immune signaling pathways. However, whether the TAK1/JNK axis harnesses the strength in regulation of signal transduction in early vertebrate adaptive immunity is unclear. In this study, by modeling on Nile tilapia (Oreochromis niloticus), we investigated the potential regulatory function of TAK1/JNK axis on lymphocyte-mediated adaptive immune response. Both OnTAK1 and OnJNK exhibited highly conserved sequences and structures relative to their counterparts in other vertebrates. Their mRNA was widely expressed in the immune-associated tissues, while phosphorylation levels in splenic lymphocytes were significantly enhanced on the 4th day post-infection by Edwardsiella piscicida. In addition, OnTAK1 and OnJNK were significantly up-regulated in transcriptional level after activation of lymphocytes in vitro by phorbol 12-myristate 13-acetate plus ionomycin (P+I) or PHA, accompanied by a predominant increase in phosphorylation level. More importantly, inhibition of OnTAK1 activity by specific inhibitor NG25 led to a significant decrease in the phosphorylation level of OnJNK. Furthermore, blocking the activity of OnJNK with specific inhibitor SP600125 resulted in a marked reduction in the expression of T-cell activation markers including IFN-γ, CD122, IL-2, and CD44 during PHA-induced T-cell activation. In summary, these findings indicated that the conserved TAK1/JNK axis in Nile tilapia was involved in adaptive immune responses by regulating the activation of lymphocytes. This study enriched the current knowledge of adaptive immunity in teleost and provided a new perspective for understanding the regulatory mechanism of fish immunity.

Inducible IL-2 production and IL-2+ cell expansion are landmark events for T-cell activation of teleost.

As a multipotent cytokine, interleukin (IL)-2 plays important roles in activation, differentiation and survival of the lymphocytes. Although biological characteristics and function of IL-2 have been clarified in several teleost species, evidence regarding IL-2 production at the cellular and protein levels is still scarce in fish due to the lack of reliable antibody. In this study, we developed a mouse anti-Nile tilapia IL-2 monoclonal antibody (mAb), which could specifically recognize IL-2 protein and identify IL-2-producing lymphocytes of tilapia. Using this mAb, we found that CD3+ T cells, but not CD3- lymphocytes, are the main cellular source of IL-2 in tilapia. Under resting condition, both CD3+CD4-1+ T cells and CD3+CD4-1- T cells of tilapia produce IL-2. Moreover, the IL-2 protein level and the frequency of IL-2+ T cells significantly increased once T cells were activated by phytohemagglutinin (PHA) or CD3 plus CD28 mAbs in vitro. In addition, Edwardsiella piscicida infection also induces the IL-2 production and the expansion of IL-2+ T cells in the spleen lymphocytes. These findings demonstrate that IL-2 takes part in the T-cell activation and anti-bacterial adaptive immune response of tilapia, and can serve as an important marker for T-cell activation of teleost fish. Our study has enriched the knowledge regarding T-cell response in fish species, and also provide novel perspective for understanding the evolution of adaptive immune system.

Dietary restriction to optimize T cell immunity is an ancient survival strategy conserved in vertebrate evolution.

Recent advances highlight a key role of transient fasting in optimizing immunity of human and mouse. However, it remains unknown whether this strategy is independently acquired by mammals during evolution or instead represents gradually evolved functions common to vertebrates. Using a tilapia model, we report that T cells are the main executors of the response of the immune system to fasting and that dietary restriction bidirectionally modulates T cell immunity. Long-term fasting impaired T cell immunity by inducing intense autophagy, apoptosis, and aberrant inflammation. However, transient dietary restriction triggered moderate autophagy to optimize T cell response by maintaining homeostasis, alleviating inflammation and tissue damage, as well as enhancing T cell activation, proliferation and function. Furthermore, AMPK is the central hub linking fasting and autophagy-controlled T cell immunity in tilapia. Our findings demonstrate that dietary restriction to optimize immunity is an ancient strategy conserved in vertebrate evolution, providing novel perspectives for understanding the adaptive evolution of T cell response.

Porcine Epidemic Diarrhea Virus nsp7 Inhibits Interferon-Induced JAK-STAT Signaling through Sequestering the Interaction between KPNA1 and STAT1.

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus that causes high mortality in piglets. Interferon (IFN) responses are the primary defense mechanism against viral infection; however, viruses always evolve elaborate strategies to antagonize the antiviral action of IFN. Previous study showed that PEDV nonstructural protein 7 (nsp7), a component of the viral replicase polyprotein, can antagonize ploy(I:C)-induced type I IFN production. Here, we found that PEDV nsp7 also antagonized IFN-α-induced JAK-STAT signaling and the production of IFN-stimulated genes. PEDV nsp7 did not affect the protein and phosphorylation levels of JAK1, Tyk2, STAT1, and STAT2 or the formation of the interferon-stimulated gene factor 3 (ISGF3) complex. However, PEDV nsp7 prevented the nuclear translocation of STAT1 and STAT2. Mechanistically, PEDV nsp7 interacted with the DNA binding domain of STAT1/STAT2, which sequestered the interaction between karyopherin α1 (KPNA1) and STAT1, thereby blocking the nuclear transport of ISGF3. Collectively, these data reveal a new mechanism developed by PEDV to inhibit type I IFN signaling pathway. IMPORTANCE In recent years, an emerging porcine epidemic diarrhea virus (PEDV) variant has gained attention because of serious outbreaks of piglet diarrhea in China and the United States. Coronavirus nonstructural protein 7 (nsp7) has been proposed to act with nsp8 as part of an RNA primase to generate RNA primers for viral RNA synthesis. However, accumulating evidence indicates that coronavirus nsp7 can also antagonize type I IFN production. Our present study extends previous findings and demonstrates that PEDV nsp7 also antagonizes IFN-α-induced IFN signaling by competing with KPNA1 for binding to STAT1, thereby enriching the immune regulation function of coronavirus nsp7.

Correlations between APACHE-II score and pressure parameters of mechanical ventilation in patients with ARDS and their value in prognostic evaluation.

Objective: To investigate the correlations between APACHE-II score and pressure parameters of mechanical ventilation in patients with acute respiratory distress syndrome (ARDS) and their value in prognostic evaluation. Methods: This was a retrospective study. The clinical data of 79 patients with ARDS treated in Shengzhou Hospital of Traditional Chinese Medicine from April 2020 to April 2022 were analyzed retrospectively. According to whether their APACHE-II scores were higher than 15, they were divided into low score group (n= 20) and high score group (n= 59). The plateau pressure (Pplat), driving pressure(ΔP) and mean airway pressure (Pmean) were compared. The correlation between APACHE-II score and pressure parameters of mechanical ventilation was analyzed. Based on the follow-up of 28-d survival, their Pplat, ΔP, Pmean and APACHE-II scores were compared. The value of APACHE-II score and pressure parameters in the prognostic evaluation of ARDS patients was analyzed. Results: Pplat, ΔP and Pmean in the low score group were significantly lower than those in the high score group(P<0.05). Pplat, ΔP, Pmean and APACHE-II score in the survival group were significantly lower than those in the control group(P<0.05). APACHE-II score showed significantly positive correlations with Pplat, ΔP and Pmean. The AUC of Pmean, Pplat, ΔP and APACHE-II score in predicting the prognosis and diagnosis of ARDS patients was 0.761, 0.833, 0.754 and 0.832, respectively. Conclusion: APACHE-II score of ARDS patients shows significantly positive correlations with pressure parameters of mechanical ventilation, and has diagnostic value for the prognosis of ARDS patients.

Porcine Deltacoronavirus Enters Porcine IPI-2I Intestinal Epithelial Cells via Macropinocytosis and Clathrin-Mediated Endocytosis Dependent on pH and Dynamin.

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes serious diarrhea in suckling piglets and has the potential for cross-species transmission. Although extensive studies have been reported on the biology and pathogenesis of PDCoV, the mechanisms by which PDCoV enters cells are not well characterized. In this study, we investigated how PDCoV enters IPI-2I cells, a line of porcine intestinal epithelial cells derived from pig ileum. Immunofluorescence assays, small interfering RNA (siRNA) interference, specific pharmacological inhibitors, and dominant negative mutation results revealed that PDCoV entry into IPI-2I cells depended on clathrin, dynamin, and a low-pH environment but was independent of caveolae. Specific inhibition of phosphatidylinositol 3-kinase (PI3K) and the Na+/H+ exchanger (NHE) revealed that PDCoV entry involves macropinocytosis and depends on NHE rather than on PI3K. Additionally, Rab5 and Rab7, but not Rab11, regulated PDCoV endocytosis. This is the first study to demonstrate that PDCoV uses clathrin-mediated endocytosis and macropinocytosis as alternative endocytic pathways to enter porcine intestinal epithelial cells. We also discussed the entry pathways of PDCoV into other porcine cell lines. Our findings reveal the entry mechanisms of PDCoV and provide new insight into the PDCoV life cycle. IMPORTANCE An emerging enteropathogenic coronavirus, PDCoV, has the potential for cross-species transmission, attracting extensive attenuation. Characterizing the detailed process of PDCoV entry into cells will deepen our understanding of the viral infection and pathogenesis and provide clues for therapeutic intervention against PDCoV. With the objective, we used complementary approaches to dissect the process in PDCoV-infected IPI-2I cells, a line of more physiologically relevant intestinal epithelial cells to PDCoV infection in vivo. Here, we demonstrate that PDCoV enters IPI-2I cells via macropinocytosis, which does not require a specific receptor, and clathrin-mediated endocytosis, which requires a low-pH environment and dynamin, while a caveola-mediated endocytic pathway is used by PDCoV to enter swine testicular (ST) cells and porcine kidney (LLC-PK1) cells. These findings provide a molecular detail of the cellular entry pathways of PDCoV and may direct us toward novel antiviral drug development.

Fish NF-κB couples TCR and IL-17 signals to regulate ancestral T-cell immune response against bacterial infection.

As fish constitute the first evolutionary group with primordial T cells, they are of importance for understanding the origin and evolution of adaptive immunity. Yet, the knowledge about how ancestral T cells function remains limited. Therefore, the teleost model Nile tilapia (Oreochromis niloticus) was used in this study to investigate the regulatory mechanisms of T-cell immunity in fish. We identified an evolutionarily conserved canonical NF-κB signaling pathway in Nile tilapia, which participates in primary adaptive immune response during Streptococcus agalactiae infection. Blockade of NF-κB activity severely impairs T-cell activation and expansion, rendering the animals more vulnerable to pathogen attack. Meanwhile, NF-κB signaling is indispensable for fish T cells to produce IL-17A during the antibacterial immune response. Moreover, IL-17A binds its receptor IL-17RA, initiates the ACT1-TRAF6-TAK1 axis, and triggers NF-κB-dependent T-cell activation, thus forming a positive feedback loop of T-cell immunity in Nile tilapia. Furthermore, IL-17A seems to promote innate immunity by regulating pro-inflammatory cytokines via TRAF6-NF-κB axis, indicating the presence of an NF-κB-dependent IL-17A signaling pathway for coordinating adaptive and innate immunity in fish. Our results suggest that fish NF-κB couples TCR and IL-17 signals to modulate ancestral T-cell immunity against bacterial infection, and the regulation of T-cell immunity by NF-κB and IL-17 is a strategy that existed prior to the divergence of the tetrapod lineage from teleost fish. This study, therefore, provides a new perspective on the evolution of adaptive immunity.

Interleukin-2 inducible T cell kinase (ITK) may participate in the anti-bacterial immune response of Nile tilapia via regulating T-cell activation.

Interleukin-2 inducible T cell kinase (ITK) plays a predominant role in the T-cell receptor (TCR) signaling cascade to ensure valid T-cell activation and function. Nevertheless, whether it regulates T-cell response of early vertebrates remains unknown. Herein, we investigated the involvement of ITK in the lymphocyte-mediated adaptive immune response, and its regulation to T-cell activation in the Nile tilapia Oreochromis niloticus. Both sequence and structure of O. niloticus ITK (OnITK) were remarkably conserved with its homologues from other vertebrates, implying its potential conserved function. OnITK mRNA was extensively expressed in lymphoid-related tissues, and with the relative highest level in peripheral blood. Once Nile tilapia was infected by Edwardsiella piscicida, OnITK in splenic lymphocytes was significantly up-regulated on 7-day post infection at both transcription and translation levels, suggesting that OnITK might involve in the primary adaptive immune response of teleost. Furthermore, upon splenic lymphocytes were stimulated by T-cell specific mitogen PHA, OnITK mRNA and protein levels were dramatically elevated. More importantly, treatment of splenic lymphocytes with specific inhibitor significantly crippled OnITK expression, which in turn impaired the inducible expression of T-cell activation markers IFN-γ, IL-2 and CD122, indicating the critical roles of ITK in regulating T-cell activation of Nile tilapia. Taken together, our results suggest that ITK takes part in the lymphocyte-mediated adaptive immunity of tilapia, and is indispensable for T-cell activation of teleost. Our findings thus provide novel evidences for understanding the mechanism regulating T-cell immunity of early vertebrates, as well as the evolution of adaptive immune system.

Activated T cells are the cellular source of IL-22 that enhances proliferation and survival of lymphocytes in Nile tilapia.

As a pleiotropic cytokine mainly secreted by CD4+ T cells, interleukin (IL)-22 plays an important role in immune regulation and infection elimination. Despite IL-22 homologues have been identified in non-mammal, whether and how IL-22 participates in the adaptive immune response of early vertebrates have not been fully addressed. In this study, we identified an evolutionarily conserved IL-22 from Nile tilapia Oreochromis niloticus (defined as OnIL-22), proved by its properties regarding sequence, gene structure, functional domain, tertiary structure and phylogeny. IL-22 was broadly expressed in lymphoid-related tissues of tilapia, and with relatively higher levels in skin, gill, intestine and liver. The expression of OnIL-22 in spleen lymphocytes was markedly induced at the adaptive immune stage after Streptococcus agalactiae infection. Moreover, once lymphocytes were activated by PMA plus ionomycin or T-cell specific mitogen PHA in vitro, OnIL-22 expression was obviously up-regulated at both mRNA and protein levels. These results thus suggest that activated T cells produce IL-22 to take part in the adaptive immune response of tilapia. Furthermore, treatment of lymphocytes with recombinant OnIL-22 increased the expression of genes related to proliferation and survival, and further promoted the proliferation and reduced the apoptosis of lymphocytes during bacterial infection or T-cell activation. These cellular effects of IL-22 seem to be associated with JAK1/STAT3 axis downstream of IL-22, because IL-22 application not only elevated the mRNA expression of JAK1 and STAT3, but also enhanced their phosphorylation in lymphocytes. Altogether, we suggest that activated T cells produce IL-22 to promote lymphocyte proliferation and survival probability via JAK1/STAT3 signaling pathway, thus participating in adaptive immune response of Nile tilapia. Our study therefore provides helpful perspective for understanding the function and mechanism of adaptive immune system in teleost.

A rare case with multiple arterial variations of the liver complicated laparoscopic pancreaticoduodenectomy.

BACKGROUND: Hepatic arterial variations were fully elaborated in anatomical monographs. Here, we aimed to present a rare case with multiple arterial variations of the liver complicated laparoscopic pancreaticoduodenectomy. CASE PRESENTATION: We report a 67-year-old woman with a periampullary tumor underwent laparoscopic pancreaticoduodenectomy. Intraoperatively, the aberrant right hepatic artery derived from the gastroduodenal artery (GDA) was observed and had accidentally sacrificed due to untimely ligature of GDA. Three-dimensional reconstruction based on preoperative contrast-enhanced CT performed to better study the anatomy. It demonstrated a replaced right hepatic artery branched from the GDA and supplied right liver lobe. Meanwhile, the middle hepatic artery derived from the common hepatic artery and supplied hepatic segment IV. Additionally, the replaced left hepatic artery emerged from the left gastric artery and fed into left liver lobe. CONCLUSIONS: The origination and course of hepatic arterial anatomy should be thoroughly assessed in planning and performing hepatopancreatobiliary surgeries. Reconstruction images of contrast-enhanced CT are helpful to visualize the vascular variations and its spatial relation with adjacent structures.

Large mechanical squeezing beyond 3dB of hybrid atom-optomechanical systems in a highly unresolved sideband regime.

We propose a scheme for the generation of strong mechanical squeezing beyond 3dB in hybrid atom-optomechanical systems in the highly unresolved sideband (HURSB) regime where the decay rate of cavity is much larger than the frequency of the mechanical oscillator. The system is formed by two two-level atomic ensembles and an optomechanical system with cavity driven by two lasers with different amplitudes. In the HURSB regime, the squeezing of the movable mirror can not be larger than 3dB if no atomic ensemble or only one atomic ensemble is put into the optomechanical system. However, if two atomic ensembles are put into the optomechanical system, the strong mechanical squeezing beyond 3dB is achieved even in the HURSB regime. Our scheme paves the way toward the implementation of strong mechanical squeezing beyond 3dB in hybrid atom-optomechanical systems in experiments.

Large and robust mechanical squeezing of optomechanical systems in a highly unresolved sideband regime via Duffing nonlinearity and intracavity squeezed light.

We propose a scheme to generate strong and robust mechanical squeezing in an optomechanical system in the highly unresolved sideband (HURSB) regime with the help of the Duffing nonlinearity and intracavity squeezed light. The system is formed by a standard optomechanical system with the Duffing nonlinearity (mechanical nonlinearity) and a second-order nonlinear medium (optical nonlinearity). In the resolved sideband regime, the second-order nonlinear medium may play a destructive role in the generation of mechanical squeezing. However, it can significantly increase the mechanical squeezing (larger than 3dB) in the HURSB regime when the parameters are chosen appropriately. Finally, we show the mechanical squeezing is robust against the thermal fluctuations of the mechanical resonator. The generation of large and robust mechanical squeezing in the HURSB regime is a combined effect of the mechanical and optical nonlinearities.

Haemophilus parasuis infection in 3D4/21 cells induces autophagy through the AMPK pathway.

Haemophilus parasuis (H. parasuis) is a common commensal in the upper respiratory tract of pigs, but causes Glässer's disease in stress conditions. To date, many studies focused on the immune evasion and virulence of H. parasuis; very few have focused on the role autophagy played in H. parasuis infection, particularly in porcine alveolar macrophages (PAMs). In this study, a PAM cell line, 3D4/21 cells were used to study the role of autophagy in H. parasuis infection. 3D4/21 cells tandemly expressing GFP, mCherry, and LC3 were infected with H. parasuis serovar 5 (Hps5). Western blot analysis and confocal and transmission electron microscopy showed that H. parasuis infection effectively induces autophagy. Using Hps strains of varying virulence (Hps4, Hps5, and Hps7) and UV-inactivated Hps5, we demonstrated that autophagy is associated with the internalisation of living virulent strains into cells. In 3D4/21 cells pretreated with rapamycin and 3-MA then infected by Hps4, Hps5, and Hps7, we demonstrated that autophagy affects invasion of H. parasuis in cells. AMPK signal results showed that Hps5 infection can upregulate the phosphorylation level of AMPK, which is consistent with the autophagy development. 3D4/21 cells pretreated with AICAR or Compound C then infected by Hps5 revealed that the autophagy induced by Hps5 infection is associated with the AMPK pathway. Our study contributes to the theoretical basis for the study of H. parasuis pathogenesis and development of novel drugs target for prevention Glässer's disease.

[Effects of MnSOD silence on in vitro tumorigenicity in NCI-H446 cells].

OBJECTIVE: To investigate the effect of manganese superoxide dismutase (MnSOD) silence on the in vitro tumorigenicity in human small cell lung cancer NCI-H446 cells and the underlying mechanisms.
 Methods: Sphere formation cells from NCI-H446 cells were obtained by suspension culture, while the expression of MnSOD and urokinase type plasminogen activator (uPAR) was analyzed by Western blot. Silence of MnSOD was performed by adenovirus infection in the second passage formation cells, and the effect of MnSOD silence on tumorigenicity in NCI-H446 cells was evaluated by sphere formation assay and soft-agar colony formation assay, while the expression of uPAR was analyzed by Western blot.
 Results: Compared with NCI-H446 cells, the sphere formation rate, colony formation rate, and the expression of MnSOD and uPAR were significantly increased in the second passage sphere formation cells in NCI-H446 cells (P<0.05). Silence of MnSOD inhibited the sphere formation rate, colony formation rate, and the expression level of uPAR in the second passage sphere formation cells in NCI-H446 cells.
 Conclusion: MnSOD may promote tumorigenicity in NCI-H446 cells by up-regulation of uPAR expression in vitro.

8-Bromo-7-methoxychrysin-blocked STAT3/Twist axis inhibits the stemness of cancer stem cell-like cell originated from SMMC-7721 cells.

Signal transducer and activator of transcription 3 (STAT3) is a member of the family of latent cytoplasmic transcriptional factors that could regulate cell proliferation, survival, and development. It has been reported that Twist is a target gene of STAT3, and STAT3/Twist signaling plays an important role in regulating cancer progress. Here, to explore whether 8-bromo-7-methoxychrysin (BrMC) inhibits liver cancer stem-like cell (LCSLC) properties via disrupting STAT3/Twist signaling, we cultured SMMC-7721 cells in vitro, and evaluated the effects of BrMC on the stemness of spheroids by determining the sphere-forming capability and migration. The sphere formation assay results showed a concentration-dependent decrease of sphere-forming capacity in LCSLCs (P < 0.05) treated with different concentrations of BrMC. Wound-healing assays results demonstrated a concentration-dependent decline in cell migration of LCSLCs treated with different concentrations of BrMC. In addition, CD133, CD44, and ALDH1 levels were decreased in LCSLCs treated with BrMC. Treatment with different concentrations of BrMC also reduced the expressions of p-STAT3 and Twist1 proteins. The effect of BrMC was substantially enhanced by co-treatment with JSI-124, a specific inhibitor of STAT3. Ectopic expression of Twist1 attenuated the inhibitory effects of BrMC on sphere formation, migration, and expression of the markers in LCSLCs. However, it had no affect on p-STAT3 expression in LCSLCs. These results demonstrated that BrMC inhibits the stemness of LCSLCs originated from SMMC-7721 cell line by inhibiting STAT3/Twist signal axis.

[Effects of apigenin on self-renewal and uPAR expression in NCI-H446 cell line].

OBJECTIVE: To investigate the effect of apigenin on self-renewal for sphere-forming cells in human small cell lung cancer cell line NCI-H446 and the underlying mechanisms.
 Methods: Sphere-forming cells from NCI-H446 cell line were cultured in stem cell-conditioned culture medium with ultra-low attachment surface plates. The rate of sphere-forming cells in the second passage sphere-forming cells was used to evaluate the inhibitory effects of apigenin on the self-renewal for sphere-forming cells. The protein level of urokinase-type plasminogen activator receptor (uPAR) in spheroids was analyzed by Western blot.
 Results: Apigenin signifcantly inhibited the self-renewal of the second passage sphere-forming cells [0, 5.0, 10.0, 20.0 µmol/L apigenin: (18.2±1.9)%, (13.6±1.7)%, (10.6±1.6)%, (6.9±1.3)%, respectively] and down-regulated uPAR expression in a concentration-dependent manner (P<0.05).
 Conclusion: Apigenin inhibits the self-renewal capacity of sphere-forming cells in NCI-H446 cells, which may be associated with down-regulation of uPAR expression.