Growth arrest and DNA damage-inducible alpha regulates muscle repair and fat infiltration through ATP synthase F1 subunit alpha.

BACKGROUND: Skeletal muscle fat infiltration is a common feature during ageing, obesity and several myopathies associated with muscular dysfunction and sarcopenia. However, the regulatory mechanisms of intramuscular adipogenesis and strategies to reduce fat infiltration in muscle remain unclear. Here, we identified the growth arrest and DNA damage-inducible alpha (GADD45A), a stress-inducible histone folding protein, as a critical regulator of intramuscular fat (IMAT) infiltration. METHODS: To explore the role of GADD45A on IMAT infiltration and muscle regeneration, the gain or loss function of GADD45A in intramuscular preadipocytes was performed. The adipocyte-specific GADD45A knock-in (KI) mice and high IMAT-infiltrated muscle model by glycerol injection (50 µL of 50% v/v GLY) were generated. RNA-sequencing, histological changes, gene expression, lipid metabolism, mitochondrial function and the effect of dietary factor epigallocatechin-3-gallate (EGCG) treatment (100 mg/kg) on IMAT infiltration were studied. RESULTS: The unbiased transcriptomics data analysis indicated that GADD45A expression positively correlates with IMAT infiltration and muscle metabolic disorders in humans (correlation: young vs. aged people, Gadd45a and Cebpa, r2  = 0.20, P < 0.05) and animals (correlation: wild-type [WT] vs. mdx mice, Gadd45a and Cebpa, r2  = 0.38, P < 0.05; NaCl vs. GLY mice, Gadd45a and Adipoq/Fabp4, r2  = 0.80/0.71, both P < 0.0001). In vitro, GADD45A overexpression promotes intramuscular preadipocyte adipogenesis, upregulating the expression of adipogenic genes (Ppara: +47%, Adipoq: +28%, P < 0.001; Cebpa: +135%, Fabp4: +16%, P < 0.01; Pparg: +66%, Leptin: +77%, P < 0.05). GADD45A knockdown robustly decreased lipid accumulation (Pparg: -57%, Adipoq: -35%, P < 0.001; Fabp4: -37%, P < 0.01; Leptin: -28%, P < 0.05). GADD45A KI mice exhibit inhibited skeletal muscle regeneration (myofibres: -40%, P < 0.01) and enhanced IMAT infiltration (adipocytes: +20%, P < 0.05). These KI mice have impaired exercise endurance and mitochondrial function. Mechanistically, GADD45A affects ATP synthase F1 subunit alpha (ATP5A1) ubiquitination degradation (ubiquitinated ATP5A1, P < 0.001) by recruiting the E3 ubiquitin ligase TRIM25, which decreases ATP synthesis (ATP production: -23%, P < 0.01) and inactivates the cAMP/PKA/LKB1 signalling pathway (cAMP: -36%, P < 0.01; decreased phospho-PKA and phospho-LKB1 protein content, P < 0.01). The dietary factor EGCG can protect against muscle fat infiltration (triglyceride: -64%, P < 0.05) via downregulating GADD45A (decreased GADD45A protein content, P < 0.001). CONCLUSIONS: Our findings reveal a crucial role of GADD45A in regulating muscle repair and fat infiltration and suggest that inhibition of GADD45A by EGCG might be a potential strategy to combat fat infiltration and its associated muscle dysfunction.

Negative Regulatory Role of the Spring Viremia of Carp Virus Matrix Protein in the Host Interferon Response by Targeting the MAVS/TRAF3 Signaling Axis.

Spring viremia of carp virus (SVCV) is a severe infectious pathogen that causes high rates of mortality in cyprinids and other fish species. Despite numerous investigations of SVCV infection, the underlying molecular mechanisms remain poorly understood. In this study, we found that the SVCV matrix protein (SVCV-M) played an inhibitory role in the host interferon (IFN) response by targeting the MAVS/TRAF3 signaling axis, thereby uncovering a previously unrecognized mechanism of SVCV escape from host innate antiviral immunity. Mechanistically, SVCV-M was located at the mitochondria independent of MAVS, which allowed SVCV-M to build an arena for competition with the MAVS platform. A microscale thermophoresis assay showed that SVCV-M had a high affinity for TRAF3, as indicated by a lower equilibrium dissociation constant (KD) value than that of MAVS with TRAF3. Therefore, the association of MAVS with TRAF3 was competitively impaired by SVCV-M in a dose-dependent manner. Accordingly, SVCV-M showed a potent ability to inhibit the K63-linked polyubiquitination of TRAF3. This inhibition was accompanied by the impairment of the IFN response, as shown by the marked decline in IFN-φ1-promoter (pro) luciferase reporter activity. By constructing truncated TRAF3 and SVCV-M proteins, the RING finger, zinc finger, and coiled-coil domains of TRAF3 and the hydrophobic-pocket-like structure formed by the α2-, α3-, and α4-helices of SVCV-M may be the major target and antagonistic modules responsible for the protein-protein interaction between the TRAF3 and SVCV-M proteins. These findings highlighted the intervention of SVCV-M in host innate immunity, thereby providing new insights into the extensive participation of viral matrix proteins in multiple biological activities. IMPORTANCE The matrix protein of SVCV (SVCV-M) is an indispensable structural element for nucleocapsid condensation and virion formation during viral morphogenesis, and it connects the core nucleocapsid particle to the outer membrane within the mature virus. Previous studies have emphasized the architectural role of SVCV-M in viral construction; however, the potential nonstructural functions of SVCV-M in viral replication and virus-host interactions remain poorly understood. In this study, we identified the inhibitory role of the SVCV-M protein in host IFN production by competitively recruiting TRAF3 from the MAVS signaling complex and impairing TRAF3 activation via inhibition of K63-linked polyubiquitination. This finding provided new insights into the regulatory role of SVCV-M in host innate immunity, which highlighted the broader functionality of rhabdovirus matrix protein apart from being a structural protein. This study also revealed a previously unrecognized mechanism underlying SVCV immune evasion by inhibiting the IFN response by targeting the MAVS/TRAF3 signaling axis.

Extensive involvement of CD40 and CD154 costimulators in multiple T cell-mediated responses in a perciform fish Larimichthys crocea.

CD40 and CD154 are well-characterized costimulatory molecules involved in adaptive humoral immunity in humans and other mammals. These two costimulatory molecules were found to be originated from teleost fish during vertebrate evolution. However, the functionality of fish CD40 and CD154 remains to be explored. In this study, we identified the CD40 and CD154 homologs (LcCD40 and LcCD154) from large yellow croaker (Larimichthys crocea), a marine species of the perciform fish family. The LcCD40 and LcCD154 share conserved structural features to their mammalian counterparts, and are widely expressed in immune-relevant tissues and leukocytes at different transcriptional levels. Immunofluorescence staining and FCM analysis showed that LcCD40 and LcCD154 proteins are distributed on MHC-II+ APCs and CD4-2+ T cells, and are significantly upregulated in response to antigen stimulation. Co-IP assay exhibited strong association between LcCD40 and LcCD154 proteins. Blockade of LcCD154 with anti-LcCD154 antibody (Ab) or recombinant soluble LcCD40-Ig fusion protein remarkably decreased the MHC-II+ APC-initiated CD4+ T cell response upon Aeromonas hydrophila stimulation, and alloreactive T cell activation as examined by mixed lymphocyte reaction (MLR). These findings highlight the costimulatory role of LcCD40 and LcCD154 in T cell activities in Larimichthys crocea. Thus, the CD40 and CD154 costimulators may extensively participate in the regulation of multiple T cell-mediated immune responses in teleost fish. It is anticipated that this study would provide a cross-species understanding of the evolutionary history of CD40 and CD154 costimulatory signals from fish to mammals.

Zbtb46 Controls Dendritic Cell Activation by Reprogramming Epigenetic Regulation of cd80/86 and cd40 Costimulatory Signals in a Zebrafish Model.

The establishment of an appropriate costimulatory phenotype is crucial for dendritic cells (DCs) to maintain a homeostatic state with optimal immune surveillance and immunogenic activities. The upregulation of CD80/86 and CD40 is a hallmark costimulatory phenotypic switch of DCs from a steady state to an activated one for T cell activation. However, knowledge of the regulatory mechanisms underlying this process remains limited. In this study, we identified a Zbtb46 homolog from a zebrafish model. Zbtb46 deficiency resulted in upregulated cd80/86 and cd40 expression in kidney marrow-derived DCs (KMDCs) of zebrafish, which was accompanied with a remarkable expansion of CD4+/CD8+ T cells and accumulation of KMDCs in spleen of naive fish. Zbtb46 -/- splenic KMDCs exhibited strong stimulatory activity for CD4+ T cell activation. Chromatin immunoprecipitation-quantitative PCR and mass spectrometry assays showed that Zbtb46 was associated with promoters of cd80/86 and cd40 genes by binding to a 5'-TGACGT-3' motif in resting KMDCs, wherein it helped establish a repressive histone epigenetic modification pattern (H3K4me0/H3K9me3/H3K27me3) by organizing Mdb3/organizing nucleosome remodeling and deacetylase and Hdac3/nuclear receptor corepressor 1 corepressor complexes through the recruitment of Hdac1/2 and Hdac3. On stimulation with infection signs, Zbtb46 disassociated from the promoters via E3 ubiquitin ligase Cullin1/Fbxw11-mediated degradation, and this reaction can be triggered by the TLR9 signaling pathway. Thereafter, cd80/86 and cd40 promoters underwent epigenetic reprogramming from the repressed histone modification pattern to an activated pattern (H3K4me3/H3K9ac/H3K27ac), leading to cd80/86 and cd40 expression and DC activation. These findings revealed the essential role of Zbtb46 in maintaining DC homeostasis by suppressing cd80/86 and cd40 expression through epigenetic mechanisms.

Regulatory role of BTLA and HVEM checkpoint inhibitors in T cell activation in a perciform fish Larimichthys crocea.

The BTLA and HVEM are two well-characterized immune checkpoint inhibitors in humans and other mammalian species. However, the occurrence and functionality of these two molecules in non-mammalian species remain poorly understood. In the present study, we identified the BTLA and HVEM homologs from large yellow croaker (Larimichthys crocea), an economically important marine species of the perciform fish family. The Larimichthys crocea BTLA and HVEM (LcBTLA and LcHVEM) share conserved structural features to their mammalian counterparts, and they were expressed in various tissues and cells examined at different transcriptional levels, with particular abundance in immune-relevant tissues and splenic leukocytes. Immunofluorescence staining and flow cytometry analysis showed that LcHVEM and LcBTLA proteins were distributed on MHC-II+ APCs and CD4-2+ T cells, and a strong interaction between LcBTLA and LcHVEM was detected in splenic leukocytes in the mixed lymphocyte reaction (MLR). By blockade assays using anti-LcBTLA and anti-LcHVEM Abs as well as recombinant soluble LcBTLA and LcHVEM proteins in different combinations, it was found that LcBTLA-LcHVEM interactions play an important inhibitory role in the activation of alloreactive T cells using MLR as a model, and APC-initiated antigen-specific CD4-2+ T cells in response to A. hydrophila (A. h) stimulation. These observations highlight the extensive functional roles of LcBTLA and LcHVEM immune-checkpoint inhibitors in allogeneic T cell reactions, and CD4-2+ T cell-mediated adaptive immune responses in Larimichthys crocea. Thus, the BTLA-HVEM checkpoint may represent an ancient coinhibitory pathway, which was originated in fish and was conserved from fish to mammals throughout the vertebrate evolution.

A Crucial But Neglected Anatomical Factor Underneath Psoas Muscle and Its Clinical Value in Lateral Lumbar Interbody Fusion-The Cleft of Psoas Major (CPM).

OBJECTIVE: To describe the anatomical feature positioned beneath the psoas muscle at the lateral aspect of the lower lumbar, and to create a new location system to identify the risk factors of lateral lumbar interbody fusion. METHODS: Six cadavers were dissected and analyzed. The anatomy and neurovascular distribution beneath the psoas major from L3 to S1 was observed and recorded, with particular focus on the L4/5 disc and below. The psoas major surface was divided homogeneously into four parts, from the anterior border of psoas major to the transverse process. The cranial-to-caudal division was from the lower edge of the psoas muscle attachment on the L4 vertebrae to the upper part of the S1 vertebrae, and was divided into five segments. Then a grid system was used to create 20 grids on the psoas major surface, from the anterior border of the muscle to the transverse process and from L4 to superior S1 , which was used to determine the anatomical structures' distribution and relationship beneath the psoas major. RESULTS: A cleft was identified beneath the psoas major, from the level of L4/5 downwards. It was filled with loose connective tissue and neurovascular structures. We termed it the cleft of psoas major (CPM). The sympathetic trunk, ascending lumbar vein, iliolumbar vessels, obturator nerve, femoral nerve and occasionally the great vessels are contained within the CPM, although there is significant interpersonal variation. The grid system on the psoas major surface helped to identify the anatomical structures in CPM. There was a considerably lower frequency of occurrence of neurovascular structures in the grids of I/II at the L4/5 level where can be considered the "safe zones" for the lateral lumbar interbody fusion. In contrast, the distribution of neurovascular structures at the L5 S1 level is dense, where the operation risk is high. CONCLUSION: The CPM exists lateral to the vertebral surface from L4 and below. Although the occurrence and distribution of neurovascular structures within the CPM is complex and varies greatly, it can provide a potential cavity for visualization during lateral lumbar interbody fusion. Using psoas major as a reference, this novel grid system can be used to identify the risk factors in CPM and thus identify a safe entry point for surgery.

Inhibitory Role of an Aeromonas hydrophila TIR Domain Effector in Antibacterial Immunity by Targeting TLR Signaling Complexes in Zebrafish.

The Toll/interleukin-1 receptor (TIR) domain is a structural unit responsible for the assembly of signal protein complexes in Toll-like receptor (TLR) and interleukin-1 receptor signaling pathways. TIR domain homologs are found in a considerable number of bacteria and enhance bacterial infection and survival in host organisms. However, whether TIR domain homologs exist in Aeromonas hydrophila, a ubiquitous waterborne bacterium in aquatic environments, remains poorly understood. In this study, a TIR domain protein (TcpAh) was identified from A. hydrophila JBN2301. TIR domain of TcpAh is highly homologous to the counterpart domains in TLRs and myeloid differentiation factor 88 (MyD88). The zebrafish infected with mutant A. hydrophila with tcpAh deletion had a remarkably lower mortality than those infected with the wild-type strain. This result suggests that TcpAh is a crucial virulence factor for A. hydrophila infection. TcpAh exhibited a strong ability to associate with MyD88, tumor necrosis factor receptor-associated factor 3 (TRAF3) and TRAF-associated NF-κB activator-binding kinase 1 (TBK1) in TIR-TIR, TIR-Death domain (DD), and other alternative interactions. This finding suggests that TcpAh extensively interferes with MyD88 and TIR domain-containing adapter inducing interferon (IFN)-ß (TRIF) signaling pathways downstream of TLRs. Consequently, CD80/86 expression was suppressed by TcpAh via attenuating TLR-stimulated NF-κB activation, which ultimately led to the impairment of the major costimulatory signal essential for the initiation of adaptive humoral immunity against A. hydrophila infection. We believe that this study is the first to show a previously unrecognized mechanism underlying A. hydrophila evades from host antibacterial defense by intervening CD80/86 signal, which bridges innate and adaptive immunity. The mechanism will benefit the development of therapeutic interventions for A. hydrophila infection and septicemia by targeting TcpAh homologs.

New Insights into IgZ as a Maternal Transfer Ig Contributing to the Early Defense of Fish against Pathogen Infection.

IgZ or its equivalent IgT is a newly discovered teleost specific Ig class that is highly specialized in mucosal immunity. However, whether this IgZ/IgT class participates in other biological processes remains unclear. In this study, we unexpectedly discovered that IgZ is highly expressed in zebrafish ovary, accumulates in unfertilized eggs, and is transmitted to offspring from eggs to zygotes. Maternally transferred IgZ in zygotes is found at the outer and inner layers of chorion, perivitelline space, periphery of embryo body, and yolk, providing different lines of defense against pathogen infection. A considerable number of IgZ+ B cells are found in ovarian connective tissues distributed between eggs. Moreover, pIgR, the transporter of IgZ, is also expressed in the ovary and colocalizes with IgZ in the zona radiata of eggs. Thus, IgZ is possibly secreted by ovarian IgZ+ B cells and transported to eggs through association with pIgR in a paracrine manner. Maternal IgZ in zygotes showed a broad bacteriostatic activity to different microbes examined, and this reactivity can be manipulated by orchestrating desired bacteria in water where parent fish live or immunizing the parent fish through vaccination. These observations suggest that maternal IgZ may represent a group of polyclonal Abs, providing protection against various environmental microbes encountered by a parent fish that were potentially high risk to offspring. To our knowledge, our findings provide novel insights into a previously unrecognized functional role of IgZ/IgT Ig in the maternal transfer of immunity in fish, greatly enriching current knowledge about this ancient Ig class.

Functional role of CD40 and CD154 costimulatory signals in IgZ-mediated immunity against bacterial infection.

CD40 and CD154 are one of the best-characterized costimulatory molecules essential for adaptive immunity, which extensively involved in T and B cell activation, IgM Ab production, isotype class switching, germinal center formation and affinity maturation. However, the functionality of CD40 and CD154 in IgZ-mediated immunity remains limited. In this study, we explored the regulatory role of Cd40-Cd154 interaction in IgZ-mediated antibacterial immunity in zebrafish. The results showed that the IgZ-mediated antibacterial response can be significantly induced in response to A. hydrophila infection. The percentage of Cd40+IgZ+ B cells and the production of IgZ Ab were substantially increased upon A. hydrophila stimulation, but these reactions were markedly declined in Cd154 blockade fish by administering anti-Cd154 Ab or recombinant sCd40-Ig protein, accompanied with the impairment of the vaccine-initiated IgZ-mediated immunoprotection of fish against A. hydrophila infection. These observations suggested the essential role of Cd40-Cd154 interaction in IgZ-mediated bacterial immunity. Notably, the Cd40 and Cd154 costimulatory signals are required for a TD antigen-induced IgZ immunity, but are not indispensable for a TI antigen-induced IgZ immune response. These findings indicated the differential role of Cd40-Cd154 interaction in bacterial TD and TI antigen-induced IgZ immunity, which suggested the existence of diverse regulatory mechanisms underlying IgZ-mediated antibacterial immune reactions. To our knowledge, this is the first report to show the functional role of Cd40-Cd154 costimulatory signaling pathway in IgZ-mediated immune defense against bacterial infection. We hope this study will improve the current understanding of the coevolution between the IgZ/IgT immunoglobins and CD40/CD154 costimulatory molecules.

Differential immune responses of immunoglobulin Z subclass members in antibacterial immunity in a zebrafish model.

Immunoglobulin Z (IgZ) or its equivalent immunoglobulin T (IgT) is a newly identified immunoglobulin (Ig) class from teleost fish. This Ig class is characterized by its involvement in mucosa-associated lymphoid tissues (MALTs) for mucosal defence against pathogen infection. Recently, several subclass members of IgZ/IgT, such as IgZ, IgZ2, Igτ1, Igτ2 and Igτ3, have been further identified from zebrafish, common carp and rainbow trout. However, the functional diversity and correlation among these subclasses remain uncertain. Here, we explored the differential immune reactions of the IgZ and IgZ2 subclasses in antibacterial immunity in a zebrafish model. IgZ was extensively distributed in the peripheral serum and skin/gill MALTs and showed a rapid induction upon bacterial infection. IgZ2 was specialized in skin/gill MALTs and showed a strong induction following IgZ production. Correspondingly, the IgZ+ B cells had a wider distribution in the systemic primary/secondary lymphoid tissues and MALTs than the IgZ2+ B cells, which were predominant in MALTs. IgZ and IgZ2 exhibited a complementary effect in antibacterial immunity by possessing differential abilities. That is, IgZ is preferentially involved in bactericidal reaction that is in part C1q-dependent, and IgZ2 participates in neutralization action through bacteria-coating activity. The production of IgZ largely depended on the αß T/CD4+ T cells, whereas that of IgZ2 did not, suggesting the different dependencies of IgZ and IgZ2 on systemic immunity. Our findings demonstrate that the functional behaviour and mechanism of the IgZ/IgT family are more diverse than previously recognized and thus improve the current knowledge about this ancient Ig class.

[Analysis on the causes and prevention strategies of the vascular injury caused by the oblique lateral lumbar fusion].

OBJECTIVE: To analyze the causes of vascular injury occurred in oblique lateral interbody fusion for treating lumbar degenerative diseases, and put forward preventive measures. METHODS: There were 235 patients analyzed from October 2014 to May 2017 in five hospitals, who were treated with oblique lateral interbody fusion with or without posterior pedicle screw fixation. There were 79 males and 156 females with an average age of (61.9±13.5) years old (ranged from 32 to 83 years). There were 7 cases of vascular injury, including 4 cases of segmental vessel injury, 1 case of left common iliac artery injury, 1 case of left common iliac veininjury and 1 case of ovarian vein injury. RESULTS: The follow up time ranged from 6 to 36 months, averagely (15.6±7.5) months. There was no pedicle screw loosen or fracture. The low back pain VAS decreased from preoperative 6.7±2.3 to 1.4±0.8 at the latest follow-up, which was statistically difference(t=7.21, P=0.033). The ODI decreased from preoperative (36.5±7.7)% to (9.4±3.6)% at the latest follow-up, which was statistically difference (t=8.11, P=0.025). CONCLUSION: Oblique lateral interbody fusion technique provides a new method for minimally invasive fusion of lumbar internal fixation. However, it has a risk of vascular injury. In order to effectively prevent the occurrence of vascular injury, the operative indications and careful and meticulous operation should be strictly grasped.

Characterization of cGAS homologs in innate and adaptive mucosal immunities in zebrafish gives evolutionary insights into cGAS-STING pathway.

Cyclic GMP-AMP synthase (cGAS) is one of the most-characterized cytoplasmic DNA sensors in humans and other mammals. However, knowledge about cGAS homologs in nonmammalian species remains limited. In this study, we report the molecular and functional identification of two cGAS homologs, namely, DrcGASa and DrcGASb, from a zebrafish (Danio rerio) model. DrcGASa and DrcGASb share the same overall conservative structural architectures and functional domains/residues to mammalian cGASs. Both homologs synthesized a 2'3'-cGAMP isomer but not a 3'3'-cGAMP isomer via oligomerization in response to DNA stimulation. Overexpression of DrcGASa/b in HEK293T cells and zebrafish embryos significantly activated NF-κB and IFN-I signaling pathways in a STING-dependent manner. Knockdown of DrcGASa or DrSTING impaired such activations, thereby reducing the host innate immunity against bacterial and viral infections. DrcGASa, but not DrcGASb, was involved in immunoglobulin Z-mediated mucosal immunity in gill-associated lymphoid tissue, suggesting differential functions between the two DrcGASs. This reaction was associated with the DrcGAS-DrSTING-IFNφ1 signaling axis in GALT's γδ T cells. Our findings provide experimental evidence that a modern cGAS-STING pathway that mainly participates in IFN-mediated immunity originated from teleost fish based on the functional constraint of cGAS and STING proteins during vertebrate evolution.

Transferrin Receptor 1-Associated Iron Accumulation and Oxidative Stress Provides a Way for Grass Carp to Fight against Reovirus Infection.

Iron is an essential element, closely linked with host immune responses. Nevertheless, the relationship between iron metabolism and virus infection is still unclear in aquatic vertebrates. To address this issue, we employed grass carp (Ctenopharyngodon idella) and its lethal virus, grass carp reovirus (GCRV), a double-strand RNA virus, as models. Our results demonstrate that GCRV infection increases the iron content and alters the expression of iron metabolism-related genes both in vivo and in vitro. Of note, the expression of C. idella transferrin receptor 1 (CiTfR1) rather than transferrin is upregulated upon GCRV infection. To clarify the implications of CiTfR1 upregulation for antiviral immunity, we proved that CiTfR1 was not a helper for GCRV invasion, but instead, it inhibited GCRV infection and promoted cell proliferation by facilitating the accumulation of intracellular labile iron pool (LIP), which increases intracellular oxidative stress. Interestingly, we found that CiTfR1 overexpression inhibited the mRNA expression of C. idella interferon 1 (CiIFN1) and CiIFN3. The present study reveals a novel antiviral defense mechanism in teleost where TfR1 induces the accumulation of LIP, leading to the suppression of virus infection and the proliferation of host cells, indicating that iron can be used as a medicated feed additive for the control of animal viral disease.

GCRV hijacks TBK1 to evade IRF7-mediated antiviral immune responses in grass carp Ctenopharyngodon idella.

TANK-binding kinase 1 (TBK1) is an important kinase that regulates the activation of interferon regulatory factor 3/7 (IRF3/7) to induce type I interferon (IFN-I) production in antiviral immune responses. However, in long-term virus-host crosstalk, viruses have evolved elaborate strategies to evade host immune defense mechanisms. In the present study, we found that grass carp (Ctenopharyngodon idella) reovirus (GCRV) hijacks TBK1 to escape IRF7-IFN-Is signaling activation. In brief, GCRV inhibited TBK1 activation by restaining K63-linked ubiquitination of TBK1 and promoting its K48-linked ubiquitination. This regulation resulted in that under low titer of GCRV infection, TBK1 overexpression specifically supressed promoter activity and phosphorylation of IRF7 and induction of downstream IFN1and IFN3. qRT-PCR data uncovered that TBK1 negatively regulated IRF7, IFN1 and IFN3 transcription levels under low viral titer infection. Along with enhancement of GCRV titers, TBK1 swiched its function to up-regulate IRF7, IFN1 and IFN3 mRNA levels. Accordingly, TBK1 promoted GCRV replication at low infected titer, but inhibited GCRV replication at high infected titer. All these results revealed a viral evasion strategy that GCRV utilizes TBK1 to block cellular IFN responses at low titers or early stages in fish species, which will lay a foundation for further researching on host-virus interactions and developing novel antiviral strategies in lower vertebrates.

Thoroughly Remold the Localization and Signaling Pathway of TLR22.

TLR22 exists in nearly all the poikilothermic vertebrates and plays a central role in the initiation of innate immunity and activation of adaptive immunity. TLR22 signaling pathway has been characterized in detail in fugu (Takifugu rubripes). Here, we thoroughly remold the localization and signaling pathways of TLR22. We characterized TLR22a and TLR22b in grass carp (Ctenopharyngodon idella), designated as CiTLR22a and CiTLR22b, and explored the ligand(s), adaptor(s), and signaling pathway(s). Results show that both CiTLR22a and CiTLR22b localize to lysosome, acidic compartment. Correspondingly, CiTLR22a and CiTLR22b directly bind and respond to dsRNA analog poly(I:C) at pH 5, but not at pH 7.4, the physiological pH. Moreover, CiTLR22a and CiTLR22b exhibit antagonistic function in signal transmission, wherein CiTLR22a facilitates the protein and phosphorylation levels of IRF7 and enhances the promoter activities of major IFNs and NF-κBs, while CiTLR22b downregulates IRF7 phosphorylation and IRF3 protein level and suppresses the IFN and NF-κB pathways. Further investigations revealed that CiTLR22a restrains grass carp reovirus (GCRV) replication and protects cells from GCRV infection, whereas CiTLR22b plays a negative role in response to GCRV infection. This is the first time to systematically clarify the signaling pathways of two isotype TLR22s; especially, subcellular localization and adaptor are different from previous TLR22 report, which results from technical limitations. The results will serve the antiviral immune mechanisms in poikilothermic vertebrates and evolutionary immunology.

ROS-induced HSP70 promotes cytoplasmic translocation of high-mobility group box 1b and stimulates antiviral autophagy in grass carp kidney cells.

Autophagy plays many physiological and pathophysiological roles. However, the roles and the regulatory mechanisms of autophagy in response to viral infections are poorly defined in teleost fish, such as grass carp (Ctenopharyngodon idella), which is one of the most important aquaculture species in China. In this study, we found that both grass carp reovirus (GCRV) infection and hydrogen peroxide (H2O2) treatment induced the accumulation of reactive oxygen species (ROS) in C. idella kidney cells and stimulate autophagy. Suppressing ROS accumulation with N-acetyl-l-cysteine significantly inhibited GCRV-induced autophagy activation and enhanced GCRV replication. Although ROS-induced autophagy, in turn, restricted GCRV replication, further investigation revealed that the multifunctional cellular protein high-mobility group box 1b (HMGB1b) serves as a heat shock protein 70 (HSP70)-dependent, pro-autophagic protein in grass carp. Upon H2O2 treatment, cytoplasmic HSP70 translocated to the nucleus, where it interacted with HMGB1b and promoted cytoplasmic translocation of HMGB1b. Overexpression and siRNA-mediated knockdown assays indicated that HSP70 and HMGB1b synergistically enhance ROS-induced autophagic activation in the cytoplasm. Moreover, HSP70 reinforced an association of HMGB1b with the C. idella ortholog of Beclin 1 (a mammalian ortholog of the autophagy-associated yeast protein ATG6) by directly interacting with C. idella Beclin 1. In summary, this study highlights the antiviral function of ROS-induced autophagy in response to GCRV infection and reveals the positive role of HSP70 in HMGB1b-mediated autophagy initiation in teleost fish.

Complications and Prevention Strategies of Oblique Lateral Interbody Fusion Technique.

OBJECTIVE: To analyze the early complications and causes of oblique lateral interbody fusion, and put forward preventive measures. METHODS: There were 235 patients (79 males and 156 females) analyzed in our study from October 2014 to May 2017. The average age was 61.9 ± 0.21 years (from 32 to 83 years). Ninety-one cases were treated with oblique lateral interbody fusion (OLIF) alone (OLIF alone group) and 144 with OLIF combined with posterior pedicle screw fixation through the intermuscular space approach (OLIF combined group). In addition, 137/144 cases in the combined group were primarily treated by posterior pedicle screw fixation, while the treatments were postponed in 7 cases. There were 190 cases of single fusion segments, 11 of 2 segments, 21 of 3 segments, and 13 of 4 segments. Intraoperative and postoperative complications were observed. RESULTS: Average follow-up time was 15.6 ± 7.5 months (ranged from 6 to 36 months). Five cases were lost to follow-up (2 cases from the OLIF alone group and 3 cases from the OLIF combined group). There were 7 cases of vascular injury, 22 cases of endplate damage, 2 cases of vertebral body fracture, 11 cases of nerve injury, 18 cases of cage sedimentation or cage transverse shifting, 3 cases of iliac crest pain, 1 case of right psoas major hematoma, 2 cases of incomplete ileus, 1 case of acute heart failure, 1 case of cerebral infarction, 3 case of left lower abdominal pain, 9 cases of transient psoas weakness, 3 cases of transient quadriceps weakness, and 8 cases of reoperation. The complication incidence was 32.34%. Thirty-three cases occurred in the OLIF alone group, with a rate of 36.26%, and 43 cases in the group of OLIF combined posterior pedicle screw fixation, with a rate of 29.86%. Fifty-seven cases occurred in single-segment fusion, with a rate of 30.0% (57/190), 4 cases occurred in two-segment fusion, with a rate of 36.36% (4/11), 9 cases occurred in three-segment fusion, with a rate of 42.86% (9/21), and 6 cases occurred in four-segment fusion, with a rate of 46.15% (6/13). CONCLUSION: In summary, OLIF is a relatively safe and very effective technique for minimally invasive lumbar fusion. Nonetheless, it should be noted that OLIF carries the risk of complications, especially in the early stage of development.

A systematic investigation on the composition, evolution and expression characteristics of chemokine superfamily in grass carp Ctenopharyngodon idella.

Chemokines are a superfamily of small cytokines and characterized based on their ability to induce directional migration of cells along a concentration gradient by binding to chemokine receptors, which have important roles in immunology and development. Due to the numerous and diverse members, systematic identifications of chemokine superfamily genes are difficult in many species. To that end, a comprehensive analysis of BLAST and scripting language was conducted to systematically identify and characterize chemokine system in grass carp (Ctenopharyngodon idella). Our results showed that C. idella chemokine superfamily consists of 81 chemokines and 37 receptors, in which, most genes possess typical structural features of the chemokine superfamily. Phylogenetic analyses confirmed the existence of three chemokine subfamilies (CC, CXC and XC) in C. idella and revealed their homologous relationships with other species. Chemokine receptors are transmembrane receptors and contains CCR, CXCR, XCR and ACKR subfamilies. mRNA expression analyses of chemokine superfamily genes indicated that many members are sustainably expressed in multiple tissues before and after grass carp reovirus (GCRV) or Aeromonas hydrophila infection, which provides in vivo evidence for the response patterns after viral or bacterial infection. Meanwhile, this study also explored the evolution of chemokine system from arthropod to higher vertebrates and then investigated the changes in gene number/diversification, gene organization and encoded proteins during vertebrate evolution. These results will serve the further functional and evolutional studies on chemokine superfamily.

Teleost-Specific TLR19 Localizes to Endosome, Recognizes dsRNA, Recruits TRIF, Triggers both IFN and NF-κB Pathways, and Protects Cells from Grass Carp Reovirus Infection.

TLRs are pivotal pattern recognition receptors in initiating innate immunity and triggering adaptive immunity. TLR pathways have been comprehensively investigated in mammals. However, the teleost-specific TLR19 pathway remains largely unknown. In this study, we identified TLR19 from grass carp (Ctenopharyngodon idella), and explored the ligand, adaptor, and signaling pathways. Pathogen-associated molecular pattern binding and luciferase activity assays indicate that TLR19 recognizes and responds to dsRNA analog (polyinosinic:polycytidylic acid). Confocal fluorescence microscopy demonstrates that TLR19 is synthesized in ribosomes not binding on endoplasmic reticulum, then transfers to early endosome post-polyinosinic:polycytidylic acid stimulation. Fluorescence colocalization and immunoprecipitation experiments confirm TLR19 interacts with adaptor TRIF, not MyD88, TIRAP, or SARM1. TLR19 facilitates protein and phosphorylation levels of IRF3, inhibits phosphorylation of IRF7. TLR19 enhances the promoter activities and mRNA expressions of major IFNs and NF-κBs; in contrast, grass carp TLR3 just significantly motivates IFN1 expression post-grass carp reovirus (GCRV) infection. Further investigations reveal that TLR19 inhibits GCRV replication by overexpression, knockdown, Western blotting techniques and virus titer assays, and protects cells from GCRV infection by flow cytometry and MTT method. Collectively, these results demonstrate that teleost-specific TLR19 recognizes dsRNA, recruits adaptor molecule TRIF, enhances IRF3 protein and phosphorylation levels, triggers both IFN and NF-κB pathways, and prevents viral proliferation. This is the first attempt to systematically clarify the TLR19 signaling pathway, which is the third TLR member recognizing dsRNA. The results will serve the antiviral immune mechanisms in teleost and evolutionary immunology.

[Case control study on two different surgical approaches combined fixation with lumbar interbody fusion for the treatment of single segmental lumbar vertebra diseases].

OBJECTIVE: To discuss the advantages and disadvantages of two different surgical approaches combined fixation with lumbar interbody fusion in treating single segmental lumbar vertebra diseases. METHODS: The clinical data of 86 patients with single segmental lumbar vertebra diseases treated from June 2011 to June 2013 was retrospectively analyzed. There were 33 males and 53 females, aged from 28 to 76 years old with an average of 53.0 years. Among them, there were 39 cases of lumbar disc degeneration, 22 cases of lumbar disc herniation complicated with spinal canal stenosis, 9 cases of huge lumbar disc herniation and 16 cases of lumbar degenerative spondylolisthesis (Meyerding degree I ). Lesion sites contained L3, 4 in 5 cases, L4, 5 in 70 cases and L5S1 in 11 cases. All the patients were treated with internal fixation and lumbar interbody fusion with 45 cases by midline incision approach (median incision group) and the other 41 cases by channel-assisted by muscle-splitting approach(channel group). Incision length, operation time, intraoperative bleeding and postoperative drainage were recorded in two groups. Visual analogue scale(VAS) was used to assess lumbar incision pain 72 h after operation. Depended on imaging results to compare the changes of the disc space height in lesion in preoperative, postoperative and final follow-up, the coronal and sagittal Cobb angle in preoperative and final follow-up, the area of multifidus and the degree of multifidus fat deposition before and after operation between two groups. Loosening or fragmentation of internal fixation, displacement of intervertebral cage and interbody fusion were observed in each group. Japanese Orthopedic Association (JOA) scoring system was used to evaluate the function before operation and at the final follow-up. RESULTS: The channel group was superior to the median incision group in incision length and postoperative drainage while the median incision group was less than the channel group in the operation time and intraoperative bleeding. The average VAS score of lumbar incision 72 h after operation was 1.50 points in median incision group and 0.97 points in channel group, and there was significant difference between two groups(P<0.05). No incision infection was found, but there were 4 cases of incisional epidermal necrosis, 1 case of incision healed badness, and 3 cases of nerve injury in channel group. The incidence of cacothesis of pedicle screw were 5.0% and 3.6% in median incision group and channel group respectively, and there was no significant difference between two groups(P>0.05). The incidence of cacothesis of translaminar facet screw were 6.6% and 12.2% in median incision group and channel group respectively, and there was significant difference between two groups(P<0.05). All the patients were followed up for 12 to 36 months with a mean of 22.8 months. The changes of disc space height had statistical difference between preoperative and postoperative(P<0.05) in all patients, but there was no significant difference between postoperative and final follow-up(P>0.05), however, there was no significant difference 3 days after operation and final follow-up between two groups(P>0.05). At final follow-up, coronal and sagittal Cobb angle were obviously improved in all patients(P<0.05), but there was no significant difference between two groups(P>0.05). One year after operation, the area of multifidus in median incision group was (789.00±143.15) mm² less than preoperative(1 066.00±173.55) mm² (P<0.05), and in channel group, was(992.00±156.75) mm² at 1 year after operation and(1 063.00±172.13) mm² preoperatively, there was no significant difference between them(P>0.05), however, there was significant difference one year after operation between two groups (P<0.05) . About the degree of multifidus fat deposition, there was significant difference between one year after operation and preoperation in median incision group (P<0.05), but there was no significant difference between one year after operation and preoperation in channel group (P>0.05), and there was significant difference at one year after operation between two groups(P<0.05). During the follow-up period, neither pedicle screw and/or translaminar facet screw loosening, displacement or fragmentation nor displacement of intervertebral cage were found. The lumbar interbody fusion rate was 95.6% in median incision group and was 95.1% in channel group, and there was no significant difference between two groups(P>0.05). No obvious adjacent segmental degeneration was observed in fixed position. JOA score in median incision group was significantly increased from 8-16 points (average: 12.77±2.56) preoperative to 21-29 points (average: 25.20±2.43) at final follow-up(P<0.05); and in channel group was significantly increased from 8-16 points (average: 12.64±2.37) preoperative to 23-29 points(average: 26.7±1.82) at final follow-up(P<0.05); there was also significant difference between two groups at final follow-up. CONCLUSIONS: Compared to the median incision approach, unilateral pedicle screw combined with contralateral translaminar facet screw fixation using channel-assisted by muscle-splitting approach has advantages of small incision, less trauma, fast recovery and so on. However, it also has shortages such as high surgical complications incidence, especially in cases that.