LINC00173 silence and estrone supply suppress ER+ breast cancer by estrogen receptor α degradation and LITAF activation.

Persistent activation of estrogen receptor alpha (ERα)-mediated estrogen signaling plays a pivotal role in driving the progression of estrogen receptor positive (ER+) breast cancer (BC). In the current study, LINC00173, a long non-coding RNA, was found to bind both ERα and lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNFα) factor (LITAF), then cooperatively to inhibit ERα protein degradation by impeding the nuclear export of ERα. Concurrently, LITAF was found to attenuate TNFα transcription after binding to LINC00173, and this attenuating transcriptional effect was quite significant under lipopolysaccharide stimulation. Distinct functional disparities between estrogen subtypes emerge, with estradiol synergistically promoting ER+ BC cell growth with LINC00173, while estrone (E1) facilitated LITAF-transcriptional activation. In terms of therapeutic significance, silencing LINC00173 alongside moderate addition of E1 heightened TNFα and induced apoptosis, effectively inhibiting ER+ BC progression.

Metabonomics and pharmacodynamics studies of Gentiana radix and wine-processed Gentiana radix in damp-heat jaundice syndrome rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Gentiana radix (GR) and wine-processed Gentiana radix (WGR) have been commonly used in folk medicine for the treatment of bile or liver disorders, including jaundice, hepatitis, swelling and inflammation for thousands of years. However, the therapeutic effects of gentian root (GR) and wine-made gentian root (WGR) treatment on damp-heat jaundice syndrome (DHJS) have not been studied in animal experiments. AIM OF THE STUDY: This study aimed to investigate the protective effects and mechanisms of GR and WGR on DHJS in rats. MATERIALS AND METHODS: In a high-fat and high-sugar diet in a humidified hot environment, hepatic injury induced by giving alpha-naphthalene isothiocyanate (ANIT) in rats were used as a DHJS model. Histological analysis, enzyme-linked immunosorbent assay (ELISA), PCR analysis, and metabolomics were used to elucidate the mechanism of GR and WGR for DHJS. RESULTS: The results indicated that GR and WGR affected DHJS by inhibiting the release of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), direct bilirubin (D-BIL), total bilirubin (TBIL), total bile acid (TBA), malondialdehyde (MDA), glutathione S-transferase (GST) (P < 0.05). In addition, they significantly reduced the gene expression levels of Na+/taurocholate cotransporting polypeptide (NTCP), bile salt export pump (BESP), multidrug resistance-associated protein 2 (MRP2) and multidrug resistance-associated protein 3 (MRP3) (P < 0.05). The WGR group improved the above function indicators better than the GR group. GR and WGR could restore 11 potential biomarkers in rats with DHJS tended to return to normal levels, these biomarkers were involved in arachidonic acid metabolism, steroid hormone biosynthesis, biosynthesis of unsaturated fatty acids, porphyrin and chlorophyll metabolism, retinol metabolism, arginine biosynthesis. The results of the metabolic pathway showed that WGR was significantly better than GR in the improvement of porphyrin and chlorophyll metabolism. CONCLUSIONS: These findings suggest that treatment with GR and WGR has a beneficial effect on DHJS in rats, the major mechanisms may be involved in improving functional indicators of the body and endogenous metabolism, and WGR is more effective than GR. It provides important evidence for the clinical application of GR and WGR in the treatment of DHJS.

LRRC8A as a central mediator promotes colon cancer metastasis by regulating PIP5K1B/PIP2 pathway.

Colorectal cancer (CRC) has been the third most common malignancy and the second cause of cancer-related mortality. As the core of volume-sensitive chloride currents, leucine-rich repeat-containing 8A (LRRC8A) contributes to tumor progression but is not consistent, especially for whom the roles in colon carcinoma metastasis were not fully elucidated. Herein, LRRC8A proteins were found highly expressed in hematogenous metastasis from human colorectal cancer samples. The oxaliplatin-resistant HCT116 cells highly expressed LRRC8A, which was related to impaired proliferation and enhanced migration. The over-expressed LRRC8A slowed proliferation and increased migration ex vivo and in vivo. The elevated LRRC8A upregulated the focal adhesion, MAPK, AMPK, and chemokine signaling pathways via phosphorylation and dephosphorylation. Inhibition of LRRC8A impeded the TNF-α signaling cascade and TNF-α-induced migration. LRRC8A binding to PIP5K1B regulated the PIP2 formation, providing a platform for LRRC8A to mediate cell signaling transduction. Importantly, LRRC8A self-regulated its transcription via NF-κB1 and NF-κB2 pathways and the upregulation of NIK/NF-κB2/LRRC8A transcriptional axis was unfavorable for colon cancer patients. Collectively, our findings reveal that LRRC8A is a central mediator in mediating multiple signaling pathways to promote metastasis and targeting LRRC8A proteins could become a potential clinical biomarker-driven treatment strategy for colon cancer patients.

Exercise ameliorates lipid droplet metabolism disorder by the PLIN2-LIPA axis-mediated lipophagy in mouse model of non-alcoholic fatty liver disease.

Excessive hepatic lipid droplets (LDs) accumulation-induced lipid metabolism disorder contributes to the development of non-alcoholic fatty liver disease (NAFLD). Exercise is a promising therapeutic strategy for NAFLD. However, the mechanism by which exercise ameliorates NAFLD through regulating the catabolism of hepatic LDs remains unclear. In the present study, we investigated the effect of perilipin2 (PLIN2)-lysosomal acid lipase (LIPA) axis mediating exercise-triggered lipophagy in a high-fat diet (HFD)-induced NAFLD mouse model. Our results showed that exercise could reduce HFD-induced hepatic LDs accumulation and change the expression of lipolysis-related enzymes. Moreover, exercise upregulated the expression of microtubule associated protein 1 light chain 3 (LC3) and autophagy-related proteins, and downregulated sequestosome 1 (P62) expression and promoted autophagosomes formation. Interestingly, exercise downregulated PLIN2 expression, upregulated LIPA expression, and increased the activity of hepatic LIPA and serum levels of LIPA in the NAFLD mouse model. Further mechanistic studies demonstrated that adenosine monophosphate-activated protein kinase (AMPK) activator-5-Aminoimidazole-4-carboxamide ribonucleoside (AICAr) treatment significantly increased mRNA levels and protein expression of LIPA and LC3II and decreased levels of PLIN2 and P62 in palmitic acid (PA)-treated HepG2 cells. PLIN2 silencing and LIPA overexpression notably increased the mRNA level and protein expression of LC3II and decreased the mRNA level and protein expression of p62, respectively. In summary, our findings reveal novel insights into the effect of exercise on improving lipid droplet metabolism disorder in NAFLD. Enhancing the PLIN2-LIPA axis-mediated lipophagy may be one of the key mechanisms involved in NAFLD alleviation by exercise.

Upregulation of exosome secretion from tumor-associated macrophages plays a key role in the suppression of anti-tumor immunity.

Macrophages play a pivotal role in tumor immunity. We report that reprogramming of macrophages to tumor-associated macrophages (TAMs) promotes the secretion of exosomes. Mechanistically, increased exosome secretion is driven by MADD, which is phosphorylated by Akt upon TAM induction and activates Rab27a. TAM exosomes carry high levels of programmed death-ligand 1 (PD-L1) and potently suppress the proliferation and function of CD8+ T cells. Analysis of patient melanoma tissues indicates that TAM exosomes contribute significantly to CD8+ T cell suppression. Single-cell RNA sequencing analysis showed that exosome-related genes are highly expressed in macrophages in melanoma; TAM-specific RAB27A expression inversely correlates with CD8+ T cell infiltration. In a murine melanoma model, lipid nanoparticle delivery of small interfering RNAs (siRNAs) targeting macrophage RAB27A led to better T cell activation and sensitized tumors to anti-programmed cell death protein 1 (PD-1) treatment. Our study demonstrates tumors use TAM exosomes to combat CD8 T cells and suggests targeting TAM exosomes as a potential strategy to improve immunotherapies.

The Role of Green Tea on the Regulation of Gut Microbes and Prevention of High-Fat Diet-Induced Metabolic Syndrome in Mice.

Green tea is a popular non-alcoholic beverage consumed worldwide and has been shown to be beneficial for human health. However, further exploration is needed to fully understand its function in reducing obesity and regulating gut microbes. Here, we investigated the modulatory effects of green tea and its functional components on high-fat diet (HF)-induced metabolic alterations and gut microbiota in obese mice. Our results showed that 1%, 2%, and 4% of green tea promotes weight loss, with the 2% and 4% groups exhibiting distinct gut microflora clusters compared to the HF group. These results were comparable to those observed in the tea polyphenols (TPP)-treated group, suggesting the TPP in green tea plays a crucial role in body weight control and gut microbiota regulation. Additionally, 32 bacteria were identified as potential obesity markers via 16S rRNA gene sequencing. The 16SrDNA gene is a chromosomal gene present in all bacterial species, highly conserved in structure and function, that can reflect the differences between different taxa. The 16S rRNA-based analysis revealed that Akkermansia, a gut-beneficial bacteria, significantly increased in the TPP group.

Patient-derived melanoma organoid models facilitate the assessment of immunotherapies.

BACKGROUND: Only a minority of melanoma patients experience durable responses to immunotherapies due to inter- and intra-tumoral heterogeneity in melanoma. As a result, there is a pressing need for suitable preclinical models to investigate resistance mechanisms and enhance treatment efficacy. METHODS: Here, we report two different methods for generating melanoma patient-derived organoids (MPDOs), one is embedded in collagen gel, and the other is inlaid in Matrigel. MPDOs in Matrigel are used for assessing the therapeutic effects of anti-PD-1 antibodies (αPD-1), autochthonous tumor infiltrating lymphocytes (TILs), and small molecule compounds. MPDOs in collagen gel are used for evaluating the chemotaxis and migratory capacity of TILs. FINDING: The MPDOs in collagen gel and Matrigel have similar morphology and immune cell composition to their parental melanoma tissues. MPDOs show inter- and intra-tumoral heterogeneity and contain diverse immune cells such as CD4+, CD8+ T, Treg, CD14+ monocytic, CD15+, and CD11b+ myeloid cells. The tumor microenvironment (TME) in MPDOs is highly immunosuppressive, and the lymphoid and myeloid lineages express similar levels of PD-1, PD-L1, and CTLA-4 as their parental melanoma tissues. Anti-PD-1 antibodies (αPD-1) reinvigorate CD8+ T cells and induce melanoma cell death in the MPDOs. TILs expanded by IL-2 and αPD-1 show significantly lower expression of TIM-3, better migratory capacity and infiltration of autochthonous MPDOs, and more effective killing of melanoma cells than TILs expanded by IL-2 alone or IL-2 with αCD3. A small molecule screen discovers that Navitoclax increases the cytotoxicity of TIL therapy. INTERPRETATION: MPDOs may be used to test immune checkpoint inhibitors and cellular and targeted therapies. FUNDING: This work was supported by the NIH grants CA114046, CA261608, CA258113, and the Tara Miller Melanoma Foundation.

HRS phosphorylation drives immunosuppressive exosome secretion and restricts CD8+ T-cell infiltration into tumors.

The lack of tumor infiltration by CD8+ T cells is associated with poor patient response to anti-PD-1 therapy. Understanding how tumor infiltration is regulated is key to improving treatment efficacy. Here, we report that phosphorylation of HRS, a pivotal component of the ESCRT complex involved in exosome biogenesis, restricts tumor infiltration of cytolytic CD8+ T cells. Following ERK-mediated phosphorylation, HRS interacts with and mediates the selective loading of PD-L1 to exosomes, which inhibits the migration of CD8+ T cells into tumors. In tissue samples from patients with melanoma, CD8+ T cells are excluded from the regions where tumor cells contain high levels of phosphorylated HRS. In murine tumor models, overexpression of phosphorylated HRS increases resistance to anti-PD-1 treatment, whereas inhibition of HRS phosphorylation enhances treatment efficacy. Our study reveals a mechanism by which phosphorylation of HRS in tumor cells regulates anti-tumor immunity by inducing PD-L1+ immunosuppressive exosomes, and suggests HRS phosphorylation blockade as a potential strategy to improve the efficacy of cancer immunotherapy.

[Short-term effectiveness of one-stage anterior and posterior cruciate ligaments and posterolateral complex reconstruction combined with medial collateral ligament repair for KD- â £ knee dislocation].

OBJECTIVE: To investigate the short-term effectiveness of one-stage anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), and posterolateral complex (PLC) reconstruction combined with medial collateral ligament repair for KD-Ⅳ knee dislocation. METHODS: Between January 2018 and June 2020, 9 patients with KD-Ⅳ knee dislocation were treated. Of 9 cases, 7 were male and 2 were female with an average age of 32.3 years (range, 23-43 years). The knee dislocation was caused by falling from height in 6 cases and traffic accident in 3 cases. The injury located at left knee in 2 cases and right knee in 7 cases. The time from injury to operation was 14-24 days, with an average of 19 days. The preoperative International Knee Joint Documentation Committee (IKDC) score was 45.6±4.2, Lysholm score was 42.4±7.0, and the knee joint active flexion range of motion was (75.2±12.3)°. The posterior drawer test, pivot-shift test, Dial test, and 0° valgus stress test were all positive. Under arthroscopy, PCL was reconstructed with the autologous tendons, ACL with allogeneic Achilles tendon, PLC with the allogeneic anterior tibial tendon by Larson enhanced reconstruction method, and MCL was repaired with anchor or simple suture. RESULTS: The operation time was 2-3 hours (mean, 2.5 hours). All incisions healed by first intention after operation. All patients were followed up12-25 months (mean, 16.1 months). After operation, 2 cases developed knee flexion disorder and pain, and 1 case had knee joint stiffness. At last follow-up, the IKDC score was 76.9±7.4, the Lysholm score was 81.6±6.4, and the knee active flexion range of motion was (122.9±7.2)°, all of which significantly improved when compared with preoperative ones ( P<0.05). During follow-up, there was no failure of the grafts. At last follow-up, there were significant differences in the posterior drawer test, pivot-shift test, Dial test, and 0° valgus stress test between pre- and post-operation ( P<0.05). The imaging review showed that the positions of the bone tunnels were satisfactory, the reconstructed ACL, PCL, and PLC structures were continuous, and MCL insertions were restored. CONCLUSION: One-stage ACL, PCL, and PLC reconstruction combined with MCL repair to treat KD-Ⅳ knee dislocation can effectively restore knee joint stability, improve joint laxity, and improve joint movement.

Costimulation of γδTCR and TLR7/8 promotes Vδ2 T-cell antitumor activity by modulating mTOR pathway and APC function.

BACKGROUND: Gamma delta (γδ) T cells are attractive effector cells for cancer immunotherapy. Vδ2 T cells expanded by zoledronic acid (ZOL) are the most commonly used γδ T cells for adoptive cell therapy. However, adoptive transfer of the expanded Vδ2 T cells has limited clinical efficacy. METHODS: We developed a costimulation method for expansion of Vδ2 T cells in PBMCs by activating γδ T-cell receptor (γδTCR) and Toll-like receptor (TLR) 7/8 using isopentenyl pyrophosphate (IPP) and resiquimod, respectively, and tested the functional markers and antitumoral effects in vitro two-dimensional two-dimensional and three-dimensional spheroid models and in vivo models. Single-cell sequencing dataset analysis and reverse-phase protein array were employed for mechanistic studies. RESULTS: We find that Vδ2 T cells expanded by IPP plus resiquimod showed significantly increased cytotoxicity to tumor cells with lower programmed cell death protein 1 (PD-1) expression than Vδ2 T cells expanded by IPP or ZOL. Mechanistically, the costimulation enhanced the activation of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB/Akt)-the mammalian target of rapamycin (mTOR) pathway and the TLR7/8-MyD88 pathway. Resiquimod stimulated Vδ2 T-cell expansion in both antigen presenting cell dependent and independent manners. In addition, resiquimod decreased the number of adherent inhibitory antigen-presenting cells (APCs) and suppressed the inhibitory function of APCs by decreasing PD-L1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression in these cells during in vitro Vδ2 T-cell expansion. Finally, we showed that human Vδ2 T cells can be expanded from PBMCs and spleen of humanized NSG mice using IPP plus resiquimod or ZOL, demonstrating that humanized mice are a promising preclinical model for studying human γδ T-cell development and function. CONCLUSIONS: Vδ2 T cells expanded by IPP and resiquimod demonstrate improved anti-tumor function and have the potential to increase the efficacy of γδ T cell-based therapies.

Small extracellular vesicles-based cell-free strategies for therapy.

Small extracellular vesicles (sEVs) are extracellular nanovesicles that contain bioactive proteins, lipids, RNA, and DNA. A variety of biological process is regulated with sEVs. sEVs are an intercellular messenger regulating recipient cell function and play a role in disease initiation and progression. sEVs derived from certain cells, such as mesenchymal stem cells and immune cells, have the potential for clinical therapy as they possess the characteristics of their parental cells. With better understanding of sEVs biogenesis, their transportation properties, extended circulatory capability, and exceptional biocompatibility, sEVs emerge as a potential therapeutic tool in the clinic. Here, we summarize applications of sEVs-based therapies in different diseases and current knowledge about the strategies in bioengineered sEVs, as well as the challenges for their use in clinical settings.

Targeting regulatory T cells for immunotherapy in melanoma.

Regulatory T cells (Tregs) are essential in the maintenance of immunity, and they are also a key to immune suppressive microenvironment in solid tumors. Many studies have revealed the biology of Tregs in various human pathologies. Here we review recent understandings of the immunophenotypes and suppressive functions of Tregs in melanoma, including Treg recruitment and expansion in a tumor. Tregs are frequently accumulated in melanoma and the ratio of CD8+ T cells versus Tregs in the melanoma is predictive for patient survival. Hence, depletion of Tregs is a promising strategy for the enhancement of anti-melanoma immunity. Many recent studies are aimed to target Tregs in melanoma. Distinguishing Tregs from other immune cells and understanding the function of different subsets of Tregs may contribute to better therapeutic efficacy. Depletion of functional Tregs from the tumor microenvironment has been tested to induce clinically relevant immune responses against melanomas. However, the lack of Treg specific therapeutic antibodies or Treg specific depleting strategies is a big hurdle that is yet to be overcome. Additional studies to fine-tune currently available therapies and more agents that specifically and selectively target tumor infiltrating Tregs in melanoma are urgently needed.

NaYF4:Yb/Tm@SiO2-Dox/Cur-CS/OSA nanoparticles with pH and photon responses.

Stimulus-triggered drug delivery systems (DDSs) based on lanthanide-doped upconversion nanoparticles (UCNPs) have attracted significant attention for treating cancers due to their merits of high drug availability, precisely controlled drug release, and low side-effects. However, such DDSs usually exhibit a single stimulus-response, which may limit the efficiency of cancer treatment. To extend response types in a single DDS, we construct NaYF4:Yb/Tm@SiO2-doxorubicin (Dox)/curcumin (Cur)-chitosan (CS)/2-Octen-1-ylsuccinic anhydride (OSA) nanoparticles with core-shell structures. Our method is based on the exploration of the synergistic effect of UCNPs and multiple drugs. In particular, the NaYF4:Yb/Tm is used to convert near-infrared light to visible light, activating Cur photosensitizers to produce singlet oxygen for photodynamic therapy, while CS/OSA responds to a low pH environment to release cancer drugs, including Dox and Cur for chemotherapy through breaking a free carboxyl group. The results show that the UCNPs with a 40 nm diameter, 23 nm thick mesoporous SiO2, and 19/1 mol% Yb3+/Tm3+concentrations could continuously release Dox and Cur at a pH value of 6.5 within 6 h after the excitation of a 980 nm-wavelength CW laser. Our study provides a promising approach for developing efficient DDSs for cancer treatment.

PPT1 inhibition enhances the antitumor activity of anti-PD-1 antibody in melanoma.

New strategies are needed to enhance the efficacy of anti-programmed cell death protein antibody (anti-PD-1 Ab) in cancer. Here, we report that inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of chloroquine derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti-PD-1 Ab in melanoma. The combination resulted in tumor growth impairment and improved survival in mouse models. Genetic suppression of core autophagy genes, but not Ppt1, in cancer cells reduced priming and cytotoxic capacity of primed T cells. Exposure of antigen-primed T cells to macrophage-conditioned medium derived from macrophages treated with PPT1 inhibitors enhanced melanoma-specific killing. Genetic or chemical Ppt1 inhibition resulted in M2 to M1 phenotype switching in macrophages. The combination was associated with a reduction in myeloid-derived suppressor cells in the tumor. Ppt1 inhibition by HCQ, or DC661, induced cyclic GMP-AMP synthase/stimulator of interferon genes/TANK binding kinase 1 pathway activation and the secretion of interferon-ß in macrophages, the latter being a key component for augmented T cell-mediated cytotoxicity. Genetic Ppt1 inhibition produced similar findings. These data provide the rationale for this combination in melanoma clinical trials and further investigation in other cancers.

A novel Adiponectin receptor (AdipoR) involved in regulating cytokines production and apoptosis of haemocytes in oyster Crassostrea gigas.

Adiponectin receptors (AdipoRs) comprise a seven-transmembrane domain-containing protein family, which specifically recognize adiponectin (APN) and play critical roles in the immunological and physiological processes in vertebrates. In the present study, a novel AdipoR is identified from oyster Crassostrea gigas (designated as CgAdipoR). The full-length cDNA of CgAdipoR is of 1209 bp encoding a polypeptide of 343 amino acids. There is an N-terminal domain, a Hly III domain, and a C-terminal domain in CgAdipoR. After the transfection of CgAdipoR, the level of intracellular Ca2+ into HEK293T cells increases significantly (1.36-fold, p < 0.05) after APN incubation. The mRNA transcripts of CgAdipoR are widely distributed in all the tested tissues, with the highest expression level in haemocytes (3.20-fold of that in hepatopancreas, p < 0.05). After lipopolysaccharide (LPS), Vibrio splendidus and polyinosinic-polycytidylic acid (poly (I:C)) stimulations, the mRNA expression of CgAdipoR in haemocytes is significantly up-regulated and reached the highest level at 24 h (15.07-fold, p < 0.01), 6 h (4.39-fold, p < 0.01) and 24 h (5.62-fold, p < 0.01) compared to control group, respectively. After CgAdipoR is interfered by specific CgAdipoR-dsRNA, the expression level of interleukins (CgIL17-1, CgIL17-2, CgIL17-3 and CgIL17-5) in haemocytes decreases significantly (p < 0.01) at 24 h post LPS stimulation, while the expression level of CgTNF-1 increases significantly (1.68-fold, p < 0.01), compared to that in the dsEGFP group. In CgAdipoR dsRNA-injected oysters, the mRNA expressions of anti-apoptotic B-cell lymphoma-2 (Bcl-2) in haemocytes significantly decreases at 24 h after LPS challenge, which is (0.58-fold, p < 0.05) of that in dsEGFP-injected oysters, while the apoptotic rate of haemocytes is significantly up-regulated (1.93-fold of that in dsEGFP group, p < 0.05). These results collectively suggest that CgAdipoR plays an important role in the immune response of oysters by regulating the expressions of inflammatory cytokines and haemocyte apoptosis.

Characterization of the First Genome of Porcine mastadenovirus B (HNU1 Strain) and Implications on Its Lymphoid and Special Origin.

Porcine adenoviruses (PAdVs) are classified into three species, PAdV-A, PAdV-B, and PAdV-C. The genomes of PAdV-A and PAdV-C have been well characterized. However, the genome of PAdV-B has never been completely sequenced, and the epidemiology of PAdV-B remains unclear. In our study, we have identified a novel strain of PAdV-B, named PAdV-B-HNU1, in porcine samples collected in China by viral metagenomic assay and general PCR. The genome of PAdV-B-HNU1 is 31,743 bp in length and highly similar to that of California sea lion adenovirus 1 (C. sea lion AdV-1), which contains typical mastadenoviral structures and some unique regions at the carboxy-terminal end. Especially, PAdV-B-HNU1 harbors a dUTPase coding region not clustering with other mastadenoviruses except for C. sea lion AdV-1 and a fiber coding region homologous with galectin 4 and 9 of animals. However, the variance of GC contents between PAdV-B-HNU1 (55%) and C. sea lion AdV-1 (36%) indicates their differential evolutionary paths. Further epidemiologic study revealed a high positive rate (51.7%) of PAdV-B-HNU1 in porcine lymph samples, but low positive rates of 10.2% and 16.1% in oral swabs and rectal swabs, respectively. In conclusion, this study characterized a novel representative genome of a lymphotropic PAdV-B with unique evolutionary origin, which contributes to the taxonomical and pathogenic studies of PAdVs.

An activating transcription factor 6 beta (ATF6ß) regulates apoptosis of hemocyte during immune response in Crassostrea gigas.

The homeostasis of immune cells during immune response is vital for hosts to defend against invaders. Activating transcription factor 6 (ATF6) is an important transcription factor in the unfolded protein response (UPR) to maintaining cellular homeostasis. In the present study, one ATF6 homologue was identified from Pacific oyster Crassostrea gigas (designated as CgATF6ß). The full length cDNA of CgATF6ß was of 2645 bp with a 1596 bp open reading frame (ORF) encoding a polypeptide of 531 amino acids. The deduced amino acid sequence of CgATF6ß was predicted to contain a transmembrane region, a conserved basic leucine zipper (bZIP) domain, a site 1 protease cleavage site, a site 2 protease cleavage site, and a Golgi localization signal. CgATF6ß mRNA was constitutively expressed in hemocytes, gill, mantle, gonad, hepatopancreas and labial palp, with a slightly higher expression level in muscle (2.45-fold of that in gill, p < 0.05). After oysters were challenged with Vibrio splendidus, the mRNA expression levels of CgATF6ß in hemocytes were significantly up-regulated at 3 h (2.68-fold of that in seawater group, p < 0.01) and peaked at 12 h (3.14-fold of that in seawater group, p < 0.01). The endogenic CgATF6ß protein was mainly located in the cytoplasm of oyster hemocytes, and it was significantly transported into the nuclei of hemocytes at 1.5 h after the challenge with V. splendidus. After an injection with CgATF6ß dsRNA, the mRNA expression of CgATF6ß was knocked down to 0.26-fold of that in dsGFP group (p < 0.01). In CgATF6ß dsRNA-injected oysters, the mRNA expressions of glucose-regulated protein 78 (GRP78), calnexin (CNX) and anti-apoptotic B-cell lymphoma-2 (Bcl-2) in hemocytes were significantly decreased at 12 h after V. splendidus challenge, which were 0.65-fold (p < 0.01), 0.54-fold (p < 0.01) and 0.17-fold (p < 0.01) of that in dsGFP-injected oysters, while the apoptotic rate of hemocytes was significantly up-regulated (1.97-fold of that in dsGFP group, p < 0.05). Collectively, these results suggested that CgATF6ß was involved in apoptosis inhibition of oyster hemocytes upon V. splendidus challenge by regulating the expression of CgGRP78, CgCNX and CgBcl-2.

Protein kinase-like ER kinase (PERK) regulates autophagy of hemocytes in antiviral immunity of Pacific oyster Crassostrea gigas.

The maintenance of cellular homeostasis is an important process for successful immune defense against pathogenic invading, in which unfolded protein response (UPR) pathway regulates endoplasmic reticulum (ER) homeostasis upon exposure to environmental changes. Protein kinase-like ER kinase (PERK) is an important ER stress sensor to be activated during the UPR to regulate cells homeostasis. In the present study, one PERK homologue was identified from Pacific oyster Crassostrea gigas (designated as CgPERK). The cDNA of CgPERK was of 4307 bp with a 3174 bp open reading frame (ORF) encoding a polypeptide of 1058 amino acids. There were two conserved protein kinases domains and two conserved autophosphorylation sites at Lys618 and Thr980 in CgPERK. The mRNA transcript of CgPERK was constitutively expressed in all the tested tissues including mantle, adductor muscle, hepatopancreas, gill, gonad and labial palp with the highest expression level in hemocytes (31.15-fold compared to mantle). The CgPERK protein was found to be located mainly in the cytoplasm of hemocytes. The mRNA expression level of CgPERK in hemocytes was significantly up-regulated and reached the highest level (5.25-fold compared to seawater group, p < 0.01) at 48 h after the oysters were stimulated with poly(I: C). Meanwhile, a significant increase of fluorescence autophagosome spots in hemocytes was also observed at 36 h post stimulation. After the mRNA expression of CgPERK was knocked down (0.49-fold compared to dsGFP group, p < 0.01) by injection of CgPERK dsRNA, the mRNA expression of autophagy related 12 (ATG12) in hemocytes was significantly decreased at 12 h post poly(I: C) stimulation, which was 0.53-fold (p < 0.01) compared to dsGFP-injected oysters. When the CgPERK was inhibited by its inhibitor GSK2656157 stimulation, the autophagosomes rate of hemocytes decreased significantly at 12 h post poly(I: C) stimulation, which was 0.34-fold (p < 0.01) of that of DMSO group. Collectively, these results suggested that CgPERK, as an UPR initiator, was involved in autophagosomes formation upon poly(I: C) stimulation by regulating the expression of ATG12, and ER stress stimulated the autophagosome formation on an ATG protein-dependent manner in oysters.

The Dicer from oyster Crassostrea gigas functions as an intracellular recognition molecule and effector in anti-viral immunity.

Dicer, as a member of ribonuclease III family, functions in RNA interference (RNAi) pathway to direct sequence-specific degradation of cognate mRNA. It plays important roles in antiviral immunity and production of microRNAs. In the present study, a Dicer gene was identified from oyster Crassostrea gigas, and its open reading frame (ORF) encoded a polypeptide (designed as CgDicer) of 1873 amino acids containing two conserved ribonuclease III domains (RIBOc) and a double-stranded RNA-binding motif (DSRM). The deduced amino acid sequence of CgDicer shared identities ranging from 18.5% to 46.6% with that of other identified Dicers. The mRNA transcripts of CgDicer were detectable in all the examined tissues of adult oysters, with the highest expression in hemocytes (11.21 ±â€¯1.64 fold of that in mantle, p < 0.05). The mRNA expression level of CgDicer in hemocytes was significantly up-regulated (36.70 ±â€¯11.10 fold, p < 0.01) after the oysters were treated with double-stranded RNA (dsRNA). In the primarily cultured oyster hemocytes, the mRNA transcripts of CgDicer were significantly induced at 12 h after the stimulation with poly(I:C), which were 2.04-fold (p < 0.05) higher than that in control group. Immunocytochemistry assay revealed that CgDicer proteins were mainly distributed in the cytoplasm of hemocytes. The two most important functional domains of CgDicer, DSRM and RIBOc, were recombinant expressed in Escherichia coli transetta (DE3), and the recombinant DSRM protein displayed significantly binding activity to dsRNA and poly(I:C) in vitro, while the recombinant RIBOc protein exhibited significantly dsRNase activity to cleave dsRNA in vitro. These results collectively suggested that CgDicer functioned as either an intracellular recognition molecule to bind dsRNA or an effector with ribonuclease activity, which might play a crucial role in anti-viral immunity of oyster.