Extracellular adenosine triphosphate (eATP) and its main metabolite adenosine (ADO) constitute an intrinsic part of immunological network in tumor immunity. The concentrations of eATP and ADO in tumor microenvironment (TME) are controlled by ectonucleotidases, such as CD39 and CD73, the major ecto-enzymes expressed on immune cells, endothelial cells and cancer cells. Once accumulated in TME, eATP boosts antitumor immune responses, while ADO attenuates immunity against tumors. eATP and ADO, like yin and yang, represent two opposite aspects from immune-activating to immune-suppressive signals. Here we reviewed the functions of eATP and ADO in tumor immunity and attempt to block eATP hydrolysis, ADO formation and their contradictory effects in tumor models, allowing the induction of effective anti-tumor immune responses in TME. These attempts documented that therapeutic approaches targeting eATP/ADO metabolism and function may be effective methods in cancer therapy. Adenosine triphosphate, also known as ATP, is actively released to the extracellular environment in response to tissue damage and cellular stress. The concentration of cellular ATP is 3 to 10 mM. However, the concentration of extracellular ATP (eATP) is only about 10 nM . CD39/ecHypoxia, acute and chronic inflammation, platelet aggregation and anticancer therapies induce tumor cells death and metabolic changes, leading to the increased concentrations of eATP and ADO in tumor microenvironment (TME) . The accCD39, CD73, ATP and ADO have been proposed as therapeutic targets in oncology. Therapeutic strategies have been developed to modulate eATP/ADO metabolism and related anti-cancer immune responses. In this review, we focused on the effects and related mechanisms of eATP and ADO in tumor immunity, and then discussed the possibility of targeting purnergic signaling in cancer therapy.Increased eATP in TME exhibits multiple functions in combination with ATP receptors. There are two P2 receptor families: P2X receptors  which are ATP-gated ion channels and P2Y receptors which are G protein-coupled receptors  . Differe2+ and Na+ increase. In hematology malignancies and solid tumors, the activation of P2X7R not only increased intracellular Ca2+ and Na+ but also decreased intracellular K+ [P2X1, 4, 5 receptors were found to express on multiple tumor cells and host cells, especially in hematological malignancies, including acute lymphocytic leukemia, acute myeloid leukemia and multiple myeloma , 15. Thelular K+ . The rollular K+ .2+ release. Three Gi/o-coupled subtypes  mainly inhibit adenylyl cyclase to regulate cyclic AMP (cAMP)/protein kinase A (PKA) pathway [Different P2YR subtypes are activated by different nucleotides. ATP is the preferred ligand only for P2Y11R. For other P2YRs, pyrimidine nucleotides and ADP are more potent agonists. The expression of P2Y11R was found on Huh7 and HepG2 hepatocellular carcinoma cells, hematopoietic stem cells (HSCs) and dendritic cells (DCs) . P2YRs a pathway , 17.Contemporary perspectives highlight the roles of TME in tumor growth and therapy. Increasing evidence indicated that targeting TME could complement traditional treatment and improve therapeutic outcomes for malignancies. As a major component, eATP in TME is at a high concentration , 19. TheTumor-associated macrophages (TAMs) are key members in TME. Macrophages (Mø) play different roles in tumor growth depending on their polarization to classically-activated macrophages ) or alternatively-activated macrophages  . M1s con+CD39+ DCs [DCs are the most important antigen presenting cells (APCs) playing a pivotal role in tumor immunity. eATP not only affects DCs maturation and migration, but also contributes to the antigen endocytosis, cytokine production and ectonucleotidase expression. eATP induces up-regulation of activation and maturation markers  and major histocompatibility complex (MHC)-II) in human peripheral blood mononuclear cell (PBMC)-derived immature DCs, possibly by stimulation of P2Y11R and P2X7R . MaturatD39+ DCs .2+ levels. All P2XRs, with the exception of P2X5, can facilitate entry of Ca2+ in response to stimulation by eATP, suggesting that P2X receptors regulate T-cell activation by increasing intracellular Ca2+ [2+ entry, nuclear factors of activated T cells (NFAT) activation and T-cell activation [+ and CD8+ T cells proliferation. eATP also acts as a potent chemoattractant for T cells through P2YRs signaling [+ T cell populations through the activation of P2X7R [Recent studies have shown that eATP regulates T cell activation, cytokine production and lymphocyte metabolism , 40. P2Xlar Ca2+ . P2XRs alar Ca2+ . In conttivation , 43–45. tivation . P2X7R aignaling . Extrace+, CD8+ lymphocytes and regulatory T cells (Tregs). Elena De Marchi et al. [+ cells and an increased number of Tregs, overexpressing the fitness markers OX40, PD-1, and CD73. However, systemic administration of the P2X7 blocker A740003 in wild-type mice showed increased CD4+ effector cells and decreased their expression of CD39 and CD73. These data illustrated that P2X7 receptor was a key determinant of TME composition.Extracellular ATP and its receptor P2X7 also exert a pivotal influence on cancer growth through modulating lymphocytes infiltration in TME, including CD4i et al.  reported+ T cells to Tregs after TCR stimulation [+ T cells to Th17 cells [In addition, eATP is a modulator for T cells function transformation. Antagonists of P2XRs can promote the cell-autonomous conversion of naive CD4mulation . The actmulation . eATP re17 cells .The participation of eosinophils in innate Th2-type immune responses is mediated by eATP . eATP alSimilar to eATP, the concentration of ADO is at a low level in extracellular media (lower than 1 μM). However, its concentration notably increases under many metabolically stressful conditions, especially in TME (over 100 μM) . The bulADO affects tumor progression directly through binding on its specific receptors expressed on cancer cells. Extracellular ADO in TME chronically activates A2BR expressed on cancer cells to suppress Ras-related protein (Rap)-1B prenylation and signaling, resulting in cell–cell contact reduction and cell scattering promotion. The major effect of A3R activation is to promote angiogenesis .Extracellular ADO in TME is also an important factor controlling antitumor immunity, conditioning both innate and adaptive immune responses Fig. .Fig. 2Ex+ T cells anti-tumor response but also reduces the population of Tregs [+ T cells differentiation to Tregs [+Gr-1+ DC subset [+ T (Tc1) cells, is latterly recognized as IL-17-producing CD8+ T (Tc17) cells. It is demonstrated that Tc17 cells stably sustain the stem-cell-like program and reject the differentiation to Tc1-like cells with the help of ADO, consequently activating A2AR, which turns Tc17 cells to long-lived memory T cells to allow the immunosuppressive properties in tumor progression [ADO is emerging as an important negative regulator of T-cell function for inhibition of mobility, migration and adhesion –61. The of Tregs . A2AR anof Tregs . For Treto Tregs , but alsto Tregs . Antagonto Tregs . Moreoveto Tregs . Meanwhito Tregs . A2AR acto Tregs . A2BR isC subset , 70. Anogression .+ and CD8+ T cells through ADO [+ T-cell responses [high B cells while A2BR activation is critically required for IL-10 production [Abundant expression of CD39 and CD73 on human B cells facilitates an efficient accumulation of ATP-generated ADO, thus contributing to T cell-B cell interactions . B cellsough ADO  and theyesponses . Functiooduction .2+ transients resulting in actin polymerization and chemotaxis, but also increases co-stimulatory molecules expression [+ T cells in a cAMP-dependent manner [ADO, an important modulator of DCs functions, has significant effects on DCs activation, maturation, as well as cytokine production. A1R, A2AR and A3R are expressed by human monocyte-derived DCs. For immature DCs, ADO not only induces Capression . For matpression . ADO is pression  and muripression . Howeverpression . ADO-cont manner . DCs frot manner . Moreovet manner . TherefoThe amount of tumor-infiltrating MCs has a close relationship with tumor aggressiveness and dissemination . A2AR, AFor NK cells, which are a type of innate immune cells, increased levels of adenosine in tumor environment inhibit the lytic activity of NK cells via binding to A2A receptors . A2AR deIncreased ADO concentration could also induce Mø recruitment in TME. Mø plays various roles in an ADO-dependent manner . A1R, A2Given the yin and yang function of eATP and ADO in tumor immunity, targeting their signaling pathways and their metabolism has attracted more and more attention in tumor therapy.Among P2Rs, P2X7R is the most potential candidate in anticancer therapy . P2X7R wP2Y12R is another important member in P2R family. Under ATP and ADP stimulation, P2Y1R initiates platelet activation followed by the ADP-P2Y12R-mediated amplification . P2Y12R Strategies targeting ADO receptors appear to be attracting approaches to block ADO immunosuppressive effects and boost anti-tumor immunity. In a sarcoma model and the poorly immunogenic LL-LCMV tumor model, pharmacologic blockade of A2AR enhanced T cell mediated tumor regression . The abiConsidering critical roles of CD39/CD73 in ATP/ADO metabolism, attempts to block CD39/CD73 activity have been tried in tumor therapy. When compared to CD39 over-expressing mice, colorectal cancer cells and melanoma cells in CD39 deficient models showed significantly slower hepatic and pulmonary metastases . InhibitPurinergic signaling has emerged as a novel mechanism to modulate anticancer immunity. The accumulations of eATP and ADO in TME play yin and yang functions in tumor immunity through their specific receptors. The metabolism from eATP to ADO is accomplished mainly through CD39 and CD73, which expressed on immune cells, endothelial cells as well as cancer cells in TME. Drugs and biological approaches blocking eATP hydrolysis, ADO formation and their effects have the potential to overcome tumor immunosuppression, allowing the induction of effective anti-tumor immune responses. This strategy could also be a new option to improve the efficacy of cytotoxic agents and checkpoint blockade inhibitors. However, given the multifaceted effects of eATP and ADO in TME, where tumor cells, host immune cells and stromal cells communicate with each other through complicated mechanisms, how to select the most suitable therapy target is still a challenge to be overcome.


In our efforts to understand the systemic features of tumors, the importance of animal models is increasing due to the recent growth in the development of immunotherapy and targeted therapies. This has resulted in increased attention towards tumor animal models using C57BL/6N, which are mainly used in immunological studies. In this study, the C57BL/6NKorl stock and two other commercial stocks (C57BL/6NA and C57BL/N6B) are evaluated by comparing the occurrence of tumors using the syngeneic model; furthermore, we compare the response to anti-cancer drugs in the syngeneic model by evaluating survival, growth of tumors, proliferation and molecular biology analysis. In the syngeneic model using LLC (Lewis lung carcinoma) cells, the survival of mice and growth of the tumor showed a better response in the C57BL/6NKorl stock, and was dependent on the cell concentration of the dosing tumor, as compared to the other C57BL/6N stocks. However, the Ki-67 staining showed only little difference in cell proliferation within the tumor tissue each mouse stocks. Comparing the sensitivity to anti-cancer drug by examining changes in growth, volume and weight revealed that cisplatin treatment for tumor-bearing C57BL/6NKorl was more dependent on concentration. The Ki-67 staining, however, showed no difference among the C57BL/6N stocks after cisplatin treatment. The expressions of p27 and p53 tumor suppressor proteins, caspase-3 and Bax showed dose-dependent increase after exposure to cisplatin, whereas the expression of Bcl-2 was reduced in a dose-dependent manner. Furthermore, the expressions of MMP-2 and VEGF involved in metastasis, as well as inflammatory genes IL-1β, IL-6 and IL-10, showed dose-dependent decrease in tumor tissue after cisplatin exposure. Differences observed among the C57BL/6N stocks were not significant. Taken together, our studies reveal that C57BL/6NKorl has the potential of being a useful biological resource established in Korea, as it does not differ from the two commercially available C57BL/6N stocks when considering response to tumor generation and sensitivity to anti-cancer drugs using the syngeneic tumor model.The online version of this article (10.1186/s42826-019-0015-z) contains supplementary material, which is available to authorized users. The global increase in cancer incidences is attributed to a rapidly aging population and numerous causes, including environmental pollutants. In Korea, cancer incidence and death rates have increased since 2002 due to advanced aging in the society, with social and economic losses ranging from 2000 to 2010 reaching $20,858 million [In general, animal model research is widely used in investigating the therapeutic efficacy and toxicity of chemicals and biological materials. Accumulated findings provide insights into the genetic mechanisms of cancer treatment and cancer prevention, malignant transformation and cancer progression. The goal of the cancer animal model is to closely reconstruct human malignancy, and apply it as a systemic tool with a greater likelihood of successfully translating the basic knowledge of the treatment in prevention of cancers in humans. The mouse has been a traditional animal model for basic and systemic cancer research, and is divided into genetically engineered models, syngeneic models and xenogeneic models –7. GenetThe C57BL/6 mouse is an inbred strain derived from the C57BL mouse, along with the lower mouse strain C57BL/10. Important line separation occurred in 1957 between C57BL/6 and C57BL/10. The well-known C57BL/6J and C57BL/6N were developed from C57BL/6 at the Jackson Institute and the National Institutes of Health in the 1940s and 1950s, respectively , 8. Sinc3)2Cl2), which exerts its anti-cancer effect by inhibiting cell division, was used in the treatment of various solid cancers including breast cancer, ovarian cancer, bladder cancer, stomach cancer and lung cancer [Cisplatin (Cis-diamine dichloroplatinum (II), Pt  supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml) and 5 or 10% fetal bovine serum , referred to here as complete DMEM. The cultured cells were kept in 100mm tissue culture dishes at 37°C in a humidified atmosphere containing 5% COad libitum access to a standard irradiated chow diet .All animal protocols used in this study were reviewed and approved by the PNU-Institutional Animal Care and Use Committee . Male three C57Bl/6N stocks (6-weeks-old) were obtained from three different sources. The C57BL/6NKorl mice were kindly provided by the Department of Laboratory Animal Resources of the National Institute of Food and Drug Safety Evaluation . The other two stocks of C57BL/6N mice (C57BL/6NA and C57BL/6NB) were purchased from vendors located in the United States (Vendor A) and Japan (Vendor B), respectively. All C57BL/6N mice were maintained and treated at the Animal Resource Center of Pusan National University, which is certified by the Food and Drug Administration (FDA) as accredited unit number 000231, and Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International according to the National Institutes of Health guidelines . During the experiment, all mice were maintained in a specific pathogen-free (SPF) state under a strict light cycle (lights on at 08:00 h and off at 20:00 h), at 23 ± 2°C and 50 ± 10% relative humidity. Animals were provided with 2 x length)/2. For examining the tumors, growth of the tumor was observed up to 30 days, measuring the volume every 2 days, after which the mice were euthanized and samples collected. To measure the effect of anticancer drugs, tumors were induced by subcutaneous injection of LLC1 cells (5 × 105 cells) in C57BL/6NKorl, C57BL/6NA and C57BL/6NB mice, followed by administering three different dose (100 ug/kg (LCP), 1 mg/kg (MCP), 5 mg/kg (HCP)) of an anti-cancer drug (Cisplatin), thrice a week. Malignant tissues, were subsequently collected and analyzed. When mice showed signs of morbidity, defined by the animal study protocol , they reached their endpoint and were euthanized for further study.Mice were randomly divided into five groups for one C57BL/6N stock and LLC Cells (indicated cell numbers) in 1x PBS (100 μL) were subcutaneously injected into the right flanks of mice. All mice were dissected after natural death. For the survival curve, the mouse reached natural death, and tumor volumes were measured once every 2 days, using the formula , and their histopathological features were examined by light microscopy .2O2 for 10 min, washed with 1x PBS, and blocked using 10% BSA. The fixed sections were then probed with Ki-67 anti-body  prepared in 1% BSA at a concentration of 1:10, incubated overnight at 4°C, and subsequently treated with a secondary antibody (Biotinylated goat anti-rabbit IgG) and HRP streptavidin . Finally, the amount of Ki-67 peptide was observed using a BX50F-3 microscope  after inducing a color reaction using stable 3,3′-diaminobenzidine .Immunohistochemical analysis (IHC) for measuring cell proliferation was assessed in malignant tissues excised from experimental animals and fixed in formaldehyde for 48 h. Fixed cancer tissues were embedded in paraffin blocks after dehydration, sliced to a thickness of 4 μm using Leica Microtome (Leica Microsystems), and collected on slides. For immunohistochemistry, slides were subjected to xylene and ethanol dehydration, followed by 3% H2HPO4, and 0.05% Tween 20) and incubated with HRP-conjugated goat anti-rabbit IgG (Invitrogen) and HRP-conjugated goat anti-mouse IgG (Invitrogen) at a 1:1,000 dilution, at room temperature for 1 h. Membrane blots were developed using Amersham ECL Select Western Blotting detection reagent .Total proteins prepared from the tumor tissue were separated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 2 h, after which the resolved proteins were transferred to nitrocellulose membranes for 2 h at 40 V. Each membrane was then incubated separately, overnight at 4°C, with the following primary antibodies: anti-p53 (Sigma-Aldrich Co.), anti-p27 (Sigma-Aldrich Co.), anti-bax , anti-Bcl2 (Abcam), caspase-3 , anti-MMP2 , anti-VEGF (Abcam), and anti-β-actin . The membranes were then washed with washing buffer . After quantification of the RNA concentration, the complement DNA (cDNA) was synthesized using a mixture of oligo-dT primer , dNTP and reverse transcriptase . Q-PCR was then conducted using a cDNA template and 2× Power SYBR Green . The reaction cycle at which PCR products exceeded this fluorescence intensity threshold during the exponential phase of PCR amplification was considered as the threshold cycle (CT).P value < 0.05 is considered as significant.Statistical analyses were performed with SPSS for Windows, release 10.10, standard version . One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons was performed to identify significant differences between groups. All values are reported as the mean ± S.E.M, and a In order to compare the responses of subcutaneously inoculated LLC tumors among the three C57BL/6N stocks, the mouse survival rate was employed to determine the response after inoculating four different concentrations of LLC cell numbers. Figure Cisplatin (cis-diamine dichloroplatinum II) is a platinum-based anti-cancer drug widely used for the treatment of many solid tumors . To evalIt is well known that administration of anti-cancer drugs induces DNA damage and destroys cancer cells by activating apoptosis in fast-growing cancer cells. In particular, increasing the expression of tumor suppressor proteins p27 and p53 following the administration of anti-cancer drugs is one of the key factors that results in cancer cell death. Therefore, the reactivity to cisplatin of C57BL/6N mice inoculated with LLC cells was compared by determining changes in the levels of p27 and p53. As shown in Fig. Cancer metastasis is the main reason resulting in a serious prognosis for cancer patients. Hence, administering anti-cancer drugs that inhibit metastasis is also critical. We, therefore, examined the effects of the anti-cancer drug on the expression of MMP2 and VEGF, a factor associated with cancer metastasis, in LLC-inoculated C57BL/6N mouse stocks. Additional file In this study, we undertook to identify differences between C57BL/6N mouse stocks among the commonly used mouse syngeneic models. We believe that this study will help to understand the differences within C57BL/6N stocks by comparing the tumor growth patterns and response to anti-cancer drugs, thereby facilitating in choosing the appropriate mouse model. The syngeneic mouse model has the following limitations: 1) the use of established tumors rather than spontaneous generation, 2) differences in tumor microenvironment, and 3) rapid growth of transplanted tumors . Thus, lCisplatin is an effective anti-cancer drug discovered in 1965, proven to have anti-cancer activity in mouse models and used in the treatment of solid tumors such as testicular cancer, ovarian cancer, cervical cancer and parietal cancer since its approval by the Food and Drug Administration in 1978 –20. It iIt is well-known that the tumor microenvironment has an inflammatory mediator accumulation and mechanism for cell penetration. In particular, tumor sites feature chronic inflammatory conditions and there exists a significant correlation between chronic inflammation and tumor invasions, metastasis and chemotherapy . As expeTaken together, we evaluated the growth and survival of tumors in syngeneic mouse models of C57BL/6N stocks, established conditions for building a syngeneic mouse model in the C57BL/6N stocks by evaluating the growth and survival of the tumor, administered cisplatin to each C57BL/6N stock model to establish the responsiveness to cancer drugs, such as tumor growth and molecular biological aspect including expression of p27 and p57, apoptosis related genes, metastasis factors and inflammatory proteins, in tumor bearing C57BL/6N stocks. Taken together, our results indicate that for all three C57BL/6N stocks, this syngeneic model has similar functionality and reactivity to anti-cancer drugs. Our results suggest that the C57BL/6NKorl mouse and C57BL/6N mouse from other commercial suppliers can be widely applied to produce a syngeneic model through tumor transplants.Additional file 1:Figure S1. Comparison of the three C57BL/6N stocks on tumor mass of mouse in LLC tumor cell transplant model. Four different numbers  of LLC cells were subcutaneous injected in C57BL/6NKorl (a), C57BL/6NA (c), and C57BL/6NB (c). Bottom paragraph represents optic observation of isolated tumors mass. Isolation of tumor mass from each stock mice at 27 days after LLC (indicated cells number/200 μl/body) transplantation. Each group consisted of 8 mice. The mouse growth was evaluated as described in Materials and Methods. Data represents the mean ± S.E.M of n = 8/group (*P < 0.05 versus no group). (TIF 106 kb)Additional file 2:Figure S2. Comparison of the three C57BL/6N stocks on tumor cell proliferation of mouse in LLC tumor cell transplant model using Ki-67 staining. Four different numbers  of LLC cells were subcutaneous injected in C57BL/6NKorl, C57BL/6NA, and C57BL/6NB, respectively. The graph presents the relative expression level of Ki-67 on tumor tissue from C57BL/6NKorl, C57BL/6NA and C57BL/6NB stocks, respectively. Each group consisted of 8 mice. Data represents the mean ± S.E.M of n = 8/group (*P < 0.05 versus No group). (TIF 300dpi)Additional file 3:Figure S3. Comparison of the three C57BL/6N stock for tumor cell proliferation in mice with anti-cancer drug administration in a LLC tumor cell transplant model using ki-67 staining. LLC cell 5x10 5 was treated with indicated concentrations of cisplatin after subcutaneous injections in C57BL/6NKorl, C57BL/6NA, and C57BL/6NB. (d) The graph presents the relative expression level of Ki-67 on tumor tissue from C57BL/6NKorl, C57BL/6NA and C57BL/6NB stocks, respectively. Each group consisted of 8 mice. Data represents the mean ± S.E.M of n = 8/group (*P < 0.05 versus no group). (TIF 755 kb)Additional file 4:Figure S4. Comparison of metastasis-related proteins expression in tumor tissue collected from LLC cell bearing C57BL/6N mice stocks. Western blotting represent relative level of MMP2 and VEGF protein in the tumor tissue from each C57BL/6N stock. The graph display the relative level of protein expression in tumor tissues from cisplatin-treated LLC cells bearing C57BL/6NKorl, C57BL/6NA, and C57BL/6NB stocks, respectively. *P < 0.05 versus LLC+Ve group (TIF 300dpi)Additional file 5:Figure S5. Differing inflammatory responses of cisplatin in tumor tissue collected from LLC cell bearing C57BL/6N mice stocks. The mRNA levels of inflammation related proteins  were measured by real-time PCR using specific primers. Each panel represents the mRNA expression level of inflammation related proteins among tumor bearing C57BL/6NKorl (a), C57BL/6NA (b), and C57BL/6NB (c) mice after treatment with cisplatin or vehicle (*P < 0.05 versus LLC+Ve group). (TIF 300dpi)


Human infections with H7N9 avian influenza A virus can result in severe diseases with high mortality. Developing an effective vaccine is urgently needed to prevent its pandemic potential. Vaccine delivery routes via mucosal surfaces are known to elicit mucosal immune responses such as secretory IgA antibodies in mucosal fluids, thus providing first-line protection at infection sites. PEG-b-PLACL (PELC) is a squalene-based oil-in-water emulsion adjuvant system that can enhance antigen penetration and uptake in nasal mucosal layers with enhanced mucin interactions. In this study, intranasal immunizations with recombinant H7 (rH7) proteins with a PELC/CpG adjuvant, as compared to the use of poly (I:C) or bacterial flagellin adjuvant, elicited higher titers of H7-specific IgG, IgA, hemagglutination inhibition, and neutralizing antibodies in sera, and increased numbers of H7-specific IgG- and IgA-antibody secreting cells in the spleen. Both PELC/CpG and poly (I:C) adjuvants at a dose as low as 5 μg HA provided an 80% survival rate against live virus challenges, but a lower degree of PELC/CpG-induced Th17 responses was observed. Therefore, the mucosal delivery of rH7 proteins formulated in a PELC/CpG adjuvant can be used for H7N9 mucosal vaccine development. Influenza A viruses pose a constant threat to human public health. In 2013, a novel H7N9 influenza virus emerged and caused severe diseases, such as pneumonia and/or acute respiratory distress syndrome . These v® 85 (sorbitan trioleate) and poly (ethylene glycol)-block-poly (lactide-co-ε-caprolactone), which has a composition similar to MF59, except for the replacement of Tween 80 with a biodegradable PEG-b-PLACL polymer [Mucosal vaccinations via mucosal routes such as nasal, sublingual, oral, rectal, or vaginal can elicit mucosal immune responses such as secretory IgA antibodies, thus providing first-line protection at infection sites ,12. Nasa polymer .In this study, we investigated intranasal rH7 immunizations in mice using a combination of PELC and K3 CpG oligodeoxynucleotides (ODN) as a mucosal adjuvant. We previously reported that inactivated H5N1 virions formulated with PELC/CpG elicited more potent immune responses than PELC alone . In paraTo develop rH7-based mucosal vaccines, groups of BALB/c mice were intranasally immunized with three doses of 5 or 20 μg rH7 proteins over a 3-week interval with or without the use of (i) PELC/CpG, (ii) poly (I:C), or (iii) FliC adjuvant A. AnalysSplenocytes were collected at 3 weeks following the third immunization of the mice, stimulated with rH7 proteins, and analyzed with the enzyme-linked immunospot (ELISPOT) assay for IgG- and IgA-ASCs. At the 5 μg dose for rH7 immunizations, the use of the PELC/CpG adjuvant significantly increased the numbers of IgG-ASCs but maintained similar numbers of IgA-ASCs in the spleen, followed by the use of poly (I:C) and FliC adjuvants A,B. At tTo determine T cell responses elicited by intranasal rH7 immunizations, with or without the PELC/CpG, poly (I:C) or FliC adjuvant, splenocytes and CLNs were collected 3 weeks after the third dose immunizations, stimulated with rH7 proteins, and analyzed using the enzyme-linked immunosorbent assay (ELISA) to determine IFN-γ, IL-4, and IL-17A production by T cells. Our data indicate significant increases in IFN-γ levels in splenocytes from all mice immunized with 5 μg or 20 μg rH7 with the use of the PELC/CpG, poly (I:C), or FliC adjuvant compared to the rH7 only control A. In CLNTo examine the levels of IL-17A in lung tissues intranasally immunized with the use of the PELC/CpG, poly (I:C), or FliC adjuvant, we conducted IHC staining to examine IL-17A production in the lung tissues of immunized mice one week after the third dose of intranasal immunizations. The results showed that the use of the poly (I:C) adjuvant, administered intranasally, induced higher IL-17A levels in lung tissue cells than that by the use of the PELC/CpG adjuvant when administered intranasally . The useTo investigate whether the use of the PELC/CpG, poly (I:C), or FliC adjuvant via the intranasal route may cause acute inflammation at the administration site, we examined the nasal cavity tissues of immunized mice using H&E staining. One week after the third dose of intranasal immunizations, mice were sacrificed and their nasal cavity sections were collected and stained (H&E) for histopathological examination. Our results revealed no inflammation (in the absence of cellular infiltration) in the nasal tissues following the three-dose intranasal rH7 immunizations with the use of the PELC/CpG, poly (I:C), and FliC adjuvants . 50) of A/Taiwan/01/2013(H7N9) viruses 3 weeks after their third dose of immunizations. The results indicate that 20 μg rH7 plus either the PELC/CpG or poly (I:C) adjuvant provided a 100% survival rate of the immunized mice following live virus challenges  adjuvant, immunized mice were challenged with 10 lethal dose 50%  PELC/CpG, (ii) poly (I:C), and (iii) the natural TLR5 ligand bacterial flagellin FliC adjuvants. Our findings indicate that the use of the PELC/CpG adjuvant, as compared to the use of the poly (I:C) or FliC adjuvant, elicited higher IgG and IgA and neutralizing antibody titers in sera, increased numbers of IgG-, and IgA-ASCs in the spleen. However, the use of the PELC/CpG adjuvant for intranasal rH7 immunization elicited a less potent Th17 cellular response in both the spleen and CLNs compared to the use of the poly (I:C) adjuvant. Both the PELC/CpG and poly (I:C) adjuvants with a 5 μg rH7 dose provided a more than 80% survival rate for the immunized mice following live H7N9 virus challenges.We have previously demonstrated that intramuscular immunizations with HA proteins formulated with PELC/CpG K3 CpG oligodeoxynucleotides (ODN) can elicit more potent neutralizing antibodies and protective immunity against H5N1 and H7N9 viruses ,21. IntrWe also measured T cell responses in the spleen and CLNs, and the results indicated that using PELC/CpG, compared to poly (I:C), for rH7 intranasal immunizations, induced less potent Th1, Th2, and Th17 cellular responses, particularly in CLNs . We alsoIntranasal immunization with rH7 plus the PELC/CpG adjuvant at a dose as low as 5 μg HA provided an 80% survival rate, similar to that offered by the use of the poly (I:C) adjuvant . These rIntranasal rH7 immunizations with the use of PELC/CpG adjuvant, as compared to poly (I:C) or FliC adjuvant, induced higher IgG and IgA and neutralizing antibody titers in sera, increased numbers of IgG-, and IgA-ASCs in the spleen. Both PELC/CpG and poly (I:C) adjuvants at a dose as low as 5 μg HA provided an 80% survival rate against live virus challenges but a lower degree of PELC/CpG-induced Th17 responses was observed. Therefore, the mucosal delivery of rH7 proteins formulated in a PELC/CpG adjuvant may be used to develop H7N9 mucosal vaccines.6 cells/mL, and then infected with a specific recombinant baculovirus at a multiplicity of infection (MOI) of 3. Culture supernatants were collected 72 h post infection, and then rH7 proteins were purified with nickel-chelated affinity chromatography , dialyzed with PBS, and stored at −20 °C.The HA cDNA sequence of the A/Shanghai/2/2013 (H7N9) virus strain was used to construct a plasmid for soluble rH7 proteins. An insect cell codon-optimized H7HA coding sequence was obtained from Genomics BioSci & Tech. Ltd. . The C terminal cytoplasmic and transmembrane domains of full-length HA were deleted and replaced with a GCN4-pII leucine zipper (MKQIEDKIEEILSKIYHIENEIARIKKLIGEV) for trimerization, followed by a thrombin cleavage site, ending with a His-tag for purification as previously described [For the vaccine preparations, 5 or 20 μg of rH7 proteins formulated with PELC/CpG (10% PELC + 10 μg CpG) , 2 μg po0–27 × 104 and 20–27 × 102, respectively) were added to each well, followed by a 1 h incubation at RT. After three washes with 300 µL PBST, the plates were incubated with 100 µL horseradish peroxidase (HRP) conjugated anti-mouse IgG antibody  for 1 h at RT. After three additional washes with 300 µL PBST, 100 µL TMB substrate  was added to each well and kept in darkness for 15 min. The reactions were stopped with 100 µL 2 N H2SO4. The optical density at 450 nm was measured with a TECAN spectrophotometer .Plates (96-well) were coated with 100 µL of purified rH7 protein (2 µg/mL), and incubated overnight at 4 °C. The plates were washed three times with 300 µL PBST (PBS with 0.05% Tween-20), and then blocked with 200 µL PBS buffer plus 1% BSA for 2 h at room temperature (RT), followed by three additional washes with 300 µL PBST. Two-fold serially diluted serum or BALF samples  treated serum samples were serially diluted two-fold, and incubated with 4 HA units of H7N9 virus (A/Taiwan/01/2013). The treated serum samples were co-incubated with 0.5% turkey red blood cells (RBCs), and HI titers were defined as the reciprocal of the highest dilution completely inhibiting hemagglutination. For the MN assay, two-fold serially diluted serum samples were mixed with equal volumes of H7N9 virus diluent  incubated at 4 °C for 1 h, and added to the prepared MDCK cells. Infectivity was defined as the presence of a cytopathic effect observed on day 4. Neutralizing titers were defined as the reciprocals of the highest serum dilutions neutralizing H7N9 virus infectivity in 50% of wells compared to uninfected cells.5 splenocytes or CLN cells were added to each well and incubated at 37 °C for 48 h. After three washes with PBST, HRP-conjugated anti-mouse IgG or HRP-conjugated anti-mouse IgA was added to each well for IgG- or IgA-ASCs, respectively. After overnight incubation at RT, and PBST and two PBS washes, 3-amino-9-ethylcarbazole substrate  was added, followed by incubation at RT for 0.5–1 h before washing with double-distilled water. Immunospots were determined using an ELISPOT plate reader .ELISPOT assays were used to determine H7HA-specific IgG and IgA ASCs. Splenocytes and CLNs were collected from immunized mice 3 weeks after the final vaccination. Multiscreen 96-well filtration plates  were coated with rH7 proteins (1 µg per well) and incubated overnight at 4 °C followed by blocking with 200 µL/well of complete RPMI 1640  at RT for 1 h. Next, 5 × 106 splenocytes per well were stimulated with 20 µg/mL of rH7 proteins for 48 h. Cells were harvested and fixed with 1% formaldehyde for 30 min at 4 °C. Stimulated splenocytes were suspended in staining buffer (2% FBS and 0.01% NaN3 dissolved in PBS), simultaneously stained with anti-B220-APC antibodies  and FITC-conjugated peanut agglutinin (PNA) , and kept for 30 min at 4 °C. The B220+ PNA+ cells (GC B cells) were gated and analyzed using Accuri C6 software (BD Biosciences).Flow cytometry analysis  was used to determine the proliferation of germinal center (GC) B cells as previously described . In brie6 cells/well and stimulated with 10 μg/mL rH7 proteins for 3 days at 37 °C prior to measuring cytokine levels in culture supernatants collected from stimulated cells. An enzyme-linked immunosorbent assay (ELISA) was used to determine H7HA-specific IFN-γ-, IL-4-, and IL-17A-secreting T cells in splenocytes and CLNs as described in the manufacturer’s instructions. In brief, 96-well plates were coated with anti-mouse IFN-γ, IL-4, or IL17A capture antibodies , followed by blocking with PBS buffer plus 1% BSA for 2 h. After washing three times, the coated wells were incubated for 1 h with biotin-conjugated IFN-γ, IL-4, or IL17A detection antibodies, respectively, and another 1 h incubation with avidin-HRP. After this, TMB substrate was added to each well and kept in darkness for 15 min. The reactions were stopped by adding 100 µL 2N H2SO4. Optical density (450 nm) was measured with a TECAN spectrophotometer.Splenocytes and CLN cells were seeded at 1 × 1050 of the H7N9 virus (A/Taiwan/01/2013). PBS-immunized mice were used as a mock control. Mouse survival and body weights were recorded daily for 14 days. Based on the Institutional Animal Care and Use Committee (IACUC) guidelines, body weight loss >25% was used as an end point. All procedures were approved by the IACUC of Academia Sinica. Per the IACUC guidelines, carbon dioxide was used to sacrifice the mice that survived the challenge experiments to ameliorate suffering. Three weeks after the final immunizations, the mice were anesthetized and intranasally challenged with 50 μL of 10 LDGroups of mice (N = 3) were sacrificed one week after the final vaccination. The nasal tissues were harvested, fixed with 10% buffered formalin, and embedded in paraffin. Sections of 5 μm were stained with H&E by the Pathology Core Laboratory of NHRI for histological examination using an Olympus DP70 microscope  at ×400 magnification.2O2 (in methanol) for 15 min, blocking with 2.5% goat serum (in PBS) for 1 h, and washing with PBS three times, the lung sections were stained with anti-IL-17A (BioLegend) in the dark at RT for 1 h. Finally, the sections were incubated with Simple Stain MAX PO  for 1 h, and then treated with DAB Quanto substrate  for 5 min followed by hematoxylin  counterstaining for 5 min. The sections of each group were observed using an Olympus DP70 microscope at ×400 magnification.Lung tissues were harvested from immunized mice (N = 3) one week after the final immunization. The 5 μm paraffin-embedded sections on silane-coated slides were dewaxed in xylene for 5 min three times, hydrated sequentially in 100%, 95%, 90%, 80%, and 60% ethanol for 5 min each, and then immersed in Trilogy  at 95°C for 30 min for antigen retrieval. After treating with 3% Hp < 0.05 indicating statistical significance. All experiments were performed at least two times each.One-way ANOVA with the Holm-Sidak method  was used to analyze the results, with


Despite the possible relationships between tracheal infection and concomitant infection of the terminal part of the lower respiratory tract , the behavior of avian influenza viruses (AIVs), such as H5N1, in the conducting airways is unclear. To examine the tropism of AIVs for cells lining the conducting airways of humans, we established human tracheal epithelial cell clones (HTEpC-Ts) and examined their susceptibility to infection by AIVs. The HTEpC-Ts showed differing susceptibility to H5N1 and non-zoonotic AIVs. Viral receptors expressed by HTEpC-Ts bound all viruses; however, the endosomal pH was associated with the overall susceptibility to infection by AIVs. Moreover, H5N1 hemagglutinin broadened viral tropism to include HTEpC-Ts, because it had a higher pH threshold for viral–cell membrane fusion. Thus, H5N1 viruses infect human tracheal epithelial cells as a result of their higher pH threshold for membrane fusion which may be one mechanism underlying H5N1 pathogenesis in human airway epithelia. Efficient replication of H5N1 in the conducting airways of humans may facilitate infection of the lower respiratory tract. Since the first case of human infection in Hong Kong in 1997, H5N1 viruses have remained a serious threat to public health worldwide, even though recent reports show that other subtypes of avian influenza viruses (AIVs), such as H5N6, H6N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, H10N7, and H10N8, can also infect humans ,14,15,16Collectively, these studies suggest that cells lining the human conducting airways  are susceptible to infection by H5N1. They also suggest a possible relationship between tracheal infection and concomitant infection of the terminal part of the lower respiratory tract (LRT) ; this is because the replication of respiratory viruses  in the conducting airways can lead to infection of the lower respiratory region which can result in acute respiratory distress syndrome (ARDS). Therefore, to fully understand the pathogenesis of AIVs in humans, we need to know the extent to which AIVs infect the conducting airways of the human respiratory tract. However, the behavior of AIVs, such as H5N1, in the conducting airways is unclear.Usually, cell lines, such as Madin–Darby canine kidney (MDCK) cells and A549 cells, both of which are susceptible to influenza viruses, are used to examine viral tropism and related pathogenesis ,27,28,29In this study, we transformed primary human tracheal epithelial cells (HTEpCs) with SV40 large T-antigen and isolated several clonal cell lines (named HTEpC-Ts). We then used these lines to evaluate the tropism of AIVs for human conducting airways and examined the resulting pathogenic effects. We focused on the susceptibility of cells to infection by currently circulating H5N1 (zoonotic) and previously circulating (non-zoonotic) AIVs. In addition, we identified the mechanism responsible for the susceptibility of HTEpC-Ts to infection by current H5N1 and non-zoonotic AIVs.Primary human cells (HTEpCs) were purchased from PromoCell Corp., Heidelberg, Germany see . All dong for 20 min and then passed through a syringe filter (0.45 μm pore size). Finally, the virus in the allantoic fluid was purified by ultracentrifugation  on PBS  (PBS (−)) containing 20% sucrose (w/v). The resulting pellets were suspended in PBS (−), and the titer was measured in focus-forming assays on MDCK cells (results are expressed as the number of focus-forming units (FFU)/mL) [The A/Beijing/262/95 (H1N1) (Beijing (H1N1)), A/Panama/2007/99 (H3N2) (Panama (H3N2)), A/crow/Kyoto/53/04 (H5N1) (Cw/Ky (H5N1)), A/chicken/Egypt/CL6/07 (H5N1) (Ck/Eg (H5N1)), A/duck/Hong Kong/820 (H5N3) (Dk/Hk (H5N3)), and A/turkey/Ontario/7732/66 (H5N9) (Tk/Ont (H5N9)) were described previously . These vFFU)/mL)  followinFFU)/mL) .The MDCK cells were cultured in minimum essential medium supplemented with 10% fetal bovine serum (FBS) and standard antibiotics (penicillin (100 units/mL), streptomycin (100 μg/mL), and amphotericin B (250 ng/mL)). The HTEpCs were cultured in Airway Epithelial Cell Growth Medium (AECGM) (PromoCell) according to the manufacturer’s instructions. The SAEC-Ts and HTEpC-Ts were cultured in D/M medium (DMM) which is based on Dulbecco’s modified Eagle’s medium (DMEM) and MCDB153 (1:1); both media were supplemented with growth factors (bovine pituitary extract (30 μg/mL), hydrocortisone (0.5 μg/mL), epidermal growth factor (0.5 ng/mL), epinephrine (0.5 μg/mL), transferrin (10 μg/mL), insulin (5 μg/mL), triiodothyronine (6.5 ng/mL), retinoic acid (0.1 ng/mL), or cholera toxin (0.1 μg/mL)), 5% FBS, and antibiotics (penicillin (100 units/mL), streptomycin (100 μg/mL), and amphotericin B (250 ng/mL)), as described previously . The HTEThe MDCK cells, in 96 well plates, were washed three times with PBS  (PBS (+)) and then inoculated for 1 h at 37 °C with sample fluid containing virions. After that, the virus inoculum was removed and the cells were washed three times with PBS (+) and overlaid with 1% methylcellulose in minimum essential medium supplemented with 0.2% bovine serum albumin and standard antibiotics (penicillin (100 units/mL), streptomycin (100 μg/mL), and amphotericin B (250 ng/mL)). At 16 h post-infection, the cells were fixed with 4% paraformaldehyde in PBS (−). After washing three times with PBS (−), the cells were stained, as described in g for 2 h) on PBS (−) containing 20% sucrose (w/v). The resulting pellets were suspended in PBS (−). Next, a monolayer of primary HTEpCs was exposed to medium (AECGM supplemented with polybrene (8 μg/mL)) containing the recombinant retrovirus harboring the SV40 large T-antigen gene. After 24 h, the medium was replaced with fresh medium. After another 48 h, G-418 sulfate (100 μg/mL) was added to the medium to isolate the immortalized cells. After 2 weeks of culturing the immortalized cells with G-418 sulfate, single-cell clones were isolated using Scienceware® cloning discs  or isolated by limiting dilution in a 96 well microplate to establish HTEpC-T clones. The HTEpC-T clones were then cultured in DMM prior to their use in experiments. The HTEpCs were immortalized by transformation with the SV40 large T-antigen gene as described previously . BrieflyRecombinant viruses were generated using a reverse genetics system based on previously described methods ,37,38. B4 to 3.0 × 104 cells/well), washed twice with PBS (+), and infected with viruses at a multiplicity of infection (m.o.i.) of 10, 1, and 0.1. The m.o.i. was calculated on the basis of the cell number and the titer of the viruses used . At 16 h post-infection with the virus, cells were fixed for 30 min at room temperature with PBS (−) containing 4% paraformaldehyde/0.1% Triton X-100, followed by washing three times with PBS (−). Viral antigens were detected by staining the cells with a rabbit polyclonal antibody raised against A/duck/Hong Kong/342/78 (H5N2) (1:1000 dilution in PBS (−)/1% bovine serum albumin) which recognizes the NP and M1 proteins of both avian and human influenza strains. Binding of the primary antibody to viral proteins was detected by an Alexa Fluor 488-conjugated secondary antibody  diluted 1:500 in PBS (−). Cell nuclei were counterstained with Hoechst 33342 (Merck). An IN Cell Analyzer 2200  apparatus was used to determine the percentage of each HTEpC-T/SAEC-T clone and primary HTEpC infected with the virus (presented as “% infectivity” in the figures). Images of infected cells were acquired by the IN Cell Analyzer 2200, and the number of antigen-positive cells and cell nuclei visible in the same field was calculated  by IN Cell Developer Toolbox software . The percentage of cells infected by the virus was calculated by dividing the number of antigen-positive cells by the total number of nuclei in the same field (×100). The number of cells counted per test well was >500 (at an m.o.i. of 10), >1000 (at an m.o.i. of 1), and >1500 (at an m.o.i. of 0.1) (the number of countable cells decreased in line with the strength of the cytopathic effects against infected cells). The results are expressed as the mean ± SD of at least three independent cultured wells. Cells were cultured in 96 well plates (2.0 × 10Sambucus nigra (SNA)-FITC (Vector Laboratories) in PBS (−) containing 0.1% bovine serum albumin to detect sialic acid α2,6-galactose  moieties or with 2.5 μg/mL Maackia amurensis (MAA)-I-FITC (Vector Laboratories) in PBS (−) containing 0.1% bovine serum albumin to detect SAα2,3Gal  GlcNAc) moieties. In addition, the cells were incubated with 2.5 μg/mL biotinylated MAA-II  in PBS (−) containing 0.1% bovine serum albumin to detect SAα2,3Gal  GalNAc) moieties, followed by washing three times with PBS (−) and blocking for 1 h at 4 °C with PBS (−) containing 1% bovine serum albumin. After washing once with PBS (−), the cells were incubated with streptavidin-FITC (1 h at 4 °C) (Vector Laboratories). Finally, after washing each batch of cells three times with PBS (−), 10,000 events were acquired by a flow cytometer  to measure the fluorescence intensity. The data were analyzed using CellQuest software (BD Biosciences). The MDCK cells were treated or mock-treated for 4 h at 37 °C with Arthrobacter ureafaciens sialidase  prepared in PBS (+) (pH 6.8) before lectins were stained using MAA-I, MAA-II, and SNA.The HTEpC-T monolayers were detached from dishes by exposure to 0.025% trypsin/EDTA and then fixed for 30 min at 4 °C with 4% paraformaldehyde. After washing twice with PBS (−) containing 10 mM glycine and once with PBS (−), the cells were blocked for 1 h at 4 °C with PBS (−) containing 1% bovine serum albumin. After washing once with PBS (−), the cells were incubated for 1 h at 4 °C with 2.5 μg/mL 5 cells/well). Briefly, cells were washed twice with PBS (+), inoculated with viruses at an m.o.i. of 10, and then incubated at 4 °C for 1 h to prevent endocytosis. The cells were then washed five times with ice-cold PBS (+) and lysed in PBS (−) containing 2% SDS. The protein concentration of the resulting cell lysates was measured using the bicinchoninic acid protein quantification kit (Thermo Fisher Scientific). Next, 10 μg of protein per lane was subjected to electrophoresis in a 10% SDS-polyacrylamide gel. The proteins were then transferred to PVDF membranes and blocked overnight at 4 °C in PBS (−) containing 5% non-fat milk and 0.1% Tween 20. The membrane was then exposed for 1 h at room temperature to the aforementioned rabbit polyclonal antivirus antibody (diluted 1:1000 in PBS (−) containing 5% non-fat milk and 0.1% Tween 20), followed by horseradish peroxidase-conjugated donkey anti-rabbit IgG  containing 0.1% Tween 20; Jackson ImmunoResearch, West Grove, PA, USA). Between steps, the membranes were washed three times with PBS (−) containing 0.1% Tween 20. Reactive bands were visualized using a chemiluminescence system (Thermo Fisher Scientific) and Fuji XR film.Viruses were allowed to bind to established human cell clones cultured in 12 well plates  and SAEC-T (1A5) clones was measured by flow cytometry analysis as described previously . BrieflyThe effect of increasing the endosomal pH on virus infection was examined by incubating HTEpC-T (LA-9) and SAEC-T (1A5) clones for 2 h in DMM containing 5% FBS plus bafilomycinA1 (BafA1)  prior to virus infection . After two washes with PBS (+), cells were infected with Cw/Ky (H5N1). The percentage of infected LA-9 and 1A5 cells was determined using an IN Cell Analyzer 2200  after immunostaining at 12 h post-infection as described above.The HTEpCs were infected (in triplicate) with viruses at an m.o.i. of 0.1. The cells were then incubated at 37 °C for 1 h with virus suspended in PBS (+). The viral solution was removed, and the cells were washed twice with PBS (+). The HTEpCs were cultured in DMM containing 0.2% bovine serum albumin and trypsin (1 μg/mL). At the indicated times post-infection, viral RNA (vRNA) titers in the cell culture supernatants of HTEpCs were measured by quantitative real-time RT-PCR as described below. Infectious viral titers in the cell culture supernatant of both HTEpCs and the HTEpC-T clone were also determined in focus-forming assays on MDCK cells as described in Progeny vRNA was extracted from the culture supernatant of infected cells using a QIAamp Viral RNA Mini Kit  and measured by quantitative real-time RT-PCR  using primers targeting the M gene as described previously . The prit-tests were used for statistical analysis. One-way analysis of variance (ANOVA) was used, followed by Dunnett’s post-hoc test for multiple comparisons. A p-value < 0.05 or < 0.01 was considered significant. Statistical analysis was performed using GraphPad Prism Version 8 software (GraphPad Software Inc.).All data are expressed as the mean and standard deviation of at least three determinations per experimental condition. Student’s To examine the mechanism(s) underlying the susceptibility of cells in the human conducting respiratory tract to AIV infection, we generated cell clones by transforming primary HTEpC with SV40 large T-antigen and then established HTEpC-Ts after cell cloning. We isolated seven HTEpC-T clones, although one of these died out within ten passages. The other six HTEpC-T clones were sub-cultured successfully for ten passages and stably expressed the SV40 large T-antigen . The morTo examine the susceptibility of each HTEpC-T clone to infection by AIVs, we infected them with highly pathogenic H5N1 viruses Cw/Ky (H5N1) and Ck/Eg (H5N1) or with previously circulating (non-zoonotic) AIVs Dk/Hk (H5N3) and Tk/Ont (H5N9). Human influenza viruses (Beijing (H1N1) and Panama (H3N2)) were used as controls. After infection by avian or human viruses, HTEpC-Ts expressed viral antigens ; howeverIn addition, to examine differences within different regions of the human airway, we compared the susceptibility of HTEpC-Ts with that of other clones . Hence, 1A5 and 21E5 cells were also transformed by the SV40 large T-antigen and named SAEC-Ts; the susceptibility of 1A5 and 21E5 cells to non-zoonotic AIVs is “adequate” and “inadequate”, respectively , althougWe focused on the observation that all clones showed uniformly highly susceptibility to infection by H5N1 viruses. The presence of multiple basic amino acids within the HA cleavage site correlates with the virulence of highly pathogenic AIVs in both birds and mammals ,43. To eTo assess the cell characteristics associated with susceptibility to viral infection, we also examined the expression of viral receptors SAα2,3Gal and SAα2,6Gal on HTEpC-Ts by staining cells with MAA-I/II and SNA. Both SAα2,3Gal and SAα2,6Gal were expressed by all HTEpC-T clones as well as SAEC-Ts (1A5 and 21E5 cells) and MDCK cells (virus-susceptible controls) .5 cells were inoculated with the virus at an m.o.i. of 10, lysed, and then immunoblotted with an antibody specific to the viral antigen  to the cells. Representative HTEpC-T (LA-9) and SAEC-T (1A5) clones  that may affect infectivity. Because the infection of cells requires viral–cell membrane fusion, which is induced by a pH-dependent HA conformational change, we investigated whether the acid stability of the HA molecule affects the susceptibility of HTEpC-Ts to viral infection. Previously, we reported that H5N1 viruses undergo HA-dependent viral–cell membrane fusion at higher pH values than non-zoonotic AIVs  the pH . TherefoTo examine whether the effect of HA acid stability on the susceptibility of HTEpC-Ts can be replicated in HTEpCs, which are primary cultured cells, we infected the latter with recombinant Dk/Hk (H5N3) harboring the H5N1 HA genes described above. The susceptibility of HTEpCs to infection by these AIVs was similar to that of HTEpC-Ts . The HTEIn the final series of experiments, we examined the possible relationship between susceptibility to infection and viral growth kinetics in both HTEpCs and their established clones. We infected HTEpCs with H5N1, non-zoonotic virus, and its recombinant AIVs at a low m.o.i. (0.1). The replication kinetics of the H5N1 virus and recombinant Dk/Hk (H5N3) harboring H5N1 HA genes were significantly higher than those of Dk/Hk (H5N3), regardless of the H5N1 strain . The kinHere, we report four main findings with respect to the mechanism underlying the susceptibility of human tracheal cells to infection by AIVs: (i) HTEpC-Ts showed differing susceptibility to infection by H5N1 and non-zoonotic AIVs; (ii) viral receptors expressed by HTEpC-Ts were able to bind viral particles at similar levels; (iii) the pH in the endosomes of HTEpC-Ts determined their susceptibility to infection by AIVs; and (iv) the H5N1 HA protein, with a higher pH threshold for HA-mediated membrane fusion, broadened the viruses’ tropism for HTEpC-Ts.Both SAEC-T clone 21E5 and HTEpC-Ts were highly susceptible to infection by human influenza and H5N1 viruses, but they were less susceptible to infection by non-zoonotic AIVs, such as H5N3 and H5N9, regardless of the presence of multiple basic amino acids in the HA cleavage site . The susThe patterns of viral receptor expression in the human airways could be associated with differences in susceptibility within different regions of the human airway. Some studies have reported that the avian receptor SAα2,3Gal (stained by MAA-II) is expressed mainly in the terminal part of the human LRT ,55, suggThe HTEpC-Ts maintained in liquid culture expressed high levels of SA  residues; this explains the high numbers of viral particles bound to the cells in the virus-binding assay . The anaThis marked ability of H5N1 to infect HTEpC-Ts prompted us to undertake functional analyses of the H5N1 HA protein which may be associated with broader tropism. Recombinant H5N3 viruses possessing the HA protein from H5N1 were able to infect HTEpC-T clone LA-9 as were wild-type H5N1 viruses . These rRecent reports, including our own, have shown that the acid stability of the H5N1 HA protein is more vulnerable to pH  than that of non-zoonotic AIVs ,69,70,71We also evaluated the susceptibility of primary HTEpCs to infection by AIVs. The susceptibility of primary HTEpCs to infection by AIVs broadly reflected that of HTEpC-Ts , suggestEven if the difference in infectivity between H5N1 and non-zoonotic AIVs in primary HTEpCs is not as marked as that in HTEpC-Ts, the difference seems to reflect the amount of virions released from infected HTEpCs upon long-term cultivation at a low m.o.i; this is because influenza viruses exhibit exponential growth as a result of multiple cycles of viral infection during long-term cultivation. During the long-term cultivation of virus within primary HTEpCs, the amount of H5N1 virions released at each time point was significantly higher than the amount of non-zoonotic AIV virions . In addiBecause viral replication in the tracheal region of the respiratory tract may result in infection of the terminal part of the LRT  which can result in ARDS, it is important to know the degree to which H5N1 infects tracheal epithelial cells. The results presented herein suggest that the susceptibility of tracheal epithelial cells to infection by H5N1 is determined by the balance between the acid stability of the HA molecule  and the pH of the endosomal compartment. Further research into the HA-governed mechanisms underlying membrane fusion will help us to develop effective strategies for the surveillance and control of AIVs.


Schizosaccharomyces pombe, TORC2 and its target, the AGC kinase Gad8 (an orthologue of human AKT or SGK1) are required for viability under stress conditions and for developmental processes in response to starvation cues. In this study, we describe the isolation of gad8 mutant alleles that bypass the requirement for TORC2 and reveal a separation of function of TORC2 and Gad8 under stress conditions. In particular, osmotic and nutritional stress responses appear to form a separate branch from genotoxic stress responses downstream of TORC2-Gad8. Interestingly, TORC2-independent mutations map into the regulatory PIF pocket of Gad8, a highly conserved motif in AGC kinases that regulates substrate binding in PDK1 (phosphoinositide dependent kinase-1) and kinase activity in several AGC kinases. Gad8 activation is thought to require a two-step mechanism, in which phosphorylation by TORC2 allows further phosphorylation and activation by Ksg1 (an orthologue of PDK1). We focus on the Gad8-K263C mutation and demonstrate that it renders the Gad8 kinase activity independent of TORC2 in vitro and independent of the phosphorylation sites of TORC2 in vivo. Molecular dynamics simulations of Gad8-K263C revealed abnormal high flexibility at T387, the phosphorylation site for Ksg1, suggesting a mechanism for the TORC2-independent Gad8 activity. Significantly, the K263 residue is highly conserved in the family of AGC-kinases, which may suggest a general way of keeping their activity in check when acting downstream of TOR complexes.The Target of rapamycin (TOR) protein kinase forms part of TOR complex 1 (TORC1) and TOR complex 2 (TORC2), two multi-subunit protein complexes that regulate growth, proliferation, survival and developmental processes by phosphorylation and activation of AGC-family kinases. In the fission yeast,  Protein kinases catalyze the transfer of phosphate from high-energy, phosphate-donating molecules, such as ATP, to their substrates. This process is pivotal for regulation of almost any aspect of cellular biology. Many human diseases are associated with aberrant functions of protein kinases due to mutations. Accordingly, there is a growing number of kinase inhibitors that have been approved for clinical use. A better understanding of how protein kinases become active and how their activity is relayed to regulate their cellular functions is much needed for rational design of kinase inhibitors and for their optimal use in the clinic. The AGC-family of protein kinases play key roles in regulating cellular growth, proliferation and survival. In human cells, as well as in the fission yeast, our cellular model system, a subgroup of the AGC kinases is activated by the TOR protein kinases. Here we report the isolation of mutations in the AGC kinase Gad8 (AKT or SGK1 in human) that bypass the requirement for activation by TOR. Analyses of how these mutations affect cellular growth revealed separate branches of stress response mechanisms downstream of Gad8, while computer simulation methods suggested a molecular mechanism that keeps the activity of Gad8 in check. Saccharomyces cerevisiae [Saccharomyces cerevisiae and the fission yeast, S. pombe [S. pombe TORC2 has been localized to the plasma membrane, but it is also found in the nucleus (reviewed in [Target of rapamycin (TOR) is an atypical serine/threonine protein kinase that belongs to the family of phosphatidylinositol-3 kinase related kinases. TOR kinases are highly conserved throughout evolution and control several aspects of metabolism, cellular growth and survival. Consequently, deregulation of TOR activity is associated with pathological outcomes, including cancer, diabetes and neurological defects –3. TOR krevisiae , 5. The revisiae . These TS. pombe . The mamS. pombe , 8. The S. pombe . The subiewed in ).S. cerevisae orthologue of S6K1 is Sch9 [S. pombe TORC1 [S. cerevisiae, there are three AGC kinases that act downstream of TORC2, Ypk1, Ypk2 and Pkc1, which are involved in a wide variety aspects of membrane homeostasis, regulation of the actin cytoskeleton, cell cycle progression and genome integrity , the cGMP-dependent protein kinase (PKG) and the protein kinase C (PKC) families . mTORC1  is Sch9 , while tbe TORC1 . mTORC2 be TORC1 –18, SGK1be TORC1  and PKCαbe TORC1 , 21. In tegrity . In. InS. cease Gad8 , therebyase Gad8 –38. Disre stress  or genote stress . The actc stress , but thic stress , 42. TORc stress , 44.S. cerevisiae [S. pombe [The majority of members of the AGC kinase that lie downstream of TOR complexes share a common mechanism of activation that involves phosphorylation at three major conserved sites revisiae  or Ksg1 S. pombe , 48. PhoS. pombe –51. SeveS. pombe , 53. TheS. pombe , 55.S. pombe TORC2, Tor1, is responsible for phosphorylation of S546 in the HM and S527 in the TM [S. pombe PDK1) to phosphorylate Thr387 in the activation loop [Similar to other AGC-kinases, full activation of Gad8 requires phosphorylation at all three sites. The catalytic subunit of n the TM . Previouion loop . In thisS. pombe TORC2, is responsible for the phosphorylation (and therefore activation) of the Gad8 kinase [gad8+ suppressed phenotypes associated with Δtor1 [gad8+ under the regulation of the nmt1+ promoter from the multicopy plasmid pREP1 [tor1 cells - independent gad8 mutant alleles.Tor1, the catalytic subunit of 8 kinase . Consistth Δtor1 , strong id pREP1  rescued promoter  is unablgad8 by random mutagenesis using error-prone PCR with pREP81-gad8+ as template. We then co-transformated the resulting PCR fragments together with linearized pREP81 plasmids into yeast cells , while the T260C mutation occurred only once (pREP81-gad8-m3)  . The K26gad8-m3) . The K26gad8-m3) .gad8-T260C or gad8-K263C to suppress additional defects associated with loss of TORC2. Cells disrupted for tor1+ are unable to grow under low-glucose conditions [gad8-T260C or gad8-K263C mutant alleles suppressed low-glucose stress sensitivity in Δtor1 cells  or sin1+ (mSin1), also results in pleiotropic defects under stress conditions . The αC-helix and the C-terminal extension regions were modeled based on AKT1 and PKCα kinases . The final model is presented in 2+ ion. The point mutation K263C is located at the small N-lobe between the αB-helix and the β4 beta-sheet, that together with the αC helix delimit the PIF-pocket. As described above, Gad8-K263C bypasses the need for phosphorylation by Tor1 at the TM and HM motifs, S527 and S546, respectively. However, there is still a need for a phosphorylation by Ksg1 at the activation loop (Thr387) to get a full activation of Gad8. Therefore, we speculated that the mutation K263C may alter the protein structure in a manner similar to that affected by Tor1 phosphorylation. In order to get insights into the effect of the mutation on the protein structure, three constructs, namely un-phosphorylated Gad8, Gad8-K263C, and Gad8 phosphorylated at both the HM and TM (Gad8-pS527/pS546) were subjected to molecular dynamics (MD) simulations. Each construct was simulated three times for 50 ns. Each replica was started from a different random seed. The stability of the resulted trajectories was tested based on the root mean square deviation (RMSD) of the backbone atoms with respect to the equilibrated structures. Next, the root mean square fluctuation (RMSF) was calculated in order to determine the degree of flexibility of each residue in each construct. RMSF plots of protein backbone atoms of un-phosphorylated Gad8, Gad8-K263C and Gad8-pS527/pS546 during the simulations are shown in gad8-Q298L, partially suppressed the mating deficiency of Δtor1 cells when expressed from the pREP81 plasmid  or camptothecin (CPT). HU generates replication stress by depleting the dNTP pools, while CPT stabilizes covalent DNA topoisomerase-I complexes, leading to collision of DNA replication forks with the drug-enzyme-DNA complex and inducing double strand breaks [gad8-K263C or gad8-T260C expressed from the pREP81 plasmid failed to suppress the sensitivity of Δtor1  complex [cdc22+, cdc18+, cdt2+, revealed that these are also poorly induced in response to replication stress (HU) in gad8-K263C, or Δtor1 gad8-K263C mutant strains , resulting in the activation of the downstream effector kinases Chk1 or Cds1 . In respe cells  recognition motif or docking interaction with substrate(s) that are specifically required for the genotoxic stress response. The observed dominant negative phenotype could result from higher affinity of a defective Gad8 mutant protein to its substrate. Further studies identifying the substrates of Gad8 are required to support such speculations.TOR (Target of Rapamycin), which can be found in two structurally and functionally distinct complexes, is a major regulator of growth, proliferation and survival –3. Whileand SGK1 . Here, w kinases , 46. We Based on our MD analyses, we introduced another mutation in the PIF pocket of Gad8, Q298L. The Q298L mutation could partially suppress the mating deficiency and genotoxic stress sensitivities of cells lacking TORC2, but did not suppress the high temperature or osmotic sensitivities. The Q298L appears to confer a dominant negative effect under condition of low glucose. These findings suggest that while mutations in the PIF pocket can render Gad8 independent of TORC2, they may also impair the protein function under specific stress conditions, possibly by interfering with interactions with specific substrates.in vitro kinase activity of Gad8-K263C is maintained in the absence of Tor1 and that combining the K263C mutation together with mutations in the phosphorylation sites of TORC2, S527A and S546A results in a mutant allele, gad8-K263C/S527A/S546A, that is able to grow at high temperature or in the presence of osmotic stress. In contrast, combining the K263C mutation with mutations in the phosphorylation sites of Ksg1 results in a mutant allele that is sensitive to these stresses. Thus, our genetic and biochemical analyses suggest that the K263C mutation bypass the requirement for TORC2-dependent phosphorylation at the HM and TM, but not the phosphorylation at the activation loop by Ksg1. How, then, does the mutation K263C mutation in the PIF pocket of Gad8 bypass the requirement for TORC2? Our MD studies suggest that the K263C mutation causes a higher flexibility of the activation loop and the PIF region. The higher flexibility of the activation loop may allow it to explore wider conformational space, thus facilitating the phosphorylation of Thr387 by Ksg1. MD analysis of Gad8 phosphorylated at both the HM and TM similarly resulted in higher flexibility at the PIF domain and the activation loop, further suggesting an intramolecular activation path that involves the C-terminal phosphorylation sites, the PIF domain and the activation loop. Additionally, our MD analysis indicated the presence of an H-bond between K263 and Q298 in the predicted non-phosphorylated structure of Gad8. This H-bond may contribute to the rigidity of the PIF pocket. Mutating the Q298 residue into leucine resulted in partial suppression of the sexual development defect or genotoxic stress sensitivity of Δtor1 cells, suggesting that Q298 is important to render Gad8 activity dependent on TORC2. Our ability to predict mutations that bypass the requirement of TORC2 under stress conditions supports the validity of our MD analyses and calls for future studies of the role of the PIF pocket in rendering Gad8 dependent on activation by TORC2. Since the PIF pocket and the mode of activation of AGC kinases by TOR complexes are both highly conserved in evolution, similar mutations in AGC kinases may also result in TORC2-independet activities.Similar to other AGC kinases, Gad8 requires at least three types of phosphorylation events; by TORC2 at its hydrophobic  and turn-motif  and by the PDK1-like protein, Ksg1, at its activation loop (T387) . We demoThe PIF pocket residues K263 and Q298 are highly conserved in the family of AGC kinases. The SGK1-K131 residue, which is equivalent to K263, was suggested to play a role in localization of SGK1 as part of a nuclear localization sequence (NLS) . HoweverS. pombe strains used in this paper are listed in 4Cl), as described in [S. pombe cells was assessed by streaking or spotting on YES or EMM plates supplemented with KCl, hydroxyurea (HU) or camptothecin (CPT), or on plates with reduced levels of glucose, or plates that are incubated at a relatively high temperature (37°C). Strains that contained plasmids were assessed on EMM plates in order to select for the plasmid, while strains that contained chromosomal integrations were assessed on YES plates. Unless otherwise specified, plates were incubated at 30° for 4 days before being photographed.ribed in , 67. EMMgad8 mutant alleles by using random PCR-based mutagenesis. 5 ng of pREP81-gad8+ plasmid was taken for PCR amplification of the gad8+ ORF with primers: #1324 (5'TCAATTGAATAAGTTGAATTAATTATTTCAATCTCATTCTCACTTTCTGACTTATAGTCGCTTTGTTAAATCATATGTCCTGGAAACTTACAAAGAGTATGTATTCCATA) and #1282 (5'ATAGTTTGAAAGAAAAACCCTAGCAGTACTGGCAAGGGAGACATTCCTTTTACCCGGGGATCCGCTAGCCCATGGGTCGATCACCTAATGACACTTCCAGGTGCT). The underlined sequences are homologous to pREP81 sequences and the rest of the primer is homologous to gad8+ sequences. Four separate PCR reactions were performed, and in each reaction the concentration of a different dNTP was decreased from the standard 2mM concentration to 2μM. Using Phire Hot Start II DNA Polymerase (ThermoFisher #F-122L), four cycles of PCR were performed with the following temperature profile: 94°C 1 min, 54°C 1 min, 72°C 2 min. The PCR products of the four separate PCR reactions were mixed together and 34 cycles of PCR were performed with the following temperature profile: 94°C 1 min, 48°C 2 min, 72°C 2 min. The resulting PCR fragments were co-transformed together with the pREP81 plasmid into tor1-L2045D cells (TA3110) using the lithium acetate procedure. Transformant cells were incubated at 37°C for 6 days. Approximately 23,000 colonies were screened. Plasmid DNA was isolated from cells that grew at 37°C and then used to transform bacterial cells for plasmid amplification. Plasmid DNA extracted from bacteria was retransformed into tor1-L2045D (TA3110) cells and into Δtor1 (TA1291) cells. Plasmids that suppressed the temperature sensitive phenotype of Δtor1 and tor1-L2045D cells were subjected to DNA sequence analysis.We obtained gad8+ using standard techniques based on homologous recombination. We first obtained a fragment containing the K263 residue using wild-type genomics DNA as template for two separate PCR reactions. One reaction used primer #1388 (5'TATCTATGCTTTAAAAACTATGAAATGCGCCCACATTGTATCTCGCAGTGAA) together with primer #252 (5'TCC CCCGGG TCACCTAATGACACTTCCAGG) and a second reaction using primer.We integrated the K263C mutation into the chromosomal locus of #251 (5' GGAATTC CAT ATGTCCTGGAAACTTACAAAGAG) together withGCATTTCATAGTTTTTAAAGCATAGATA). The underlined sequences indicate the site encoding for K263C. For the PCR reaction, 34 cycles of PCR were performed with the following temperature profile: 94°C 1 min, 54°C 1 min, 72°C 2 min. The resulting two PCR fragments were stitched together using 1μl of PCR product from each PCR reaction as template and primers #251 and #252. PCR amplification was achieved by 34 cycles of PCR with the following temperature profile: 94°C 1 min, 48°C 1 min, 72°C 2 min. The PCR product was transformed into the gad8::ura+ (TA1029) strain and plated onto 5-FOA plates for selection against ura4+ cells. Genomic DNA was isolated from cells that grew in the presence of 5-FOA and were further subjected to DNA sequence analysis to ensure the presence of the K263C mutation at the correct locus.#1389 . Cells were broken for 45 min with glass beads, centrifuged for 10 min at 10,000 × g and the supernatant was collected. 20 μg of total protein extract was resolved on SDS-PAGE using 10% acrylamide gels. For immunoprecipitation, 1,000 μg of proteins were prepared and pre-cleared with 20 μl of protein A-Sepharose and protein G-Sepharose beads mixture . 2 μl of hemagglutinin (HA) antibodies were added to the cleared extract and incubated overnight at 4 0C. The beads were washed once with lysis buffer, once with lysis buffer containing 0.5 M NaCl, and twice with buffer A . The resulting immunoprecipitates were used for in vitro kinase assays.Cells were grown to mid-logarithmic phase, washed once with water, and resuspended in lysis buffer  containing 0.1 μg of GST-Fkh2. After incubation for 10 min at 30°C, the reaction was terminated by addition of 7 μl of 5 × SDS-PAGE sample buffers and incubated for 5 min at 80°C. The reaction was detected by Western blot analysis using anti-phospho-AKT substrate antibody .We applied a nonradioactive for Gad8 , based ofor Gad8 . For theProteins were resolved by SDS-PAGE 10–15% acrylamide gels and transferred to nitrocellulose membranes, blocked with 5% milk in TBST and immunoblotted with the indicated antibodies. Detection was carried out using the ECL SuperSignal detection system (Thermo Scientific). Gad8 Thr387 phosphorylation events were detected using total protein extracts by phosphospecific antibodies. Antibodies against Gad8 Thr387-P were raised against the Gad8 phosphopeptide CRFANWpSYQRPT . To follact1+ and relative expression levels were quantified using the comparative method and calculated as 2−ΔΔCt. The amount of expression was expressed relative to the expression level of wild-type cells grown in minimal medium . Oligonucleotides used for qRT-PCR analyses are listed in RNA extractions and qRT-PCR analysis were performed as described in . 50 ml oS. pombe Gad8 (accession number: Q9P7J8) was downloaded from the UniprotKB/Swiss-Prot server [+ ion. The models were ranked using DOPE energy score, which was calculated by the "evaluate model" python script in MODELLER. The highest ranked model was tested for stereochemical quality using Procheck [The sequence of t server . Availabt server . The cryt server  was fount server  and PKCαt server . After vt server . The modProcheck  and was The mutation K263C and the phosphorylation at positions S527 and S546 were introduced into Gad8 using Schrodinger’s Maestro 11.2. All the structures were prepared using the Protein Preparation Wizard as implemented in Maestro 11.2. This protocol adds missing hydrogen atoms considering a pH value of 7.0 ± 1.0, optimizes the hydrogen bond network, and performs restrained minimization. For further calculations, the ATP analogue (AMP-PNP) was replaced by ATP. Next, each construct  were submerged in TIP3P water model in octahedron box with an additional 15-Å extension along each axis of the protein. Sodium and chloride ions were added to the water phase in order to neutralize the system and to obtain a salt concentration of 0.15 M.All MD simulations were performed with the GROMACS 5.0.6 package  using thThe resulting trajectories were visually inspected using VMD 1.9.3 software . The staS1 FigA.gad8-m1/m2/m3, suppressed the temperature or osmotic stress sensitivities of tor1-L2045D or Δtor1 cells. Stress sensitivities were evaluated by growth on solid EMM media. Empty pREP81 vector or pREP81-gad8+ plasmids were used as negative control. pREP1-gad8+ was used as positive control. B. Schematic representation of the ORF of gad8 mutant alleles that were isolated in a screen for Tor1-independent alleles.(TIF)Click here for additional data file.S2 FigA-B, Stress sensitivities were evaluated by serial dilutions of the indicated transformant strains on solid EMM media. C, Mating efficiencies of wild type (WT), Δtor1 or Δste20 strains transformed with the indicated plasmids. The results are the mean values of three independent experiments.(TIF)Click here for additional data file.S3 FigA-B, Western blot analysis of protein extracts isolated from strains expressing no-tag Gad8, Gad8-HA or Gad8-K263C-HA. Cells were grown to mid-log phase and left untreated in YES medium, or shifted for 1 h to EMM containing no carbon source (-Glu), YES containing 1M KCl or 1 M NaCl (A), or shifted for 1 h to YES containing 12 mM hydroxyurea (HU) or 40 μM camptothecin (CPT) (B). Phosphorylation of Gad8 at S456 was detected with anti-Gad8-S546-P phsophospecific antibodies (S546-P). Anti-actin antibody was used as loading control.(TIF)Click here for additional data file.S4 FigS. pombe, S. cerevisiae and human. The tor1-independent gad8 mutations are in red.Alignment of the amino acid sequences surrounding the point mutations in several members of the AGC kinases in (TIF)Click here for additional data file.S5 FigA. Cells carrying a gad8 kinase-dead allele, gad8-K259R are sensitive to DNA damaging conditions. Stress sensitivities were evaluated by growth on solid YES media. Plates were incubated for 3 days. B. Cells carrying gad8 mutant alleles that cannot be phosphorylated by TORC2 are sensitive to genotoxic stress. Stress sensitivities were evaluated by serial dilutions as above.(TIF)Click here for additional data file.S6 FigA, Plasmids containing gad8-K263C or gad8-T260 are unable to suppress genotoxic sensitivity in TORC2 mutant cells. Stress sensitivities were evaluated by serial dilutions of cells onto EMM media B,gad8-K263A or gad8-K263R confer genotoxic stress sensitivity in wild type cells, while suppressing high temperature, osmotic stress or low-glucose sensitivities in Δtor1 cells. in wild type cells. Stress sensitivities were evaluated by serial dilutions of cells onto EMM media. C, Mutant alleles of gad8 that mimic constitutive phosphorylation at S527 and S546 suppress high temperature, osmotic stress, low glucose stress, as well as genotoxic stress. pR3C-gad8-S546D and pR3C-gad8-S527D/ S546D are a kind gift from A. Yamashita, National Institute for Basic Biology, Okazaki, Japan.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Paracoccus versutus MAL 1HM19 is a mixotrophic nitrate-reducing sulfide-oxidizing bacterium which plays a crucial role in hydrogen sulfide (H2S) and nitrate (NO3−) removal. In this study, we report the draft genome sequence of P. versutus MAL 1HM19. Paracoccus versutus MAL 1HM19 is a mixotrophic nitrate-reducing sulfide-oxidizing bacterium which plays a crucial role in hydrogen sulfide (H2S) and nitrate (NO3−) removal. In this study, we report the draft genome sequence of P. versutus MAL 1HM19. Paracoccus, phylogenetically classified in the class Alphaproteobacteria, comprises Gram-negative, nonmotile bacteria which belong to the facultative chemolithotrophic sulfide-oxidizing bacteria , was reported for its unique ability to simultaneously convert H2S and NO3− to sulfate (SO42–) and dinitrogen (N2) via the H2S oxidation and NO3− reduction pathways (The genus bacteria . They habacteria –8. RecenP. versutus strain MAL 1HM19 was cultured in modified Coleville synthetic brine (mCSB) under the following conditions: temperature, 35°C; pH 7.0; and incubation time, 72 h. Then, the genomic DNA of P. versutus MAL 1HM19 was extracted using the DNeasy PowerSoil pro kit . The quality and quantity of the DNA were tested, and the DNA was sequenced using an Illumina HiSeq 4000 instrument by CD Genomics , according to Mira et al. (P. versutus DSM 582) using the Burrows-Wheeler Aligner (BWA) software , while 3.7% of the total genes were predicted to be pseudogenes. Moreover, the genome also contained 53 RNA genes  and 54 tRNA genes. The N50 and L50 values were 65,835 bp and 26, respectively, while the largest contig from the draft genome sequence was 279,639 bp long.The genome of P. versutus MAL 1HM19 has been deposited in GenBank under the accession number QPML00000000 and BioProject number PRJNA482279. The raw sequencing reads have been deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRR10902179.The draft genome sequence of


Serratia marcescens strains isolated as contaminants from platelet concentrates by Canadian Blood Services and designated CBS2010/11 (CBS11) and CBS2010/12 (CBS12). Genomic sequence analyses showed that CBS11 has one chromosome and one plasmid (pAM01), whereas CBS12 has no plasmids.In this report, we present the genome sequences of two  Serratia marcescens strains isolated as contaminants from platelet concentrates by Canadian Blood Services and designated CBS2010/11 (CBS11) and CBS2010/12 (CBS12). Genomic sequence analyses showed that CBS11 has one chromosome and one plasmid (pAM01), whereas CBS12 has no plasmids.In this report, we present the genome sequences of two  Serratia marcescens is a common bacterial contaminant found in platelet concentrates (PCs) and has been associated with adverse transfusion reactions in recipient patients  were assembled de novo with the PacBio SMRT analysis software (7.0.0.63985) for the CBS11 genome and SMRT analysis software (6.0.0.47841) for CBS12, using the HGAP4 protocol (SMRTLink v6.0.0). A subset of reads were subjected to BLAST analyses to ensure quality control and authenticity of the genome assembly. Contig end trimming and circularization with Circlator (v1.5.5) were performed, followed by polishing with Quiver by the Canadian Center for Computational Genomics (C3G). Once polished, sequences were annotated with Prokka (v1.12) (ASM78391v2) produced by the University of Maryland School of Medicine and Institute for Genome Science (IGS). In all tool applications, default parameters were used except where otherwise noted. The CBS11 genome sequence resolved into 2 contigs, namely, a 5,352,713-bp contig and a 139,857-bp contig, representing a chromosome and a plasmid, respectively, with a G+C content of 59.39%, that combined included 4,950 genes, 22 rRNAs, 101 tRNAs, and 1 noncoding RNA. The CBS12 genome sequence resolved into 1 5,642,069-bp contig ascribed to the sole chromosome containing 5,204 genes, 22 rRNAs, 93 tRNAs, and 1 noncoding RNA.The genomes of strains CBS2010/11 (CBS11) and CBS2010/12 (CBS12) were sequenced and assembled using Pacific Biosciences (PacBio) technology . Briefly, the strains were grown overnight in Luria-Bertani (LB) broth (Difco), and genomic DNA was extracted using the Purelink genomic DNA kit (ThermoFisher) in accordance with the manufacturer’s instructions. The purity and quantity of DNA were evaluated by a Nanodrop instrument and by an Invitrogen Qubit 4 fluorimeter, respectively, prior to submission to the Génome Québec facility. A total of 5 μg of high-molecular-weight genomic DNA  was sheared using g-TUBEs  at 4,000 rpm for 60 seconds on each side, on the centrifuge 5424 (Eppendorf). The DNA libraries were prepared in accordance with the PacBio 20-kb template preparation kit using the BluePippin size selection system protocol that includes the use of the SMRTbell template prep kit 1.0 reagents for DNA damage and end repair and ligation steps. The DNA library was size selected on a BluePippin system  using a cutoff range of 12 kb to 50 kb. The sequencing primer was annealed at a final concentration of 0.8333 nM, and the P6 v2 polymerase was bound at 0.500 nM. The libraries were sequenced on a PacBio RS II instrument at a loading concentration (on plate) of 140 pM using the MagBead OneCellPerWell loading protocol, DNA sequencing kit 4.0 v2, and single-molecule real-time (SMRT) cells v3 with 240-minute movie collection. The combined data consisted of 110,975 reads with an S. marcescens plasmid PWNM146 (GenBank accession number LT575492)  with CP053927 (chromosome of CBS11), CP053926 (plasmid pAM01), and CP053925 (chromosome of CBS12). Accession numbers for raw reads for the CBS11 chromosome and plasmid pAM01 and for the CBS12 chromosome are PRJNA634532 and PRJNA634544, respectively.This genome project has been deposited in GenBank under accession numbers


Glutathione (GSH) is an endogenous tripeptide antioxidant that consists of glutamate-cysteine-glycine. GSH content is limited by the availability of glutamate and cysteine. Furthermore, glutamine is involved in the regulation of GSH synthesis via the glutamate–glutamine cycle. P2X7 receptor (P2X7R) is one of the cation-permeable ATP ligand-gated ion channels, which is involved in neuronal excitability, neuroinflammation and astroglial functions. In addition, P2X7R activation decreases glutamate uptake and glutamine synthase (GS) expression/activity. In the present study, we found that P2X7R deletion decreased the basal GSH level without altering GSH synthetic enzyme expressions in the mouse hippocampus. P2X7R deletion also increased expressions of GS and ASCT2 (a glutamine:cysteine exchanger), but diminished the efficacy of N-acetylcysteine  in the GSH level. SIN-1 , which facilitates the cystine–cysteine shuttle mediated by xCT (a glutamate/cystein:cystine/NAC antiporter), did not affect basal GSH concentration in WT and P2X7R knockout (KO) mice. However, SIN-1 effectively reduced the efficacy of NAC in GSH synthesis in WT mice, but not in P2X7R KO mice. Therefore, our findings indicate that P2X7R may be involved in the maintenance of basal GSH levels by regulating the glutamate–glutamine cycle and neutral amino acid transports under physiological conditions, which may be the defense mechanism against oxidative stress during P2X7R activation. Glutathione (GSH) is an endogenous tripeptide antioxidant that consists of glutamate-cysteine-glycine. Glutamate cysteine ligase (GCLC) converts glutamate and cysteine  to the dipeptide γ-glutamylcysteine (γGluCys), which is the rate-limiting step in cellular GSH synthesis. Thereafter, GSH synthetase (GSS) generates GSH by adding glycine (derived from exogenous glycine or serine) to γGluCys in an ATP-driven reaction . In the alanine, serine and cysteine (termed ASC). Among them, SLC1A4 and SLC1A5 are known as ASCT1 and ASCT2, respectively. Although ASCT1 and ASCT2 have high affinity for these amino acids, ASCT2 differs from ASCT1 by also accepting glutamine and asparagine as high affinity substrates [The solute carrier 1 (SLC1) family includes neutral amino acid exchange proteins that preferentially transfer the substrates bstrates ,9. In adbstrates ,11. Thusd-serine release from astrocytes [The P2X7 receptor (P2X7R) is one of the cation-permeable ATP ligand-gated ion channels which is involved in neuronal excitability, neuroinflammation and astroglial functions ,16,17,18trocytes ,20,21. Ttrocytes ,6,7, butN-acetylcysteine  in GSH synthesis induced by SIN-1. Thus, these findings suggest that P2X7R may modulate GSH levels by regulating the glutamate–glutamine cycle and neutral amino acid transports via ASCT2 and xCT under physiological conditions.Here, we demonstrate that P2X7R deletion decreases the basal GSH level in a mouse hippocampus, although it did not influence GCLC, GSS and GLS expression levels. However, P2X7R deletion increased GS and ASCT2 expressions without altering xCT expression. Furthermore, P2X7R deletion prevents the diminished efficacy of +/+, wild type, WT) and P2X7R−/−  mice  in the present study. Animals were given a commercial diet and water ad libitum under controlled conditions . All experimental protocols described below were approved by the Institutional Animal Care and Use Committee of Hallym University . Every effort was made to reduce the number of animals employed and to minimize animal discomfort. All reagents were obtained from Sigma-Aldrich , except as noted.We used male C57BL/6J . Coronal sections (300 μm thickness) were cut on a vibratome  and slices were subsequently transferred to oxygenated ACSF  at room temperature [Five hours after NAC  or vehicle treatment, animals were sacrificed by cervical dislocation and decapitated. Brains were rapidly removed and placed in ice-cold cutting solution  were randomly selected within the hippocampus . Thereafter, mean fluorescence intensity of 4-HNE signals on each section was measured by using AxioVision Rel. 4.8 software. Intensity measurements were represented as the number of a 256 gray scale. The intensity of each section was standardized by setting the threshold level (mean background intensity obtained from five image inputs). Manipulation of the images was restricted to threshold and brightness adjustments to the whole image.Brain slices were immersed into 4% paraformaldehyde in 0.1 M PB (pH 7.4) overnight. The brain tissues were cryoprotected by infiltration with 30% sucrose overnight. Thereafter, the slices were frozen and sectioned with a cryostat at 30 μm. Free-floating sections were washed 3 times in PBS  and incubated with 3% bovine serum albumin in PBS for 30 min at room temperature. Later, sections were incubated with glial fibrillary acidic protein  or a cocktail solution containing MAP1 and 4-HNE antisera  in PBS cn = 7 in each group). The primary antibodies used in the present study are listed in Animals were decapitated under urethane anesthesia . Animal protocols were approved by the Institutional Animal Care and Use Committee of Hallym University . The hippocampus was rapidly dissected out and homogenized in lysis buffer. The protein concentration in the supernatant was determined using a Micro BCA Protein Assay Kit . Thereafter, Western blot was performed by the standard protocol  and an intact band   . P2X7R dmembrane , our finp < 0.05 vs. WT animals; n = 7; ASCT2-mediated glutamine release from astrocytes is required for alanine, serine or cysteine in extracellular space . Considep < 0.05 vs. vehicle; n = 7; Next, we applied NAC  to validate whether P2X7R deletion affects the efficacy of NAC in GSH synthesis. In WT mice, NAC elevated GSH concentration to 4.53 ± 0.09 μg/mg protein  signals in both WT and P2X7R KO mice A,B. SincThe major findings in the present study are that P2X7R deletion reduced the basal GSH level, accompanied by increased GS and ACST2 expressions. In addition, the lack of P2X7R prevented the reduced efficacy of NAC in GSH synthesis induced by SIN-1, suggesting the relevance between P2X7R and xCT system .N-glycosylated ASCT2 expression that is an indicative of its cell surface expression [Glutamate is one of the excitatory neurotransmitters and substrates for GSH as well as bioenergetics. In astrocytes, the up-taken glutamate is converted to glutamine via GS ,3. Interpression . Since tpression , our finpression .On the other hand, glutamine is involved in GSH synthesis via the glutamate–glutamine cycle, mediated by GLS ,6,7. ConIn the present study, we found that P2X7R deletion abrogated the GSH-increasing capacity of NAC without altering xCT expression. Since the GSH conversion from NAC is required for subphysiological glutamine level , it is l2+-dependent PKC isoforms, such as PKCα and PKCβI, but not the Ca2+-independent isoform PKCδ in osteoclasts [2+-dependent PKCα, PKCβI, PKCβII, and PKCγ) and Gö6979 (an inhibitor of Ca2+-dependent PKCα and PKCβI) [2+-independent, diacylglycerol-dependent PKC isoform) in glial cells also causes a marked decrease in the expression of GS [There is no experimental evidence or literature concerning P2X7R interactions with xCT. However, over 50 different proteins have been identified to physically interact with P2X7R, since P2X7R contains a long intracellular C-terminus that constitutes 40% of the whole protein . In parteoclasts . Howevereoclasts ,50. Furteoclasts . In the eoclasts . Indeed,d PKCβI) . In addion of GS . With reRecently, the decrease of GSH level has been shown to induce cognitive decline and neuronal death during aging and neurodegenerative diseases ,54. InteThe present study demonstrates for the first time the P2X7R-mediated regulation of GSH levels in the hippocampus. Under physiological conditions, P2X7R deletion reduced the basal GSH level and the efficacy of NAC in GSH synthesis by modulating GS and ASCT2 expression, and presumably by xCT inhibition. In addition, P2X7R deletion prevented the decrease in the efficacy of NAC in GSH production induced by SIN-1. Therefore, we suggest that P2X7R may be involved in the regulation of GSH metabolism under physiological conditions.


We report detection of a highly pathogenic avian influenza A(H5N8) clade 2.3.4.4b virus in Europe. This virus was generated by reassortment between H5N8 subtype virus from sub-Saharan Africa and low pathogenicity avian influenza viruses from Eurasia. A sudden increase in deaths was observed, accompanied by neurologic signs such as trembling, inability to walk, paralysis of the wings, and pedaling movements of the legs. A total of 3,000–5,000 birds died during the first 3 days after the onset of clinical signs. Organ samples submitted to the National Reference Laboratory for Avian influenza at the National Veterinary Research Institute, Pulawy, Poland, were positive for avian influenza virus and were characterized as HPAI H5N8.We conducted antigenic characterization of the virus isolate by using the hemagglutination inhibition (HI) assay, which showed that the H5N8 virus in Poland had higher antigenic reactivity with European Union Reference Laboratory reference HPAI H5N8 A/turkey/Italy/7898/2014 (IT-7898) chicken antiserum  compared with reactivity determined for European Union Reference Laboratory HPAI H5N1 A/chicken/Scotland/1/59 (SCOT-59) and LPAI H5N3 A/teal/England/7394–2805/06 (ENG-7394) antiserum.A comparison of HI titers obtained with the IT-7898, SCOT-59 and ENG-7394 homologous antigens and those recorded against the strain from Poland showed differences of 2 log2, 4 log2, and 5 log2, respectively. The H5N1 and H5N3 strains belong to the H5 Eurasian lineage and are unrelated to the GS/Gd lineage, which supports the marked difference in reactivity. Pathotyping of the virus from Poland by using the intravenous pathogenicity index recorded a value of 3.0, confirming the highly pathogenic phenotype in chickens.Anser albifrons) in Germany, near the border with Poland, was also found to be infected  and 1 case in a wild bird (a dead goshawk found near the index farm) had been detected in different regions from eastern to western Poland. Since the reassortant virus was detected in Poland, the World Organisation for Animal Health has been notified about similar outbreaks in Slovakia, Hungary, Romania, Germany, and the Czech Republic. A greater white-fronted goose (https://www.gisaid.org) under isolate nos. EPI_ISL_405278 and EPI_ISL_402134, respectively. Phylogenetic analysis (The genomes of HPAI H5N8 strains from index cases from Nigeria (A/guinea fowl/Nigeria/OG-GF11T_19VIR8424–7/2019) and Poland (A/turkey/Poland/23/2019) have been sequenced  and submanalysis  of the hanalysis .Topology of the polymerase basic protein 2, polymerase acidic protein, HA, neuraminidase, matrix, and nonstructural protein gene phylogenies showed that the viruses from Poland and Nigeria clustered together (nucleotide identity 98.9%–99.5%)  and alsoFor the polymerase basic protein 1 and nucleoprotein genes, the virus from Nigeria grouped with the 2018 viruses from South Africa as for the other gene segments, whereas the virus from Poland clustered with LPAI viruses identified in recent years in the Chany and Kurgan regions of Russia. This finding indicates that the strain from Poland is a reassortant virus derived from LPAI viruses identified in wild birds in those areas of Asia , which rOur data describe a novel HPAI H5N8 genotype of clade 2.3.4.4b in Europe, in which 6 gene segments originated from sub-Saharan Africa HPAI H5N8 clade 2.3.4.4b viruses and 2 gene segments from Eurasia LPAI viruses. It has been shown that Africa might serve as a strong epidemiologic area of circulation for Gs/GD H5 , compared with the epidemic wave during 2016–17 (October 2016), might be explained by unusually mild temperatures in molting areas in Russia during November and December 2019  virus, Europe, December 2019.