WT) but not cap-mutant eIF4E (eIF4Ecap mutant) increased the activation of the eIF4F complex. Treatment with the active-site dual mTOR inhibitor CC214-1 reduced the level of the eIF4F complex by decreasing the cap bound fraction of eIF4G and increasing the levels of 4E-BP1. CC214-1 inhibited both the cap dependent and global protein translation. CC214-1 inhibited c-Myc, and cyclin D3 translation by decreasing polysomal fractions from lymphoma cells. Inhibition of eIF4E with shRNA further decreased the CC214-1 induced inhibition of the eIF4F complex, c-Myc, cyclin D3 translation, and colony formation. These studies demonstrate that the eIF4F complex is deregulated in aggressive lymphoma and that dual mTOR therapy has therapeutic potential in these patients.Deregulated mRNA translation has been implicated in disease development and in part is controlled by a eukaryotic initiation complex eIF4F . We demonstrate here that the cap bound fraction from lymphoma cells was enriched with eIF4G and eIF4E indicating that lymphoma cells exist in an activated translational state. Moreover, 77% (110/142) of diffuse large B cell lymphoma tumors expressed eIF4E and this was associated with an inferior event free survival. Over-expression of wild-type eIF4E (eIF4E Non-Hodgkin lymphoma (NHL) is the 7th most common cause of cancer in the USA with diffuse large B-cell lymphoma (DLBCL) being the most common type in the US. With staIn vitro studies have attributed this partial and transient response to rapalogs to additional changes that occur, including high expression of mTORC2 and subsequent Akt and eIF4E phosphorylation.[eIF4E is controlled by 4E-binding proteins (4E-BPs) that are one of the important downstream targets of the mTOR pathway. Hyper-phrylation. To improrylation. We recenrylation. The presWe assessed the formation of the active eIF4F (m7GTP-eIF4E-eIF4G) translation initiation complex by a pull down assay using an agarose-immobilized m7GTP cap analog to capture eIF4E and its binding partners eIF4G and eIF4A in MCL cell line cells. The relative amount of captured eIF4G or eIF4A serves as an indicator of the integrity of the eIF4F translation complex. Cell lysates from Jeko, Mino, Granta, JVM2 and CD19+ normal B cells were incubated with m7GTP and analyzed by immunoblotting for the level of eIF4A and eIF4G. Our data demonstrate that the cap-bound fraction from normal B cells contained very little eIF4G and eIF4A. However, all the MCL cell lysates were enriched with eIF4G, eIF4E and eIF4A Figure . In ordeeIF4E/shRNA and HEK293con shRNA stable cells were immunoprecipitated with m7GTP beads and immunoblotted with eIF4G, 4E-BP1 and eIF4E antibodies. eIF4E inhibition reduced the association of eIF4G and 4E-BP1 without affecting whole cell lysate (10% input)  Figure .eIF4E/WT), but not in cap mutant (HEK293cap mutant) cells  was not able to increase the global protein translation (Data not shown). Colony formation in HEK293eIF4E/shRNA was significantly reduced and a 70% reduction was observed in the eIF4E-depleted cells  is responsible for the activated eIF4F complex in aggressive lymphoma.p-value = 0.12)   tumor subtypes  or positive (>10% tumor cells eIF4E positive). Overall, 77% (110/142) patient samples were eIF4E positive Figure . The eIF) Figure . eIF4E e) Figure  or ABC  cell lines. All four MCL cell lines over-expressed hyper-phosphorylated 4E-BP1 at all the 3 sites; however, normal B cells have hypo-phosphorylated 4E-BP1 . CC214-1 inhibited the cap-dependent translation in a dose dependent manner with limited effect on the cap-independent translation as shown by luciferase assay Figure .in-vitro. Similar to the cell lines, CC214-1 treatment caused inhibition of patient tumor cell survival  as compared to control shRNA cells (50%) Figure . To evals Figure . Westerns Figure , right.n = 150) express eIF4E by IHC and that this expression predicts shorter EFS. These results are consistent with other studies in solid tumors that demonstrated inferior patient outcome when the tumor cells overexpressed eIF4E.[The eIF4F complex, an important downstream target of the mTOR pathway, plays a critical role in the regulation of cap-dependent translation. Herein we show that the active translation initiation eIF4F complex (eIF4E-eIF4G-eIF4A) bound to cap is deregulated in aggressive lymphoma cells whereas expression of active eIF4F assembly in normal B cells is low. In the present study we demonstrated that 76% of tumors from newly diagnosed DLBCL patients  lines Jeko, Mino, Granta and JVM2 were purchased from ATCC and grown in Roswell Park Memorial Institute medium with 10% fetal bovine serum (FBS). HEK293T cells were grown in DMEM medium with 10% FBS.Antibodies and reagents. CC214-1, a dual mTORC1/mTORC2 inhibitor, was provided by Celgene Pharmaceuticals.[Th37/46, phospho-4E-BP1Th70, phospho-4E-BP1Ser65, antibodies were purchased from the Cell Signaling Technologies . Antibodies for eIF4E, eIF4G, 4E-BP1, cyclin D3, c-Myc and Mcl-1 were also purchased from Cell Signaling Technologies; antibody to Actin was purchased from Santa Cruz . 7-Methyl guanosine triphosphate-Sepharose 4B (m7GTP) beads were purchased from GE Healthcare .euticals. Phospho-Patient samples. Tissue microarrays (TMA) were constructed using triplicate 0.6-mm cores from paraffin-embedded DLBCL tissue blocks (n = 142) and included 10 nonmalignant tonsil controls. The TMA was obtained through the University of Iowa/Mayo Lymphoma SPORE. All patients provided written consent for use of their samples and outcome information and the master protocol was approved by the Institutional Review Boards of the Mayo Clinic and University of Iowa. Normal B cells were isolated using CD19 microbeads (Miltenyi Biotec) from peripheral blood mononuclear cells from healthy donors.Cap affinity assay (m7GTP). Briefly eight to ten million cells were washed with ice-cold 1x PBS  followed by lysis with cap binding buffer  using four freeze-thaw cycles. 7-Methyl GTP-Sepharose beads were added to the lysates and incubated at 4°C for 3 hours with rotation. Subsequently, the beads were washed to dissolve the protein bound to the beads and western blot was performed with specific antibodies.RNA extraction, cDNA synthesis and RT-PCR. RNA extraction, cDNA synthesis, qualitative and quantitative RT-PCRs (Q-PCR) were performed as previously described.[escribed. The follTransient transfection for adherent cells. For plasmid transfection, cells were transfected with 5 μg of plasmids such as PCDNA3.1, eIF4EWT and eIF4Ecap mutant using lipofectamine 2000 reagent  and Opti-MEM medium. For small interfering RNA (siRNA) experiments, cells were transfected with 50 nM c-Myc siRNA and control siRNA using lipofectamine 2000 reagent.Western blotting. Western blotting was performed as described before.[d before. DensitomColony formation assay. HEK293T cells were treated accordingly and were incubated for 6 days. Cells were washed, fixed and stained with a staining solution . Colonies were counted using colony count program on Alphaimager IS-3400 .3H leucine incorporation assay. 1 × 105 cells were treated accordingly for 48 hours. Then cells were incubated for 24 h in the presence of 2 μCi/ml leucine. 10% trichloroacetic acid was added and cells were incubated for another 30 min at 4ºC. Cells were lysed with 0.4N Sodium hydroxide and transferred in triplicates in a 96 well plate. 3H leucine radioactivity was determined by liquid scintillation spectrometer.Cell survival by annexin V/Propidium iodide. Annexin V/PI binding was assayed as described.[escribed.Luciferase cap dependent transfection. Jeko cells were transfected with luciferase plasmid (pRF) using Amaxa Cell Line Nucleofector kit as per manufacturer's instructions. Plasmid pRF was a kind gift from Dr. Gregory Goodall.['s instruction. Goodall. The luciThymidine incorporation.3H-thymidine incorporation assay as described before.[d before.Polysome analysis. Polysome analysis was performed as described earlier with some modifications.[ications., 39 70–9Immunohistochemistry (IHC). DLBCL cases (n = 132) were classified into GCB or non-GCB molecular type based on the Hans algorithm applied to paraffin-embedded tumor samples.[ samples. IHC on t samples. A 10% cuStatistics. Event-free survival (EFS) was defined as time from diagnosis to progression, relapse, re-treatment, or death due to any cause. Associations between eIF4E status and EFS were assessed using Cox proportional hazards models and Kaplan Meier curves. The p-value for in-vitro data was calculated using the means from 3 different experiments (two-tailed unpaired Student's t test).


RAS. AE9a encodes a C-terminally truncated A/E protein of 575 amino acids that retains the ability to recruit histone deacetylases (HDACs). Expression of AE9a leads to rapid development of leukemia in experimental mouse systems. We have recently shown that treatment of mice bearing A/E9a;NrasG12D tumors with the histone deacetylase inhibitor (HDACi) panobinostat leads to degradation of the A/E9a fusion protein, cell cycle arrest, differentiation of AML blasts into mature granulocytes and prolonged survival. Herein, we sought to enhance this therapeutic effect.One of the most frequently found abnormalities in acute myeloid leukemia (AML) is the t translocation, which is seen in around 15% of patients. This translocation results in the production of the AML1/ETO (A/E) fusion protein and commonly involves cooperating activating mutations of A/E9a;NrasG12D leukemias with panobinostat and arsenic trioxide (ATO) resulted in a significant survival advantage compared to mice treated with either agent alone. Moreover, some of the mice treated with the panobinostat/ATO combination showed complete tumor responses and remained in remission for over 220 days. Panobinostat caused differentiation of A/E9a;NrasG12D cells while ATO induced apoptosis of the leukemic cells, an effect that was enhanced following co-treatment with panobinostat.Combined treatment of mice bearing Our results indicate that leukemic blast differentiation mediated by panobinostat combined with induction of apoptosis by ATO could be therapeutically beneficial and should be considered for patients with t AML.The online version of this article (doi:10.1186/s13148-014-0034-4) contains supplementary material, which is available to authorized users. A/E (AE9a) and NrasG12D that recapitulates the genetics and pathology of human t AML [A/E9a;NrasG12D tumors with the HDACi panobinostat leads to degradation of the A/E9a fusion protein, cell cycle arrest, differentiation of AML blasts into mature granulocytes, and prolonged survival [One of the most frequently found abnormalities in acute myeloid leukemia (AML) is the t8;21) translocation, which is seen in around 15% of patients [survival .A/E9a;NrasG12D tumors with panobinostat resulted in significant therapeutic efficacy [A/E9a;NrasG12D leukemia cells were treated with either vehicle, panobinostat alone, ATO alone, or the combination of panobinostat and ATO -conjugating enzyme 9, resulting in enhanced SUMOylation and degradation of PML [A/E9a;Nras tumor cells treated in vitro for 24 hours with either vehicle (DMSO) or panobinostat  acute promyelocytic leukemia (APL) has resulted in cure rates of up to 90% in both newly diagnosed and relapsed APL patients with few side effects ;17 acute. The mecAlthough HDAC-inhibitors have been minimally successful in the clinic when used as single agents, it is likely that they will be best utilized when combined with other anti-cancer agents. At the onset of this investigation, we performed toxicity studies on mice not bearing tumors to determine the maximum tolerated dose (MTD) of ATO as a single agent or in a combination. Upon establishing our dosing schedule, we observed an initial but not significant weight loss and no significant additional hematological toxicity  and ATO results in degradation of the PML-RARα fusion oncogene, differentiation of tumor cells, and apoptosis in leukemic blasts. The similarities between our results using panobinostat in combination with ATO in mice bearing A/E9a;NrasG12D tumors and the effects of retinoids and ATO in models of PML-RARα-driven APL indicate that, as differentiation therapy combined with ATO has been so successful in curing patients with PML, the combination of panobinostat and ATO for relapsed t patients may have similar success.We propose that patients with t AML may benefit from this type of targeted combination therapy. Indeed, a similar strategy has been extraordinarily successful in improving the treatment for acute promyelocytic leukemia resulting from the t translocation. In that subtype of AML, treatment with all-2O (D5W). Arsenic trioxide (Phenasen™) was obtained from the Peter MacCallum Cancer Centre and further diluted in phosphate-buffered saline to a 0.5 mg/mL solution.All reported research involving the use of animals was approved by the Animal Experimentation Ethics Committee (AEEC) at the Peter MacCallum Cancer Centre (ref. E472). C57BL/6 mice were purchased from the Walter and Eliza Hall Institute of Medical Research. Panobinostat was provided by Novartis and prepared as a 2 mg/mL solution in 5% dextrose/dHG12D  were generated as previously described [Mouse tumors expressing MSCV-AML1/ETO9a-IRES-GFP (A/E9a) and MSCV-luciferase-IRES-Nrasescribed . At the A/E9a;NrasG12D leukemia cells (1 × 106) were then serially transplanted into sublethally irradiated mice by intravenous injection. Treatment was initiated once the leukemic burden reached between 5% and 20% GFP-positive cells in peripheral blood. Mice were treated daily for 5 consecutive days per week via intraperitoneal injection over 4 weeks. Treatment cohorts were either (1) ATO (2.5 mg/kg); (2) panobinostat (25 mg/kg) for 1 week followed by 15 mg/kg for 3 weeks; (3) panobinostat (25 mg/kg) for 1 week followed by panobinostat (15 mg/kg) combined with ATO (2.5 mg/kg); or (4) control mice receiving an equivalent volume of vehicle.To perform therapeutic studies, A/E9a;NrasG12D leukemia cells were cultured ex vivo at 37°C under 5% CO2 in RPMI-1640  containing 20% FCS supplemented with 10 ng/mL IL-3, 10 ng/mL IL-6 and 50 ng/mL SCF . Cells were treated with panobinostat (16 nM) and/or ATO (2.5 μM) for 24 hours or the equivalent volume of vehicle.Whole cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes . Membranes were blocked with 5% nonfat milk in Tris-buffered saline/0.1% Tween-20 at room temperature and incubated overnight with antibodies against AML1 , acetyl-histone H3 , PML , or β-actin  at 4°C. Membranes were developed using appropriate horseradish peroxidase–coupled secondary antibodies  and enhanced chemiluminescence .4Cl, 10 mM KHCO3, 0.1 mM EDTA) and washed twice in fluorescence-activated cell sorter (FACS) staining buffer . Cells were pre-incubated with blocking anti-CD16/CD32 (2.4G2) and stained on ice with antibodies specific for c-Kit (CD117), Sca1 (Ly6A/E), Mac-1 (CD11b) and Gr1  in FACS staining buffer for 30 minutes. Data were collected on a FACSCanto II flow cytometer (BD Biosciences) and analyzed using FlowJo software .Cell suspensions were incubated in red cell lysis buffer (150 mM NHhttp://www.moleculardevices.com/metamorph).Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining was performed using the Apoptag Peroxidase In Situ Apoptosis Detection Kit (Millipore). Sections were counterstained using hematoxylin, and images were recorded with a Zeiss microscope and a × 60 lens. Quantitation of positive TUNEL staining was performed using MetaMorph™ Microscopy Automation & Image Analysis Software (2x2H2O) to detect dead or dying cells by flow cytometry. Data were collected on a FACSCanto II flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).Cells were isolated from the femurs of treated mice and stained with APC-conjugated AnnexinV (BD Biosciences) and 1 μg/ml propidium iodide in AnnexinV binding buffer . All other statistical analyses (using a 2-tailed unpaired Student t test) were also performed with this software.Kaplan-Meier survival curves were created, and survival of mice (using a log-rank test) was statistically analyzed with GraphPad Prism software (


Type 1 diabetes mellitus (T1D) is usually diagnosed when >80% of the pancreatic beta-cells are destroyed by the immune system. The autoimmune destruction of these cells is slow, providing a potentially long lag of time to identify individuals at risk, and maybe prevent T1D development. We can predict T1D development through the determination of some islet autoantibodies. However, these antibodies appear fairly late in the course of T1D, not being ideal biomarkers of the initial destruction of beta-cells. Therefore, new biomarkers are needed to improve the identification of at risk-individuals. MicroRNAs (miRNAs) are a class of small noncoding RNA molecules that negatively regulate gene expression by inducing target mRNA cleavage or by inhibiting protein translation. Changes in their expression were described in several pathological conditions, including autoimmune diseases. Circulating miRNAs are attractive biomarker candidates as they can be easily collected, are stable under different storage conditions and can be measured using specific assays.To investigate circulating candidate miRNAs as potential biomarkers for T1D diagnosis.We analyzed 25 T1D patients  and 20 age- and gender-matched nondiabetic controls. Expressions of 48 miRNAs were investigated in the plasma using Stem-loop RT-PreAmp Real-time PCR and TaqMan Low Density Array cards (Life Technologies). Array data was analyzed using Symphony (Life Technologies) and SPSS 18.0 programs.Seventy-seven percent (37/48) of the miRNAs analyzed were detected in plasma of the sample, some described as being involved in immune regulation or beta-cell function. Among these 37 miRNAs, 35.1% (13/37) were differentially expressed between controls and T1D patients with <5 yrs. of diagnosis: 15.4% (2/13) miRNAs were 2-5 n-fold downregulated (miR-93* and miR-146a) while 84.6% (11/13) miRNAs were 2-40 n-fold upregulated  in T1D patients. On the other hand, no differences were detected between controls and T1D patients with >5 yrs. of diagnosis.Our data demonstrate that some circulating miRNAs are differentially expressed in T1D patients in the first yrs. of the diagnosis. Ongoing studies will further explore the role of these miRNAs as novel biomarkers for T1D prediction.


Sun exposure is the main source of vitamin D. Due to many lifestyle risk factors vitamin D deficiency/insufficiency is becoming a worldwide health problem. Low 25(OH)D concentration is associated with adverse musculoskeletal and non-musculoskeletal health outcomes. Vitamin D supplementation is currently the best approach to treat deficiency and to maintain adequacy. In response to a given dose of vitamin D, the effect on 25(OH)D concentration differs between individuals, and it is imperative that factors affecting this response be identified. For this review, a comprehensive literature search was conducted to identify those factors and to explore their significance in relation to circulating 25(OH)D response to vitamin D supplementation. The effect of several demographic/biological factors such as baseline 25(OH)D, aging, body mass index(BMI)/body fat percentage, ethnicity, calcium intake, genetics, oestrogen use, dietary fat content and composition, and some diseases and medications has been addressed. Furthermore, strategies employed by researchers or health care providers  and environment (season) are other contributing factors. With the exception of baseline 25(OH)D, BMI/body fat percentage, dose and type of vitamin D, the relative importance of other factors and the mechanisms by which these factors may affect the response remains to be determined. According to the Endocrine Society’s definition of vitamin D insufficiency and deficiency, 25(OH)D levels of 50–75 and below 50 nmol/L, respectively , it has Traditionally, severe vitamin D deficiency, deficiency and insufficiency were defined as 25(OH)D concentrations <12, <25 and 25–50 nmol/L, accordingly . There iTo prevent or combat vitamin D deficiency/insufficiency, vitamin D supplementation is an easy, effective and cost-effective strategy. However, in response to a given dose of vitamin D supplement, the increase in 25(OH)D concentration has been reported to differ between individuals ,12,13,14et al. (2014)  vs. 50.0  nmol/L (p = 0.03), respectively  and 25(OH)D2 increased in those receiving daily 1000 IU vitamin D2 for 25 weeks [2 was 32 nmol/L per 1000 IU vitamin D2 daily. The decline in 25(OH)D3 could be explained by lower availability of substrate of vitamin D3 for hepatic hydroxylation. A drop in mean total 25(OH)D was reported in both vitamin D3 and placebo groups by approaching colder months which is consistent with what is known about the effect of season on 25(OH)D concentrations. Lehmann et al. (2013) [3 in both vitamin D2 and placebo groups, however, the decline in vitamin D2 group was 2.5 times more than that of the placebo group  suggesting other mechanisms.Logan 25 weeks . The abs. (2013)  also rep2 may affect the metabolism of vitamin D3 are not clear. Some scientists suggest that physiologic doses of vitamin D2 does not interfere with vitamin D3 metabolism and may increase total 1,25(OH)2D 2D2 accompanied by a slight decrease in 1,25(OH)2D3, mirroring the increase in 25(OH)D2 and decrease in 25(OH)D3, respectively) [et al., on the other hand, suggested that the up regulation of mechanisms involved in vitamin D2 metabolism may lead to an increase in the degradation of 25(OH)D3 [3 than vitamin D2 [3 is hydroxylated in liver mitochondria is five times more than that of vitamin D2  [3 is preferentially 25-hydroxylated vitamin D2 is 24-hydroxylated and deactivated [3; this molecule first undergoes 25-hydroxylation, then 24-hydroxylation and then an additional side chain oxidation to be biologically deactivated [3 and its metabolites than vitamin D2 may explain the higher potency of vitamin D3 [The mechanistic pathways by which vitamin Dctively) ,92. Arma25(OH)D3 . There itamin D2 . The ratctively) . While vctivated . This isctivated . Houghtotamin D3 .2 for VDBP than that of 25(OH)D3 results in a shorter half-life of 25(OH)D2 [2 metabolites might be more evident at the pharmacological doses. Furthermore, if 25(OH)D2 is not recognised the same as 25(OH)D3 by kidney and acts as an additional substrate, as was proposed by Biancuzzo et al. (2013), pharmacological doses of vitamin D2 may result in an increase in total 1,25(OH)2D. High concentrations of 1,25(OH)2D decrease the production and excretion of PTH [The lower affinity of 25(OH)D25(OH)D2 . The shon of PTH ,97, inhi2, in clinical settings, have been proven to be effective in maintaining serum 25(OH)D above 50 nmol/L in people with vitamin D deficiency [It should be noted that despite the evidence (intervention and mechanistic studies) of differences in potency between the two isoforms, pharmacologic doses of vitamin Dficiency ,99.et al. (2014) reported a dose-dependent increase in 25(OH)D concentration by using placebo or 30,000 and 60,000 IU vitamin D monthly and an incremental change of 2.2 nmol/L and 1.8 nmol/L per 100 IU vitamin D input in the 30,000 IU and 60,000 IU groups, respectively [Evidence shows that higher doses are associated with smaller increases in 25(OH)D concentrations per unit of vitamin D compared with lower doses of vitamin D ,46,100. ectively  and 4000ectively  have beeHepatic hydroxylation is a saturable process and with an input above physiological norm of vitamin D, serum vitamin D concentration increases, and the reaction switches from the first order to zero order . It is pet al. (2011) also reported a higher compliance rate in subjects receiving monthly regimen  than daily regimen  [The dosing frequency may influence the response to supplementation through its effect on compliance rate ,87. An iet al. (2013) showed that 25(OH)D response to supplementation was better in oral than IM form  [vs. 43.6%, respectively, p = 0.06) [Vitamin D supplements administered intramuscularly (IM) are often given in bolus dosages, and are useful for patients with absorption disorders and with low compliance and in areas where oral supplements are not available. However, there are some concerns about the safety  and effe = 0.03) . It shou = 0.03) . However = 0.06)  and the  = 0.06)  at the fet al. (2007) assigned healthy post-menopausal women to receive either daily 800 IU for two years followed by daily 2000 IU vitamin D for one year or placebo through the entire study period [Several trials have shown that 25(OH)D response to vitamin D supplementation peaks at three-months ,15,104, y period . Mean 25p < 0.001) [In another study, however, 25(OH)D concentrations increased and PTH levels decreased significantly at six months compared with three months (< 0.001) . Further< 0.001) ,54. Unre< 0.001) .et al. (2012) [3 and vitamin D3 and converts them into inactive photoproducts including lumisterol, tachysterol and 7-dihydroxy cholesterol [Seasonal effect on 25(OH)D concentrations has been reported in several trials. Circulating 25(OH)D concentration has been frequently reported to be lower in winter than summer months due to the vitamin D input from sun exposure . The sea. (2012) . Holick lesterol .The relationship between 25(OH)D concentration and vitamin D supplementation is not straightforward and is influenced by a large number of factors. Some of these factors such as basal 25(OH)D concentration are well documented. Evidence is emerging for others such as BMI/body fat% and season, while for calcium intake, dietary fat content and composition, and genetics the evidence is either mixed or in its infancy. The mechanisms by which these factors may affect the response are not well understood. Accordingly, there is an urgent need for more well-designed studies: (1) to establish the significance of these factors; (2) to identify other unknown factors; (3) to determine the mechanistic pathways by which these factors may exert their roles and (4) to strengthen our knowledge and understanding to inform the dose of supplementation required. It should be noted that increasing 25(OH)D concentration alone is not meaningful if it is not accompanied by improved clinical outcomes, or at least biomarkers. Clinical trials investigating the impact of improving vitamin D status on various health and disease outcomes are also warranted. Finally, chronic diseases are multifactorial in origin and many variables that contribute to the development of these diseases, such as unhealthy dietary habits, aging and physical inactivity, are risk factors for vitamin D deficiency . Therefo


Separation of low boiling gas mixtures is widely concerned in process industries. Now their separations heavily rely upon energy-intensive cryogenic processes. Here, we report a pseudo-absorption process for separating low boiling gas mixtures near normal temperature. In this process, absorption-membrane-adsorption is integrated by suspending suitable porous ZIF material in suitable solvent and forming selectively permeable liquid membrane around ZIF particles. Green solvents like water and glycol were used to form ZIF-8 slurry and tune the permeability of liquid membrane surrounding ZIF-8 particles. We found glycol molecules form tighter membrane while water molecules form looser membrane because of the hydrophobicity of ZIF-8. When using mixing solvents composed of glycol and water, the permeability of liquid membrane becomes tunable. It is shown that ZIF-8/water slurry always manifests remarkable higher separation selectivity than solid ZIF-8 and it could be tuned to further enhance the capture of light hydrocarbons by adding suitable quantity of glycol to water. Because of its lower viscosity and higher sorption/desorption rate, tunable ZIF-8/water-glycol slurry could be readily used as liquid absorbent to separate different kinds of low boiling gas mixtures by applying a multistage separation process in one traditional absorption tower, especially for the capture of light hydrocarbons. The operation temperature of cool tank for separating hydrogen (H2) with methane (CH4) is as low as 113 K and the separation temperature for CH4 with C2H4 also reaches 163 K. Motivated by the staggering energy costs associated with these deep cool separations, developing the substitute technologies has become a very active area3456Separation of low boiling gas mixtures is widely involved in process industries, especially in petrochemical industry. Now their separations heavily rely upon energy-intensive distillation-based cryogenic technologies, which present a class of the most important and also the most costly processes in the chemical industry12 adsorption/storage and carbon dioxide (CO2) capture in the past decades14151617182021222324252627282930As an emerging class of porous materials, metal-organic frameworks (MOFs)910111213The enthusiasm of the scientific community about MOFs and other nano-porous materials as solid-adsorbents, however, does not yet resonate in the process engineering community232Membrane process is another approach for the separation of low boiling gas mixtures at normal temperature3134363738392 remained, while that for other gas species  decreased dramatically. Thus the ZIF-8/glycol slurry could be used for CO2 capture with high selectivityIn order to take the advantages of both MOFs and distillation, we recently proposed a so called absorption-adsorption hybrid methodIn this work we demonstrate that the key factor affecting the separation selectivity of absorption-adsorption hybrid method is the penetration selectivity of liquid membrane formed by solvent molecules around the ZIF-8 particles for different gas species. Green solvents like water and glycol were used to adjust the penetration selectivity of liquid membrane aiming at enhancing the separation selectivity of ZIF-8 slurry for different kinds of low boiling gas mixtures. By this way, ZIF-8/water-glycol slurry could be tuned to integrate absorption, membrane, and adsorption into a pseudo-absorption process with enhanced separation efficiency over single absorption, membrane or adsorption process and easily applied in traditional multistage separation process like distillation for separating low boiling gas mixtures near normal temperature.4, C2H6, C2H4, C3H6, N2, and H2) in ZIF-8 suspended in glycol and water, respectively. By comparing them with the isotherms on pure solid ZIF-8 . Lighter molecules such as N2 and H2 have very high osmotic pressures. As shown by 3H6, C2H6, C2H4 and CH4, in lower gas uptake range, while there are still high osmotic pressures for lighter species N2 and H2. This phenomenon implies that ZIF-8/water slurry could be used to separate light hydrocarbons from N2 and H2 promisingly.Obviously, there are big differences in osmotic pressures between lighter and heavier components for both ZIF-8/glycol slurry and ZIF-8/water slurry. There is no observable osmotic pressure for propene , coal bed gas (CH4/N2), pyrolysis gas (CH4/C2H4/C2H6/C3H6/H2) involved in the production of C2H4, tail gas of synthesis ammonia apparatus (H2/N2/CH4/Ar), and refinery dry gases (CH4/C2H4/C2H6/N2/H2/CO2) is widely involved in energy and petrochemical industries. As stated above, most gas species involved in these gas mixtures have certain osmotic pressures on ZIF-8/glycol slurry except for C3H6. For CO2, we can eliminate the osmotic pressure by adding methylpyrazole (mIm) to glycol and it could then be separated from other gas species with high efficiency2H4 or C2H6 in both ZIF-8/glycol slurry and ZIF-8/glycol-mIm slurry are very low unless their partial pressures are high enough. This means they cannot be removed from gas mixture sufficiently using these slurries unless the operation pressure is very high. C3H6 might be separated from gas mixtures containing lighter components  by using ZIF-8/glycol slurry sufficiently. However, it can also be separated from them by using ZIF-8/water slurry or ZIF-8/water-glycol slurry efficiently. As two later slurries have lower viscosities  are in simple thermodynamic equilibrium without the presence of liquid membrane and the liquid absorbent and solid adsorbent in the slurry do not affect each other. In this case, the selectivity of the slurry is the weighted average of the selectivity of the (pure) liquid absorbent and that of (pure) solid adsorbent2H4 is very important raw material for the production of large number of chemicals, the low cost separation of C2H4 is strongly concerned by petrochemical industries. In most cases, C2H4 is accompanied by C2H6 in gas mixture, e.g., pyrolysis gas and catalytic cracking (FCC) dry gas. As C2H6 is the best raw material for producing C2H4, its capture is also very important. As an example of practical use of ZIF-8 slurry, here we put emphasis on the capture of C2 (C2H4 + C2H6) from catalytic cracking dry gas CH4/C2H6/C2H4/N2/H2. As pyrolysis gas CH4/C2H6/C2H4/N2/H2 is the sub system of FCC dry gas, the results obtained here are also applicable for separation of pryolysis gas.As C4/C2H6/C2H4/N2/H2 (24.83/7.24/19.82/26.71/21.40 mol%) was synthesized according to the typical composition of catalytic cracking dry gas. At first, we compared the C2 capture efficiency of different slurry materials, ZIF-8/glycol, ZIF-8/water, ZIF-8/water-glycol with a glycol mass fraction of 0.2 in aqueous phase. The detailed experimental results were shown in SC2) and the highest capture selectivity (S) of C2 over other components. As expected, the ZIF-8/glycol slurry has very low sorption capacity because of higher osmotic pressures, i.e. the sorption impetus is too low for gas molecules to enter the pores of ZIF-8.The feed gas CHSC2) on ZIF-8/water slurry decreases with the increase of its partial pressure. For practical application, the higher gas/absorbent ratio is always critical for decreasing the separation cost. The gas/absorbent ratio is determined by the solubility coefficient of object gas species. Thus how to increase SC2 is very important. SC2 increased more than three times when temperature decreased from 293.15 K to 269.15 K under 29 bar or so. The separation selectivity also increased obviously. However, SC2 increases little when temperature changes from 303.15 K to 283.15 K. This is consistent with As shown by 4/C2H6/C2H4/N2/H2 (25.06/2.99/8.03/34.19/29.78 mol%) and CH4/C2H6/C2H4/N2/H2 (25.12/17.89/42.51/12.99/1.49 mol%) were synthesized according to the gas compositions of gas phase and slurry phase produced in the separation of feed gas CH4/C2H6/C2H4/N2/H2 (24.83/7.24/19.82/26.71/21.40 mol%) at 269.15 K and 2.98 MPa. The former one was separated under same pressure of 2.98 MPa and a little lower temperature of 267.15 K. The later one was separated under lower pressure of 1.08 MPa and higher temperature of 293.15 K. In fact, this separation represent a flash of slurry produced in the initial separation of feed gas CH4/C2H6/C2H4/N2/H2 (24.83/7.24/19.82/26.71/21.40 mol%) under 2.98 MPa. The experimental results for three equilibrium stage separation were shown in For evaluating multistage separation efficiency of ZIF-8/water-glycol slurry, we performed separation experiments simulating a three-stage separation involving both temperature swing and pressure swing with respect to the capture of C2 from FCC dry gas. To do so, another two feed gas samples CHEssential to any gas capture material is the energy required for gas release. Here the regeneration properties of both ZIF-8/water and ZIF-8/water-glycol slurry were also investigated. The separation ability of these two kinds of slurries that regenerated under atmosphere pressure and that regenerated by vacuuming at room temperature were shown in 2 and H2. When using the mixed solvent composed of water and glycol, the osmotic pressure changes with the increase of glycol concentration. By this way, the apparent separation selectivity of ZIF-8 slurry becomes tunable. When tuning the separation selectivity of ZIF-8 slurry, other factors should also be taken into account. In positive aspect, besides increasing separation selectivity, adding glycol to water makes the slurry more stable and finer. It can also inhibit the emergence of serious foaming during desorption. In addition, it can decrease the freezing point of slurry and allow the slurry to work below the ice point (273.15 K) where dramatic increase of sorption capacity may occur. In negative aspect, adding glycol to water results in an increase of viscosity and a decrease of sorption/desorption rate. One should balance the positive factors and negative ones when determining the content of glycol.Several important insights emerge in this work. When porous material like ZIF-8 suspended in suitable solvents whose molecules are too large to enter the pores of solid particles, solvent molecules self-assemble liquid membrane surrounding solid particles. Compared with traditional solid membrane, the self-assembly liquid membrane will never be destroyed and its permeability is fully tunable by adjusting the composition of solvent. The strength of the membrane is determined by the interaction between the solvent molecules and the function group on the surface of solid particles. Just like traditional solid membrane, there also exist obvious and different osmotic pressures between two sides of liquid membrane for different gas molecules. This is attributed to the large difference between the concentration of gas molecules in solvent phase and solid phase, which results in a reverse gas concentration gradient across the liquid membrane, i.e., the gas concentration at the inner boundary might be larger than that at the outer boundary of the membrane. Hence a positive excess pressure, i.e., osmotic pressure, is required for the transfer of gas molecules through the liquid membrane from outside to inside. The strength or permeability of liquid membrane for specific gas species could be tuned aiming at increasing the apparent separation selectivity of the slurry formed by porous material and solvent. Surrounding ZIF-8 particles, glycol molecules form tighter membrane, indicated by higher osmotic pressures for most gas species lighter than propene. Water molecules form looser film because of the hydrophobic behavior of ZIF-8 and osmotic pressures emerge only for very light gas species like N2H4, C2H6, etc.) at temperature only 4 K below the ice point, ZIF-8/water-glycol slurry has high sorption capacity and selectivity, indicating capture of light hydrocarbons might become cheaper if using ZIF-8/water-glycol slurry compared with traditional separation approaches like PSA, middle cooling oil absorption, cryogenic separation, etc. We think ZIF-8/water-glycol slurry approach may revolutionize the separation of low boiling gas mixture because it takes the advantages of distillation, membrane, and adsorption together.When using ZIF-8/solvent slurry as absorbent for gas mixture separation, three single separation processes, i.e. absorption, membrane, and adsorption, are integrated into one pseudo-absorption process. Due to the membrane effect of liquid film, enhanced separation could be achieved compared with simple absorption or simple adsorption. Our separation experiments on different kinds of gas mixtures show that ZIF-8/water slurry always manifests remarkable higher separation selectivity than solid ZIF-8 and it can be tuned to further enhance the capture of light hydrocarbons by adding suitable quantity of glycol to water. ZIF-8/water-glycol slurry is really liquid-like as diameters of most ZIF-8 particles are 500 nm or so. When the glycol concentration in liquid is controlled to be below than 20 wt%, its viscosity is obviously lower than that of pure liquid glycol. More importantly, it has high sorption/desorption rate. We believe it can be readily used to separate different kinds of low boiling gas mixtures under or near normal temperature, by applying traditional absorption towers with multiple gas-liquid equilibrating stages. Especially, we found for light hydrocarbons  was used to analyze the composition of the prepared gas mixtures.Materials used in this work include ZIF-8, glycol, water, and feed gases. Among them, ZIF-8 was purchased from Sigma-Aldrich. Glycol was purchased from Beijing Chemical Reagents Company, China. Analytical grade nitrogen (99.99%), methane (99.99%), ethylene (99.99%), ethane (99.99%), propylene (99.99%) and hydrogen (99.999%) were purchased from Beijing AP Beifen Gas Industry Company, China. The feed gas mixtures CH3 and that of the blind cell plus tubes connecting to it is 112 cm3. The maximum working pressures of these two cells are designed to be 20 MPa. To directly observe samples in the cell, a luminescence source of type LG100H is mounted on the outside of the cell. A secondary platinum resistance thermometer (type-pt100) is used as the temperature sensor. A calibrated Heise pressure gauge and differential pressure transducers are used to measure the system pressure. The uncertainties of pressure and temperature measurements are ±0.01 MPa and ±0.1 K, respectively. Real-time readings of the system temperature and pressure are recorded by a computer.All the ab(d)sorption measurement experiments were performed using the experimental apparatus as schematically illustrated in PE. Gas mixture in the equilibrium gas phase of the sapphire cell was sampled under constant pressure by pushing the connected hand pump and analyzed by a HP 7890 gas chromatograph. The volume of the slurry in the sapphire cell can be obtained by measuring the height of the equilibrium liquid phase. The inner radius of the sapphire cell is known to be 1.27 cm. In this work, the sorption amount of one gas species in the slurry was determined through mass balance as described below.Before the experiments, the sapphire cell was dismounted from the apparatus, washed with distilled water and dried, then loaded with a known quantity of dry porous material. After that, a known amount of solvent was immersed into the sapphire cell slowly and evenly. Both the used dry porous material and solvent were weighed by an electrical balance with a precision of ±0.1 mg. The mixture of porous material and liquid solvent was stirred to form a suspension mixture . Subsequently, the cell was installed back onto the apparatus. The system (sapphire cell + blind cell + tubes connecting two cells) was then purged through vacuuming. Enough amount of feed gas was injected into the blind cell, then the desired value of temperature was set through the air-bath. Once both temperature and pressure of the blind cell were kept constant, the pressure of gas mixture in the blind cell was recorded as the initial pressure nt) of the feed gas mixture that was injected into the sapphire cell is calculated by the following formula:The total mole number  in the gas phase of the sapphire cell after sorption equilibrium is determined by:where PE is the equilibrium pressure of the sapphire cell and ZE is the compressibility factor corresponding to T , PE, and equilibrium gas composition. Vg is the volume of equilibrium gas phase in the sapphire cell. The total sorption amount of one gas species i in the slurry is calculated as,where zi and yi are the mole fractions of gas species i in the feed gas and equilibrium gas phase, respectively. Its apparent mole fraction in the equilibrium slurry phase then can be obtained by the following formula:where m is the component number of gas phase. The net adsorption amount  of the slurry is calculated with,where 2H6 + C2H4), Sc2, in the slurry and the apparent selectivity (S) of C2 over all other gas components (CH4 + N2 + H2) together are used to characterize the ability of the slurry recovering C2 from multi-component feed gas mixtures, CH4 (1)/C2H6 (2)/C2H4 (3)/N2 (4)/H2 (5),In this work the apparent volume solubility coefficient of C2 (CThe adsorbents (solid ZIF-8) were characterized by XRD (SIMADU XRD 6000) with Cu Kα radiation  at a scanning rate of 2 °C per minute. The morphologies and energy dispersive X-ray spectroscopy measurements were obtained using a FEI Quanta 200F scanning electron microscope.The size distribution of samples in ZIF-8/liquid slurry was measured by virtue of the Zetasizer Nano-ZS laser nanoparticle-size analyzer manufactured by the Britain Malvern Instruments. The viscosity of ZIF-8 slurry were measured by using a modified Cannon-Ubbelohde suspended level capillary viscometers of (0.8 mm) in diameterHow to cite this article: Liu, H. et al. Tunable integration of absorption-membrane-adsorption for efficiently separating low boiling gas mixtures near normal temperature. Sci. Rep. 6, 21114; doi: 10.1038/srep21114 (2016).


As a vaccine vector the E1B55K deletion may allow for the insertion of a transgene nearly 1,000 base pairs larger than now possible. This has the potential of extending the application for which the vectors are clinically known. However, the immune priming ability of E1B55K-deleted vectors is unknown, undermining our ability to gauge their usefulness in vaccine applications. For this reason, we created an E1B55K-deleted Ad5 vector expressing full-length single chain HIVBaLgp120 attached to a flexible linker and the first two domains of rhesus CD4 (rhFLSC) in exchange for the E3 region. In cell-based experiments the E1B55K-deleted vector promoted higher levels of innate immune signals including chemokines, cytokines, and the NKG2D ligands MIC A/B compared to an E1B55K wild-type vector expressing the same immunogen. Based on these results we evaluated the immune priming ability of the E1B55K-deleted vector in mice. The E1B55K-deleted vector promoted similar levels of Ad5-, HIVgp120, and rhFLSC-specific cellular and humoral immune responses as the E1B55K wild-type vector. In pre-clinical HIV-vaccine studies the wild-type vector has been employed as part of a very effective prime-boost strategy. This study demonstrates that E1B55K-deleted adenoviruses may serve as effective vaccine delivery vectors.Type 5 human adenoviruses (Ad5) deleted of genes encoding the early region 1B 55-kDa (E1B55K) protein including Onyx-015 ( ΔE3) is being developed for both HIV and influenza vaccines [E1B55K gene is deleted a defect allows the vector to replicate in and selectively kill cancer cells. Thus E1B55K-deleted vectors such as Onyx-015 (dl1520) have made it to phase 3 clinical trials in the US as cancer therapeutics. A similar vector (H101) has been released to treat cancer in China [E1B55K-deleted vectors may be suited for vaccine delivery purposes as well. Such vectors may permit the insertion of larger transgenes and/or immune modulators not currently possible with the E1B55K wild-type vector, however their immune priming ability remains unknown. To address this, we created a replication-competent E1B55K-/E3-deleted Ad5 host-range mutant (Ad5hr)-recombinant encoding full-length single chain HIVBaLgp120 attached to a flexible linker and the first two domains of rhesus CD4 (rhFLSC). This rhFLSC HIV immunogen was previously described [E1B55K wild-type vector for immunization of mice and nonhuman primates [E1B55K-deleted vector were able to promote similar specific immune responses as the wild-type vector, then it might stand as a better vaccine delivery vector.To facilitate the goal of an effective preventative HIV vaccine, we are developing an approach involving priming with replicating adenovirus (Ad) recombinants and boosting with envelope protein. This approach has elicited potent transgene-specific humoral and cellular immune responses  capable vaccines –10. Replin China , 12. E1Bescribed  and usedprimates , 15. TheE1B55K deletion may display innate immune signals that differ from an E1B55K wild type vector. In E1B55K-deleted virus compared to those infected with the E1B55K wild-type virus.Ad5 E1B55K is reported to control expression of immune response genes . Therefo+ T cells, some CD4+ T cells and γδ T cells [E1B55K deletion on NKG2D ligands we infected HeLa cells that are known to express MIC A/B with MAd5rhFLSC or ΔE1B55KrhFLSC. At early times post-infection Ad5 seems to repress surface expression of MIC A/B. By 48 hours post infection (hpi) surface expression levels of MIC A/B increased in a fraction of ΔE1B55KrhFLSC-infected cells to above that observed for both mock and MAd5rhFLSC-infected cells  band was observed in lanes containing DNA from the ΔE1B55KrhFLSC vector and a 1759 bp band in lanes containing DNA from the MAd5rhFLSC vector . In our previous study we observed only very low levels of Ad5-specific T cells in mice immunized with the MAd5rhFLSC vector . Thus thE1B55K deletion on the levels of IgG binding antibodies induced against the vector and the HIV immunogen, sera from immunized mice were evaluated by ELISA. Both the ΔE1B55KrhFLSC and MAd5rhFLSC vectors induced high-titered binding antibodies against rhFLSC and the HIVBaLgp120 subunit  by binding of the inoculum virus to B-cells, macrophages and or dendritic cells; 2) by viral proteins produced and secreted into the surrounding milieu; 3) by the uptake of infected dead or dying cells by surrounding cells. All of these lead to processing and antigen presentation. The transgene by contrast, only engages the immune system by the latter two mechanisms. Consequently, there might be a limit beyond which immunogen expression levels make no immunological difference. In that case the different levels of rhFLSC promoted by the two vectors  would noesponses , 27. For viruses  supporti viruses . While tBaLgp120 and Ad5 in the immunized mice  was approved by the NCI-Bethesda Animal Care and Use Committee (ACUC). All efforts were made to minimize suffering.2.All cell lines were obtained from the American Type Culture Collection. Cervical carcinoma-derived HeLa cells, human embryonic kidney-derived 293 cells, human lung adenocarcinoma epithelial derived A549 cells, and African green monkey kidney-derived CV-1 cells were maintained in Dulbecco’s modified Eagle’s medium (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies). Mouse lung epithelial cells TC1 and LA4 were maintained in RPMI (Life Technologies) supplemented with 10% newborn calf serum (HyClone Laboratories). Cells were maintained at 37°C in a humidified atmosphere with 5% COE1B55K) is described elsewhere [ΔE1B55KrhFLSC, BamHI digested, gel isolated pBRAd5hrΔE3 (TPL-rhFLSC-pA) shuttle plasmids [dl1520 [ΔE1B55KrhFLSC recombinant candidates were screened by PCR using the following forward and reverse primer pairs: TTTTCTGCTGTGCGTAACTT; ATCTTCATCGCTAGAGCCAA. This yielded a 1759 base pair fragment in the wild-type but an 827 base pair fragment in viruses lacking E1B55K (E1B55K-deleted virus (6.0x1010 particles/5.0x1010 PFU) was 1.2 and for MAd5rhFLSC 1.3.The MAd5rhFLSC virus . The animal protocol was reviewed and approved by the Animal Care and Use Committee prior to implementation. Mice  were inoculated intraperitoneally at weeks 0 and 4 with 5.0x10escribed .6) were treated and stained as previously described [+ and CD8+ populations, were subdivided into CD44high cells. This gating groups the central memory (CM) and effector memory (EM) cells as previously described [Balgp120 peptides was determined following subtraction of the values obtained with non-stimulated samples.Splenocytes (2 × 10escribed  except hescribed . The per9 PFU Ad5hr or 100ng per well of either HIVBaLgp120 (ABL) or rhFLSC (Profectus BioSciences). The plates were exposed to 1% BSA blocking solution (KPL) for 2 hours at room temperature. The serum samples were serially diluted and applied in duplicate to the 96-well plates and incubated at 37°C for 1 hour. The plates were washed with PBS–Tween, exposed to either peroxidase-conjugated goat anti-mouse IgG (H + L), IgM, IgA, or rat anti-mouse IgG1 or IgG2a, and thereafter incubated for another hour. After washing the plates were developed with TMB  peroxidase substrate solution. The reaction was stopped by adding 1M H3PO4 and the plates were read at 450nm within 30 min. Endpoint titers were defined as two times the OD corresponding to the background value of the plate as the cutoff.Antibody binding titers were assayed by enzyme-linked immunosorbent assay (ELISA). Ninety-six well plates were coated with 1.0x10Cells were infected in serum free media, lysed with RIPA buffer (Life Technologies), and chemokines/cytokines were captured using a 25-plex Multiplex ELISA according to the manufacturer’s specifications (Life Technologies). The plates were read and analyzed using a Bio-Plex 100 instrument and software (BioRad).HeLa cells were infected at a MOI of 50 for 48, 72 or 96 hours. The cells were washed twice with PBS and surface stained for 30 min at RT with an isotype control or MIC A/B-APC antibodies (Biolegend) at concentrations determined by titration, and resuspended in 1% paraformaldehyde in PBS. Approximately 20,000 cells were acquired for analysis using a FACS Calibur Cytometer. Data were analyzed using FlowJo version 9.5.2 (Tree-Star Inc.) and graphs and statistics were obtained using Prism 6.0 (GraphPad Software Inc.).Initial statistics were obtained using Prism v6.0 (GraphPad) and confirmed using SAS/STAT Softward version 9.3 (SAS Institute Inc.). Differences between measures were assessed using either a Mann-Whitney-Wilcoxon paired test or one-way ANOVA. Logarithmic or arcsine transformation of raw data was applied when needed for consistency with distributional assumptions.


We determined the prevalence of influenza A virus in dogs in Taiwan and isolated A/canine/Taiwan/E01/2014. Molecular analysis indicated that this isolate was closely related to influenza A(H6N1) viruses circulating in Taiwan and harbored the E627K substitution in the polymerase basic 2 protein, which indicated its ability to replicate in mammalian species. Infections with influenza viruses are rare in dogs. However, interspecies transmission of an equine influenza A(H3N8) virus to dogs was identified during a respiratory disease outbreak in Florida, USA, in 2004  dogs at the National Taiwan University Veterinary Hospital in Taipei. The remaining 193 serum specimens were obtained from free-roaming dogs in rural areas.All serum specimens were tested for antibodies against influenza A virus by using a species-independent blocking ELISA . All antibody-positive serum specimens were further tested by using a hemagglutination inhibition (HI) assay. HI was determined according to procedures recommended by the World Organisation for Animal Health. Chicken erythrocytes (1%) were used. Serum samples were treated with receptor-destroying enzyme  before conducting the assay to destroy nonspecific inhibitors , and RNA was extracted by using a commercial kit  according to the manufacturer’s instructions. A 1-step RT-PCR was then performed by using the One-Step RT-PCR Kit (QIAGEN). A primer set (M52C/M253R) specific for a highly conserved region of matrix (M) gene was used for detection of influenza A virus nucleotides (http://platform.gisaid.org/epi3/frontend#185d95) and GenBank. Multiple sequence alignments and phylogenetic analyses were performed by using MEGA6 software . Trees were constructed by using the maximum-likelihood method and analysis with 1,000 bootstrap replications.Phylogenetic trees were constructed with complete nucleotide sequences obtained from the Global Initiative on Sharing All Influenza Data  household dogs and 6/193 (3.1%) free-roaming dogs were positive by ELISA for influenza A virus. The HI assay showed that 1 of the 9 virus-positive dogs had antibodies against influenza A(H6N1) virus (titer = 20). This dog was from Yunlin, Taiwan, a rural area that is a major site for poultry production. No serum samples had antibodies against influenza A(H5N2) virus.Nasal swab specimens from 4/185 (2.1%) dogs were positive by RT-PCR for influenza virus M gene. All 4 RT-PCR positive dogs had nasal discharges or coughing. Three of these dogs were <6 months of age and adopted from an animal shelter (n = 1) or rescued from the streets (n = 2). The fourth dog was a 15-year-old household pet.Influenza A virus was isolated from a 4-month-old dog co-infected with canine distemper virus. The virus influenza isolate was designated A/canine/Taiwan/E01/2014 (GenBank accession nos. KM20333–KM203344). This dog was rescued from the streets and had severe purulent nasal discharge, cough, and fever. Chest radiographs showed a severe bilateral air bronchogram in the lung field. Serum specimens  from this dog were assessed by using ELISA and HI assay, but no seroconversion was observed.Sequence homology of 8 influenza virus gene segments from A/canine/Taiwan/E01/2014 (H6N1) was compared with segments in the Global Initiative on Sharing All Influenza Data . Hemaggl,,Only 1 basic amino acid (PQIATR*G) was found at the HA cleavage site of A/canine/Taiwan/E01/2014. G228S substitution (H3 numbering) on the receptor binding site for HA was also observed for this virus, which indicated increased virus binding ability for the α2–6 sialic acid receptor  isolated from humans ,,.Phylogenetic analysis of HA and NA gene segments indicated that A/canine/Taiwan/E01/2014 belongs to the H6N1 lineage that has been circulating in chickens in Taiwan since 1997 .–Avian influenza A(H6N1) viruses have been widespread in chickens in Taiwan since 1972  dogs had RT-PCR−positive results for this virus. A/canine/Taiwan/E01/2014 was isolated from 1 dog that was co-infected with canine distemper virus. On the basis of molecular analysis of A/canine/Taiwan/E01/2014, HA, NA, PB1, PB2, NP, and NS genes showed high homology (>97% nucleotide identity) with avian H6N1 subtype virus isolates that are currently prevalent in Taiwan. PA and M genes of A/canine/Taiwan/E01/2014 showed 99% nucleotide identity with A/chicken/Taiwan/2593/2013 (H5N2).Phylogenetic analysis showed that 8 eight virus genes were derived from H6N1 subtype viruses isolated in Taiwan. All 8 influenza virus genes found in the dog probably originated from avian sources. We speculate that a complete avian influenza virus had infected this dog. However, additional analysis is required to verify this hypothesis.Phylogenetic relationship between influenza A(H6N1) virus A/canine/Taiwan/E01/2014 isolated from dogs in Taiwan and other influenza A virus lineages.


Avian influenza A (H7N9) virus has emerged recently and continues to cause severe disease with a high mortality rate in humans prompting the development of candidate vaccine viruses. Live attenuated influenza vaccines (LAIV) are 6:2 reassortant viruses containing the HA and NA gene segments from wild type influenza viruses to induce protective immune responses and the six internal genes from Master Donor Viruses (MDV) to provide temperature sensitive, cold-adapted and attenuated phenotypes.LAIV candidate A/Anhui/1/2013(H7N9)-CDC-LV7A (abbreviated as CDC-LV7A), based on the Russian MDV, A/Leningrad/134/17/57 (H2N2), was generated by classical reassortment in eggs and retained MDV temperature-sensitive and cold-adapted phenotypes. CDC-LV7A had two amino acid substitutions N123D and N149D (H7 numbering) in HA and one substitution T10I in NA. To evaluate the role of these mutations on the replication capacity of the reassortants in eggs, the recombinant viruses A(H7N9)RG-LV1 and A(H7N9)RG-LV2 were generated by reverse genetics. These changes did not alter virus antigenicity as ferret antiserum to CDC-LV7A vaccine candidate inhibited hemagglutination by homologous A(H7N9) virus efficiently. Safety studies in ferrets confirmed that CDC-LV7A was attenuated compared to wild-type A/Anhui/1/2013. In addition, the genetic stability of this vaccine candidate was examined in eggs and ferrets by monitoring sequence changes acquired during virus replication in the two host models. No changes in the viral genome were detected after five passages in eggs. However, after ten passages additional mutations were detected in the HA gene. The vaccine candidate was shown to be stable in the ferret model; post-vaccination sequence data analysis showed no changes in viruses collected in nasal washes present at day 5 or day 7.Our data indicate that the A/Anhui/1/2013(H7N9)-CDC-LV7A reassortant virus is a safe and genetically stable candidate vaccine virus that is now available for distribution by WHO to vaccine manufacturers. Vaccines are still the best countermeasure against emerging influenza virus infections with pandemic potential. Antigenic analysis of influenza A(H7N9) viruses isolated indicates that the hemagglutinin (HA) and neuraminidase (NA) genes of the viruses remain similar, and all viruses are antigenically close to A/Anhui/1/2013 (H7N9) virus which was recommended for vaccine development by the WHO (http://www.who.int/influenza/vaccines/virus/candidates_reagents/summary_a_h7n9_cvv_20150317.pdf?ua=1).The emergence of avian influenza A (H7N9) viruses in China with a high mortality rate in humans poses a significant global health concern –3. WhileLive attenuated influenza vaccine (LAIV) are based on cold-adapted, temperature-sensitive vaccine virus strains that replicate in the nasopharynx but poorly in the lower respiratory tract. LAIVs are 6:2 reassortant viruses containing the HA and NA gene segments from wild type influenza viruses to induce protective immune responses and the six internal gene segments from a Master Donor Viruses (MDV) to provide temperature sensitive, cold-adapted and attenuated phenotypes. Two types of LAIVs are available commercially. The first, licensed as FluMist , is based on A/Ann Arbor/6/60 influenza A and B/Ann Arbor/1/66 influenza B; it is currently produced using seed viruses made by reverse genetics . LAIVs bwt) A/Anhui/1/2013 (H7N9) virus after a single dose, another study showed protective efficacy of similar LAIV virus in a mouse model [http://www.who.int/phi/Day1_Session3_PATH_Marks.pdf).Several studies have shown that A(H7N9) influenza virus appears to be capable of evading human cellular and humoral immune response –16. Therse model . The clise model –28. A rese model . The devse model , 31. Howwt A/Anhui/1/2013, and A(H7N9)RG-LV2, which had mutations in HA and NA similar to CDC-LV7A. The egg selected mutations in candidate vaccine virus were also evaluated for their effect on antigenicity. The final live vaccine candidate was evaluated for its homogeneity, genetic stability and safety in the ferret model.In this study, we report the generation of a live attenuated influenza vaccine against A/Anhui/1/2013 (H7N9) virus using classical reassortment The vaccine candidate contained the HA and NA genes derived from A/Anhui/1/2013(H7N9) and six internal genes of MDV A/Leningrad/134/17/57 virus. The vaccine candidate generated by classical reassortment in embryonated chicken eggs acquired two amino acid substitutions in HA  and one in NA T10I. To evaluate the effect of these mutations on the replication capacity of the reassortants in eggs, two recombinant viruses were generated by reverse genetics—A(H7N9)RG-LV1, which contained the HA and NA identical to original wt influenza virus strain A/Anhui/1/2013 (H7N9) and other A(H7N9) viruses, A/Netherlands/219/2003, A/Mallard/ Netherlands /12/2000, A/Shanghai/1/2013, A/Shanghai/2/2013, A/Taiwan/1/2013, A/Hong Kong/5942/2013, A/Jiangsu/1/13, A/Zhejiang/2/2013, A/Shanghai/7/2013, A/Hong Kong/ 2212982/2014, A/Hong Kong/734/2014, A/Hong Kong/56/2015, A/Hong Kong/2550/2015, A/British Columbia/1/2015 were obtained from the CDC repository. Passage history E2/E1, where E#/ means number of egg passages in submission laboratory and /E# means number of egg passages at Center for Disease Control (CDC), Atlanta, GA. A/Leningrad/134/17/57 (H2N2) Master Donor Virus (MDV) was provided by Institute of Experimental Medicine (IEM), Russia/ BioDiem, Australia. The viruses used for vaccine candidate generation were propagated in 9 to 12-day-old specific pathogen free (SPF) eggs . All reassortment experiments between influenza A (H7N9) virus and MDV were performed in an approved biosafety level 3 (BSL3) containment laboratory. 293T human embryonic kidney (HEK) cells were obtained from American Type Culture Collection (ATCC) and were maintained in DMEM High Glucose  supplemented with 10% fetal bovine serum (Life Technologies), 1x GlutaMAX (Life Technologies) and 40 μg/ml Neomycin .Egg adapted wt A/Anhui/1/2013 was prepared according to the method developed by IEM, St. Petersburg, Russia [wt virus were inoculated into 10-day-old SPF eggs and incubated at 32°C for 2 days. HA-positive allantoic fluids (AF) were combined and diluted 1:10 using antiserum prepared against the MDV in ferrets. The virus-serum mixtures were incubated overnight at 4°C and then passaged once in SPF eggs at 25°C for 6 days, following the blind passage at 32°C. HA-positive AF were analyzed by HI assay for antigenic specificity with antiserum to MDV and wt influenza virus. AF which exhibited antigenic specificity of wt virus were combined and a cloning by limiting dilution procedure was carried out in the presence of antiserum at 25°C. 6:2 reassortants were selected as described previously [A reassortant influenza virus that possesses the internal genes of MDV and the surface antigen genes of , Russia , 32, 33 eviously .RNA was isolated from influenza virus-containing allantoic fluids and purified on the MagnaPure LC  using the MagNA Pure Total Nucleic Acid Kit (Roche) following the manufacturer’s instructions. Clarified allantoic fluid of infected eggs (200 μL) was used for RNA isolation. RNA was eluted in a final volume of 50 μL of water.Genomic composition of the reassortant influenza viruses was assessed by pyrosequencing analysis of NA and the 6 internal genes. Pyrosequencing analysis was performed using the PyroMark Q96 ID Platform  following the manufacturer’s instructions. RT-PCR with biotinylated primers, gel analysis of the RT-PCR product, sample preparation, pyrosequencing reaction, and data analysis were done as described previously . The priwt A/Anhui/1/2013 (H7N9) virus using PrimerExpress 3.0 Software , the sequences are listed in The genetic homogeneity of the final seed virus was confirmed by real time RT-PCR test as described previously . Strain 50). For sensitivity assay RNA was extracted from AF with a known viral titer, in EID50, and serial 10-fold dilutions of extracted RNA were used for amplification with designed primer/probe sets. The sensitivity of the assays was determined as the lowest concentration at which positive signal was obtained.Specificity and sensitivity of the real time RT-PCR assays were evaluated relative to the value of 50% egg infected dose (EIDwt influenza viruses [The HI assay was used to determine the origin of HA genes of reassortant virus clones. HI assays were performed in 96-well V-microtiter plates using 0.5% turkey red blood cells (tRBC) and antiserum against MDV or  viruses , 37.ca and ts phenotypes of the viruses were assessed by evaluating viral replication in embryonated chicken eggs at permissive (32°C), restrictive (38°C) and low temperatures (25°C). Infectious titer of wt and MDVs at different temperature were measured by 50% egg infectious dose per milliliter (EID50/ml). Briefly, ten-fold serial dilutions of allantoic fluid were made in PBS  and 0.2 ml of each dilution was inoculated into 10-day-old SPF eggs. Five eggs were infected with each virus dilution and incubated at 32°C and 38°C for 48 h. and 6 days at 25°C. Harvested allantoic fluids were tested for virus presence by hemagglutination using 0.5% tRBC. The virus titer was calculated using Reed and Muench method [10 reductions in viral titers at the restrictive temperature compared with that observed at the permissive temperature were considered ts. Viruses that displayed ≤ 3 log10 reductions in viral titers at low temperature were considered ca.The trictive °C and loDNA from a set of eight plasmids was transfected into HEK293T cells using Lipofectamin2000 transfection reagent (Life Technologies) according to the manufacturer's protocol. Two days after transfection 0.4 μg/ml TPCK-treated trypsin (Sigma-Aldrich) was added to culture supernatants and after an additional 24 hours transfected cells were re-suspended in culture media and inoculated into embryonated chicken eggs. Allantoic fluid was harvested two days after inoculation and the presence of virus was revealed by agglutination of tRBC . The serThe complete viral cDNAs for each segment  of the cloned viruses were synthesized from purified viral RNA using SuperscriptTM III One-Step RT-PCR System with Platinum Taq High Fidelity (Life Technologies). Viral genome fragments  were amplified using gene segment specific primers. RT-PCR products were resolved by the 2% E-Gel agarose electrophoresis system  and were purified by ExoSAP-IT system . Sanger sequencing of the cDNA was performed using the BigDye Terminator v3.1 Cycle Sequencing kit . The sequencing extension products were purified using the BigDye Xterminator Purification kit (Life Technologies) and analyzed using an ABI 3500xL DNA Analyzer . Trace files were assembled in Sequencer . BioEdit Sequence Alignment Editor  software was used to align consensus sequences of gene segments with the corresponding reference sequences. The sequences of HA and NA were uploaded to Global Initiative on Sharing All Influenza Data (GISAID) EpiFlu database.4 or 106 EID50 of either CDC-LV7A or A(H7N9)RG-LV1 virus, respectively). Ferrets were boosted in the hind limb with concentrated virus and adjuvant at 14 days post-infection (dpi) and were exsanguinated at 28 dpi. Ferret antisera against other H7 viruses tested by HI, including those previously identified as candidate vaccine viruses, were generated using the same strategy with a range of i.n. inoculation doses depending on the strain. Serum was stored at -20°C until further use. As previously described for the HI assay, viruses were standardized to 8 HAU/50ul and added to serially diluted, receptor destroying enzyme (RDE)-treated antisera  followed by incubation at room temperature and agglutination with tRBC [Antigenic characterization was performed using a panel of representative H7 wild type viruses/antisera as well as antisera generated against WHO candidate vaccine viruses. To generate ferret antisera against the LAIVs used for HI testing, serologically naïve, male ferrets greater than six months of age  were inoculated intranasally (i.n.) with 500 μl of diluted virus per nare  that were serologically negative for currently circulating influenza viruses were used to determine the pathogenicity of the vaccine candidate. Ferrets were inoculated intranasally (1 ml) with virus diluted in PBS. For each virus, three animals were euthanized on day 3 post-inoculation (p.i.) to determine the extent of virus replication in multiple organs. Anesthesia occurred through an intramuscular injection of a ketamine-xylazine-atropine cocktail . For euthanasia, ferrets were heavily sedated with the above cocktail, exsanguinated, and euthanatized via intracardiac injection of Euthanasia V solution (1 ml/kg). The heartbeat was monitored after delivery of Euthanasia V solution until the cessation of beating, at which time euthanasia was confirmed. The remaining 3 ferrets were monitored daily for clinical signs including body temperature, weight loss, respiratory symptoms, and lethargy for 14 days p.i. Clinical signs of infection did not exceed humane endpoints as measured by weight loss, lethargy, or appearance of neurologic symptoms. Nasal washes were collected from anesthetized ferrets on days 1, 3, 5, 7 and 9 (or 10) p.i., rectal swabs were collected on days 1, 3, 5 and 7 p.i. No analgesics were given during the course of infection as this would impact the ability to identify certain clinical signs of infection such as body temperature and lethargy or malaise. No ferrets experienced an unintended death or were humanely euthanized due to excessive illness observed during the course of the study.wt A/Anhui/01/2013 (E2/E1) and MDV and genotyped using pyrosequencing. Based on the pyrosequencing analysis eggs 23 and 24 were selected for further cloning procedure (data not shown). The AF from eggs 23 and 24 were cloned by limiting dilution in the presence of antiserum to MDV, and twelve 6:2 reassortants were identified. The sequencing analysis of surface antigen genes of these clones revealed the variety of mutations in HA and NA genes. Clone 2383 with minimal number of changes in HA and NA genes (Live attenuated A(H7N9) influenza candidate vaccine virus was generated by classical reassortment in eggs by co-infection of NA genes  was pickwt A/Anhui/01/2013 (E2/E1) was performed. The alignment of sequences for the six internal genes showed that all sequences were identical to those of MDV. The nucleotide sequence of HA gene of A/Anhui/1/2013 (H7N9) egg isolate E2/E1, which was used for reassortment, had a mixed population of N and D at position 123 (H7 numbering) compared to the HA sequence of wt A/Anhui/1/2013 from a human specimen (GISAID accession number EPI439507) which had N123. The HA gene of CDC-LV7A vaccine candidate had two egg adapted mutations, N123D and N1149D. The NA sequence of wt parental E2/E1 virus had a mixed population at position 10 (T or I) compared to original wt NA sequence (GISAID accession number 439509) which had 10T. The NA sequence of CDC-LV7A was identical to one of the wt parental E2/E1 virus with fixed T10I amino acid substitution. The sequences of the HA and NA genes of CDC-LV7A were deposited to GISAID EpiFlu Database—HA accession number EPI516488, NA accession number EPI516487.Complete genome sequence analysis of CDC-LV7A followed by alignment of the six internal protein gene segments  with those of MDV A/Leningrad/134/17/57 and the two surface antigen segments (HA and NA) with the corresponding segments of the wt Anhui/01/2013 E1 isolate and A(H7N9)RG-LV2 with mutations N123D and N129D in HA and T10I in NA (same as in CDC-LV7A) using the reverse genetics approach. The HA and NA genes of the A/Anhui/1/2013 virus were amplified by PCR from synthetic DNA amplicons, and cloned into a reverse genetics vector flanked by human polymerase I promoter and mouse RNA polymerase I terminator element [The mutations present in the HA of a vaccine candidate seed virus could potentially affect its antigenicity and effectiveness , 40. To  element , 42. The element  and HA ats) and cold adapted (ca) phenotype. In this study we analyzed the phenotypic characteristic of candidate vaccine virus CDC-LV7A and evaluated the selected-from-eggs mutations in HA and NA genes on their effect of virus replication. Ts and ca phenotypes of CDC-LV7A, A(H7N9)RG-LV1 and A(H7N9)RG-LV2 were analyzed by propagating viruses in SPF eggs at permissive (32°C), restrictive (38°C) and low (26°C) temperatures. Replicative efficiency of candidate vaccine virus CDC-LV7A and A(H7N9)RG-LV2 at the permissive temperature was similar to that detected for wt A/Anhui/1/2013 and MDV A/Leningrad/134/17/57  (wt virus (107.3 and 106.9 compare to 102.2 EID50/ml) and was not different from MDV (107.7 EID50/ml) confirming its ca phenotype. At restrictive temperature (38°C) replication efficiency of CDC- LV7A and A(H7N9)RG-LV2 was significantly reduced. It was seven log10 lower than at 32°C (10 2.9 and 103.1 EID50/ml compared to 1010 EID50/ml), five log10 lower than at 26C (10 2.9 and 103.1 EID50/ml compared to 107.3 EID50/ml) and it was similar to replication efficiency of MDV (102.2 EID50/ml). These results showed that recombinant H7N9-RG-LAIV2 virus was phenotypically indistinguishable from the CDC-LV7A reassortant—they both exhibited ca and ts phenotypes similar to MDV—i.e., replicated efficiently at low temperature (26°C), but not at 38°C. (10 at 26°C (10 4.4 compare to 107.7and 10 6.9 EID50/ml) and 38°C (10 3.1 compare to 0 EID50/ml) and more than two log10 at 32°C (107.7 compare to 1010 EID50/ml).The important properties of live attenuated influenza vaccine include the limitation of replication of the vaccine viruses above 37°C and the effective replication at low temperatures , thus providing temperature sensitive (ctively) . Howeverat 38°C. . Recombi50 per reaction. Homogeneity of CDC-LV7A seed candidate vaccine virus was analyzed using RNA corresponding to105 EID50/reaction. MDV and wt viruses were also tested as controls. The positive signals were detected only with primers/probe specific for internal genes of MDV and HA and NA genes for A/Anhui/01/2013 , A/Netherlands/219/2003 (H7N7), A/Mallard/Netherlands/12/2000 (H7N3) and A(H7N9) reference viruses isolated from 2013 to 2015. The HI titers of antisera generated against reassortant viruses were within a two-fold difference to the wild type reference viruses, suggesting that both reassortants were antigenically related to the wild type A/Anhui/1/2013 and could be considered as candidate vaccine viruses RG-LV1 to assess their antigenicity. Two-way HI cross-reactivity of reassortant viruses were compared to one another, to the  viruses . Antigen viruses , indicat6 plaque forming units (PFU) of wt A/Anhui/1/2013 or 106.1 PFU of A/Anhui/1/2013 (H7N9)-CDC-LV7A virus. Morbidity was reduced in ferrets infected by the A/Anhui/1/2013 (H7N9)-CDC-LV7A vaccine candidate compared to the wt A/Anhui/1/2013 virus. Peak virus shedding in nasal washes was observed on day 1 p.i. for both groups of ferrets but was 83-fold lower in animals infected by the candidate vaccine virus (Wt A/Anhui/1/2013 virus replicated efficiently throughout the respiratory tract of ferrets while A/Anhui/1/2013 (H7N9)-CDC-LV7A virus was limited to the upper respiratory tract of 3/3 animals. No virus was detected in any extrapulmonary tissues tested from animals infected by the A/Anhui/1/2013 (H7N9)-CDC-LV7A candidate vaccine virus . To date all A(H7N9) reassortants available for distribution by WHO were generated by reverse genetics.The ongoing A(H7N9) influenza virus outbreak in China has renewed concerns about zoonotic influenza viruses with pandemic potential. WHO regularly updates antigenic and genetic characteristics of circulating zoonotic influenza viruses and recommends development of candidate vaccine viruses (CVVs) for pandemic preparedness. CVVs available for distribution to manufacturers of inactivated influenza vaccine are listed and updated twice annually (wt A/Anhui/1/2013(H7N9) virus. Since this vaccine seed virus was prepared by classical reassortment, it was important to confirm the absence of any genetic material of undesired origin and to demonstrate that there were no contaminating wt internal genes present that may allow efficient lower respiratory replication in vaccinees that may cause illness. Likewise, it was critical to demonstrate that HA or NA genes from the MDV were not present which might influence vaccine effectiveness. CDC-LV7A seed virus was shown to be homogeneous by real time RT-PCR analysis. To ensure the quality of the vaccine seed virus and to demonstrate the stability of the vaccine reassortant genome composition and sequence, sequencing of the reassortants was performed after five and ten passages in eggs. No mutations in any genes were detected after five passages. However, after ten passages a number of new egg adapted mutations was detected. This suggested that passaging of the vaccine seed virus during the manufacturing process should not exceed five passages since the HA gene might acquire additional egg adapted changes.The current report describes the generation of A(H7N9) LAIV obtained by classical reassortment in eggs. The vaccine candidate A/Anhui/1/2013(H7N9)-CDC-LV7A virus was shown to grow to high titers in eggs and was antigenically similar to wt A/Anhui/1/2013 HA and NA gene sequences replicated poorly in eggs compared to the classically generated reassortant CDC-LV7A (or A(H7N9)-RG-LV2)—i.e., only up to 107.7 EID50/ml at 32°C; these levels are analogous to a H7N9 LAIV candidate based on A/Ann/Arbor/6/60 [8.5 EID50/ml [10 EID50/ml), which is very significant for manufacturing, when from the same amount of eggs allantoic fluid almost 100 times more doses of live vaccine could be produced.Since eggs are the only currently approved substrate for vaccine production, reassortants generated by reverse genetics must still be passaged several times in eggs, hence acquiring egg adapted mutations. These mutations, which usually improve vaccine virus yield, might affect vaccine immunogenicity. Thus, continuous analysis for mutations in viruses used for vaccine production is critical. During the vaccine manufacturing process the seed virus undergoes four to five additional passages before blending the pool to formulate LAIV . The lacbor/6/60  and PR8-bor/6/60 . An H7N9wt, parental virus at titers equivalent to or within a two-fold reduction to the homologous virus titer and vice versa. Post-infection ferret antisera were raised against CDC-LV7A and A(H7N9)RG-LV1 viruses to assess their antigenicity. Two-way HI testing showed that sera against both reassortants efficiently inhibited hemagglutination by the homologous A(H7N9) virus as well as the parental virus indicating that mutations in HA and NA of CDC-LV7A did not affect antigenicity of the vaccine candidate. Moreover, the antisera against both reassortants also inhibited hemagglutination (within four-fold of homologous titers) by all wt A(H7N9) isolates tested including viruses collected in 2013, 2014 and 2015, indicating that CDC-LV7A seed virus is an ideal LAIV candidate vaccine.Critical vaccine properties, however, are antigenic relatedness to circulating viruses and protective immunogenicity. Therefore, vaccine seed viruses must pass a two-way HI test to be considered for vaccine production , 46. In The high LAIV yield in eggs and ‘needle-free’ nasal administration makes a LAIV a desirable vaccine for use. Since 2009, WHO was licensed to develop, register, manufacture, use and sell seasonal and pandemic LAIV based on Russian MDVs. It transferred the technology to produce LAIV by classical reassortment in eggs to manufacturers in India, Thailand and China –12. The S1 Table(DOCX)Click here for additional data file.


Arhgap35, which introduces a Leu1396 to Gln substitution in the GAP domain. This results in decreased GAP activity for the prototypical Rho-family members, RhoA and Rac1, likely due to disrupted ordering of the Rho binding surface. Consequently, Arhgap35-deficient animals exhibit hypoplastic and glomerulocystic kidneys. Investigation into the cystic phenotype shows that p190A is required for appropriate primary cilium formation in renal nephrons. P190A specifically localizes to the base of the cilia to permit axoneme elongation, which requires a functional GAP domain. Pharmacological manipulations further reveal that inhibition of either Rho kinase (ROCK) or F-actin polymerization is able to rescue the ciliogenesis defects observed upon loss of p190A activity. We propose a model in which p190A acts as a modulator of Rho GTPases in a localized area around the cilia to permit the dynamic actin rearrangement required for cilia elongation. Together, our results establish an unexpected link between Rho GTPase regulation, ciliogenesis and glomerulocystic kidney disease.Rho family GTPases act as molecular switches regulating actin cytoskeleton dynamics. Attenuation of their signaling capacity is provided by GTPase-activating proteins (GAPs), including p190A, that promote the intrinsic GTPase activity of Rho proteins. In the current study we have performed a small-scale ENU mutagenesis screen and identified a novel loss of function allele of the p190A gene  Arhgap35), which is associated with aberrant primary ciliogenesis. The primary cilium is a microtubule-based signalling center involved in cell differentiation and homeostasis. A role for the actin cytoskeleton in ciliogenesis has recently emerged, but the underlying regulatory mechanisms remain poorly understood. In this study we identify a requirement for the local modulation of Rho GTPase by p190A RhoGAP during primary cilium formation, which constitutes the first demonstration of ciliogenic regulation by RhoGAP proteins. Together, this work identifies deficiencies in actin cytoskeletal dynamics as an underlying cause for ciliary and glomerulocystic malformations.Glomerulocystic kidney disease occurs either in isolation or in combination with other cystic diseases. To date, the paucity of mouse models have impeded our progress in understanding the molecular mechanisms leading to glomerular cyst development. Using an ENU mutagenesis approach, we present here a novel mouse model of glomerular cyst formation caused by a point mutation in p190A RhoGAP ( The dynamic regulation of actin polymerization is crucial for cell physiology and closely associated with cellular processes such as migration, survival, polarization and cytokinesis ,2,3. AmoArhgap35, Grlf1) is a potent regulator of Rho GTPases. It is composed of a N-terminal GTPase domain necessary for its activity, a series of conserved FF domains, and a C-terminal RhoGAP domain [The RhoGAP protein p190A  mutagenesis screen [Arhgap35). We further demonstrate a crucial role for p190A in modulating actin dynamics during ciliogenesis.Glomerulocystic disease is a subtype of polycystic kidneys that exhibit primarily glomerular cysts, characterized by severe dilation of Bowman’s capsule and progressive glomerular atrophy . It manis screen  to identTo uncover novel regulators of mammalian urogenital system development, we undertook a recessive ENU mutagenesis approach in which a total of 28 G1 C57BL/6J males  were out-crossed to wild type C3H/HeNCrl females to generate G2 mice . PhenotyArhgap35 (p190A RhoGAP gene), which was located within the 18cM interval and occurred in all affected embryos. To further investigate the association between the altered allele and the kidney phenotype, we Sanger-sequenced Arhgap35 in 59 additional embryos. As expected, the vast majority of affected kidneys were homozygous for the altered Arhgap35 allele (Single nucleotide polymorphism (SNP)-based linkage analysis was undertaken to identify the genetic locus associated with kidney hypodysplasia in affected embryos. The characterization of 76 samples identified a single peak of approximately 18 cM on chromosome 7 (rs3675839-rs31924991) . Additio5 allele  suggestiArhgap35D34) result from a loss-of-function mutation in the Arhgap35 gene, we obtained the previously generated Arhgap35tmjset1 mutant line [Arhgap35- mice) and generated homozygous mutant and compound heterozygous mutant animals for both alleles. Gross histological characterization of the urogenital system of Arhgap35D34/D34 kidneys at embryonic day 17.5 (E17.5) revealed a defined renal cortex and medulla, similar to control animals  showed early perinatal lethality, neural tube closure defects  or D34 mutant GAP (p190A GAPL1396Q) domains as GST-fusion proteins for in vitro GAP activity assays. As expected, each small GTPase possessed little intrinsic GTP-hydrolytic activity alone, while addition of p190A GAPWT significantly enhanced the rate of GTP hydrolysis , which reproducibly form cilia upon serum withdrawal. Whereas approximately 60% of wild type MEFs formed cilia, less than 20% of ype Figs  and S7A.rameters , furtherrameters .WT construct in wild type MEFs revealed an enrichment of p190A co-localizing with γ-tubulin at the base of cilia in 12% of cells (L1396Q) (R1284A)  , suggest(R1284A) . We furt(R1284A) . TogetheArhgap35D34/D34 MEFs to wild-type levels  have been identified that together define a shallow Rho-binding pocket on p190A comprised of two α-helices and the catalytic arginine loop [Using a combination of classical single nucleotide polymorphism (SNP) mapping and exome sequencing approaches, we have identified a point mutation in t strain , and by  alleles ,18 identine loop ,49. InteArhgap35-deficient cells suggest that both Rac1/2 and RhoA may be substrates for p190A during cilia formation; however, while Rac inhibition only partially rescues ciliogenesis in the absence of p190A activity, ROCK inhibition completely rescues both cilia number and length. Together, these results raise the intriguing possibility that p190A plays a role at the docked basal body to downregulate active RhoA (and possibly Rac1) to allow the localized actin cytoskeleton rearrangement required for cilia elongation  analysis was used to genotype animals by PCR-amplifying 600bp fragments around the single nucleotide polymorphisms (SNPs) rs31200925 and rs31924991. Mice carrying the C3H/HeNCrl (wild-type) allele at these SNPs have restriction sites for Bgl II and Hind III, respectively, while animals with the C57BL/6J (mutated) allele do not. Restriction digest of the PCR product results in fragments of 600bp (C57BL/6J) and/or 400bp and 200bp (C3H/HeNCrl) allowing for genotype determination. When appropriate, Arhgap35 was also PCR-amplified for Sanger sequencing. All primers used are provided in N-ethyl-N-nitrosourea  was administered as 3 weekly doses by intraperitoneal injections to 8-week old male C57BL/6J mice (Jackson Laboratories) to induce germline mutations as described previously . G0 founSNP Array screening was performed on the Low-density Mouse Linkage Panel . R-QTL mapping was performed using the R software platform . Whole EL1396Q and GFP-p190AR1284A constructs were generated from the full-length wild-type construct by site directed mutagenesis. For the GFP-p190A domain constructs, the appropriate domains were PCR amplified from the full-length wild-type construct. For in vitro GAP assays, the GAP domain (nt 4417–5143) was PCR amplified from the either the full length GFP-p190AWT or GFP-p190AL1396Q constructs using specific primers containing BglII and EcoRI restriction sites, which allowed for cloning in the BamHI and EcoRI sites of the pGEX-6P1 bacterial expression vector. All constructs were sequence verified. All primers used are provided in Full length p190A-pEGFP-C1 mammalian expression vector was generated as described . The GFPRecombinant GST-tagged p190A GAP protein was produced in BL21(DE3) bacteria by conventional methods, and purified using Glutathione-agarose beads (Life Technologies). Cleavage of the GST tag, and elution of free p190A GAP protein, was achieved using PreScission protease (GenScript). Protein was quantified so that 1.5ug of GAP was added per GAP assay reaction. The in vitro GAP assay (Cytoskeleton) was performed according to manufacturer’s instructions.The amount of activated RhoA (RhoA-GTP) was assessed using the Rho Activation Assay Biochem Kit (Cytoskeleton) following manufacturer’s instructions. Briefly, control and mutant MEFs were grown to 70% confluence, and serum starved for 16hr to assess the basal level of RhoA activation. Cells were lysed on ice, clarified by centrifugation and snap frozen. 400ug of each lysate was used for pulldown assay using the Rho binding domain from rhotekin conjugated to sepharose beads. The amount of activated RhoA was assessed by western blot, following standard procedures. The primary antibodies used were mouse anti-RhoA  and rabbit anti-GAPDH . Secondary antibodies used were HRP-conjugated anti-mouse  and HRP-conjugated anti-rabbit .To visualize the potential impact of the L1396Q substitution on the structure of p190A RhoGAP, the Pymol software program was used to image the human p190A GAP domain (PDB: 3FK2). Substitution of glutamine for leucine 1396 was done using the mutagenesis function of Pymol, and the rotamer position of the glutamine side chain and potential steric clashes with surrounding amino acids were assessed for all possible conformations.Gata3, Ncc, and Wt1 have been described [Arhgap35 probe was generated from E12.5 whole embryo cDNA using specific primers containing a T7 promoter sequence  were formalin-fixed, paraffin embedded and processed according to standard methods for Hematoxylin and Eosin staining at the McGill University Histology Facility. Section immunofluorescence and in situ hybridizations were performed on dissected UGS, fixed in 4% paraformaldehyde and cryo-sectioned as described previously . Probes escribed ,64,65. Tsequence . Probe wArhgap35+/+ and Arhgap35D34/D34 embryos. Cells were maintained in DMEM (Wisent Inc.) supplemented with 10% FBS (Gibco), 1% Penicillin-Streptomyosin (Wisent Inc.). Ciliogenesis was stimulated by serum withdrawal for 20 hours before addition of Y27632 , GSK 429286A , or NSC 23766  for an additional 4 hours. Alternatively, for experiments involving Blebbistatin , Latrunculin A  or Cytochalasin D , ciliogenesis was stimulated for 23.5 hours prior to treating with the inhibitor for an additional 30 minutes. Cells were then fixed with 4% paraformaldehyde, and processed for immunofluorescence. P190A localization during ciliogenesis was examined by transfecting the indicated full length GFP-p190A constructs in mouse embryonic fibroblasts (MEFs) using the Nucleofector technology (Lonza). Twenty-four hours post transfection, ciliogenesis was stimulated by serum withdrawal for an additional 24 hours. Cells were then fixed with 4% paraformaldehyde and processed for immunofluorescence. A minimum of 50 cells were assessed in each sample.Mouse embryonic fibroblasts were derived as described previously  from Arh510 META Axiovert 200M inverted microscope at the Advanced BioImaging Facility of McGill University . Cilia length and basal body positioning were determined using the measure function of the ImageJ image analysis program [t-tests (as indicated) using GraphPad Prism 6 software. Data are presented as mean ± SEM; p<0.05 was considered significant.Images were acquired on a Confocor LSM  program  from conS1 FigN-ethyl-N-nitrosurea (ENU)-mutagenized male C57BL/6J mice were outcrossed to wild-type C3H/HeNCrl females to produce first generation (G1) offspring. Sons from this cross were outbred to wild-type C3H/HeNCrl females to produce the second generation (G2), from which daughters were backcrossed to their father to recover recessive mutations in the third generation (G3). G3 progeny were dissected at embryonic day 17.5 (E17.5) for gross characterization that identified the D34 line.(TIF)Click here for additional data file.S2 Fig(A-B) Section in situ hybridization on control and D34-mutant kidneys shows no significant difference in collecting ducts and ureteric tips marked by Gata3 expression (A), nor in distal tubule density and differentiation marked by Ncc (B) expression. (C) Section immunohistochemistry for Lotus Tetragonolobus Lectin (LTL) on control and Arhgap35D34/D34 kidneys shows no difference in proximal tubule formation. (D) Arhgap35D34/D34 kidneys contain significant increases in glomerular and proximal tubule cysts compared to wild-type E17.5 kidneys. Scale bars, 100μm *p<0.05, **p<0.01, ***p<0.005 (TIF)Click here for additional data file.S3 Fig(A-C) Whole E17.5 embryos that are Arhgap35-/- or compound heterozygous with the Arhgap35D34 allele exhibit spina bifida (closed arrowhead) and exencephaly (open arrowhead). (D) Active RhoA in mouse embryonic fibroblasts derived from wild type or Arhgap35D34/D34 animals was assessed by pulldown with Rho binding domain (RBD) bound beads. GAPDH was used for normalization. (E) Denisometric analysis of (D) was performed using ImageJ to reveal an increase in active RhoA in D34-mutant cells.(TIF)Click here for additional data file.S4 Fig(A) In situ hybridization of E17.5 kidney section for Wt1 shows a normal progression of podocyte development. (B) Quantification of the average density of H&E-stained glomeruli normalized to kidney area shows no difference between control and Arhgap35D34/D34 animals . (C-D) Immunofluorescence staining for podocyte markers  and structural markers  show no obvious misorganization in Arhgap35D34/D34 glomeruli, either pre- or post-cyst formation (arrowheads). (E) Section immunofluorescence for cilia (acetylated α-tubulin) and the podocyte lineage  shows normal cilia in Bowman’s capsule cells . (F) Quantification of cilia number per glomerulus from (F) shows no significant difference between control and Arhgap35D34/D34 animals, irrespective of glomerular dilation . Scale bars, 10μm(TIF)Click here for additional data file.S5 Fig Section in situ hybridization (20X magnification) for Arhgap35 on wild type and D34-mutant animals shows weak expression in distal tubules (DT), collecting ducts (CD), and nephrogenic zone.  Section in situ hybridization (63X magnification) for Arhgap35 on wild type and Arhgap35D34/D34 embryos shows strong expression in proximal tubules (PT) but only weak expression in the glomerulus (G). Scale bars, 50μm(TIF)Click here for additional data file.S6 Fig(A) Immunofluorescence staining for γ-tubulin  and acetylated α-tubulin (axoneme) reveal a defect in cilia elongation (closed arrowheads) in the S-shaped body (dotted lines) of Arhgap35D34/D34 animals. Scale bars, 10μm (B) Immunofluorescence staining for acetylated α-tubulin (axoneme) of E14.5 proximal tubules  shows a defect in cilia elongation (open arrowheads) in Arhgap35D34/D34 animals that precedes cystogenesis. Scale bars, 5μm(TIF)Click here for additional data file.S7 Fig(A) Immunofluorescence staining for γ-tubulin  and Arl13b (axoneme) reveal a defect in cilia elongation in Arhgap35D34/D34 mouse embryonic fibroblasts compared to control. (B-C) Treatment with the ROCK1/2 inhibitor, Y27632, rescues the defects in cilia number (B) and cilia length (C) in Arhgap35D34/D34 mouse embryonic fibroblasts. (D) Immunofluorescence for phalloidin and acetylated α-tubulin in control and Arhgap35D34/D34 mouse embryonic fibroblasts shows a normal F-actin cytoskeleton in ciliated cells. Staining for phalloidin in mouse embryonic fibroblasts overexpressing full length p190AWT-GFP shows no noticeable effect on F-actin organization. (E-F) Treatment with the myosin II inhibitor Blebbistatin does not rescue the defects in cilia number (D) and cilia length (E) in Arhgap35D34/D34 mouse embryonic fibroblasts. (G-H) Treatment with the actin polymerization inhibitor Latrunculin A rescues the defects in in cilia number (G) and cilia length (H) in Arhgap35D34/D34 mouse embryonic fibroblasts. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 (one-way ANOVA)(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.