We have shown that Dicer processes 7SL RNA into different fragments ranging from ∼20 to more than 200 nucleotides. Here we addressed the molecular functions of these 7SL RNA fragments and found that some of them functioned as dominant-negative regulators of the full-length 7SL RNA, interfering with signal recognition particle (SRP) complex formation. Transfection of these 7SL RNA fragments inhibited the expression of cell surface glycoproteins, the targeting of a reporter protein to the endoplasmic reticulum, and the secretion of secreted alkaline phosphatase. These results suggest that some Dicer-processed 7SL RNA fragments interfered with SRP-mediated protein targeting. Moreover, we showed that Dicer knockdown enhanced SRP-mediated protein targeting and that transfection of a mixture of the 7SL RNA fragments partially restored this effect. Our data indicate that Dicer can fine-tune the efficiency of SRP-mediated protein targeting via processing a proportion of 7SL RNA into fragments of different lengths. The signal recognition particle (SRP) is a key component of the cellular machinery that couples the ongoing synthesis of proteins to their proper subcellular compartments and is essential for cotranslational insertion of membrane and secretory proteins into the lumen of the endoplasmic reticulum (ER) The eukaryotic SRP complex comprises six distinct polypeptides  bound to an RNA molecule (the 7SL RNA) 7SL RNA was first detected in Rous sarcoma virus particles In the present study, we investigated the cellular functions of the Dicer-processed 7SL RNA fragments and found that some function as dominant-negative regulators of the full-length 7SL RNA by interfering with the formation of the SRP complex and inhibiting SRP-mediated protein targeting.AAGGCTTACCTTCTCCAGGCT), Control (AATTCTCCGAACGTGTCACGT). RNA transfection was performed as described HepG2.2.15 and HEK293T cells were cultured in RPMI 1640 medium , RKO and T29 cells were cultured in DMEM (Hyclone), and HCT116 cells were cultured in McCoy's 5A medium . All media were supplemented with 10% fetal bovine serum. We obtained siRNAs from Invitrogen  and constructed plasmids expressing shRNAs (shDCR and shCon) by inserting the corresponding hairpin oligonucleotides into the pSUPER.neo+GFP vector . The target sequences of siRNAs and shRNAs were as follows: Dicer , 5′-AAGCTTAATTATAATACGACTCACTATAG-3′ and 5′-GACCTGCTCCGTTTCCGACCTGG-3′; 7SL(97-299), 5′-AATTATAATACGACTCACTATAGGGAGACCGCGCCCGGCCTGATGAGTCCGTGAGGACGAAACGGTACCCGGTACCGTCTGCCGATCGGGTGTCCGCAC-3′ and 5′-AGAGACGGGGTCTCGCTATGTTG-3′.The control RNA (a fragment of LacZ) was previously described Cells were lysed in RIPA buffer and equal amounts of denatured protein were subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes . Membranes were then incubated with primary antibody, followed by horseradish peroxidase-linked secondary antibody, and immune complexes were detected using ECL plus reagent (Millipore). The primary antibodies were as follows: Dicer and SRP19 , SRP9 and SRP14 , SRP54 and SRP68 , SRP72 , γ-tubulin and β-actin .GAPDH mRNA served as an internal control. Primer sequences used to detect Dicer and GAPDH mRNAs were as described 5′-GGCTTCCTTGGCTACCATAA-3′ and GGCATTCTGATGGGAACTTG; SRP14, 5′-CAGCCCATCCATGTTAGCTC-3′ and AGCTACCGATGGGAAGAAGA-3′; SRP19, 5′-AGACTTTGGTCAGCACCTCC-3′ and AAACAGGAAGATGGGAGCCT-3′; SRP54, 5′-TTCCAAGGTCTGCTAGAACCA-3′ and CTCCCAGCTTTCTTGGGTTT-3′; SRP68, 5′-CGCTCCTTAGTTTCTTCGCTT-3′ and AGAGGGAGAACGGTTCCAGT-3′; SRP72, 5′-AGCCATCTTTCTGGATCTGG-3′ and TACATTCGGAAGAAGGGTGG-3′.Total RNA was prepared using TRIzol® reagent (Invitrogen) and incubated with RNase-free DNase I  for 30 min. The DNA-free RNA was reverse transcribed using a M-MLV reverse transcription kit  according to the manufacturer's instructions. Samples prepared in the absence of reverse transcriptase served as negative controls. SYBR green real-time PCR was performed using the ABI PRISM 7300 Sequence Detection system (Applied Biosystems). The level of 2, 0.01% octaethylene glycol dodecyl ether , 1 mM dithiothreitol, 30 U/ml RNase inhibitors (Promega), 0.25% Triton X-100, and a cocktail of protease inhibitors). The homogenized cells were incubated at 0°C for 10 min, potassium acetate was added to 500 mM, and nuclei were isolated by centrifuging the homogenate at 800×g for 10 min. The postnuclear fraction (1 mg) was incubated with 30 pmol 7SL RNA fragments or control LacZ RNA at 0°C for 10 min, 37°C for 60 min, layered onto a 10–40% glycerol gradient, and centrifuged at 5°C for 8 h at 40,000 rpm in a Beckman SW41Ti rotor. Ten equal fractions were collected from the top to the bottom of the gradient. One-third of each fraction was used for RNA extraction using TRIzol LS (Invitrogen), and the level of 7SL RNA was measured by real-time RT-PCR using the following primers, 5′-GGAGTTCTGGGCTGTAGTGC-3′ and 5′-ATCAGCACGGGAGTTTTGAC-3′ to specifically detect the full-length 7SL RNA. The remainder of each fraction was treated with trichloroacetic acid to precipitate proteins for western blot analysis.Fractionation of postnuclear extracts was carried out as described with modifications http://facs.scripps.edu/software.html).Cells were washed three-times with phosphate-buffered saline (PBS) and stained with Alexa Fluor® 488 conjugate-lectin GS-II  at room temperature for 30 min. The cells were then washed three-times and resuspended in PBS. Fluorescence was detected using FACSAria (BD Biosciences) and analyzed using WinMDI software (HEK293T cells were transfected with the plasmid pECFP-ER (Invitrogen). This plasmid encodes enhanced cyan fluorescent protein (ECFP) fused at its 5′ and 3′ ends to the endoplasmic reticulum (ER) targeting sequence of calreticulin and the ER retrieval sequence, KDEL, respectively. A cell line stably expressing pECFP-ER was transfected with specific RNAs as indicated, and images of fluorescent cells were obtained using a Leica DMI3000 B microscope. When compared with the corresponding control, the images were taken with the same light intensity. However, to present the results more clearly, different light intensities were applied in different experiments.Cells were co-transfected with a plasmid encoding SEAP  and different RNAs or shRNAs as indicated. The growth medium was changed every 24 h and collected at indicated time points. SEAP activity was determined using the Great EscAPe SEAP kit (BD Biosciences) following the manufacturer's instructions.Small RNAs less than 200 nt were extracted from HepG2.2.15 cells using the E.Z.N.A. Micro RNA Kit  and polyadenylated using the Poly(A) Polymerase Tailing Kit . Reverse transcription was then performed using the SMARTScribe M-MLV Reverse Transcriptase System  with CDS III/3′ PCR Primer and SMART IV Oligonucleotide. Next, the 7SL RNA derived-cDNA was annealed with a biotin-labeled 7SL sRNA5cd oligonucleotide (biotin-GAGTTCTGGGCTGTAGTGCGCTA) and purified using Dynabeads® M-280 Streptavidin magnetic beads (Invitrogen) according to the manufacturer's instructions. The purified DNA was PCR-amplified using CDS III/3′ PCR and 5′ PCR primers and cloned into the pGEM-T Easy Vector (Promega). Primer and oligonucleotide sequences are as follows:ATTCTAGAGGCCGAGGCGGCCGACATGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3′); SMART IV Oligonucleotide (5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3′); and 5′ PCR Primer (5′-AAGCAGTGGTATCAACGCAGAGT-3′).CDS III/3′ PCR Primer , 7SL RNA is also processed by Dicer into longer fragments We then tested whether these 7SL RNA fragments interfere with the formation of the SRP complex. Postnuclear extracts incubated with the control RNA or 7SL RNA fragments were fractionated on glycerol gradients. As shown in We reported that Dicer-processed 7SL RNA fragments are reduced in Dicer-knockdown cells To further investigate whether enhanced SRP function in Dicer knockdown cells is due to reduction of Dicer-processed 7SL RNAs, we transfected Dicer-knockdown cells with a mixture of the 7SL RNA fragments, including 7SL(1-96), 7SL(1-212), and 7SL(97-299). The 7SL RNA fragment mixture not only partially inhibited the secretion of SEAP , but alsDicer is essential for the biogenesis of microRNAs that may target SRP subunits, thus decreased Dicer expression may result in elevated levels of SRP proteins. To exclude the possibility that the increased efficiency of ER-mediated protein targeting in Dicer knockdown cells was mediated by increased levels of SRP proteins, the expression of SRP subunits was monitored using real-time RT-PCR and western blotting analyses. We found that knockdown of Dicer did not alter the expression of SRP proteins .We previously demonstrated that Dicer processes 7SL RNA into fragments of different lengths Of note, 7SL RNA fragments longer than 200 nt or lacking the 7SL sRNA5cd sequence cannot be detected by our cloning method. Another limitation of this method is that any RNA shorter than 200 nt that contains a sequence similar to that of 7SL sRNA5cd can be cloned. Therefore, further effort is necessary to develop a more specific and efficient method to clone all of the 7SL RNA fragments.Our data indicate that Dicer can fine-tune the efficiency of SRP-mediated protein targeting via processing a proportion of 7SL RNA into fragments of different lengths that interfere with the function of the full-length 7SL RNA.Figure S17SL sRNA5cd does not modulate SRP-mediated protein targeting. (A) HEK293T cells were co-transfected with the pSEAP2-control plasmid and different concentrations of 7SL sRNA5cd or the control small RNA. SEAP activity was determined 48 h post-transfection. (B) HEK293T cells stably expressing ECFR-ER were transfected with 7SL sRNA5cd or the control small RNA. Fluorescence was measured 48 h after transfection. (C) HepG2.2.15 cells were transfected with 7SL sRNA5cd or the control small RNA. Cell surface glycoproteins were measured 48 h later. Horizontal and vertical axes denote intensity of fluorescence and number of events, respectively. The filled histogram represents unstained cells, the black line represents cells transfected with the control small RNA, and the red line represents cells transfected with 7SL sRNA5cd.(TIF)Click here for additional data file.Figure S2Flow chart for cloning long 7SL RNA fragments. Small RNAs shorter than 200 nt were polyadenylated and reverse transcribed. The cDNAs derived from 7SL RNA fragments were purified using a biotin-labeled 7SL sRNA5cd oligonucleotide and PCR-amplified. The purified PCR products were cloned into the pGEM-T easy vector and sequenced.(TIF)Click here for additional data file.Figure S3Dicer-processed 7SL RNA fragments. (A) 7SL RNA Dicer cleavage sites  were predicated according to the sequences of 7SL sRNA5cd and 7SL sRNA8b. (B) Diagram of the synthetic Dicer-processed 7SL RNA fragments. Red arrows indicate the PCR primers that can only amplify the full-length 7SL RNA but not any of the synthetic 7SL RNA fragments(TIF)Click here for additional data file.Figure S47SL(1-96) inhibits secretion of SEAP in a concentration-dependent manner. HEK293T cells were co-transfected with the pSEAP2-control plasmid and different amounts of 7SL(1-96) or LacZ RNA. SEAP activity was determined 48 h post-transfection, *p<0.05, **p<0.01 as compared with LacZ RNA.(TIF)Click here for additional data file.Figure S57SL(1-212) inhibits the secretion of SEAP in different human cell lines. The pSEAP2-control plasmid was co-transfected with 7SL(1-212) or LacZ RNA, and SEAP activity was determined 48 h post transfection. **p<0.01 compared with LacZ RNA.(TIF)Click here for additional data file.Figure S6Knockdown of Dicer in different human cell lines. Representative western blot of Dicer. Detection of β-actin was used as the loading control. Con: control cells, K/D: Dicer knockdown cells.(TIF)Click here for additional data file.Figure S7Dicer knockdown enhances the expression of cell surface glycoproteins. Cells were transfected twice with siDCR or siCon, and cell surface glycoproteins were measured 96 h later. Horizontal and vertical axes denote intensity of fluorescence and number of events, respectively. The filled histogram represents unstained cells, the thick line represents Dicer knockdown cells, and the dashed line represents the control cells.(TIF)Click here for additional data file.Figure S8Dicer knockdown does not alter the expression of SRP proteins. HepG2.2.15 cells were transfected twice with siDCR or siCon, the expression of SRP proteins were measured by real-time RT-PCR (upper panel) and western blotting (lower panel) 96 h after transfection. Detection of β-actin was used as loading control. Con: control cells, K/D: Dicer knockdown cells.(TIF)Click here for additional data file.


H9N2 influenza viruses have been circulating worldwide in multiple avian species and have repeatedly infected humans to cause typical disease. The continued avian-to-human interspecies transmission of H9N2 viruses raises concerns about the possibility of viral adaption with increased virulence for humans. To investigate the genetic basis of H9N2 influenza virus host range and pathogenicity in mammals, we generated a mouse-adapted H9N2 virus (SD16-MA) that possessed significantly higher virulence than wide-type virus (SD16). Increased virulence was detectable after 8 sequential lung passages in mice. Five amino acid substitutions were found in the genome of SD16-MA compared with SD16 virus: PB2 , HA (L226Q) and M1 (R210K). Assessments of replication in mice showed that all of the SD16-MA PB2, HA and M1 genome segments increased virus replication; however, only the mouse-adapted PB2 significantly increased virulence. Although the PB2 E627K amino acid substitution enhanced viral polymerase activity and replication, none of the single mutations of mouse adapted PB2 could confer increased virulence on the SD16 backbone. The combination of M147L and E627K significantly enhanced viral replication ability and virulence in mice. Thus, our results show that the combination of PB2 amino acids at position 147 and 627 is critical for the increased pathogenicity of H9N2 influenza virus in mammalian host. H9N2 influenza viruses circulate worldwide and are endemic in multiple terrestrial avian species in Asia To date, some H9N2 viruses have demonstrated increased virulence for mammals. In 2007 to 2009, Bi et al isolated six H9N2 viruses from chickens in northern China that were highly lethal to mice with properties of systemic spread because they replicated well in multiple organs without prior adaptation The pathogenesis of influenza A viruses is a polygenic trait Mice are ideal animal models for investigating pathogenic mechanisms and host range determinants of influenza A viruses All animal research was approved by the Beijing Association for Science and Technology, the approve ID is SYXK (Beijing) 2007–0023, and complied with the guidelines of Beijing laboratory animal welfare and ethical of Beijing Administration Committee of Laboratory Animals.Human embryonic kidney (293T) and Madin-Darby canine kidney (MDCK) cells (Peking Union Cell Center) were maintained in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (FBS).The H9N2 virus A/chicken/Shandong/16/05 (SD16) was isolated from diseased chicken in Shandong, China and propagated in 10-day-old embryonated chicken eggs (ECE) at 35°C for 72 h. Virus titers were measured by plaque assay in MDCK cells and were expressed as plaque forming units (pfu). All experiments with live viruses and transfectants generated by reverse genetics were carried out in a biosafety level 3 conditions with investigators wearing appropriate protective equipment and compling with general biosafety standard for microbiological and biomedical laboratories of Ministry of Health of the People's Republic of China (WS 233–2002).Groups of five to seven week-old BALB/c mice  were lightly anesthetized with Zoletil 50  and inoculated intranasally with 50 μl of allantoic fluid containing SD16 virus. At 3 days post-inoculation (dpi), mice were euthanized and their lungs were harvested and homogenized, and 50 μl of supernatant from the centrifuged homogenate was used as inoculum for the next passage. After 8 passages, the infected mice died within 6 dpi. Then the virus in the lung homogenate was cloned twice by plaque purification in MDCK cells, and the cloned virus was passaged once in the allantoic cavities of 10-day-old ECE at 35°C for 72 h to prepare a virus stock. The experimental protocol was evaluated and approved by the Animal Pathogenic Microbiology Laboratory Bio-safety Committee, College of Veterinary Medicine, China Agricultural University.Viral RNAs were extracted from the allantoic fluid of ECE infected with plaque-purified mouse-adapted SD16 (SD16-MA) virus and the eight viral genes were amplified by Reverse transcription-PCR (RT-PCR). The amplified cDNAs were inserted into the genomic expression plasmid PHW2000 The eight gene segments of SD16 and SD16-MA were amplified by RT-PCR and cloned in the genomic expression plasmid, PHW2000. Mutations of interest in the PB2 gene were introduced by PCR-based site-directed mutagenesis with primer pairs containing point mutations. All of the constructs were sequenced to confirm the site-directed mutations.2. Six hours post-transfection, the supernatants were replaced with 2 ml of OPTI-MEM containing 2 μg/ml TPCK-trypsin (Sigma–Aldrich). Forty-eight hours post-transfection, the supernatants were harvested and propagated in 10-day-old ECE to produce stock viruses. Virus titers were determined by plaque assay on MDCK cells.SD16, SD16-MA, reassortant viruses between SD16 and SD16-MA, and SD16 PB2 mutants were generated by reverse genetics as described previously 5 pfu of viruses in 50 μl phosphate buffered saline (PBS). Three mice in each group were euthanized at 3 dpi, lungs, hearts, livers, spleens, kidneys and brains were collected for virus titration in MDCK cells. The remaining five mice in each group were monitored for weight loss and mortality for 14 days. Mice that lost more than 25% of their body weight were humanely euthanized.Groups of 5-week-old female BALB/c mice  were anesthetized with Zoletil 50  and inoculated intranasally with 1050), groups of five 5-week-old female mice anesthetized with Zoletil 50 and inoculated intranasally with 50 μl of 10-fold serial dilutions of viruses in PBS. The mice were monitored for 14 days. MLD50 was calculated and expressed in pfu.To determine the fifty percent mouse lethal dose .For cytokine quantification, mouse lungs collected at 3 dpi were stored in single use aliquots at −70°C. Lungs (n = 3) were homogenized individually in 500 μl tissue lysis buffer. Cytokine concentration in lungs were measured using the Bio-Plex Mouse Cytokine 8-Plex panels  with beads specific for mouse IL-1β, IL-2, IL-4, IL-5, IL-10, IFN-γ, TNF-α and GM-CSF. Array analysis was performed by the Bio-Plex Protein Array System .Confluent MDCK or A549 cells were infected with SD16, SD16-MA or rescued viruses at a multiplicity of infection (MOI) of 0.01, overlaid with serum-free DMEM containing 2 μg/ml TPCK-trypsin (Sigma–Aldrich) and incubated at 37°C. Cell supernatants were harvested every 12 hours until 60 hours post infection (hpi) and titrated by plaque assay on MDCK cells.The PB2, PB1, PA and NP gene segments of SD16, SD16-MA and SD16 PB2 mutants were inserted into pCDNA3.1 plasmid. The PB2, PB1, PA and NP plasmids (125 ng each) were transfected to sixty percent confluent 293T cells, together with fire-fly luciferase reporter plasmid pYH-Luci (10 ng) and internal control plasmid expressing renilla luciferase, Renilla (2.5 ng). After 24 hours of transfection, cell lysate was prepared with Dual-Luciferase Reporter Assay System (Promega) and luciferase activity was measured using GloMax 96 microplate luminometer (Promega).P-value less than 0.05 was considered statistically significant.Statistically significant differences between experimental groups were determined using analysis of variance (ANOVA) with the GraphPad Prism software package . A 6 pfu of virus per mouse. Survival of infected animals was monitored and virus titers of lung homogenates were determined after each passage. The mice infected with wild-type SD16 did not show any clinical symptom of disease and virus titer in the lung was 7.3 log10 pfu/ml. However, after 5 to 6 passages, virus titers increased to 8.5 to 8.8 log10 pfu/ml (data not shown). After 8 passages, mice showed a extensive weight loss and died within 6 dpi, indicating that mouse-adapted variants of H9N2 virus had acquired mutations that profoundly affected virulence.Most H9N2 influenza viruses are avirulent for mice P>0.05), the SD16-MA grew faster than SD16, reaching its maximum titers of 7.8 log10 pfu/ml at 24 hpi, versus virus titer of 5.7 log10 pfu/ml at 24 hpi (P<0.05) and reaching peak yield at 48 hpi for SD16 (10 pfu higher than SD16 before 60 hpi (P<0.05), while the peak yield of SD16-MA was similar to SD16 (P>0.05)  . These r50 value showed that SD16-MA was >103.5-fold more virulent than SD16 virus (5 pfu), all mice infected with SD16-MA exhibited clinical signs of disease, including decreased activity, huddling, bunched posture, and ruffled fur. Mice in this group lost 20.8% weight and began to die at 6 dpi ; IL-1β and IL-5 levels in lungs of mice infected with SD16 and SD16-MA were similar (P>0.05); while the levels of IL-10 and IFN-γ in lung homogenates of mice inoculated with SD16-MA were higher than those of SD16 infected mice (P<0.05). In summary, no obvious correlation was found between the production of inflammatory cytokines and chemokines and the viral pathogenicity.Inflammatory cytokines and chemokines have been suggested to be involved in the pathogenesis of animal and human infected with influenza viruses 10 pfu/g, P<0.05) than wild-type SD16 virus (∼5.7 log10 pfu/g)  . Both viTo identify the molecular markers responsible for the virulence of mouse-adapted variant in BALB/c mice, the genomes of the wild-type and mouse-adapted virus were sequenced. Sequence analysis identified five mutations among 3 genes that were involved in adaptation of SD16 strain to mice; specifically PB2 , HA (L226Q), and M (R210K) . The PB2P<0.05) was ∼achieved by PB2 E627K mutation that was similar for all mutant combinations that contained this mutation, PB2 E627K/M147L, PB2 E627K/V250G and PB2 E627K/M147L/V250G.To evaluate whether the observed mutations in the PB2 protein of mouse-adapted H9N2 variant affect polymerase activity, we measured the activity of reconstituted RNP complexes in 293T cells by a luciferase minigenome assay. SD16-MA carrying PB2 M147L, V250G, and E627K had polymerase activities that was ∼630% relative to wild-type SD16 virus (100%) . To assiin vitro, six single or double gene substituted viruses and six PB2 mutants were generated using the SD16 backbone. The inherent growth characteristics of these rescued viruses in MDCK and A549 cells were studied. Although the SD16-MA virus replicated faster in MDCK cells than SD16, none of the SD16 viruses containing SD16-MA genome segments grew to higher titers than the SD16 parental strain in MDCK cells. Whereas many reassortants generated lower yields than SD16, those reassortants containing SD16-MA PB2 and/or M genes replicated to the highest yields that were better than other single or double gene reassorted viruses (P<0.05), with the maximum titers ranging from 7.80–7.95 log10 pfu/ml (P<0.05), reaching maximum titers of more than 7.7 log10 pfu/ml at 48 hpi  and SD16:MA PB2-M (MLD50  = 103.6 pfu) were less virulent than SD16:MA PB2 (MLD50  = 103.5 pfu), indicating negative gene interaction effects for the combinations. These results indicated that mutations in PB2 alone were responsible for the greatest increases in replication and lethality of H9N2 virus in mice.To determine the role of each SD16-MA mutational gene in the virulence phenotype, six reassortant viruses  were gen50 and viral replication in lungs of these viruses in BALB/c mice were evaluated. Single amino acid substitution PB2 M147L or V250G did not increase the pathogenicity of SD16 in mice with MLD50 >106.5 pfu, while PB2 E627K slightly increased pathogenicity with MLD50 reduced more than 10-fold compared to wild-type virus (50  = 103.8 pfu ) to SD16:MA PB2 (MLD50  = 103.5 pfu)  , indicat16 virus  and 2. TViral adaptation is considered to be one of the major mechanisms for generating pandemic influenza viruses in vitro, but only the mouse-adapted PB2 gene significantly increased viral virulence. Thus all of the 3 mutant genes were demonstrated to possess increased replicative functions in mice indicating the functional basis for their selection on mouse adaptation. The PB2 gene has multiple functions, such as binding host capped mRNAs and initiating viral mRNA synthesis, making it an important pathogenic determinant et al adapted a wildtype duck H9N2 virus in quail and chickens through serial lung passages. The adapted viruses were readily infect mice and resulted in a quick selection of PB2 E627K mutation In the study, viruses containing SD16-MA PB2, HA or M gene all grew better in lungs of mice than SD16, whereas mutations in PB2 and M1 protein demonstrated increased viral replication ability Most of the previous studies found major contributions of single amino acid mutations to viral adaptation and virulence, such as PB2 E158G et al reported that PA N383D contributed to the difference in the polymerase activities of two H5N1 viruses, but could not increase viral virulence, which may be affected by high level of polymerase accumulation in the nucleus of influenza virus-infected cells. Here, PB2 E627K increased the polymerase activity and replication of virus, but did not significantly increase morbidity, as did the combination of V250G and E627K. Combining mutations at 147 and 627 in PB2 could confer increasing polymerase activity and virulence to the adapted virus. These results suggested that high polymerase activity was a prerequisite for the high virulence.Increased polymerase activity was considered to play a role in the adaptation of influenza viruses, because it corresponded to the optimal growth in mammalian cells, replicative fitness and virulence in mice in vivo and in vitro, but were not lethal for mice. These results suggested that high virus load was a prerequisite for the high virulence of virus in mice, but not sufficient. Other factors, such as polymerase activity and regulation of the proinflammatory response, also contribute to virulence of influenza viruses Viral replication is thought to be an important and characteristic prerequisite for virulence. Some highly pathogenic H5N1 avian influenza viruses and wild-type H9N2 viruses that were lethal to mice without adaptation all replicated well in lungs, and even replicated systemically In summary, using a highly lethal mouse-adapted H9N2 virus, we demonstrated mutations in PB2 protein were critical for the adaptation and virulence of H9N2 virus in mice. Although the E627K mutation on its own enhanced replication and polymerase activity, it did not significantly increase pathogenicity of virus in mice. The combination of M147L and E627K in PB2 was critical for the high virulence of mouse-adapted H9N2 virus.


Raltegravir (RAL) is an HIV-1 integrase strand-transfer inhibitor that has exhibited substantial efficacy and a favorable safety profile in HIV-1 infected patients. The goal of this study was to explore the presence of natural polymorphisms and primary mutations related to RAL resistance among HIV-1 patients failing to multiple antiretroviral agents.25 plasmas from HIV-1 infected patients with HAART failure were studied. Genetic analysis was performed amplifying and sequencing DNA encompassing 288 amino acids of HIV-1 integrase gene. Drug resistance mutations and polymorphisms were examined following Low et al, 2009. Genetic subtypes were analyzed using REGA HIV Subtyping Tool (http://www.bioafrica.net/subtypetool/html/subtypinghiv.html).Of the 25 patients, 15 were males and 10 females. All of them are more than 18 years old and 19 patients born in Sao Paulo city. 22 patients were infected by HIV-1 subtype B, 1 by subtype F and 2 by B/F recombinants. No Raltegravir resistance related mutations were observed, however we identified following polymorphisms: V72I (44%), T97A (4%), Q146K (4%), V151I (28%), V201I (52%), T206S (8%), I203M (12%), S230N (4%), M154L (4%), K156N (16%) e K156R (4%). Furthermore, we observed amino acid substitutions at codons 163 in two patients (G163E e G163V) and 138 in one patient (E138N).Despite the absence of RAL primary resistance mutations, we found a high frequency of polymorphisms that were related to in vitro reduced susceptibility to RAL. Furthermore, substitutions at codons 163 (G163R) and 138 (E138K) are called secondary mutations, which are capable to restore viral fitness due to the presence of primary mutations. Further studies are needed to determine the importance of these polymorphisms in reducing the genetic barrier to RAL resistance among treated individuals.


The mimotopes of viruses are considered as the good targets for vaccine design. We prepared mimotopes against multiple subtypes of influenza A and evaluate their immune responses in flu virus challenged Balb/c mice.The mimotopes of influenza A including pandemic H1N1, H3N2, H2N2 and H1N1 swine-origin influenza virus were screened by peptide phage display libraries, respectively. These mimotopes were engineered in one protein as multi- epitopes in Escherichia coli (E. coli) and purified. Balb/c mice were immunized using the multi-mimotopes protein and specific antibody responses were analyzed using hemagglutination inhibition (HI) assay and enzyme-linked immunosorbent assay (ELISA). The lung inflammation level was evaluated by hematoxylin and eosin (HE).Linear heptopeptide and dodecapeptide mimotopes were obtained for these influenza virus. The recombinant multi-mimotopes protein was a 73 kDa fusion protein. Comparing immunized infected groups with unimmunized infected subsets, significant differences were observed in the body weight loss and survival rate. The antiserum contained higher HI Ab titer against H1N1 virus and the lung inflammation level were significantly decreased in immunized infected groups.Phage-displayed mimotopes against multiple subtypes of influenza A were accessible to the mouse immune system and triggered a humoral response to above virus. Influenza A can cause significant morbidity and mortality levels in human. The human influenza A pandemics killed about millions of people worldwide over the past  and seasonal influenza A killed more than 250,000 each year ,5. ThereThe viruses mimotopes are considered to be good targets for the vaccine design since they can induce antibodies against both viral original and mutant antigen . ProtectC179 monoclonal antibody (A/H2N2 subtype) was purchased from Takara Bio Inc ; Mouse monoclonal antibody (IV.C102) against influenza virus A strain H1N1 was from Santa Cruz ; Purified H3N2 goat polyclonal IgG specific to influenza A/Texas 1/77 was from Virostat ; SIV sera were prepared from patients hospitalized by swine-origin influenza virus A/2009 and their binding activities were tested by ELISA. Endotoxin was removed by purification with polymyxin B chromatography. Endotoxin levels were < 0.1 unit/μg of protein by the Limulus Amebocyte Lysate QCL-1000 pyrogen test (Cambrex).Ph.D.-7, Ph.D.-12 and Ph.D.-C7C were produced by New England Biolabs, Inc , with random linear 7-mer, 12-mer or cyclic 7-mer peptides fused to minor coat proteins (pIII) of M13 filamentous phages.11 pfu), Ph.D.-12 (1.5 × 1011 pfu) and Ph.D.-C7C (2 × 1011 pfu) libraries diluted with 0.2 ml TBST were dropped into the coated wells respectively. The incubation wells were rocked gently for 30 min, followed by discarding nonbinding phages. The binding phages were eluted with 0.2 M Glycine-HCl (pH 2.2), 1 mg/ml BSA and neutralized with 1 M Tris•HCl (pH 9.1). The eluted phages were used to infect log-phase bacteria ER2738, concentrated by PEG precipitation and submitted to the second round of selection. The following selections were performed as above except that the Tween-20 concentration was raised from 0.1% to 0.5% in the wash steps. After 3 rounds of selection, the eluted phages were used for plaque isolation. 32 plaque clones were amplified for ELISA test and single-strand DNA preparation.C179  was coated on three wells of 24-wells microplate at 4°C overnight. The coated wells were blocked with 2% bovine serum albumin (BSA) at 37°C for 1 h, then washed with Tris•HCl buffer solution (TBS) containing 0.1% Tween-20 (TBST) for 6 times. Ten microliter Ph.D.-7  was coated on three wells of 24-wells microplate at 4°C overnight. The blocking and bio-spanning procedures were carried as did in C179 antibody, except that the binding phages were eluted with IV.C102 . After 3 rounds of selection, the eluted phages were used for plaque isolation.H3N2 polyclonal antibody (30 μg/ml) and goat IgG (100 μg/ml) were coated respectively. After blocking with BSA and washing with TBST for 6 times, Ph.D.-7, Ph.D.-12 and Ph.D.-C7C libraries were added into the goat IgG-coated wells for 30 min incubation. Then, the nonbinding phages were transferred to H3N2 polyclonal antibody-coated wells for additional 30 min incubation. After that, the non-binding phages were discarded, and the binding phages were eluted with H3N2 polyclonal antibody. The eluted phages were amplified in bacteria ER2738 and used in the following selections as above.Goat anti-human IgG (100 μg/ml) was coated on six wells. After blocking, three wells were added with diluted SIV sera (1:20), the others were added with human IgG (100 μg/ml). Then they were placed at 4°C overnight and washed with TBST for 6 times. Peptide phage display libraries were added into the IgG wells for 30 min incubation. Subsequently, the nonbinding phages solutions were transferred to the sera wells for 30 min incubation. Then, the binding phages at the sera wells were eluted with goat anti-human IgG. The eluted phages were used for plaque isolation at the end of the 3rd selection.Ninety-six-well plates were coated with mAb and BSA  by incubation at 4°C overnight, and blocked with 5% BSA in TBS. Affinity-selected phage were added to the wells and allowed to bind at 37°C for 1 h. After the unbounded phages were removed with 5% TBST, the bound phages were detected by incubation with peroxidase-labelled murine anti-M13 antibodies (Pharmacia). The bound peroxidase was determined by incubation with Opheny lenediamine dihydrochloride  in buffer . When the reaction was stopped by the addition of 3 N HCl, A450 nm was determined with an ELISA reader (BioRad). All the assays were carried out in triplicate.The phage from the 3rd biopanning eluate was cloned for immune-analysis. The nucleotide sequence of the gene III insert was determined as the instruction manual. The amino acid sequence of the insert was deduced from the nucleotide sequence and was compared with native influenza A. The sequences that appeared > 3 times among the selected phage clones were classified as the consensus sequence. The aligned amino acid sequences shared by three or more identical amino acids within the dodecapeptides (heptapeptides) were determined as the mimotopes of the matched protein sequences.EcoR I/BamH I enzyme site by Sangon .The 7- and 12-mer mimotopes of H1N1, H2N2, H3N2 and SIV were linked by GSGGS with the mimotope sequences of SIV7-SIV12-H1N17-H1N112-H3N27- H3N212-H2N27-H2N212. Each mimotope represents the peptide with the highest frequency on phage surface. The codon usage was optimized by species preference and GC content. The gene was synthesized with EcoR I and BamH I endonucleases, and ligated separately into precut pGEX-2 T-1, pGEX-4 T-1 and pET43a (+) plasmids. The ligation products were used to transform competent Trans 109 E. coli cells, which were selected on LB-AMP agar plates at 37°C for 12 h. Three AMP-resisitant clones were picked randomly for plasmids extraction, EcoR I/BamH I digestion and gene sequencing. The confirmed plasmids with correct insert were transformed into competent BL21 (DE3) E. coli cells for protein expression. Four transformed bacteria BL21 (DE3) clones were picked from LB-AMP agar plates for culturing overnight in LB-AMP medium. The resultant bacteria were inoculated into 5 ml fresh medium, cultured to mid-log growth phase for protein expression induction with 1 mM IPTG. After 12 h induction, the bacteria sample was aliquot for SDS-PAGE analysis.The multi-mimotope gene was cut with E. coli cells containing pET43a (+)-multi-mimotope plasmids were induced with IPTG for 12 h. The resultant medium was centrifuged with 8000× g for 10 min and the pellet was resuspended into 10 ml of 20 mM TBS (pH 7.9). The cells were broken by ultrasounding with 100 W for 100 s, followed by centrifuging to remove the cell debris. The supernatant was filtered through 0.22 μm membrane and then loaded onto pre-equilibrated Ni2+-NTA-resin. Then, the resin was rinsed by TBS containing 5 mM imidazole; the binding protein was eluted by TBS containing variable imidazole.One hundred milliliters of BL21 (DE3) The recombinant multi-mimotope was coated on 96-well microplate with concentration of 10 μg/ml at 4°C overnight, followed by blocking with 2% BSA at 37°C for 2 h. The bait antibodies including C179, H1N1 monoclonal antibody, H3N2 polyclonal antibody and SIV sera were added into wells separately to incubate with coated protein at 37°C for 2 h. The wells were washed with PBST for 6 times. Then, HRP-conjugated secondary antibodies were added for binding at 37°C for 2 h. The TMB solution was then added into the wells for color development, which was stopped with 3 N HCl. The unrelated protein BSA was coated as control protein to determine the binding specificity of multi-mimotope to the antibodies.To evaluate the potential of the selected mimotopes as experimental vaccine candidates, purified phage mimotopes were used to immunise female inbred specific-pathogen-free BALB/c mice through intraperitoneal administration.3 f.f.u A/Puerto Rico/8/1934 (H1N1) by intranasal inoculation of 50 μl per mouse. Mice were weighed on the day of virus challenge and then every three days for two weeks. Two weeks after the challenge, lungs were removed for pathological examination. Blood samples were taken to measure serum Ab titers by ELISA. Animals were conducted and approved by the Institutional Animal Care and Use Committee of the Academy of Military Medical Science, under protocol number 0054921. All experiments were performed according to institutional guidelines.The multi-mimotope protein was concentrated to 1 mg/ml and injected intraperitoneally  or subcutaneously (100 μg per mouse) into BALB/c mice (9 per group) as emulsion (1:1) with complete Freund's adjuvant (CFA) for the first immunization and with incomplete Freund's adjuvant (IFA) for the booster injection at 14 days later. The control group were injected with PBS. Ten days after the booster injection, except for control group, other 5 groups were challenged with 2 × 10The concentrations of IgG Abs against H1N1 influenza virus were measured by ELISA. Purified antigen was coated on the microtitre plates   and incubated at 4°C overnight. Serial 2-fold dilutions of sera (100 μl/well) from each group of unimmunized or immunized and immunized infected were incubated for 1 h at 37°C. Goat anti-mouse IgG HRP  was used to detect IgG Abs and O-phenylenediamine dihydrochloride  was used as substrate for HRP and the reaction was monitored at an absorption of 492 nm using an ELISA reader .Lung tissue samples were fixed in 10% formalin and embedded with paraffin,sections were cut at 5 μm thickness and were stained with hematoxylin eosin (HE).t-test. The survival rates of mice in test and control groups were compared by using Fisher's exact test. All differences were considered significant at P values0.05.The data from test groups were evaluated by Student'sWith H2N2 monoclonal antibody C179 as bait protein, binding phages from Ph.D.-7, Ph.D.-12 and Ph.D.-C7C peptide phage-display libraries were enriched by three rounds of binding-elution-amplification. Thirty-two binding phage clones were picked up randomly from every library for ELISA testing with C179 coated and uncoated wells (BSA blocked). Twelve phage clones with higher ELISA signal ratio (C179 to BSA) were chosen for single-strand DNA preparation and DNA sequencing. As shown in Figure The dominant sequence displayed on linear heptopeptide phages binding to IV.C102 H1N1 monoclonal antibody was QWTWTQY, whereas the linear dodecapeptide was DCWQMDRKTCPL, cyclic heptopeptide was PLHARLP. All sequences and their frequencies were listed in Table The dominant sequence displayed on linear heptopeptide phages binding to swine-origin influenza virus A sera was ETKAWWL, whereas the linear dodecapeptide was QAHNWYNHKPLP, cyclic heptopeptide was PLHARLP. All sequences and their frequencies were listed in Table The dominant sequence displayed on linear heptopeptide phages binding to influenza A virus A/H3N2 polyclonal IgG was WPWHNHR, whereas the linear dodecapeptide was VWSTPPHADGPA, cyclic heptopeptide was LGALSHT. All sequences and their frequencies were listed in Table The multi-mimotope SIV7-SIV12-H1N17-H1N112-H3N27-H3N212-H2N27- H2N212 of influenza A covered the mimotopes of H2N2, H1N1, H3N2 and SIV subtypes. Each subtype contained heptopeptide and dodecapeptide mimotopes. The whole amino acids sequence and nucleotide sequence were shown in Table 5EcoR I/BamH I digestion and gene sequencing. After transforming into BL21 (DE3) E. coli cells and inducing with IPTG, the multi-mimotope genes were expressed as GST-fused protein with pGEX-2 T-1, pGEX-4 T-1 plasmids or as Nus/His6-fused protein with pET43a (+) plasmid. The proteins were of 42KD and 73KD in respective fused forms.The multi-mimotope gene was subcloned into pGEX-2 T-1, pGEX-4 T-1 and pET43a (+) expression plasmids, respectively. Their reading frames were confirmed by E. coli cells containing pET43a (+)-multi-mimotope plasmids were chosen for induction with IPTG. The Nus/His6-fusion multi-mimotope with endogenous His6 tag was bound by Ni2+-NTA-resin and eluted with TBS containing 60 mM imidazole  e Figure . The imie Figure . The eluy Figure .The results Figure  showed tFourteen days after A/Puerto Rico/8/1934 (H1N1) virus challenge, 9 of the 9 mice in PBS group, 7 of the 9 mice in 50 μg multi-mimotope i.p. group, 5 of the 9 mice in 100 μg multi-mimotope i.p. group and 8 of the 9 mice in 100 μg multi-mimotope s.c. group died. However, only 2 of the 9 mice in 200 μg multi-mimotope i.p. group died Figure . Six daySera were collected and pooled from mice infected with A/PR8 (H1N1) influenza virus. The ELISA method was used to detect IgG Ab titers against H1N1 influenza viruses. As shown in Figure Gross lesions were observed in the lungs of mice of the unimmunized infected groups,including pulmonary hyperemia,hemorrhage and consolidation. Importantly, the lung inflammation levels of the 200 μg multi-mimotope immunized infected group were significantly decreased compared with those of the matched unimmunized infected groups. Necrotizing interstitial pneumonia was found with light microscope in all cases of death in the unimmunized infected groups. The pulmonary tissue exhibited hyperemia, emorrhage and inflammatory exudation, leading to consolidation. The lumina of alveoli and bronchioles were variably filled with protein-rich edema fluid, fibrin, erythrocytes and cell debris, admixed with many neutrophils and lymphocytes in the unimmunized infected groups Figure .Current trivalent influenza vaccines can elicit production of neutralization antibody to benefit human beings ,19-23. Hin vivo [In the case of mimotope immunisation, several studies have shown effective responses in vivo ,31. Furtin vivo -14.E. coli, which was confirmed by SDS-PAGE [Monoclonal antibody C179, antigen binding fragment (Fab) CR6261 and single-chain variable fragment antibody (scFv) F10 recognize conserved epitope of hemagglutinin across different subtypes of influenza A viruses ,15. To mSDS-PAGE .In recent years, the universal influenza vaccines have been under investigation worldwide, including conserved epitope of surface M2 -36. HoweThe authors declare that they have no competing interests.YWZ, DPX, HBS conceived the study and revised the manuscript critically for important intellectual content. YWZ, JC and CFZ made substantial contributions to its design, acquisition, analysis and interpretation of data. XYX, EQQ, JH performed experiments. PYM, JC, KL, SSZ participated in the design, analysis and interpretation of data. All authors read and approved the final manuscript.


GC, VDR, CYP2R1, CYP24A1, CYP27B1, C10or88 and DHCR7/NADSYN1 genes were analysed in 758 participants. Genotype distributions were in Hardy–Weinberg equilibrium for the adult population for all the studied polymorphisms. Four SNPs in CYP2R1  and six SNPs in GC  were statistically significantly associated with serum 25(OH)D concentrations in children, adults and all combined. Several of the SNPs were in strong linkage disequilibrium, and the associations were driven by CYP2R1-rs10741657 and rs10766197, and by GC-rs4588 and rs842999. Genetic risk score analysis showed that carriers with no risk alleles of CYP2R1-rs10741657 and rs10766197, and/or GC rs4588 and rs842999 had significantly higher serum 25(OH)D concentrations compared to carriers of all risk alleles. To conclude, our results provide supporting evidence that common polymorphisms in GC and CYP2R1 are associated with serum 25(OH)D concentrations in the Caucasian population and that certain haplotypes may predispose to lower 25(OH)D concentrations in late summer in Denmark.Environmental factors such as diet, intake of vitamin D supplements and exposure to sunlight are known to influence serum vitamin D concentrations. Genetic epidemiology of vitamin D is in its infancy and a better understanding on how genetic variation influences vitamin D concentration is needed. We aimed to analyse previously reported vitamin D-related polymorphisms in relation to serum 25(OH)D concentrations in 201 healthy Danish families with dependent children in late summer in Denmark. Serum 25(OH)D concentrations and a total of 25 SNPs in  Vitamin D deficiency is a widespread problem in developed countries 3, which immediately undergoes a thermal isomerization to vitamin D3. Dietary sources provide two forms of vitamin D: Vitamin D2  derived from invertebrates (plants and fungi) and vitamin D3  derived from animal sources. Ingested vitamins D2 and D3 are absorbed in the small intestine and transported with chylomicrons and lipoproteins to the liver, whereas dermally synthesized vitamin D3 diffuses via the blood to the liver tightly bound to group-specific complement (GC) In humans, vitamin D is produced mainly in the skin during exposure to solar ultraviolet blue (UVB) radiation (270–300 nm) 2 and D3) undergoes a series of enzymatic conversions in the liver and kidneys to become biologically active. The hepatic enzyme 25-hydroxylase (CYP2R1) converts vitamin D to 25-hydroxyvitamin D (25(OH)D). This is the major circulating form of vitamin D in the blood. To become biologically active, 25(OH)D is converted to 1,25-dihydroxyvitamin D 2D). This occurs mainly in the kidneys, but also in other tissues expressing the enzyme 25(OH)D-1α-hydroxylase (CYP27B1). The biological effect of vitamin D is mediated when 1,25(OH)2D binds to the vitamin D receptor (VDR). To prevent excessive vitamin D signalling in the target organs, 1,25(OH)2D limits its own activity by inducing 24-hydroxylase (CYP24A1) converting 1,25(OH)2D to the biologically inactive water-soluble calcitroic acid which is excreted in the bile Dietary or dermally synthesized vitamin D (hereafter “D” refers to DThe best biomarker of vitamin D concentration is the serum 25(OH)D concentration. Approximately 25% of the inter-individual variability in plasma 25(OH)D concentrations can be explained by external factors such as diet, regular use of vitamin D supplements and exposure to sunlight (dependent on season and latitude) GC, CYP24A1, CYP2R1, CYP27B1 and VDRGC and CYP2R1 genes. Furthermore, nicotinamide adenine dinucleotide synthetase-1/7-dehydrocholesterol reductase (NADSYN1/DHCR7), and the region harbouring the open-reading frame 88 (C10orf88) on chromosome 10q26.13 were also found to be associated with vitamin D concentrations in blood.Genetic epidemiology of vitamin D is in its infancy and a better understanding of how genetic variation influences vitamin D concentrations is needed. A growing number of studies have uncovered polymorphisms associated with vitamin D concentrations. By candidate gene analysis, five genes have been found, including In Denmark, low vitamin D status is common during the winter due to inadequate dietary intakes and lack of solar radiation from September to April In this study, we analysed previously reported vitamin D-related polymorphisms in relation to serum 25(OH)D concentrations in 201 healthy Danish families with dependent children to confirm previous findings and thus help identifying individuals that may have increased risk of developing vitamin D insufficiency.http://clinicaltrials.gov (NCT01184716). All adult participants and guardians on the behalf of the children participants gave written consent to participate.The present cross-sectional study used baseline data from the VitmaD intervention study described in detail elsewhere et al. DNA was extracted from peripheral blood leukocytes as described by Miller CYP2R1  CYP24A1  CYP27B1 (rs10877012) C10orf88 (rs6599638) DHCR7/NADSYN1  GC  VDR (rs731236 (TaqI), rs757343 (TruI); rs7139166; rs10783219).All SNPs were located in or near genes involved in vitamin D synthesis, activation or degradation. The following SNPs were selected on the basis of evidence of significant association in previous studies: http://www.broadinstitute.org/mpg/snap/ldsearchpw.php) and Haploview software version 4.2 for the adult population.Deviation from Hardy–Weinberg equilibrium (HWE) was tested for the adult population using Chi-square test with Bonferroni’s correction . No significant deviation from HWE was observed. Linkage disequilibrium (LD) between polymorphisms was evaluated using Pearsons’ r, SNAP version 2.2 (2 and 25(OH)D3 and were conducted by isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) at Clinical Biochemical Department, Holbæk Hospital, Denmark. As primary calibrator the standard reference material, vitamin D in humans (SRM 972) from the National Institute of Standards and Technology was used. The analytic quality of 25(OH)D assay was assured by Vitamin D External Quality Assessment Scheme certification and the mean bias was –3.2%. The Inter-assay CVs for 25(OH)D2 were 7.6% and 4.6% at 43 and 150 nmol/L, respectively, and for 25(OH)D3 2.2% and 2.8% at 30 and 180 nmol/L, respectively Measurements of serum 25(OH)D concentrations are described in detail elsewhere CYP2R1 and GC genes, respectively. Among the children the inferred haplotype combinations described 100% and 96% of the observed genotypes for CYP2R1 and GC genes, respectively. Each derived haplotype was assigned a number. Homozygote haplotype combinations were numbered with two identical numbers e.g. 11. The combinations of heterozygote haplotypes were given by the combination of the number of each haplotype e.g. 1+ 2 = 12.Statistical analyses were performed using SAS Enterprise Guide 4.3 . Serum 25(OH)D concentrations were log transformed to approximate a normal distribution and all means are presented as geometric means. A nominal P-value of 0.05 was considered statistically significant. Linear mixed models with family as a random factor were applied to account for the possible dependence between the participants. Furthermore, in the linear mixed models the following categorical variables were included: age , sex , BMI  according to standards for children Genetic risk scores were calculated as the sum of risk alleles and included as risk factors in linear mixed models adjusted for family and confounding variables. The correlation coefficient for rs10741657, rs10766197, rs4588 and rs842999 were very similar and therefore it was not necessary to weight the score by effect size. All the analyses were performed separately for children, adults and for all combined.Genotyping and serum 25(OH)D concentrations were available for 758 participants. CYP2R1 were statistically significantly associated with serum 25(OH)D concentrations in all three groups. Furthermore, for all three groups none of the analysed SNPs in CYP24A1, CYP27B1, C10orf88 and DHCR7/NADSYN1 were statistically significantly associated with serum 25(OH)D concentration. For all three groups all analysed SNPs in GC, except rs2298849  and rs222020 , were statistically significantly associated with serum 25(OH)D concentration. The VDR rs731236 was only statistically significantly associated with 25(OH)D concentration in all combined and rs757343 was statistically significant in children and all combined. Only SNPs that were statistically significantly associated with 25(OH)D concentrations in children, adults and all combined were included in further analyses.Associations between genotypes and serum 25(OH)D concentrations are shown for children, adults and all combined in In the adult population, rs10741657-rs7116978 (Pearson’s r  = 0.90), and rs1076697-rs1562902  were in strong LD. To establish which of the SNPs had the strongest association to serum 25(OH)D concentrations, we assess the association between one SNP and serum 25(OH)D concentrations while adjusting for the other SNPs, family and confounding factors in a linear mixed model. After adjustment, rs10766197 (p = 0.0846) had the strongest association compared to rs1562902 (p = 0.8211), and rs10741657 (p = 0.2545) had the strongest association compared to rs7116978 . In further analysis only rs1076697 and rs10741657 were included.CYP2R1 variants rs10741657 and rs7116978 formed four haplotypes, where haplotype 1 and 2 were most frequent  as the sum of the number of G-alleles of rs10741657 and A-alleles of rs10766197 , A. AfteGC-variants were in LD.In the adult population, rs4588 was in strong LD with rs2282679 (Pearson’s r = 0.997), rs17467825 (Pearson’s r = 0.997) and rs16846876 (Pearson’s r = 0.805). Furthermore, rs17467825-rs2282679 (Pearson’s r = 1.00), and rs2282679-rs16846876  were also in strong LD. To establish which of the 4 SNPs had the strongest association to serum 25(OH)D concentrations, we assess the association between one SNP and serum 25(OH)D concentrations while adjusting for the other SNPs, family and confounding factors in a linear mixed model. The strongest association was observed for rs4588 (p = 0.0099) compared to rs2282679 (p = 0.0230), rs17467825 (p = 0.0230) and rs16846876 . Further analyses only included rs4588. None of the other GC-variants  formed five haplotypes, where haplotype 1 and 2 were the most frequent (GC (data not shown). The association between haplotype combinations and serum 25(OH)D concentrations was statistically significant in children (p = 0.0344), and all combined (p = 0.0018) but not in adults (p = 0.1541).The three significant frequent . The comCarriers of haplotype combination 22 encompassing the variant alleles of rs4588 and rs842999 had low serum 25(OH)D concentrations. Conversely, carriers of haplotype combination 11 encompassing the variant allele of rs12512631 had high serum 25(OH)D concentration. Thus, the variant allele of rs12512631 was associated with high low serum 25(OH)D concentrations and the variant alleles of rs4588 and rs842999 were associated with low serum 25(OH)D concentrations. Since the lowest serum 25(OH)D concentrations were observed for haplotype combination 22 carriers, this could indicate that rs4588 is the biologically relevant polymorphism rather than rs842999 since haplotype combination 44 encompassing the C-allele of rs842999 is associated with higher serum 25(OH)D concentrations.The genetic risk score (range 0–4) was calculated as the sum of the number of A-alleles of rs4588 and C/A-alleles of rs842999 , B. AfteFor the tri-allelic variant rs842999, there was a dose-dependent relationship between serum 25(OH)D concentrations and carriage of none, one or two copies of the G-allele . Thus, cCYP2R1 (rs10741657 and rs10766197) and GC (rs4588 and rs842999)  , C. The s842999) , C. AfteCYP2R1 and GC genes were statistically significantly associated with serum 25(OH)D concentrations.In this present study, we studied the association of 7 prominent vitamin D-related genes with serum 25(OH)D concentrations in 201 Danish families with dependent children in late summer in Denmark, and found that common variants in CYP2R1 gene encodes the key enzyme that converts vitamin D to 25(OH)D in the liver CYP2R1 variants rs1562902, rs7116978, rs10741657 and rs10766197, were significantly associated with serum 25(OH)D concentrations in both children, adults and all combined. Furthermore, rs10741657-rs7116978, and rs10766197-rs1562902 were in strong LD. The association appeared to be driven by rs10741657 and rs10766197, which are located in the promoter region of the CYP2R1 gene. We found that non-carriers of rs10741657 and rs10766197 risk alleles had the highest mean serum 25(OH)D concentrations.The 2 = 1, HapMap Data Rel 24/phase II Nov 08), was associated with serum 25(OH)D concentrations. Furthermore, these findings were successfully replicated in 2,221 subjects. Wang et al. CYP2R1 gene predicts 25(OH)D concentrations.Our results are consistent with previous findings. In the study of Wjst et al. GC gene encodes the vitamin D binding protein (DBP) that binds and transports blood 25(OH)D and other vitamin D metabolites to their target organs. Less than 0.04% of blood 25(OH)D circulates in free form (bioavailable). Most is bound with high affinity to DBP (83–85%) and with lower affinity to albumin (12–15%) GC gene may affect the DBP binding and bioavailability of 25(OH)D and other vitamin D metabolites. Thus, there may be a relationship between phenotype and blood 25(OH)D concentrations.The GC gene are associated with 25(OH)D concentrations. The most studied GC-variants are rs4588 and rs7041, giving three common GC-isoforms, GC1F , GC1S , and GC2 , which differ by amino acid substitutions and/or by glycosylation . Several studies have shown that vitamin D status differs significantly depending on rs4588 and/or rs7041 genotype, where the A-allele of rs4588 and the T-allele of rs7041 are consistently associated with lower 25(OH)D concentrations 2D is higher for the C-allele of rs4588 than for the A-allele GC2 phenotypes that is associated with low vitamin D concentrations should be metabolized faster. Kawakami et al. observed that the metabolic rate was indeed higher in GC2-2 individuals than in GC1-1 individuals GC2 genotype, which is associated with low 25(OH)D concentrations, is also associated with low mean DBP GC2 genotype is more frequent in populations living in northern climates There is accumulating evidence that variants in the GC variant rs2282679 is in high LD with rs4588. Wang et al. 2 = 0.97) were significantly associated with 25(OH)D concentrations in 3,210 Han Chinese. In the study by Berry et al. 2 = 0.98), and rs4588 was significantly associated with 25(OH)D concentrations in 6,551 subjects from the British birth cohort. Zhang et al. 2 = 0.98) and that the association with serum 25(OH)D concentrations is most likely driven by rs4588. Zhang et al. Since the two GWAS studies GC variants were in strong LD and the strongest associations with serum 25(OH)D concentrations were observed for rs4588 and rs842999. We observed a dose-dependent relationship between carrying none, one or two copies of the G-allele of the tri-allelic rs842999 and 25(OH)D concentrations. Furthermore, genetic risk score analysis for rs4588 and rs842999 showed that non-carriers of the risk alleles of rs4588 and rs842999 had the highest serum 25(OH)D concentrations.In our study, several of the significant CYP2R1-rs10741657 and rs10766197, and GC-rs4588 and rs842999), and found the largest%-range in mean serum 25(OH)D concentrations  compared to genetic risk score analysis of CYP2R1  or GC  indicating an additive effect. In general, there was a better association between genetic risk score and serum 25(OH)D concentrations in children than in adults. We speculate that the more risk alleles in CYP2R1 and GC genes a subject carries, the more prone the subject will be for having a low serum 25(OH)D concentration. In Denmark, sufficient serum 25(OH)D concentrations are defined as >50 nmol/L We made a joint genetic risk score analysis for all four risk variants D concentrations. Furthermore, VDR-rs731236 was only statistically significant in all combined and rs757343 was statistically significant in children and all combined. False-positive (type 1 errors) results, which are common in studies of the association between genetic markers and outcomes, and the relative small sample size, resulting in statistical reduced power might explain these findings. We consider children and adults as two natural subpopulations due to biological differences, difference in lifestyle, eating patterns and use of multivitamins In our study population, none of the investigated SNPs in 3-fortification of bread and milk reduced the decrease in serum 25(OH)D concentrations seen during winter and ensured 25(OH)D >50 nmol/L in healthy Danish families. Whether such a dietary intervention program could ensure adequate serum 25(OH)D concentrations in subjects with genetic predisposition for vitamin D deficiency warrants further study.Genetic variants may accelerate or protect against vitamin D deficiency and the genetic effect is life-long. We speculate that individuals with genetically determined low vitamin D concentrations may need different health recommendations in order to improve their serum 25(OH)D concentrations thereby avoiding adverse health outcomes. A study by Engelman et al. GC and CYP2R1 may contribute to the variation of serum 25(OH)D concentrations in a healthy population. Notably, genetic risk score analysis revealed that non-carriers of risk alleles of CYP2R1 rs10741657 and rs10766197, and/or GC rs4588 and rs842999 had statistically significantly higher serum 25(OH)D concentrations compared to carriers of all risk alleles.In conclusion, our results support the current evidence that common genetic variation in


Influenza A viruses are well characterized to antagonize type I IFN induction in infected mammalian cells. However, limited information is available for avian cells. It was hypothesised that avian influenza viruses (AIV) with distinct virulence may interact differently with the avian innate immune system. Therefore, the type I IFN responses induced by highly virulent and low virulent H5N1 AIV and reassortants thereof were analysed in chicken cells.A144V) and with the attenuated H5N1 AIV A/duck/Hokkaido/Vac-1/04 (Vac) carrying the haemagglutinin (HA) of the Yama virus (Vac-Yama/HA), that both induced type I IFN in these cells. The substitution of the NS segment from Yama with that from Vac in the Yama backbone resulted in induction of type I IFN secretion in HD-11 cells. However, vice versa, the Yama NS segment did not prevent type I IFN induction by the Vac-Yama/HA virus. This was different with the PB1/PB2/PA segment reassortant Yama and Vac-Yama/HA viruses. Whereas the Yama virus with the Vac PB1/PB2/PA segments induced type I IFN in HD-11 cells, the Vac-Yama/HA virus with the Yama PB1/PB2/PA segments did not. As reported for mammalian cells, the expression of H5N1 PB2 inhibited the activation of the IFN-β promoter in chicken DF-1 fibroblast cells. Importantly, the Yama PB2 was more potent at inhibiting the IFN-β promoter than the Vac PB2.The highly pathogenic (HP) AIV A/chicken/Yamaguchi/7/04 (H5N1) (Yama) did not induce type I IFN in infected chicken HD-11 macrophage-like cells. This contrasted with an NS1 mutant Yama virus (Yama-NS1The present study demonstrates that the NS1 protein and the polymerase complex of the HPAIV Yama act in concert to antagonize chicken type I IFN secretion in HD-11 cells. PB2 alone can also exert a partial inhibitory effect on type I IFN induction. In conclusion, the control of type I IFN induction by H5N1 HPAIV represents a complex phenotype that involves a particular viral gene constellation rather than a single viral protein. Collectively, these findings contribute to understand the high virulence of HPAIV H5N1 viruses observed in the chicken host. Type I interferons (IFN) exert key functions in the innate immune defence against influenza A virus infections by limiting viral spread and replication . Host cein vitro study, viral genetic elements controlling type I IFN induction in H5N1 AIV-infected chicken cells were identified.The HPAIV H5N1 A/chicken/Yamaguchi/7/04 (Yama) ,18 inducA144V) corresponding to that described for A/goose/Guangdong/1/96 (H5N1) at position 149 [This study employed multiple H5N1 AIV viruses  disrupted the block of type I IFN induction Figure . HoweverWe next assessed the contribution of the polymerase complex in the control of type I IFN induction in virus infected HD-11 cells. Interestingly, the Yama virus with Vac-derived PB2/PB1/PA induced type I IFN, despite of the expression of Yama/NS1 Figure . Vice veNext, reassortant Yama viruses carrying either the Vac PB1 or PB2 segments were analysed. The failure of Yama with Vac PB2 but not with Vac PB1 to control type I IFN, indicated a role of the Yama PB2 in type I IFN antagonistic activity of the virus Figure . The inhIn mammalian cells, PB2 was also found to inhibit IFN-β induction besides NS1 ,14. ConsThe present study revealed that type I IFN production in infected embryonated chicken eggs and HD-11 cells was related to the virulence of the virus. In particular, infection of HD-11 cells with the HP Yama virus did not result in type I IFN secretion, as opposed to infection with the less virulent Vac-Yama/HA virus. The viral factors determining type I IFN production were identified with mutational analyses, revealing the involvement of NS1 and the PB2 proteins of the Yama virus in the prevention of type IFN induction.in vivo. These cells may be analogous to the mammalian plasmacytoid dendritic cells (pDCs), supported by the observation that their activation was independent of virus replication and therefore of NS1 expression, as it is the case for mammalian pDCs.The fact that the A144V mutation within NS1 of the Yama virus results in strong induction of type I IFN indicates that NS1 functions as an inhibitor of the Yama virus-mediated type I IFN induction. A critical role of alanine 144 of H5N1 NS1 in the counteraction of type I IFN induction was also reported for the corresponding alanine at position 149 of the A/goose/Guangdong/1/96 H5N1 NS1 protein . InteresIn order to elaborate on the gene segments that may be involved in the inhibition of type I IFN induction by the Yama virus, various reassortants of the Yama and Vac-Yama/HA viruses were analysed for type I IFN induction in HD-11 cells. The fact that the Vac NS segment in the Yama backbone abrogated the capacity of the virus to prevent type I IFN induction, but not vice versa, indicated that additional viral determinants were involved in the control of type I IFN induction. As expected from previous results with mammalian cells ,14, we din vivo contributes to the rapid establishment of infection and fast dissemination in the infected chicken host [The inhibition of type I IFN induction by HPAIV in HD-11 is a complex property that involves the NS1 and the PB2 of the viral polymerase complex. This inhibition is restricted to particular cell types. From these data one may speculate that the capacity of the HP Yama virus as opposed to the LP Vac virus to counteract early type I IFN induction at the primary replication sites ken host ,25.2. The chicken macrophage-like cell line HD-11 (ATCC) [2. The quail fibroblast cell line CEC-32 carrying a luciferase gene controlled by the chicken Mx promoter [2. Human embryonic kidney (HEK) 293T cells (ATCC) were cultured in DMEM + GlutaMAX-I supplemented with 10% FBS at 37°C with 5% CO2. Madin-Darby canine kidney (MDCK) cells were grown in minimal essential medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS), nonessential amino acids (Invitrogen), and 1 mM sodium pyruvate (Invitrogen) at 37°C with 5% CO2. Chicken splenocytes were isolated by cutting spleen into small pieces and disrupting cell aggregates through a 40 μm cell strainer (Becton Dickinson) followed by purification of the cells using Ficoll gradient centrifugation . After two washes with PBS , the cells were cultured in DMEM, 10% FBS and 2% autologous chicken serum in 96-well plates at 2.5 × 105 cells/well, and stimulated with viruses for 24 h.DF-1 chicken fibroblast cells  were propagated in DMEM + GlutaMAX-I  in the presence of 10% heat-inactivated fetal bovine serum  at 39°C and 5% CO1 (ATCC)  was cultpromoter  was kind50)/ml according to the Reed-Muench formula . For particular experiments, virus was inactivated chemically using 2-bromoethylamine hydrobromide as previously described [The viruses HPAIV H5N1 A/chicken/Yamaguchi/7/04 (Yama), HPAIV H5N1 A/whooper swan/Mongolia/3/05 and LPAIV H5N1 A/duck/Hokkaido/Vac-1/04 (Vac) were generated by reverse genetics using pHW2000-derived plasmids with the permission of Robert G. Webster . The Vac virus is composed of the NA and NS gene segments from A/duck/Mongolia/47/01 (H7N1) and the PB2, PB1, PA, HA, NP, and M segments from A/duck/Mongolia/54/01 (H5N2) .The alanine to valine substitution at the amino acid position 144 of the Yama NS1 protein was introduced with PCR-based site-directed mutagenesis using the 5' phosphorylated oligonucleotides Y-NS1mutAV-R (5'-TCCTTCTTCTGTGAAAACTCTAAG-3') and Y-NS1mutAV-F (5'-GCAATCGTGGGAGAAATCTCAC-3'). The PCR was performed according to standard protocols using the pHW2000-derived plasmid carrying the Yama NS segment as template and the Accuprime Pfx polymerase (Invitrogen). The resulting DNA fragment was religated, and subsequent plasmid DNA was purified from transformed 5 cells per well. The next day, the cells were infected at a MOI of 0.05 TCID50/cell with selected viruses produced on MDCK cells. Chicken splenocytes were seeded in 96-well cell culture plates at a density of 2.5 × 105 cells per well in 0.1 ml of DMEM + GlutaMAX-I supplemented with 16% FBS and 4% chicken serum. The splenocytes were immediately infected with 5-fold serial dilutions of virus in 0.1 ml Opti-MEM I + GlutaMAX-I. At different times after infection, the virus titre in the supernatant was determined in MDCK cells as described above, and type I IFN bioactivity was quantified with the bioassay described below. In order to monitor the survival of the HD-11 cells after infection, the cells were fixed and stained with a crystal violet solution containing 1% formaldehyde, 0.5% (w/v) crystal violet and 30% ethanol. The fixed and stained cells were washed extensively with tap water and treated with lysis solution composed of 0.6% SDS and 40 mM HCl in Isopropanol. The optical density of the crystal violet was measured at 595 nm.HD-11 macrophages and DF-1 fibroblasts were seeded in 24-well cell culture plates at a density of 5 × 10Cells were lysed in a hypotonic buffer containing 20 mM morpholinepropanesulfonic acid, 10 mM NaCl, 1.5 mM MgCl2, 1% Triton X-100 (pH 6.5), and protease inhibitor cocktail (Sigma). Proteins were separated by SDS-PAGE under reducing conditions and analysed by Western blotting (41) using anti-V5 (Invitrogen), anti-HaloTag (Promega), or anti- -actin (C4) antibodies (Santa Cruz) and IRDye-labeled secondary antibodies (Li-Cor Biosciences). The signals were acquired and quantified using the Odyssey infrared imaging system (Li-Cor Biosciences).4 cells per well. The next day, dilutions of recombinant chicken IFN-α  and virus-inactivated samples were added to the CEC-32 cells. The samples containing infectious virus were inactivated at 65°C for 30 minutes prior to the addition to the CEC-32 cells. Chicken IFN-α was resistant to this treatment. After 6 h of incubation at 39°C with 5% CO2, the CEC-32 cells were lysed and luciferase activity was determined using the Centro LB 960 luminometer (Berthold technologies). The data were expressed as type I IFN units per ml of sample. For the transient chicken IFN-β promoter reporter assay, DF-1 cells seeded at a density of 2-4 × 104 cells/well in a 96-well plate were co-transfected with 25-50 ng/well of the reporter plasmid pGL3-P-chIFN-β-luc expressing firefly luciferase under the control of the chicken IFN-β promoter [The type I IFN bioactivity in the supernatants of stimulated cells was measured with an Mx promoter-driven luciferase reporter bioassay described elsewhere  and adappromoter  and withpromoter . The celThe authors declare that they have no competing interests.Conceived and designed the experiments: ML, NR, AS, YS. Performed the experiments: ML, HM. Analysed the data: ML, NR. Wrote the paper: ML, NR, AS, YS.All authors have read and approved the final manuscript.


OPTN gene is involved in several functions including regulation of endocytic trafficking, autophagy and signal transduction. Certain missense mutations in the gene OPTN cause normal tension glaucoma. A glaucoma-causing mutant of optineurin, E50K, induces death selectively in retinal cells. This mutant induces defective endocytic recycling of transferrin receptor by causing inactivation of Rab8 mediated by the GTPase-activating protein, TBC1D17. Here, we have explored the mechanism of E50K-induced cell death. E50K-OPTN-induced cell death was inhibited by co-expression of a catalytically inactive mutant of TBC1D17 and also by shRNA mediated knockdown of TBC1D17. Endogenous TBC1D17 colocalized with E50K-OPTN in vesicular structures. Co-expression of transferrin receptor partially protected against E50K-induced cell death. Overexpression of the E50K-OPTN but not WT-OPTN inhibited autophagy flux. Treatment of cells with rapamycin, an inducer of autophagy, reduced E50K-OPTN-induced cell death. An LC3-binding-defective mutant of E50K-OPTN showed reduced cell death, further suggesting the involvement of autophagy. TBC1D17 localized to autophagosomes and inhibited autophagy flux dependent on its catalytic activity. Knockdown of TBC1D17 rescued cells from E50K-mediated inhibition of autophagy flux. Overall, our results suggest that E50K mutant induced death of retinal cells involves impaired autophagy as well as impaired transferrin receptor function. TBC1D17, a GTPase-activating protein for Rab GTPases, plays a crucial role in E50K-induced impaired autophagy and cell death.The protein optineurin coded by  MYOC, OPTN and WDR36OPTN are associated mainly with normal tension glaucoma, a subset of POAG, where intraocular pressure is within normal limits (10–20mm Hg) but retinal ganglion cell death is observed leading to glaucoma OPTN were shown to cause amyotrophic lateral sclerosis Glaucoma is a heterogeneous group of optic neuropathies characterized by the death of retinal ganglion cells and its axons leading to permanent blindness OPTN, codes for the protein optineurin, which is involved in a variety of functions including regulation of endocytic trafficking, autophagy, immune response, mitosis and NF-κB signal transduction The gene, et al have further revealed that E50K transgenic mice show severe retinal degeneration where all the retinal cell layers are affected The mutant E50K, where glutamic acid at position 50 is mutated to lysine, is a severe disease causing mutation which is located in the conserved bZIP region It has been suggested that E50K-induced death of retinal cells involves autophagy, a quality control mechanism that is used by the cells to remove damaged proteins and organelles through lysosomal degradation In vitro, it acts on several Rabs, but in the cells it acts on Rab8 to regulate endocytic trafficking of TFR TBC1D17, a GAP for Rab GTPases, was identified as an optineurin-interacting protein in a yeast two-hybrid screen for novel optineurin-interacting proteins Here, we have explored the role of TBC1D17 and autophagy in E50K-induced cell death. For this purpose, we have used a retinal cell line, earlier known as retinal ganglion cell line RGC-5 which was the only ganglion cell line available for studies pertaining to glaucoma 5′-GGCTCCTCAGAAGATTCCGCTGTTGAAATTAGGATGGC-3′. Reverse primer- 5′-GCCATCCTAATTTCAACAGCGGAATCTTCTGAGGAGCC-3′. Human TBC1D17 expression plasmids with HA and GFP tags have been described previously Plasmid vectors HA-OPTN/E50K, GFP-OPTN/E50K and the double mutant, E50K-D474N have been described earlier Mouse monoclonal and rabbit polyclonal HA, mouse monoclonal GFP and rabbit polyclonal cdk2 antibodies were from Santa Cruz Biotechnology. Rabbit polyclonal cleaved caspase-3 antibody was from Cell Signaling Technology. Mouse monoclonal TFR antibody and rabbit polyclonal TBC1D17 antibody were obtained from Invitrogen. Rabbit polyclonal p62/SQSTM1 antibody was from Sigma-Aldrich and mouse monoclonal LC3 antibody was from Enzo Life Sciences. Secondary antibodies Cy-3 conjugated anti-mouse and anti-rabbit IgG, and HRP conjugated anti-mouse and anti-rabbit IgGs were from Amersham. Alexa-488 and Alexa-633 conjugated anti-mouse and anti-rabbit IgGs were from Molecular Probes.ShRNA vectors targeting mouse TBC1D17 (shRNA1 and shRNA2) were constructed using pmU6 vector, which is an U6-promoter based vector as explained previously 2 at 37°C in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin. Transient transfections in these cells were performed using Qiagen column  purified plasmids and Lipofectamine 2000  reagent according to the manufacturer’s instructions.All the experiments were carried out using a cell line previously reported as retinal ganglion cell line, RGC-5 The cells grown on coverslips were transfected with required plasmids and allowed to overexpress proteins for the required time. The cells were then fixed and indirect immunofluorescence of cells was carried out as described previously To study colocalization, stained coverslips were observed under LSM 510 NLO Meta Confocal Microscope (Carl Zeiss) and images were captured using 63X/1.4 NA oil immersion objective. For imaging purpose, a 488 nm argon laser, 561 nm DPSS laser and 633 nm HeNe633 laser were used for GFP, Cy3 and Alexa633 respectively. Colocalization was determined by observing the staining pattern and two middle sections were projected to observe colocalization using LSM 510 (version 3.2) software. Colocalization was quantitated by calculating Pearson’s correlation coefficients using the LSM 510 software.Quantitative analysis of cell death was carried out essentially as described earlier To induce autophagy, cells were incubated with amino acid and serum-free media  for 2 hours. To study autophagy flux, a reporter construct mCherry-GFP-LC3B was co expressed with the required plasmids and images taken using LSM 510 Meta NLO Confocal Microscope with 63X oil immersion objective lens (NA 1.4). Optical Z-sections of 0.33 µm were taken from top to the bottom of the cell. These images were then analyzed using Imaris software  for measuring total number of green dots (or yellow dots/autophagosomes) and total number of red dots. Only red dots, which represent autolysosomes were calculated by subtracting green/yellow dots from total red dots for each cell.The proteins were resolved by SDS-PAGE and transferred to nitrocellulose membrane (Hybond-ECL from Amersham) using semi-dry transfer apparatus (Amersham). Western blot analysis was done as described previously t test. P values less than 0.05 were considered statistically significant. The level of significance was shown at 3 levels, viz., *0.05, **0.01 and ***0.001.Statistical significance of differences among experimental groups was assessed by Student’s E50K mutant of optineurin causes death in retinal cells but the mechanism of induction of this cell death is not clear The E50K mutant impairs TFR recycling that is mediated by TBC1D17 Optineurin functions as an autophagy receptor through its ability to interact with LC3 (a protein present in autophagosomes) and ubiquitin Previously, it has been shown that the E50K mutant forms large vesicular structures which are mostly positive for transferrin receptor LC3, an autophagosome marker, exists in two forms where the cytosolic LC3-I form converts to lipidated LC3-II form which gets inserted into the autophagosomal membranes. The changes in the level of the LC3-II form is correlated with the degree of autophagosome formation and is used as an indicator to study changes in autophagy levels Function of optineurin in autophagy depends on its ability to interact with LC3 and ubiquitin through well defined sites  Colocalization of Rab12 (green), WT OPTN/E50K (blue) and endogenous TBC1D17 (red) is shown in merged panels. Scale bar: 10 µm. (B) Bar diagram showing correlation coefficient of colocalization of Rab12 with WT-OPTN and E50K mutant. (C) Bar diagram showing correlation coefficient of colocalization of Rab12 with endogenous TBC1D17 in cells expressing WT-OPTN and E50K mutant. n = 15 cells. *** p<0.001(TIF)Click here for additional data file.Figure S2Colocalization of optineurin and TFR with autophagosomes. Cells plated on cover slips were transfected with GFP-LC3 and HA-WT/E50K and stained with HA (blue) and TFR (red) antibodies. Images show TFR colocalization with GFP-LC3B structures (autophagosomes) in WT or E50K expressing cells. Scale bar: 10 µm(TIF)Click here for additional data file.


Using the four-factor-three-level Box–Behnken design, 29 runs were designed considering the effective ranges of the influential factors. The optimized values for the operational parameters under the respective constraints were obtained at PH of 7.2, Fe3O4/ZnO concentration of 1.78 g/L, initial MTBE concentration of 89.14 mg/L and [H2O2]/ [MTBE] molar ratio of 2.33. Moreover, kinetics of MTBE degradation was determined under optimum condition. The study about core/shell magnetic nanoparticles (MNPs) recycling were also carried out and after about four times, the percentage of the photocatalytic degradation was about 70%.The degradation of methyl tert-butyl ether (MTBE) was investigated in the aqueous solution of coated ZnO onto magnetite nanoparticale based on an advanced photocatalytic oxidation process. The photocatalysts were synthesized by coating of ZnO onto magnetite using precipitation method. The sample was characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), and vibration sample magnetometer (VSM). Besides, specific surface area was also determined by BET method. The four effective factors including pH of the reaction mixture, Fe In the end, kinetics of MTBE degradation was determined in optimum condition.In this work Festewater. The fou3_6H2O), hydrogen peroxide (35% w/w), ferrous sulfate (FeSO4_7H2O), zinc acetate (ZnAC2_2H2O), aqueous ammonia (NH3_H2O) HNO3 and NaOH were purchased from Merk. ammonium carbonate ((NH4)2CO3) purchased from Dae jung.Methyl tert-butyl ether (99.9%), ferric chloride , SEM , BET , 2 lamps , gas choromatography (GC) equipped with a helium ionization detector (HID) , vibration sample magnetometer , magnetic stirrer  and digital pH meter .3O4 magnetic nanoparticles (MNPs). Co-precipitation is a facile and convenient way to synthesize MNPs from aqueous salt solutions. This is accomplished by addition of ammonia to mixture of ferric chloride (0.5 M) and ferrous sulfate (0.5 M) with molar ratio of 1.75:1 under inert argon protection until pH value reached to 9. After 30 min stirring, the precipitate collected using a magnet and washed with deionized water until pH reached to 7. The modification process has been carried out via sonication of 4 g Fe3O4 and 200 ml sodium citrate (0.5 M) mixture for 20 min, which was then stirred for 12 h at 60°C under Ar protection. Afterwards, the precipitate collected and rinsed with acetone. The Fe3O4/ZnO core/shell MNPs were obtained by coating the modified Fe3O4 MNPs with direct precipitation using zinc acetate and ammonium carbonate. The modified Fe3O4 added to 100 ml of deionized water and sonicated for 20 min to make a stable ferrofluid. Then, 30 ml of this ferrofluid was added into a flask to form Fe3O4/ZnO composite. Two solutions were made by adding 12.16 g ZnAC2_2H2O and 7.6 g (NH4)2CO3 respectively into 100 ml of deionized water, and then, these two solutions were dropped slowly into the flask. Then the precipitate was collected and washed with water, aqueous ammonia (pH 9) and ethanol. Thereafter, the precipitate was dried under vacuum in 12 h and calcined according to desired calcination temperature and time/[MTBE] molar ratio of 5. However, higher concentration of H2O2 can have a negative effect. This may be due to the formation of HO2o, a species that is significantly less reactive than HOo/[MTBE] molar ratio of 2.33. In addition, according to the Langmuir-Hinshelwood kinetic model, the apparent pseudo-first-order constant was 0.033 (1/min) for experimental results under optimum conditions. Also the recycling and reuse of MNPs was significantly successful.In this study, FeXRD: X-ray diffraction; SEM: Scanning electron microscopy; VSM: Vibration sample magnetometer; RSM: Response surface modeling; MNP’s: Magnetic nanoparticles; GAC: Granular activated carbon; OFAT: Optimizing one factor at a time; GC: Gas chromatography; HID: Helium ionization detector; AVOVA: Analysis of variance; ZPC: Zero point charge; AOP’s: Advanced oxidation processes.The authors declare that they have no competing interests.MS and MHR designed and performed experiments, analyzed data and wrote the paper; MA designed and performed experiments; AA and SAAN performed the experiments and prepared the final manuscript; RA designed the kinetic model, revised and edited the manuscript. All authors read and approved the final manuscript.