The human antigen R (HuR) stabilizes many mRNAs of proto-oncogene, transcription factors, cytokines and growth factors by recognizing AU-rich elements (AREs) presented in their 3’ or 5’ untranslated region (UTR). Multiple lines of experimental evidence suggest that this process plays a key role in cancer development. Thus, destabilizing HuR/RNA interaction by small molecules presents an opportunity for cancer treatment/prevention. Here we present an integrated approach to identify inhibitors of HuR/RNA interaction using a combination of fluorescence-based and NMR-based high throughput screening (HTS). The HTS assay with fluorescence polarization readout and Z’-score of 0.8 was used to perform a screen of the NCI diversity set V library in a 384 well plate format. An NMR-based assay with saturation transfer difference (STD) detection was used for hits validation. Protein NMR spectroscopy was used to demonstrate that some hit compounds disrupt formation of HuR oligomer, whereas others block RNA binding. Thus, our integrated high throughput approach provides a new avenue for identification of small molecules targeting HuR/RNA interaction. RNA bin2β4 fold ,20, the 2β4 fold ,22, wher2β4 fold ,23. RRM32β4 fold .1H-15N HSQC is unparalleled in its ability to identify the binding site, even though its utility is limited to smaller proteins that yield good NMR spectra [1H-15N protein HSQC spectra revealed that one of the compounds (C10) disrupts HuR/RNA interaction, whereas another compound (C11) impedes HuR oligomerization. Our results suggest that this integrated approach can be a valuable strategy for screening compound libraries for inhibitors of the HuR function.Elevated expression of HuR is linked to carcinogenesis in many human tumors and correlates with poor outcome –29. For  spectra . 12 comp5’-FAM labeled RNA oligonucleotide (AUUUUUAUUUU) derived from the 3’ UTR of the c-Fos proto-oncogene was purchased from MWG operon (HPLC purified and RNase free). Unlabeled c-Fos RNA oligo was purchased from IDT (HPLC purified and RNase free).E.coli. BL21 Rosetta cells. The culture was grown to OD260~0.6 at 37°C, and the protein expression was induced by addition of 1mM IPTG. The incubator temperature was reduced to 18°C immediately following IPTG addition and the cells cultured for additional 16–18hrs. The cells were harvested by centrifugation for 20 min at 6000rpm and sonicated in the presence of protease inhibitors and 15% Glycerol in 50mM Tris pH8.0. Cell lysate was spun down at 18000rpm and the supernatant was collected. The protein was purified using metal chelation chromatography, followed by size exclusion chromatography on the G75  or G200 (for full-length construct) resin. Size exclusion chromatography buffer contained 50mM phosphate buffer, 100mM NaCl, 5mM TCEP at pH7.0. 13C/15N or 15N labeled proteins were expressed in 13C/15N or 15N-enriched M9 minimal media respectively and purified using the same protocol.Full length (residue 1–326), RRM1-2 (residue 1–190), RRM1 (residue 1–98) and RRM2 (residue 102–186) constructs of HuR was PCR amplified from pMal-c2x plasmid containing full length HuR (gift from Dr. Gorospe) and cloned into pET14b vector. They were both expressed in Fluorescence polarization measurements were performed in the 384-well plate format on a Biotek Synergy2 plate reader using 480/20nm excitation and 520/20nm emission filters. The NCI diversity set V library containing 1597 compounds was screened in this study. All samples were dissolved in phosphate buffer . The assay mixture contained 50nM HuR and 10nM RNA to yield FP values approximately 80% of the maximum value observed for fully bound RNA. NCI diversity set V library in 96-well format (10mM stock in DMSO) was transferred to 384-well plate, and then diluted into 50uM in phosphate buffer. 5ul of 50uM compound stock was added to 20 ul assay mixture to yield 10uM final compound concentration.2O or H2O. Saturation transfer difference (STD) NMR spectra were acquired at 298K on Avance III Bruker 500 MHz spectrometer equipped with a 1.7mm TCI cryogenic probe and an automated SampleJet sample changer. Compound concentration in the samples was 0.5 mM and the HuR concentration was 10uM. Protein saturation was achieved using the STDDIFFESGP.3 pulse. The on resonance was set for -500Hz (-1ppm) and off resonance was set for 25000Hz (50ppm), the saturation time is 3s. 16 scans were acquired with 1.5s relaxation delay between the scans. The data was processed using nmrPipe and plotted using Xmgrace. 2D 1H-15N HSQC was acquired with 2048 and 128 complex points in the direct and indirect dimensions, respectively. Protein concentration was 0.1~0.2mM. Backbone assignment of RRM1-2 was derived from the analysis of 3D HNCACB, CBCACONH.Protein samples were prepared in 50mM phosphate buffer at pH 7.0 containing 100mM NaCl, 5mM TCEP in DSedimentation velocity analytical ultracentrifugation was performed on a Beckman XLA analytical ultracentrifuge using an AN50 Ti rotor with standard Epon 2-channel centerpieces. The samples were spun at 25000rpm for~12hrs at 20 C. 25 scans measuring absorbance at 280nm were collected. The data were analyzed using Ultrascan III software.We first characterized the binding of the full-length HuR with 11-mer polyribonucleotide derived from the 3’ UTR of the mRNA of the C-fos oncogene . The binTo evaluate the assay readiness, we performed competition assay with unlabeled RNA. For FP based competitive HTS, Z’ score is the quality factor for the evaluation of an assay: the higher of Z’ value, the more robust of the assay is . We calcWe then performed initial screening using NCI diversity set V, containing 1597 compounds as described in the Material and Methods section. The standard deviation for FP measurement in the screen was 18.2 in unfiltered raw data . CompounNMR-based assays are good secondary assays for HTS, because they provide evidence for direct interaction of the hit compounds with the target protein, thus removing the possibility of the interaction with the RNA or another false-positive effect arising from the direct influence of the hit compound on the fluorescent tag. STD-NMR is a powerful way of monitoring small-molecule/protein interaction because of the ease to implement, the need for low concentration of protein (less than 10uM protein), and the no-limitation for molecular weight. 4 out of 12 compounds from the primary screens displayed STD difference effects  and thos1H-15N HSQC spectrum of the 15N-labeled full length protein reveals that very few signals are visible and they display poor spectral dispersion (1H-15NHSQC spectra of a shorter HuR construct containing only domains RRM1-2. Only compound C10 was observed to have an effect on the spectrum of RRM1-2 (13C/15N labeled RRM1-2 (maximum concentration we can get is about 0.15mM due to the precipitation above 0.15mM), the assignment of RRM1-2 becomes more challenging task. At the end, we have managed to obtain 85% backbone assignment  is one of most robust, reliable and reproducible binding assays used today : CSP is spersion . This inspersion . In the spersion . This isspersion , althougspersion . We thenf RRM1-2 . Upon adf RRM1-2 . The crysignment . The chesignment , which wsignment , indicat15N HSQC  Figs. ThRNA binding proteins (RBPs) that associate with specific mRNA and function as mRNA turnover and translation regulatory RNA binding protein (TTR-RBP) have emerged as pivotal post-transcriptional regulators of gene expression in mammalian cells –4. The aHuR contains three distinct modules known as RNA recognition motifs (RRMs): the N-terminal tandem RRM1-2 and the C-terminal RRM3 . RRM1 anE. coli expression system and a fluorescently-labeled RNA oligo. We also show that NMR-based assays can be used as a secondary filter to remove false positives arising in the primary screening assay by selecting compounds that directly interact with the target protein. During the time of preparing manuscripts, a paper using FP to screen HuR inhibitor was published [1H-15N HSQC are commonly used for studying the protein-ligand binding, which provide the information for residue specific interaction as illustrated in Previous efforts to discover inhibitors of HuR/RNA interaction yielded a low molecular weight inhibitor using confocal fluctuation spectroscopic assay , in whicublished . SPR wasublished  and is nublished ,42. NMR ublished –39. Thus1H-15N HSQC experiments can be used to evaluate effects of the compounds on protein oligomerization and protein/RNA interaction as is demonstrated in this study. HSQC spectra revealed that compound C11 restores signals that are not observed in the spectra of the full-length HuR protein. This striking effect most likely arises from the inhibition of HuR oligomerization caused by C11 binding to the protein, since there is no domain-domain interaction among three RRMs 1H-15N HSIn summary, our preliminary studies reveal that small molecules can inhibit HuR/RNA interaction either by directly competing for the RNA binding site on the RRM1-2 segment  or indirS1 Fig(A) The standard deviation is 18.2 in unfiltered raw data. The average FP value is 171.22 (red solid line). Compounds producing FP readings significantly higher than the average  were ignored due to protein precipitation and/or fluorescence quenching caused by compounds. The hits having 5-standard deviation  below the average were also removed due to their intrinsic fluorescence. (B) The standard deviation was 12.4 with the average of fluorescence reading at 171.40 (red dotted line) after the removal of 18 compounds from (A). The hits having 3-standard deviation  below the average were selected for further verification. (C) The selected hits from (B) were subjected retesting using original condition. The cut-off is same as initial hits pick-up.(TIF)Click here for additional data file.S2 FigCompounds at 250uM in final concentration in the assay volume of 25uL were added to the mixture of 10nM RNA and 50nM HuR in the first well in 384 format plate followed by a serial dilution into next well where the concentration of RNA and HuR was kept constant.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig15N-labeled RRM1. The end-point spectrum (RRM2/RRM1 = 9:1) (red) was overlaid with that of free RRM1 (blue). Two spectra overlaid very well, indicating that RRM1 did not interact with RRM2.Unlabeled RRM2 was titrated into 0.1mM (TIF)Click here for additional data file.S5 Fig13  using Ultrascan III software, while our AUC data indicated that only ~10% monomer existed in solution, the majority of species was dimer/oligomer. We tested 4 compounds from secondary screening. 30% and 50% fraction of boundary were in orange. 10uM HuR and 20uM compounds were used in AUC experiments in the total volume of 500ul. We found that both C10 and C11 shifted the boundary of species toward monomer, while C2 and C8 did not, indicating that C10 and C11 partially disrupted the formation of HuR dimer/oligomer.Sedimentation velocity studies are informative of the overall shape of molecule. The predicted moomer of full length HuR (36 kDa) is ~2.5x10(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 FigRRM1 and RRM2 are colored green. RNA is in orange. The residues interact with RNA was shown in sticks and colored red.(TIF)Click here for additional data file.S8 FigThe panel A-F represents compounds C1-6 respectively.(TIF)Click here for additional data file.S9 FigThe panel A-F represents compounds C7-12 respectively.(TIF)Click here for additional data file.S1 Table0-X)*p)).IC50s were obtained by fitting to the equation of ΔP = ΔPmax/(1+10^((Logx(DOC)Click here for additional data file.


Suppressor of cytokine signaling (SOCS) family is an important negative regulator of cytokine signaling and deregulation of SOCS has been involved in many types of cancer. All cervical cancer cell lines tested showed lower expression of SOCS1, SOCS3, and SOCS5 than normal tissue or cell lines. The immunohistochemistry result for SOCS proteins in human cervical tissue also confirmed that normal tissue expressed higher level of SOCS proteins than neighboring tumor. Similar to the regulation of SOCS in other types of cancer, DNA methylation contributed to SOCS1 downregulation in CaSki, ME-180, and HeLa cells. However, the expression of SOCS3 or SOCS5 was not recovered by the inhibition of DNA methylation. Histone deacetylation may be another regulatory mechanism involved in SOCS1 and SOCS3 expression, however, SOCS5 expression was neither affected by DNA methylation nor histone deacetylation. Ectopic expression of SOCS1 or SOCS3 conferred radioresistance to HeLa cells, which implied SOCS signaling regulates the response to radiation in cervical cancer. In this study, we have shown that SOCS expression repressed by, in part, epigenetically and altered SOCS1 and SOCS3 expression could contribute to the radiosensitive phenotype in cervical cancer. Members of the suppressor of cytokine signaling (SOCS) family of proteins play key roles in the negative regulation of cytokine signal transduction. These proteins act in a negative feedback loop, inhibiting the cytokine-activated Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway to modulate cellular responses . SOCS1 aIt is generally accepted that cervical cancers are radiosensitive and treatment outcomes are still promising even after the tumor is diagnosed too late for treatment using radical hysterectomy. In Korea, cervical cancer is a major health concern for women, accounting for 9.8% of new female cancer cases. Although incidence and mortality rates have been decreasing, the incidence of cervical cancer in the elderly is increasing . Howeverde novo human cervical cancersthat are radiosensitive. We have shown that SOCS1, SOCS3, and SOCS5 expression is repressed, in part, epigenetically by DNA hypermethylation and histone deacetylation. We show that altered SOCS1 and SOCS3 expression may contribute to the radiosensitive phenotype in cervical cancer.In the present study, we have analyzed SOCS1, SOCS3, and SOCS5 gene expression in a panel of cell lines representing primary, 2 at 37°C. For inhibition of DNA methyltransferase, cells were seeded at a density of 2–5 × 105/100-mm dishes and treated with 5-azacytidine (5-AzaC) at 10 μM for 72 h. Media and 5-AzaC were replaced every 24 h. For inhibition of histone deacetylase, cells were treated with Trichostatin A (TSA) at 100 or 200 nM for 16 h. Both inhibitors were purchased from Sigma Chemical Co. . A normal cervix tissue was obtained from a patient who underwent hysterectomy for myoma uteri. We obtained a written informed consent from a patient before surgery. The Institutional Review Board approved the consent procedure and the study protocol (K-1103-003-016) at the Korea Institute of Radiological and Medical Science.The human cervical carcinoma cell lines CaSki, HeLa, ME-180, and SiHa were obtained from the Korean Cell Line Bank . Normal human fibroblast cell lines CCD-18Lu, CCD-18Co, and WI-38 were purchased from the American type culture collection . The CaSki and ME-180 cell lines were maintained in RPMI-1640  and the HeLa, SiHa, and normal fibroblast cell lines were maintained in DMEM (PAA Laboratories), supplemented with 10% fetal bovine serum  and 100 units of penicillin and streptomycin (PAA Laboratories). All cells were cultured in a humidified incubator with 5% COACG GCG AGC CCT TGG; antisense, TTT CTG CTG TCT TTG GGA CCT), SOCS1 , SOCS3 , and SOCS5 . Fluorogenic probes (6-carboxyfluorescein) were: CypA, CGC GTC TCC TTT GAG CTG TTT GCA; SOCS1, CCT CGG GAC CCA CGA GCA TCC; SOCS3, TTG CGC ACG GCG TTC ACC AC; SOCS5, AGG ATG ATT TTG TGA ACC GTG AAG TAC GTG A. DNMT1 gene expression was measured using SYBR premix Ex Taq II kit (Takara Bio Inc) with primers . The relative quantification of gene expression was calculated with the ddC(t) method, where CypA mRNA was used for normalization. The PCR program was as follows: after an initial pre-incubation step at 95°C for 3 min, there were 45 cycles, each consisting of 95°C for 15 s and 60°C for 60 s. The last amplification cycle was followed by a melt curve analysis to make sure about the specificity of the qRT-PCR.Total RNA from cells and tissue was isolated using the Hybrid-R Total RNA Purification Kit . A total of 1 μg of total RNA was reverse transcribed using the PrimeScript RT Reagent Kit  with 25 pmol of oligo-dT primer and 50 pmol of random hexamer. cDNA was diluted 1/10 with EZ Dilution Buffer (Takara Bio Inc), and 2 μL was used as template in a 20 μL PCR reaction. Quantitative PCR was performed using Premix Ex Taq (Takara Bio Inc) in a CFX-96 thermocycler . The following primers were used for PCR: Cyclophilin A (CypA) . Immunohistochemical staining for SOCS1, SOCS3, and SOCS5 were performed by an automated immunohistochemical stainer  on formalin-fixed paraffin embedded tissue sections of squamous cell carcinomas obtained from uterine cervix. Sections were deparaffinized with xylene for 15 min and pretreated in a microwave oven using 0.01 M citrate buffer (pH 6.0) for 30 min. Sections were incubated with primary antibodies directed against SOCS1 , SOCS3  and SOCS5  for 60 min at room temperature and detected using UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen (Thermo Fisher Scientific).2, 0.25 mM dNTPs, 0.5 μM specific primer mix and 1 unit Ex-Taq Hot Start enzyme (Takara BioInc) was used. The PCR products were electrophoresed on a 2% agarose gel, stained with RedSafe Nucleic Acid Staining Solution  and visualized under UV illumination. Primer lists and PCR conditions for MSP are summarized in MSP was carried out to investigate the methylation status of the promoter regions of the SOCS1, SOCS3, and SOCS5 genes. After bisulfite treatment to modify the DNA, PCR was performed to distinguish methylated from unmethylated DNA as described by Herman et al . GenomicBisulfite-treated genomic DNA was amplified by the primers listed in GCCGAATACATTCTGATGGATCTCGAGATCCATCAGAATGTATTCGGC (TRCN0000021890) and GCCCAATGAGACTGACATCAACTCGAGTTGATGTCAGTCTCATTGGGC (TRCN0000021891). The control scrambled shRNA expressing plasmid was obtained from Addgene with the following hairpin sequence: CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG. For packaging, pCMV-dR8.2 dvpr and pCMV-VSVG were used, both of which were purchased from Addgene . The production of viral particles and transduction of target cells was conducted following the pLKO.1 protocol from Addgene. Supernatants from 293T cells transfected by the shRNA and packaging vectors were collected 48 and 72 h post transfection followed by concentration of the virus. Concentrated virus was used for infections in the presence of 8 μg/mL polybrene. One day after infection, puromycin (1 μg/mL) was applied for 4 days to select transduced cells, which were harvested for realtime PCR analysis.The lentiviral shRNA expression plasmids targeting DNMT1 were generated by cloning oligonucleotides into the pLKO.1 puro vector. The sequences of hairpins targeting DNMT1 are: BamHI fragment containing SOCS1 cDNA tagged with Myc at C-terminus or SOCS3 cDNA tagged with FLAG at C-terminus was subcloned into BglII site of pLNCX2. Clones with correct orientation were screened with EcoRI digestion and then confirmed by sequencing. The SOCS5 gene of mouse origin was purchased from Openbiosystems (MMM1013-9202191) and amplified with sense (5’-AAACTCAGATCTACCATGGATGGATAAAGTGGGGAAAATG-3’) and antisense(5’-AGATATGGATCCCTCGAGCTACTACTTATCGTCATCATCTTTATAATCGATCTTTGCCTTGACTGGTTCTC-3’) primers. A BglII and SalI fragment containing SOCS5 with FLAG at C-terminus was subcloned into the BglII and XhoI site of pLNCX2 retroviral vector.Constructs for overexpressing SOCS1, SOCS3, and SOCS5 were made by cloning the SOCS1, SOCS3, and SOCS5 ORF into the pLNCX2 retroviral vector (Clontech). The SOCS1 and SOCS3 genes of mouse origin were kindly provided from Dr. Cacalano NA . The SOCS overexpressing retroviral vectors and packaging plasmids, pCMV-Gag-Pol and pCMV-VSVG, were cotransfected into 293T cell. Supernatants from 293T cells were collected 48 and 72 h post transfection followed by concentration of the retrovirus and used to infect HeLa cells in the presence of 8 μg/mL polybrene. One day after infection, G418 (200 ng/mL) was applied until stable colonies formed. The colonies were pooled to check the SOCS overexpression and used for clonogenic assay.Cells were lysed with SDS sample buffer  and then boiled for 10 min. Protein content was measured with the BCA Protein Assay Reagent (Intron). Equal amounts of cell lysate were separated by SDS-polyacrylamide gels, transferred to a nitrocellulose membrane, immunoblotted and detected by chemiluminescence using ECL detection reagents. Anti-myc, anti-actin and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotech  and the anti-FLAG antibody was from Sigma Chemical Co.137Cs unit at a dose rate of 3.2 Gy/min with a GC-3000 Elan irradiator . After 14 days, colonies were stained with 0.4% crystal violet . The colonies were counted and groups of >30 cells were considered as colonies. The plating efficiency (PE) represents the percentage of cells seeded that grow into colonies under a specific culture condition for a given cell line. The survival fraction, expressed as a function of irradiation, was calculated as follows: Survival fraction = colonies counted/(cells seeded × PE/100). We repeated the experiments five times to ensure reproducibility.Clonogenic survival was defined as the ability of cells to maintain clonogenic capacity and to form colonies. Briefly, cells were harvested from exponential phase cultures by trypsinization, counted and seeded for colony formation. On the following day, gamma radiation was delivered using a dual-source t-test was performed and p-values less than 0.05 were considered statistically significant.Data are given as mean ± SD. The paired Student’s To reveal the possible role of SOCS in cervical cancer development and proliferation, expression of SOCS1, SOCS3,and SOCS5 was examined in several human cervical cancer cell lines, CaSki, HeLa, ME-180, and SiHa. The mRNA level of SOCS1 , SOCS3 , and SOCTo confirm that the lower expression of SOCS1, SOCS3, and SOCS5 in cervical cancer cells is a general phenomenon observed in human tumors, immunohistochemical staining of SOCS proteins was performed in human cervical cancer tissue and in the surrounding normal tissue as a control . All thrTo determine whether DNA methylation contributes to the downregulation of SOCS proteins in cervical cancer, a methylation-specific PCR (MSP) assay was performed. SOCS1 expression was correlated with differential DNA methylation only in CaSki, HeLa, and ME-180 cells . The SOCTo confirm the MSP result, the promoter regions of SOCS1, SOCS3, and SOCS5 genes encompassing CpG islands were cloned and five independent clones were sequenced –3D. Unmein vivo,treatment with 5-AzaC was used to inhibit DNMT1 , a selective inhibitor of the class I and II mammalian histone deacetylase (HDAC) families of enzymes, increased SOCS1 gene expression in CaSki, HeLa, and SiHa but not ME-180 cells . SOCS3 gTo examine the biological effects of increased SOCS gene expression in cervical cancer cells, several HeLa cells were established by infection with retroviruses overexpressing the SOCS gene. Expression of the exogenous SOCS gene in neomycin resistant HeLa cells was confirmed by western blotting . There wSOCS is a large superfamily of proteins that are generally viewed as antagonists of cytokine-induced Janus-activated kinase-STAT signaling. However, SOCS is involved in the regulation of additional signaling pathways, including phosphatidylinositol 3-kinase and ERK MAPK . StudiesOur data showed that altered expression of SOCS1or SOCS3 affected the radiation response of cervical cancer cells. Overexpression of SOCS1 and SOCS3 improved cell survival after ionizing radiation . SOCS1 iin vivo.Wild-type cervical cancer cell lines showed repressed expression of SOCS1, SOCS3, and SOCS5. Both DNA methylation and histone deacetylation may contribute to the downregulation of SOCS 1, 3, 5 genes in cervical cancer cells. Recovery of SOCS1 and SOCS3 gene expression decreased the radiosensitivity of HeLa cells. It appears unlikely that the function of SOCS1 and SOCS3 in solid tumors act uniformly in one direction S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Flavobacterium and Chryseobacterium strains from the plant rhizosphere. It consists of Reasoner’s 2A agar (R2A) prepared by autoclaving phosphate and agar separately and supplementing with 50 mg L−1 cycloheximide and 1 mg L−1 tobramycin. A comparison was made among the following nine media: PSR2A-C/T, PSR2A-C/T supplemented with NaCl, R2A agar, R2A agar supplemented with cycloheximide and tobramycin, 1/4-strength tryptic soy agar (TSA), 1/10-strength TSA, soil-extract agar, Schaedler anaerobe agar (SAA), and SAA supplemented with gramicidin, for the recovery of Flavobacterium and Chryseobacterium strains from the Welsh onion rhizosphere. Flavobacterium strains were only isolated on PSR2A-C/T, and the recovery rate of Chryseobacterium strains was higher from PSR2A-C/T than from the eight other media. In order to confirm the effectiveness of PSR2A-C/T, bacteria were isolated from onion rhizosphere soil with this medium. Flavobacterium and Chryseobacterium strains were successfully isolated from this sample at a similar rate to that from the Welsh onion rhizosphere.An effective medium designated phosphate separately autoclaved Reasoner’s 2A supplemented with cycloheximide and tobramycin (PSR2A-C/T) has been developed for the isolation of  Bacteroidetes is one of the bacterial lineages that is abundant in the rhizospheres of various plants  effects are considered to be one of the main agricultural uses of these strains. Several Flavobacterium strains have been reported to possess PGP capabilities, such as the solubilization of inorganic phosphate and production of indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase  -Gln-Gly, casein, and keratin, and produce proteases  , 38. Som systems , 32, 56.hitinase , mannanahitinase , and amyhitinase , 49. Furroteases , 55. Strbendazim , polycycbendazim , and 2,4bendazim .Flavobacterium and Chryseobacterium have more recently been isolated from the rhizospheres of Panax notoginseng (“F. notoginsengisoli”) (F. nitrogenifigens”) (Flavobacterium sp. strain F52) (C. solani) (C. artocarpi) (C. zeae) (C. arachidis and C. geocarposphaerae)  . Although many Flavobacterium and Chryseobacterium strains have been isolated by commonly used media, such as Reasoner’s 2A (R2A) agar and tryptic soy agar (TSA)  , switchgfigens”) , bell peain F52) , eggplan solani) , cempedatocarpi) , maize (C. zeae) , and peaphaerae) , implyinar (TSA) , 52, 53,Allium fistulosum L.) cv. Kujo-hoso and onion (A. cepa L.) cv. Sharm were used as the source materials for the isolation of bacteria. The Welsh onion and onion were sown in plug trays containing commercial potting medium  and grown for 90 and 40 d, respectively. Seedlings of both plants were transplanted into plastic pots  filled with soil collected from a field on the experimental farm of the university and were grown for 70 d in a greenhouse at 20–25°C under natural light with regular watering and fertilizing. Three plants were uprooted and loosely adhering soil was removed from the roots by gentle shaking for the collection of rhizosphere soil. Three grams of the pooled roots of each plant (1 g of the root of each plant body) was then suspended in sterile distilled water (SDW) and shaken for 15 min on a rotary shaker at 150 rpm. Serial soil dilutions of the samples were prepared in SDW and used for the surface plating of agar plates as described below.Rhizosphere soils of the Welsh onion (−1) (SAA-Gr)  . R2A agaFlavobacterium and Chryseobacterium are known to produce bright yellow non-diffusible and non-fluorescent flexirubin pigments , transferred into 1.5-mL Eppendorf tubes, and stored at −80°C until used.A 100-μL aliquot of each serial dilution of Welsh onion rhizosphere soil was spread onto the surface of each medium in triplicate plates (90 mm in diameter). R2A agar and SEA plates were incubated at 25°C in the dark for 14 d. Plates of 1/4-strength and1/10-strength TSA were incubated at two different temperatures. One set of each medium plate was incubated at 25°C in the dark for 14 d and the other set was incubated at 30°C in the dark for 3 d. SAA and SAA-Gr plates were incubated at 35°C in the dark for 3 d. Well-isolated, discrete colonies showing a yellow/orange color on plates that yielded 10–100 colonies were selected and streaked using quadrant streaking on R2A agar plates (60 mm in diameter) because pigments . After sBacteroidetes bacteria with a yellow/orange color from R2A medium in isolation trial 1. Accordingly, R2A medium was selected as the basal isolation medium in subsequent isolation trials. Although colonies of Bacteroidetes bacteria appeared on R2A, this medium allowed the growth of contaminants such as fungi and orange pigment-producing sphingomonads belonging to the class Alphaproteobacteria. Accordingly, in this trial, R2A agar was supplemented with the fungicide cycloheximide (50 μg mL−1) and bacteriocide tobramycin (1 μg mL−1) (R2A-C/T). Diluted suspensions of Welsh onion rhizosphere soil were spread onto the surface of each medium in triplicate plates (90 mm in diameter) and incubated at 25°C for 14 d. After being incubated, bacteria with a yellow-orange color were isolated and stored as described above.As described below, it was only possible to isolate Flavobacterium are sensitive to inhibitory compounds in media in which phosphate compounds are autoclaved together with agar (−1), tryptone (0.5 g L−1), peptone (1.5 g L−1), starch (1.0 g L−1), glucose (1.0 g L−1), and agar (30 g L−1). Solution B consisted of K2HPO4 (0.6 g L−1), MgSO4 (48 mg L−1), and sodium pyruvate (0.6 g L−1). Solutions A and B were autoclaved separately and then mixed at the same quantity in order to prepare the basal medium, designated phosphate separately autoclaved R2A (PSR2A) medium. Two types of R2A agar were prepared: R2A-C/T and PSR2A supplemented with cycloheximide (50 μg mL−1) and tobramycin (1 μg mL−1) (PSR2A-C/T). Diluted suspensions of Welsh onion rhizosphere soil were spread onto the surface of each medium in triplicate plates (90 mm in diameter) and incubated at 25°C for 14 d. After being incubated, bacteria with a yellow-orange color were isolated and stored as described above.ith agar . Accordi−1), tobramycin (1 μg mL−1), and 1% NaCl (PSR2A-C/T/N). Diluted suspensions of Welsh onion and onion rhizosphere soil were spread onto the surface of each medium in triplicate plates and incubated at 25°C for 14 d. After being incubated, bacteria with a yellow/orange color were isolated and stored as described above.Two types of PSR2A were prepared: PSR2A-C/T and PSR2A supplemented with cycloheximide (50 μg mLhttp://ddbj.nig.ac.jp/blast/blastn) with the reference sequences in the DNA Data Bank of Japan (DDBJ) database. Information on the closest relatives of the 16S rRNA gene sequences of each strain was provided as Escherichia coli NBRC102203T of the 16S rRNA gene) obtained from 14 strains related to Flavobacterium and 19 strains related to Chryseobacterium, isolated from Welsh onion and onion rhizosphere soil on PSR2A-C/T in isolation trials 3 and 4, were aligned with reference sequences using CLUSTALW  according to the manufacturer’s instructions. The 16S rRNA gene of each isolate was amplified with 27f and 1492r primers  and TaKaCLUSTALW . NeighboCLUSTALW , and 1,0Flavobacterium and 19 strains related to Chryseobacterium, isolated from Welsh onion rhizosphere soil in isolation trials 3 and 4 and onion rhizosphere soil in isolation trial 4, on PSR2A-C/T were deposited in DDBJ with the following accession numbers: LC096252–LC096256, LC034259–LC034263, and LC034264–LC034267 , LC096244–LC096251, LC034273–LC034278, and LC034268–LC034272 .The 16S rRNA gene sequences of 14 strains related to Bacteroidetes. In contrast, three genera of Bacteroidetes, including Chryseobacterium, were found on R2A agar medium. Based on the results from isolation trial 1, R2A agar was the most suitable basal medium for the isolation of Bacteroidetes strains.Six types of agar media were used to isolate bacteria from Welsh onion rhizosphere soil. The total bacterial colony counts in the triplicate plates of each medium are summarized in Bacteroidetes-related strains (11% of yellow/orange-colored bacteria) among the six types of media tested, a significantly large number of strains associated with Proteobacteria (71%) and Actinobacteria (18%) were also retrieved. Furthermore, R2A agar medium allowed fungal contaminants (data not shown). These results led us to use the antibiotics tobramycin and cycloheximide in order to prevent the growth of Gram-negative bacteria and fungi, respectively. Tobramycin is a strong inhibitor of Gram-negative bacteria, including most Sphingomonas strains, but not most strains of Flavobacterium and Chryseobacterium , suggesting that the addition of antibiotics is a convenient way by which to prevent the proliferation of rhizosphere-associated Proteobacteria and fungi.Fourteen yellow/orange-colored colonies appeared on the triplicate plates of R2A agar and R2A-C/T media . In accopigments , 47; theet al. , was used as the basal medium in isolation trial 3.One possible way to improve the recovery rate of target microorganisms on selective medium is to reduce environmental stress factors in the medium. Tanaka et al.  previousSphingomonas strains were found on R2A-C/T medium, and all yellow/orange-colored isolates were assigned to Bacteroidetes. Although no isolates assigned to the genus Flavobacterium were recovered from R2A-C/T plates, 5 (26%) out of the 19 isolates obtained from PSR2A-C/T plates were affiliated with the genus Flavobacterium. In addition, the proportion of isolates assigned to the genus Chryseobacterium was higher in PSR2A-C/T plates (42%) than in R2A-C/T plates (24%).The colony counts of yellow/orange-colored bacteria were 17 and 19 on triplicate plates of R2A-C/T agar and PSR2A-C/T media, respectively . AccordiFlavobacterium and Chryseobacterium are often isolated from seawater  out of the 15 isolates obtained from PSR2A-C/T/N plates were affiliated with the phylum Actinobacteria and only the remaining four isolates (27%) were assigned to the phylum Bacteroidetes. The proportion of the isolates assigned to the genus Chryseobacterium was higher in PSR2A-C/T plates (30%) than in PSR2A-C/T/N plates (7%). Isolates assigned to the genus Flavobacterium may only be recovered from PSR2A-C/T plates.In this trial, two types of PSR2A media (PSR2A-C/T and PSR2A-C/T/N) were used for bacterial isolation. Serial dilutions of the rhizosphere soil of the Welsh onion were spread onto the surfaces of these media. The colony counts of the yellow/orange-colored bacteria were 20 and 15 on triplicate plates of PSR2A-C/T and PSR2A-C/T/N media, respectively . Among tFlavobacterium and Chryseobacterium , on the above two media. The results obtained showed that 23 and 19 bacterial colonies with a yellow/orange color appeared on triplicate plates of PSR2A-C/T and PSR2A-C/T/N media, respectively (Bacteroidetes phylum. The proportion of Chryseobacterium strains isolated from onion rhizosphere soil was higher in PSR2A-C/T plates (22%) than in PSR2A-C/T/N plates (16%), and Flavobacterium strains were only isolated from PSR2A-C/T plates. The reason why Flavobacterium strains did not appear on PSR2A-C/T/N medium was that rhizosphere-associated Flavobacterium may be sensitive to high concentrations of NaCl. Further studies on the physiology of Flavobacterium strains are needed in order to clarify this, and information on optimum NaCl concentrations for the growth of these strains is valuable for the selective isolation of Flavobacterium and Chryseobacterium strains from rhizosphere environments. In short, PSR2A-C/T is suitable as a medium for the isolation of Flavobacterium and Chryseobacterium from the rhizosphere. However, the isolation frequencies of the strains assigned to genus Niabella were relatively high from Welsh onion and onion rhizosphere soil. Further studies to reduce or prevent the growth of Niabella strains will be needed in order to improve the recovery rates of Flavobacterium and Chryseobacterium strains.Bacteria were isolated from onion rhizosphere soil, which is also a rich source of ectively . EighteeFlavobacterium strains were clustered with the species originating in soil   were nearly twice the size of aquatic Flavobacterium strains (2.86–3.96 Mbp), implying that terrestrial Flavobacterium strains possess higher metabolic capacities  than those of aquatic Flavobacterium strains. A comparative genomic analysis of Flavobacterium including the strains obtained in the present study is an important subject awaiting further investigation. Most Chryseobacterium strains were closely related to C. daecheongense NBRC 102008T and C. wanjuense NBRC 106394T  . Kolton   the use of R2A agar as the basal medium, 2) supplementation with tobramycin and cycloheximide in order to prevent the growth of Proteobacteria strains and fungi, 3) autoclave phosphate separately to reduce inhibitory substances, 4) an incubation at 25°C for 14 d, and 5) the selection of yellow/orange-pigmented colonies (Flavobacterium and Chryseobacterium strains.We herein succeeded in developing optimized medium for the isolation of colonies . This st


Cystatin C (Cys C) is a small, potent, cysteine protease inhibitor. An Ala25Thr (A25T) polymorphism in Cys C has been associated with both macular degeneration and late-onset Alzheimer’s disease. Previously, studies have suggested that this polymorphism may compromise the secretion of Cys C. Interestingly, we found that untagged A25T, A25T tagged C-terminally with FLAG, or A25T FLAG followed by green fluorescent protein (GFP), were all secreted as efficiently from immortalized human cells as their wild-type (WT) counterparts . Supporting these observations, WT and A25T Cys C variants also showed similar intracellular steady state levels. Furthermore, A25T Cys C did not activate the unfolded protein response and followed the same canonical endoplasmic reticulum (ER)-Golgi trafficking pathway as WT Cys C. WT Cys C has been shown to undergo signal sequence cleavage between residues Gly26 and Ser27. While the A25T polymorphism did not affect Cys C secretion, we hypothesized that it may alter where the Cys C signal sequence is preferentially cleaved. Under normal conditions, WT and A25T Cys C have the same signal sequence cleavage site after Gly26 (referred to as ‘site 2’ cleavage). However, in particular circumstances when the residues around site 2 are modified  mutation), A25T has a significantly higher likelihood than WT Cys C of alternative signal sequence cleavage after Ala20 (‘site 1’) or even earlier in the Cys C sequence. Overall, our results indicate that the A25T polymorphism does not cause a significant reduction in Cys C secretion, but instead predisposes the protein to be cleaved at an alternative signal sequence cleavage site if site 2 is hindered. Additional N-terminal amino acids resulting from alternative signal sequence cleavage may, in turn, affect the protease inhibition function of Cys C. Cystatin C (Cys C) is a small, 13.3 kDa reversible competitive inhibitor of papain-like cysteine proteases which is ubiquitously expressed throughout the body, including in the testes, liver, pancreas, brain, and retinal pigmented epithelium (RPE) . Cys C iTwo variations in the Cys C coding sequence have been associated with disease. A Leu68Gln  mutation in Cys C is associated with hereditary cerebral hemorrhage with amyloidosis –12, whilInterestingly, while the proteases that Cys C binds to are typically localized to the lysosome (reviewed in ), Cys C Cystatin C  was amplified by polymerase chain reaction (PCR) and inserted into the pENTR1A Dual Selection vector  using the BamHI and EcoRI restriction sites. Mutations (A25T and G26K) as well as FLAG-tag insertions (DYKDDDDK) were generated by the Q5 Mutagenesis Kit (New England Biolabs (NEB), Ipswich, MA). Cys C constructs were then recombined into either the pTREx-DEST30, pcDNA-DEST40, pcDNA-DEST47, or pAd/CMV/DEST (Life Technologies) destination vector by an LR Clonase II reaction (Life Technologies). All constructs described herein were expressed in a constitutive manner. Only recombination into the pcDNA47-DEST vector altered the encoded Cys C protein. In this instance, we used a pENTR1A Cys C FLAG entry construct without a stop codon. After recombination into the pcDNA-DEST47 vector, the resulting construct encoded for Cys C FLAG green fluorescent protein (GFP), containing a C-terminal FLAG tag followed by a 22 amino acid linker (WNSRPHSRYLDPAFLYKVVRSR) and then a cycle 3 variant of GFP. All constructs were verified by sequencing.2. Both cell lines were authenticated using short tandem repeat profiling .Human embryonic kidney cells  were maintained in Dulbecco’s Modified Eagle Medium  media supplemented with 10% fetal bovine serum  and penicillin/streptomycin/glutamine . Human retinal pigment epithelial cells  were maintained in DMEM/F12 (Corning) media also with FBS and P/S/Q. All cell lines were cultured at 37°C and 5% COGaussia luciferase (hGLuc) construct serving as a control was co-transfected along with Cys C plasmids (50 ng hGLuc: 1 μg of Cys C plasmid) using X-tremeGENE HP. Co-transfection did not change Cys C secretion. For ARPE-19 transfections, two days before the transfection, cells were replated into 10 cm dishes to ensure that they did not reach confluence. The day before the transfection, ARPE-19 cells were replated at a density of 100,000 cells/well of a 12 well plate. Cells were then transfected using Lipofectamine 3000 ((Life Technologies), 1 μg of DNA, 3 μL of Lipofectamine 3000, 1 μL of P3000 reagent). Transfection efficiency was assessed by transfecting an enhanced GFP (eGFP) construct  and estimating the number of eGFP-positive cells 24 h after transfection -driven constructs using either X-tremeGENE 9  1 μg DNA: 3 μL transfection reagent) or X-tremeGENE HP ((Roche) 1 μg DNA: 2 μL transfection reagent), as described previously . For a stion see .http://bioinfo.ut.ee/primer3-0.4.0/); Cys C forward primer: 5’-GCGAGTACAACAAAGCCAGC-3’, Cys C reverse primer: 5’-ATGTGGCTGGTCATGGAAGG-3’.Transcript levels of transfected cells were evaluated by extracting cellular RNA  followed by generation of the corresponding complementary DNA  and SYBR Green detection  on a QuantStudio 6 (Life Technologies). Ribosomal protein, large, P2 , asparagine synthetase , glucose-regulated protein 78  and ER-localized DnaJ homolog 4  primers were described previously . Cys C qhttp://www.cbs.dtu.dk/services/SignalP/) were used to predict the likelihood of signal sequence cleavage of the different protein variants described in this paper. The following parameters were used: Organism—Eukaryotes; D-cutoff value—default; Graphics output—PNG and EPS; Output format—standard; and Method—input sequence may include TM regions. This software is based on the following paper: [SignalP versions 2.0 to version 4.1  [w/v], 0.5% sodium deoxycholate [w/v]) with Halt protease inhibitors  followed by centrifugation. Soluble protein was normalized using a bicinchoninic acid (BCA) assay  and 40 μg of protein was denatured with 1x LDS buffer with reductant. Conditioned media or intracellular lysates were then loaded on a 4–12% BOLT gel and run for 25–30 min at 200 V using 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Proteins were transferred to nitrocellulose membranes using a G2 Blotter (Pierce). Blots were blocked in Odyssey Blocking Buffer . After blocking, blots were probed with an anti-Cys C antibody , anti-FLAG M2 , or anti-β-actin  followed by an appropriate infrared-conjugated secondary antibody . All blots were imaged and quantified on a LI-COR Odyssey Fc (LI-COR).For western blots performed on neat, conditioned media, cells were transfected for 24 h, after which media was changed and replaced with the corresponding growth media containing 2% FBS  for an additional 24 h . For analysis of secreted proteins, 40 μL aliquots of conditioned media were taken and denatured at 70°C for 10 min using 1x lithium dodecyl sulfate (LDS) buffer with reductant. For western blotting of intracellular proteins, cells were harvested by trypsinization and cells were lysed in RIPA buffer  and eluted in 1x LDS buffer without reductant. After the elution, when the sample was removed from the beads, reductant was added, and the sample was further denatured. Samples were run for western blotting as described earlier.HEK-293T cells were transfected as described above for 24 h. Media was changed 24 h after transfection with 10% FBS-containing media and allowed to condition for an additional 24 h. Media was removed and an equal amount (400 μL) was immunoprecipitated (IP’d) for ≥2 h at 4°C using 3–5 μL of either anti-FLAG M1 agarose beads (Sigma) or anti-FLAG M2 magnetic beads (Sigma). Beads were washed thrice with Hanks Balanced Salt Solution with CaAs an orthogonal approach to determine Cys C signal sequence cleavage, we validated the IP and western blotting approach using intact mass spectrometry (MS) on purified Cys C FLAG variants. Briefly, ARPE-19 cells (2–3 million cells per 10 cm dish) were infected with adenovirus (produced as described previously ) encodinThe resazurin assay  which reGaussia luciferase (hGLuc) along with Cys C demonstrated that there were similar hGLuc levels in WT and A25T Cys C transfected samples, indicating that our transfections and sampling of the conditioned media were consistent , and one set of constructs encoding for WT or A25T Cys C with a C-terminal FLAG and GFP-tag (Cys C FLAG GFP). After transfection of these constructs into HEK-293T cells, we monitored the secretion and intracellular steady state levels of each Cys C variant . Surprisnsistent . Transcrnsistent . Finallynsistent .While HEK-293T cells are easy to manipulate genetically and have served as model systems in numerous studies, human retinal pigment epithelium cells (ARPE-19) would arguably be a better system to test potential secretion differences between WT and A25T Cys C from the perspective of AMD. Therefore, we transfected WT and A25T Cys C FLAG constructs into ARPE-19 cells and followed Cys C secretion . TransfeSince we did not observe any obvious differences in WT or A25T Cys C secretion in either HEK-293T or ARPE-19 cells, we further scrutinized how, or whether, the A25T polymorphism could alter Cys C processing or function. Previously, the 26 residue WT Cys C signal sequence (predicted in ) was verTo test whether Cys C could in fact be preferentially cleaved at site 1  vs. site 2 , we altered the Cys C signal sequence by a simple point mutation; a positively charged lysine residue at the ‘-1’ position  immediately preceding the site 2 cleavage site. Charged amino acids are strongly disfavored at this position , and wouWhile the presence of charged residues such as lysine can increase the apparent molecular weight of Cys C , the LysThe G26K experiments demonstrated that Cys C can be altered such that site 1 cleavage is favored at the expense of site 2. To further probe whether the A25T polymorphism could influence signal sequence cleavage in particular scenarios when the sequence around site 2 is altered or modified, thus shifting the preference of Cys C signal sequence cleavage from site 2 to site 1, we designed a series of N-terminal FLAG-tagged Cys C constructs. Placement of the FLAG octapeptide immediately after site 1 (Ala20), designated as N20 FLAG Cys C, is predicted to retain site 1 cleavage, but eliminate the possibility of site 2 cleavage , S4C FigTo more definitively identify and quantify the cleavage site propensity for each of the Cys C variants, we immunoprecipitated (IP’d) equal amounts of conditioned media from Cys C transfected cells with either anti-FLAG M1 or anti-FLAG M2-conjugated beads. The amount of Cys C that is pulled down by the anti-FLAG M1 antibody represents the amount of protein with an extreme N-terminal FLAG tag, whereas the amount of Cys C pulled down by the anti-FLAG M2 antibody represents total FLAG-tagged Cys C. We found that the entirety of the N20 FLAG WT Cys C (99 ± 10%) or N20 FLAG A25T Cys C (100 ± 11%) that was secreted harbored an extreme N-terminal FLAG tag . These rAs an alternative approach to the biochemical antibody recognition assay described in Herein we presented a comprehensive evaluation of WT and A25T Cys C signal sequence cleavage and secretion from transfected, immortalized cells. Much to our surprise, we found that WT and A25T Cys C were both secreted efficiently from HEK-293T and ARPE-19 cells. Furthermore, A25T Cys C did not trigger the UPR, and followed the same trafficking pathway as WT Cys C. Signal sequence prediction software identified two potential Cys C signal sequence cleavage sites, one after Ala20 (site 1), and another after Gly26 (site 2). While site 2 is the default cleavage site, WT and A25T Cys C are able to be cleaved at either site 1 or 2 and still be secreted efficiently. We noted that when modifications are made near site 2, such as the mutation of Gly26 to lysine (G26K), or the addition of an N-terminal FLAG tag after residue 26 (N26 FLAG), the A25T polymorphism increased the likelihood of alternative signal sequence cleavage either at site 1, or potentially after Ala13. Ultimately, this alternative cleavage results in additional N-terminal amino acids in the mature, secreted protein. These additional N-terminal amino acids may, in turn, affect the protease function of Cys C.Our findings which demonstrate that WT and A25T Cys C have similar steady state levels of secreted and intracellular protein differ from a number of studies which focused on A25T Cys C secretion , 56 or iet al. noted that homozygosity for the A25T polymorphism still conferred significant risk for the development of late-onset AD even though levels of plasma A25T were not significantly lower when compared to WT Cys C in other AD patients [Interestingly, we are not the first group to observe that WT and A25T Cys C are present in fluids at similar levels. In agreement with our findings, Chuo patients . Additiopatients , Italianpatients , 29, Jappatients , Finnishpatients  and Dutcpatients  populatipatients . Thus, ipatients , 27–32, patients , 17, oneet al. detected that A25T Cys C enriched from cerebral spinal fluid was O-glycosylated at Ser27 or Ser28 and still maintained part of the Cys C signal sequence  compared to WT Cys C (32%). While site 1 cleavage has not been described before for WT Cys C, Nilsson  site 1, ). We beltOGlyc/, ). HowevetOGlyc/, ). Thus, et al. and Wallin et al. demonstrated that the first ten N-terminal amino acid residues of Cys C were integral for effective inhibition of cathepsin B and L and for internalization into cells [It is unclear whether alternative signal sequence cleavage resulting in additional amino acid residues would affect Cys C protease inhibition function. Previous work by Abrahamson to cells , 61. BasS1 Fig(A-F) HEK-293T cells were transfected with pEGFP-N1 , WT Cys C FLAG GFP , or A25T Cys C FLAG GFP  and imaged 24 h afterward. Bright field  and GFP-channel  images were captured. Transfection efficiencies for HEK-293T cells were consistently above 70%.(PDF)Click here for additional data file.S2 FigGaussia luciferase (hGLuc) construct along with Cys C demonstrates similar levels of another secreted protein after transfection. n = 3, mean ± S.D. (B) Cys C expression levels in HEK-293T cells 48 h after transfection. Representative data of at least three independent experiments. Mean ± 95% C.I. (C) Viability comparison of Cys C-expressing HEK-293T cells 48 h after transfection. n ≥ 3, mean ± S.D.  Representative transfection efficiency of ARPE-19 cells. ARPE-19 cells were transfected with pEGFP-N1 and imaged 24 h later using transmitted light (D) or a GFP filter set (E). Transfection efficiencies for ARPE-19 cells were consistently above 65%. (F) qPCR validation of UPR induction after BFA, Tg or Tm treatment of ARPE-19 cells. Representative data of three independent experiments, mean ± 95% C.I.(A) Co-expression of a humanized, secreted (PDF)Click here for additional data file.S3 Fig(A) SignaIP 4.1 prediction of cystatin A (a non-secreted cystatin), (B) cystatin C, (C) cystatin D, (D) cystatin F, (E) cystatin M and (F) cystatin S.(PDF)Click here for additional data file.S4 Fig SignaIP 4.1 prediction of G26K (A) or A25T/G26K Cys C (B). (C) SignaIP 4.1 prediction of N20 FLAG WT Cys C, which eliminates site 2 cleavage. (D) SignaIP 4.1 prediction of N26 FLAG WT Cys C, which maintains the two potential sites. Mutation of Ala25 to Thr did not change the predicted cleavage sites.(PDF)Click here for additional data file.S5 Fig Viability of the transfected HEK-293T cells was evaluated 48 h post transfection by the resazurin assay.  Cys C expression levels were monitored by qPCR 48 h after transfection. n ≥ 3, mean ± S.D. for all panels.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


The seasonal influenza vaccine is currently the most effective preventive modality against influenza infection. Nasopharyngeal samples of vaccinated and non-vaccinated patients presenting with Influenza-like-illness (ILI) were collected from over 20 outpatient clinics located in different geographic parts of Israel and were tested for the presence of influenza viruses (influenza A and influenza B). Here we show, that in the 2014-2015 season, the vaccine that included the A/Texas/50/2012 H3N2 virus was ineffective. Significant numbers of individuals vaccinated with the 2014-2015 vaccine, of all ages, were infected with influenza A (H3N2), manifesting similar symptoms as the non-vaccinated group. We further demonstrate that the Israeli circulating influenza A(H3N2) virus was different than that included in the 2014-2015 northern hemisphere vaccine, and that antibodies elicited by this vaccine were significantly less efficient in neutralizing influenza A(H3N2) infection. Influenza virus is a major cause of morbidity and mortality around the world , 2. In aNasopharyngeal samples of patients presenting with Influenza-like-illness (ILI) were collected from over 20 outpatient clinics located in different geographic parts of Israel Figure  and testAll ILI patients that were positive for influenza AH3N2) prior to week 49 of 2014 were not immunized. However, starting from the 2nd week of 2015 we observed that relatively large proportion  of the patients infected with influenza A(H3N2) influenza were vaccinated at least one time  circulating in Israel differs from the 2014-2015 influenza A(H3N2) vaccine strain, we isolated viruses from 22, randomly selected, 9 vaccinated and 13 non-vaccinated individuals and performed molecular characterization.As seen in the phylogenetic analysis Figure , the infMost of the patients suffered from fever, cough, rhinitis and weakness Figure  and no sTo determine the ability of antibodies elicited by the 2014-2015 influenza vaccine, to neutralize the new circulating influenza A(H3N2) virus, we obtained sera from 21 individuals, that had received the 2014-2015 influenza vaccine and tested them against the influenza A(H3N2) A/Texas/50/2012 and the influenza A(H3N2) A/Israel/P-151/2014 virus using a microneutralization assay . PositivWe show here that relatively large proportion of individuals vaccinated with the 2014-2015 northern hemisphere vaccine were infected with the influenza A(H3N2) Israeli strain. We further demonstrated that antibodies elicited by vaccination had reduced ability to neutralize the influenza (H3N2) circulating in Israel; the diseases presentation was similar in ILI individuals infected with influenza A(H3N2) irrespective of whether they were vaccinated or not.Phylogenetically we show that the influenza A(H3N2) viruses obtained from vaccinated and non-vaccinated individuals are different than the vaccinating strain. The Israeli viruses isolated from all individuals belong to the 3C.2a, while the vaccinating strain A/Texas/50/2012 virus belongs to the 3C.1 group. Similar 3C.2a viruses were observed countries in Europe and in the US , 10-12. The institutional review board (IRB) of the Sheba Medical Center approved this research (Helsinki Number 1967-15-SMC). Informed consent was signed by all participants of this study who donated sera.Nasopharyngeal samples of patients presenting with Influenza-like-illness (ILI) were collected from over 20 outpatient clinics located in different geographic parts of Israel and are tested for the presence of influenza viruses (influenza A and influenza B). The survey was anonymous and participants were recorded by serial numbers(MDCK) cells were obtained from the WHO collaborating Centre for influenza virus, the national institute for medical research (MRC), London UK. The cells were used for a maximum of 25 passages and maintained in Dulbecco's modified Eagle's medium  containing 10% fetal bovine serum , 2 mM l-glutamine, and the antibiotics penicillin and streptomycin.The extraction of viral RNA was performed with KingFisher Real-time reverse transcription-PCR (rRTPCR) was performed to determine the Influenza. Reactions were performed the Ambion Ag-Path master mix  using TaqMan Chemistry on the ABI 7500 instrumentH3 forward primer 5′-AAT GGT TGG GAG GGA ATG-3′, H3 reverse primer 5′-TTG AGT GCT TTT RAG ATC TGC-3′, H3 probe 5′-VIC-TTG GTA CGG TTT CAG GCA TCA-TAMRA-3′, where TAMRA is 6-carboxytetramethylrhodamine.To establish a phylogenetic tree, from randomly selected samples. The specific primers used were:H3N2-F6 H3N2-F6 5′ AAGCAGGGGATAATTCTATTAACC 3′H3HA-R1075 5′ AACCGTACCAACCRTCCACCATTC 3′RT-PCR products were sequenced using ABI PRISM Dye Deoxy Terminator cycle sequencing kit . Reaction mixtures were analyzed on Applied BiosystemsThe Sequencher® 5.0 program  was used to compare the nucleotide sequences. Phylogenetic trees were prepared by nearestneighbor joining analysis using Clustal X with 1000 bootstraps and trees were visualized using TreeView or NJ plot software.Selected sequences were deposit in GenBank (KT006534-KT006349).Human sera were heat inactivated, and two fold serial dilutions of 1:20-1:2560 were performed in immunoassay plates. The diluted sera were mixed with an equal volume of the same medium containing 100 TCID50/50 ml of influenza virus (A/Texas/50/2012 or the Israeli strain A/Israel/P-151/2014). After 1 hour incubation at 37° MDCK cells were added to each well. The plates were incubated for 20 hour at 37°C. The supernatant was removed, and monolayers were washed with PBS and fixed in 80% cold acetone for 10 minutes.The presence of viral protein was detected by ELISA as previously described , 14. SpeA Binomial 95% confidence interval was calculated for this rate. The chi-square test was applied to evaluate the differences. A p value <0.05 was considered to be statistically significant. All analyses were performed using SPSS , SAS  and Excel softwares.


In a screen for mutants that suppress exo70B1 resistance, we identified nine alleles of TIR-NBS2 (TN2), suggesting that loss-of-function of EXO70B1 leads to activation of this nucleotide binding domain and leucine-rich repeat-containing (NLR)-like disease resistance protein. This NLR-like protein is atypical because it lacks the LRR domain common in typical NLR receptors. In addition, we show that TN2 interacts with EXO70B1 in yeast and in planta. Our study thus provides a link between the exocyst complex and the function of a ‘TIR-NBS only’ immune receptor like protein. Our data are consistent with a speculative model wherein pathogen effectors could evolve to target EXO70B1 to manipulate plant secretion machinery. TN2 could monitor EXO70B1 integrity as part of an immune receptor complex.During exocytosis, the evolutionarily conserved exocyst complex tethers Golgi-derived vesicles to the target plasma membrane, a critical function for secretory pathways. Here we show that  exo70B1 mutant are dependent on TIR-NBS2 (TN2), an atypical intracellular immune receptor-like protein that lacks leucine-rich repeats. TN2 physically associates with EXO70B1, and TN2 transcripts accumulate at much higher levels in the exo70B1 mutant. These data are consistent with a model where activation of a receptor pathway containing TIR-NBS2 is responsible for activated defense responses and cell death in exo70B1. Our data further suggest that this, and possibly other, exocyst components could be targets of effectors that are guarded by immune receptors.Secretory pathways play an important role in the plant immune response by delivering antimicrobial compounds and metabolites to the site of infection. The evolutionarily conserved exocyst complex is involved in exocytosis, the final step in the secretory pathway. We showed that loss of the function of EXO70B1, a subunit of exocyst complex, results in activated defense responses, and enhanced resistance to a range of pathogens. We found that EXO70B1 associates with the SNARE complex protein SNAP33, which is involved in focal secretion of defense-related proteins. Enhanced disease resistance and cell death in the  Arabidopsis . Lo. LoGolovstaining . H2O2 acstaining . Callosestaining . The sam Jing411 . Assessmescribed  with minPseudomonas syringae infection was performed by leaf infiltration  as described [600 = 0.2) in 10 mM MgCl2 containing 0.025% Silwet L-77, and the infected plants were placed under plastic domes for 24 hr.escribed , unless Hyaloperonospora arabidopsidis (H. a.) Noco2 (5 × 104 spores/mL) was performed according to [Inoculation of rding to .exo70B1-3 seeds were incubated with 0.3% EMS for 16 h. Around 60,000 M2 plants derived from 5,000 M1 plants were scored by infection with G. cichoracearum. Plants showing wild type-like phenotypes were selected for further characterization.The exo70B1-3 mutant was crossed to ecotype Landsberg erecta (Ler). F2 plants displaying exo70B1-3 mutant phenotypes were selected for mapping. Using rough mapping markers [TN2 was also identified by standard map-based cloning.The  markers  and our EXO70B1 was amplified and cloned into binary vector pCAMBIA1300. The construct was then introduced into Agrobacterium tumefaciens strain GV3101, and transformed into exo70B1-3 plants by the floral dip method [For complementation analysis, a 4.3-kb genomic sequence including 1531 bp upstream of the start codon, and 835 bp downstream of the stop codon of p method . TransfoExtraction of total RNA, synthesis of first-strand cDNA and analysis of gene expression were performed as described previously .EXO70B1, including 1531 bp upstream of the start codon, was cloned into the pBI121 vector. Homozygous transgenic plants in the T3 generation were used for GUS activity analysis according to [The promoter region of rding to .SA extraction and HPLC assays were conducted as described previously ,81.EXO70B1-pGBKT7 was co-transformed into Saccharomyces cerevisiae strain AH109 with SEC3a, SEC5a, SEC6, SEC8, SEC10, SEC15b, EXO84bN , PEN1 and SNAP33 fused with pGADT7. The constructs containing SEC3a, SEC5a, or SEC15b fused with pGADT7 were kindly provided by Dr. Viktor Zárský [600 = 0.5 with sterile water, and a 10 μL drop of dilution was plated on SD/-Ade/-His/-Leu/-Trp plates to test the interaction.The MATCHMAKER GAL4 Two-Hybrid System 3 (Clontech) was used to examine the interactions between EXO70B1 and other exocyst subunits, as well as PEN1 and SNAP33. r Zárský . The conThe interaction of EXO70B1 and TN2 was assayed using Gateway-modified versions of the traditional pEG202 and pJG4-5 vectors . pDONR20EXO70B1 and TN2 were first cloned into pSY736 vector in frame with YFPN. The derived YFPN-fused-sequences were then amplified and inserted into the Gateway entry vector pDONR207 and subsequently the destination vector pMDC32. Similarly, to create YFPC fused to EXO70B1, SNAP33, VAMP721 and PEN1, the coding sequence of each gene was cloned into pSY735 vector in frame with YFPC. The inserts were subcloned into pDONR207 and pMDC32 vectors. Agrobacterium GV3101 carrying different YFPN and YFPC pairs were infiltrated into four-week-old Nicotiana benthamiana [To create the constructs used in BiFC analysis, the coding sequences of thamiana . YFP fluN. benthamiana, EXO70B1 was cloned into the pGWB11 vector with C-terminal FLAG fusion using gateway system (Invitrogen). Similarly, PEN1 was cloned into pEarleyGate 201 vector with 35S promoter and N-terminal HA fusion, while SNAP33 and TN2 were cloned into pEarleyGate 203 vector with 35S promoter and N-terminal Myc fusion. Transient expression, total protein extractions and immunoblots were performed as described previously [To create the constructs used in coimmunoprecipitation analysis in eviously . BrieflyEXO70B1 without the stop codon, was cloned into the binary vector pEGAD [EXO70B1pro:EXO70B1-GFP construct was introduced into the A. tumefaciens strain GV3101 and transformed into exoB1-3 plants using the floral dip method [Arabidopsis, we amplified SNAP33 genomic sequence (without stop codon) and fused with an HA tag, and cloned it into pMDC163 vector using gateway system (Invitrogen). And exo70B1-3 plants expressing EXO70B1pro:EXO70B1-GFP were transformed with derived SNAP33pro:SNAP33-HA construct, and the T1 plants were used for coimmunoprecipitation immunoblot assays.For immunoprecipitation, proteins prepared from NB1 buffer  were incor pEGAD  between p method . To testG. cichoracearum for 3 days, and the infected leaves were excised and quickly frozen in liquid nitrogen. Total protein was isolated and subjected to immunoblot with rabbit anti-pTEpY , anti-MPK3 or anti-MPK6 (Sigma) as the primary antibody, following the manufacturer’s instructions.To detect MAP kinase activation, 4-week-old plants were inoculated with The primers used in this work are listed in S1 FigG. cichoracearum at 7 dpi (lower panel) were photographed. Bar = 20 mm.Five-week-old uninfected control (upper panel) and plant infected with (PDF)Click here for additional data file.S2 FigFive-week-old plants grown in the standard short day conditions were photographed. Bar = 20 mm.(PDF)Click here for additional data file.S3 Fig(A) Leaves were detached from four-week-old plants and photographed at 7 dpi with G. cichoracearum. The exo70B1 ein2 and exo70B1 coi1 mutants displayed mildew-induced cell death, similar to the exo70B1 single mutant.(B) Fungal structures and dead host cells on the surface of leaves at 7 dpi were examined by trypan blue staining. Bar = 100 μm.(C) Fungal growth was assessed in plants by counting the number of conidiophores per colony at 5 dpi. Data represent mean and standard deviation (n > 30). Significant difference is indicated by asterisk . The experiments were repeated three times with similar results.(PDF)Click here for additional data file.S4 Fig(A) Four-week-old plants were inoculated with G. cichoracearum. Representative leaves were removed and photographed at 7 dpi.(B) Plant cell death and fungal structures on the infected leaf surface were stained with trypan blue at 7dpi. Bar = 100 μm.(C) Fungal growth was monitored in plants at 5 dpi by counting the number of conidiophores per colony. Lower-case letters indicate statistically significant differences .(PDF)Click here for additional data file.S5 Fig(A) The exo70B1-3 mutation was identified by standard map-based cloning. Markers and BAC clones are indicated. Structure of the EXO70B1 gene is shown at the bottom. exo70B1-3 is a T-DNA insertion mutant. The insertion site of exo70B1-3, and previous identified T-DNA lines exo70B1-1, exo70B1-2 are indicated by the triangles.(B)-(D) Complementation of exo70B1-3 by Agrobacterium tumefaciens-mediated transformation. A genomic clone containing EXO70B1 complemented the exo70B1-3 mutation. The transcript accumulation of EXO70B1 was examined by RT-PCR (B). Four-week-old plants were infected with G. cichoracearum and photographed at 7 dpi (C). Accumulation of the PR1 transcripts was examined at 3 dpi by real-time quantitative RT-PCR with ACT2 as an internal control (D). The asterisk indicates a significant difference from WT . Bars represent mean and standard deviation from three biological simples. Three independent experiments were performed with similar results.(PDF)Click here for additional data file.S6 Fig(A) Leaves of four-week-old plants inoculated with G. cichoracearum at 7 dpi.(B) Infected leaves stained with trypan blue to visualize cell death and conidiophores. Bar = 100 μm.(C) Quantification of fungal growth in plants at 5 dpi by counting the number of conidiophores per colony. Statistically significant differences are indicated by lower-case letters .(PDF)Click here for additional data file.S7 Fig(A) Yeast two-hybrid assays of interactions between EXO70B1 and other exocyst subunits. Single colonies were resuspended in 150 μL of sterile water, and 10 μL of which was dropped on SD-Ade-His-Leu-Trp plates and incubated at 28°C for 4 days.(B) Overnight culture from single yeast colonies were diluted in sterile water to OD = 0.5, and a serial of dilutions 1:30, 1:900, 1:27000 were prepared, and 10 μL of each dilution was spotted on SD-Ade-His-Leu-Trp plates and incubated at 28°C for 4 days.(PDF)Click here for additional data file.S8 FigEXO70B1 promoter-GUS expression was examined in two-week-old seedling (A), four-week-old plant (B), eight-week-old plant (C) and silique (D).(PDF)Click here for additional data file.S9 FigPonceau stain of membranes indicates equal loading.(PDF)Click here for additional data file.S10 Fig35Spro:PEN1-HA and EXO70B1pro:EXO70B1-GFP were co-expressed in N. benthamiana leaves. Total protein was extracted, and PEN1-HA was immunoprecipitated by anti-HA antibody. Proteins were analyzed by immunoblotting using anti-HA or anti-GFP antibody.(PDF)Click here for additional data file.S11 Fig(A) Four-week-old plants were infected with G. cichoracearum and photographed at 7 dpi.(B) Infected leaves were stained with trypan blue to visualize the fungal structures and dead plant cells. Bar = 50 μm.(PDF)Click here for additional data file.S12 Figtn2-1 mutation was identified by map-based cloning. Structure of the TN2 (At1g17615) gene is shown at the bottom.The (PDF)Click here for additional data file.S13 Fig(A) Plants were grown in the standard short day conditions. Uninfected four-, five- and six-week-old wild type (WT), exo70B1-3 and exo70B1-3 tn2-1 plants were photographed. Bar = 20 mm.(B) Infected leaves were stained with DAB to examine accumulation of H2O2. Bar = 50 μm.(C) Free SA was extracted from leaves of uninfected plants or plants infected with G. cichoracearum at 3 dpi.(D) Four-week-old plants were infected with G. cichoracearum at 3 dpi. MAPK activation was assessed by the immunoblot analysis using anti-pTEpY antibody, and accumulation of MPK3 or MPK6 protein was examined by immunoblot analysis using anti-MPK3 or anti-MPK6. Ponceau staining is shown as loading control.(PDF)Click here for additional data file.S14 Figtn2-6 (G222R), which is in the middle of the P-loop domain, is indicated by an arrow.The P-loop domain is indicated. TN2 contains a GRS (instead of GKT) motif in the P-loop domain. The mutation of (PDF)Click here for additional data file.S15 FigTN2 were examined by quantitative real-time PCR and normalized to ACTIN2. Bars represent mean and standard deviation from three biological experiments. The asterisk indicates statistically significant difference .The relative transcript levels of (PDF)Click here for additional data file.S16 FigTN2 was examined by quantitative real-time PCR, with ACT2 as an internal control. Bars represent mean and standard deviation from three biological experiments. The asterisks indicate statistically significant difference from the wild type .Leaves from plants of different ages were used for RNA isolation. The transcript accumulation of (PDF)Click here for additional data file.S17 Fig(A) Leaves of four-week-old wild type and tn2-1 plants inoculated with G. cichoracearum at 7 dpi. Bar = 10 mm.(B) Infected leaves were stained with trypan blue at 7 dpi. Bar = 100 μm.(C) Fungal growth was assessed in plants at 5 dpi by counting the number of conidiophores per colony.(D) Four-week-old plants of wild type and tn2-1 were inoculated with suspension of Pto DC3000 (OD600 = 0.0005) by infiltration. Bacterial growth was monitored at days 0 and 3. Bars represent means and standard deviation of three independent biological replicates.(PDF)Click here for additional data file.S18 FigPto DC3000 (OD600 = 0.2) by spray infection. Bacterial growth was monitored at days 0 and 3. Bars represent means and standard deviation of three independent biological replicates. Statistically significant difference is indicated with the asterisk .Four-week-old plants were inoculated with suspension of (PDF)Click here for additional data file.S19 Fig(A) Four-week-old plants were inoculated with G. cichoracearum. Leaves were removed and photographed at 7 dpi. Bar = 10 mm(B) Infected leaves were stained with trypan blue to show fungal structures and dead plant cells at 7 dpi. Bar = 100 μm.(C) Fungal growth was monitored in infected leaves at 5 dpi by counting the number of conidiophores per colony. Statistically significant differences were indicated by lower-case letters . Bars represent mean and standard deviation (n>30). The experiments were repeated three times with similar results.(PDF)Click here for additional data file.S20 FigPonceau stain of membranes indicates equal loading.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


The ever-expanding rollout of antiretroviral therapy in poor resource settings without routine virological monitoring has been accompanied with development of drug resistance that has resulted in limited treatment success.pol-RT genotyping for drug resistance was performed using an in-house protocol.A cross-sectional study with one time viral load was conducted during the period between 2012 and 2013 to determine treatment failure and drug resistance mutations among adults receiving first-line (44) (3TC_d4T/AZT_NVP/EFV) and second-line (20) (3TC/AZT/LPV/r) in Nairobi, Kenya. HIV-1 A total of 64 patients were recruited (mean age 36.9 yrs.) during the period between 2012 and 2013 of the 44 adult patients failing first-line 24 (40.9%) had drug resistance mutations. Eight (8) patients had NRTI resistance mutations with NAMS M184V (54.2%) and K65R (8.4%) mutations being the highest followed by TAMs T215Y and K70R (12.5%). In addition, among patients failing second-line (20), six patients (30%) had NNRTI resistance; two patients on K103N and G190A mutations while V106A, Y184V, A98G, Y181C mutations per patient were also detected. However, for NRTI two patients had TAM T215Y. M184V mutation occurred in one patient.The study findings showed that HIV-1 drug resistance was significantly high in the study population. The detected accumulated resistance strains show that emergence of HIV drug resistance will continue to be a big challenge and should be given more attention as the scale up of treatment in the country continues. The Kenyan ministry of health began providing antiretroviral (ARV) therapy to the public sector in 2003. Kenya tUntil 2009, the standard first-line regimens were stavudine (d4T) plus lamivudine (3TC) combined with a third agent: a non-nucleoside reverse transcriptase (RT) inhibitor (NNRTI) in adults and older children or ritonavir-boosted lopinavir (LPV/r) in young children who had received nevirapine (NVP) for prevention of mother-to child transmission (PMTCT), 6. By 2With the introduction of combination therapy with potent and cost-effective generic antiretroviral therapies (ARTs), quality of life has significantly improved, transmission rates reduced and survival rates increased among HIV infected individuals, 10. HowAlthough the information on drug resistance patterns of HIV-1 among treatment-exposed patients is crucial for the development of novel effective drugs, unfortunately there is no existing system that monitors patterns of drug resistance in patients failing therapy. This stFollowing WHO recommendations a cross-A standardized questionnaire was used to collect demographic data that included age, sex, ARV use , education levels, marital status and drug adherence. Those patients who had treatment interruptions at the time of blood sampling were excluded from the study. Additionally, patients who transferred from other treatment clinics and those who defaulted but came back for treatment later were also excluded from the study. In addition, participants with a previous history of ART exposure for prevention of mother to child transmission (PMTCT) or for post-exposure prophylaxis (PEP) were excluded from the study. Three ml of blood samples were collected from each participant for routine CD4 count counts and the remnants centrifuged to obtain plasma, for viral load quantification and HIVDR testing.CD4 T cell counts were performed using a FACSCalibur flow cytometer  and expressed in absolute count. Individpol gene was amplified using the primers (RT18:5′GGAAACCAAAAATGATAGGGGGAATTGGAGG3′ and RT21 5′ CTGTATTTCTGCTATTAAGTCTTTTGATGGG 3′) and second round primers (RT 1: 5′ CCAAAAGTTAAACAATGGCCATTGACAGA 3′ and RT4: 5′ AGTTCATAACCCATCCAAAG 3′) in the second round[Whole blood samples from patients failing first and second line ART were obtained and separated to obtain Peripheral blood mononuclear cells (PBMCs) by Ficoll-Hypaque density gradient centrifugation. Proviral DNA was extracted from the PBMCs using DNAzol  lysis and ethanol precipitation. Nested polymerase chain reaction (PCR) was performed using AmpliTaq Gold . Briefly, HIV- 1 nd round. Amplifind round.http://www.ncbi.nlm.nih.gov/projects/genotyping/formpagex.cgi), RIP (http://www.hiv.lanl.gov/content/sequence/RIP/RIP.html), and Jumping HMMER (http://jphmm.gobics.de/submission_hiv.html), were used in confirming subtyping[Genotypic drug resistance in the pol-RT region was defined as the presence of one or more resistance-related mutations, as specified by the consensus mutation figures of the International AIDS Society-USA. HIV sububtyping.A total of sixty four (64) patients failing first-line 44 (67.2%) and second line 20 (32.8%) therapy were evaluated. The participants were aged between 11 yrs and 70 yrs, with an average age of 37 yrs. Thirty (46.2%) were female and 34 (63.8%) males with all being residents of different regions of Nairobi. Data on length of time on the failing regimen was not available.Immunological and virological failure was determined by use of CD4 count (>350 cells/ml) and viral load (>1000 HIV RNA copies/ml). We determined whether age, marital status, WHO disease stage and education levels influenced virological and/or immunological failure  had developed drug resistance mutations. The most common NRTI mutation was M184V 12 (66.7%) of the 18 patients who were infected with drug resistance strains. The K65R mutation occurred in two patients without TAMs in combination with M184V mutations among those on TDF containing first-line drugs. Of the 6 patients who harboured TAMs mutations, only 3 of them had T215Y mutations while 3 had K70R mutations among those who were on or on a previous AZT treatment. Nevertheless, these two mutational pathways did not occur both in a single patient (Table p = 0.31).The NNRTI mutation profiles differed between patients failing EFV compared to those on NVP containing regimens. The K103N 11(61.1%) was most common NNRTI mutations with highest frequency occurring on EFV  than NVP . Nevertheless, this occurrence was not any significant different (EFV seemed to select for a wider range of mutations compared to NVP Table . Of the Of the 20 patients failing second line drugs 6 patients harboured drug resistance mutations. These mutations were M184V (n = 1), T215Y (n = 2), V106A (n = 1), A98G (n = 1), Y18C1C (n = 1), G190A (n = 1) and V108IV (n = 1). However four patients had diverse drug mutations combinations of K103N and V108IV (n = 1); Y184V, G190A and T215Y (n = 1); K103N, M184V and T215Y (n = 1); and A98G, Y181C (n = 1) mutations. Single occurring mutations of V106A occurred as expected in a patient who had NVP regimen.From the 64 samples that were successfully amplified and sequenced, analysis of the sequences showed that the majority belonged to HIV subtype A 35/64 (54.5%) followed by subtype D 17/64 (26.7%), subtype C 8/64 (12.5%) and CRF AE 3/64 (4.7%).In this study, among patients who had virological failure with known NNRTI and NRTI mutations, those on first and second-line therapy were 40.9% and 30% respectively. The mutation patterns observed in this study were similar to those obtained in subtype B. The M184V was the most predominant mutation followed K103N. These findings were similar to those obtained from previous studies in Kenya suggestiOf the patients on first-line, 45.5% of them had no mutations associated with drug resistance similar to those reported in 17% South Africa. This muThe occurrence of NNRTI mutations varied depending on the NVP and EFV regimen. Though patient were on NVP or EFV they both selected, K103N, Y181C, A190S mutations with mutations; Y188L, A190A detected on those on NVP while Y184V, K70KT mutations on those on EFV. Though mutations Y181C and G190A are mostly selected by NVP they were detected among those on EFV. These mutations Y181C and G190A are known to reduce susceptibility to Etravirine which could be used either for second or third-line regimen.However, among patients failing second-line, 70% patient harboured virus with no drug associated mutations. This also suggested better patient’s adherence. In 20% (6/20) patient harbouring drug resistance viral strains, two patients had first TAMs mutation pathway of T215Y and also with NAMs M184V mutations. For NNRTI mutations, two patients had K103N and A190A. The observed low rates of acquired drug resistance among patients failing second-line suggested the currently used NRTI regimens are still effective. It could be argued that the detected mutations among patients on second line could be associated with the effect of persistent of acquired HIV-1 drug resistance mutations before switching to therapy.pol gene that that is targeted by currently used drugs. Early Warning Indicators are often required as indicated by for efficient follow out of the ART and limitations of the development of HIV drug resistance. However, this study did not capture this data, including on length of time the patients were put on the failing regimen.Despite these findings, this study had limitations. The cross-sectional study and single sampling approach could not confirm virological failure or rule out blips, which can occur even during effective treatment–24. BaseThis study detected minority complex drug resistance profiles that are predictive of resistance to currently used second-line NRTIs and NNRTIs regimens. Though the currently used drugs are still effective, the accumulated resistance strains observed in this study clearly shows that emergence of HIV drug resistance will continue to remain a priority while scaling up ART coverage.


We found that the virus was non-pathogenic in mice and was stable after serial passaging in eggs. Furthermore, we found that a monovalent influenza A (H7N9) split vaccine prepared from the virus was immunogenic in mice and ferrets. When given intramuscularly, the vaccine (two doses of at least 15-µg) completely protected mice from normally lethal wild-type H7N9 virus challenge. In summary, our H7N9 vaccine, developed over a short time, is a potential candidate for further clinical evaluation and human use.In early spring 2013, the emergence of the influenza A (H7N9) virus in humans in Eastern China raised concerns of a new influenza pandemic. Development of a safe and effective H7N9 influenza vaccine is urgently needed. To this end, we first synthesized the hemagglutinin (HA) and neuraminidase (NA) genes of the influenza A (H7N9) virus A/AnHui/1/2013. Using reverse genetics, we rescued a reassortant virus (H7N9/PR8) that contained the HA and NA genes from wild-type H7N9 and six genes encoding internal proteins from the A/Puerto Rico/8/34 (PR8) virus. Next, the pathogenicity of the reassortant virus was evaluated both  On 31 March 2013, the Chinese National Health and Family Planning Commission announced the occurrence of three cases of human infection with influenza A (H7N9) Infection caused by influenza H7N9 virus is a zoonosis, and the animal reservoir of the virus is the global aquatic bird population http://www.who.int/influenza/vaccines/virus/candidates_reagents/a_h7n9/en/), but the experimental data on H7N9 influenza vaccines were incomplete.Currently, several strategies are being used to develop vaccines that protect humans against influenza virus infection. Inactivated, subunit, and live attenuated vaccines are under development. As the influenza genome is segmented, laboratories in the WHO influenza network have generated a reassortant virus used for annual influenza vaccinations. This virus contains surface protein-encoding genes from the currently circulating influenza virus, but the remaining six genes, encoding internal proteins, are derived from PR8, a laboratory-adapted H1N1 strain. Thus, the reassortant virus has the antigenic characteristics of the circulating strain and the high-yield properties of PR8 In the present paper, we report on the creation of an H7N9 influenza vaccine candidate virus using reverse genetics. All experiments employed Vero cells approved by the WHO for the manufacture of human vaccines. Using an eight-plasmid system, we (rapidly) rescued a reassortant H7N9 influenza vaccine virus from such cells, and assessed the pathogenicity thereof both in vivo and in vitro. Next, the immunogenicity and protective efficacy of the candidate vaccine were evaluated in animal models. Our work shows that reverse genetics can be used to prepare for influenza pandemics that may threaten China.All experiments using infectious influenza viruses, including animal studies with live viruses, were conducted under biosafety level 3 containment. Mice were housed at an appropriate temperature under a standard light/dark cycle. All animal experiments were conducted in accordance with the Guidelines for Animal Experiments of the Beijing Institute of Microbiology and Epidemiology and were approved by the Institutional Animal Care and Use Committee of that institute . All infections and sample collections were performed under sodium pentobarbital anesthesia (60–80 mg/kg) and all efforts were made to minimize animal suffering.The human influenza A H7N9 viruses (A/AnHui/1/2013), isolated in China in 2013 5′-TATTCGTCTCAGGGAGCAAAAGCAGGGG-3′) and Bm-HA-2 (5′ -ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTT-3′). The neuraminidase gene was amplified using the primer pair Ba-NA-1 (5′-TATTGGTCTCAGGGAGCAAAAGCAGGAGT-3′) and Ba-NA-2 (5′-ATATGGTCTCGTATTAGTAGAAACAAGGAGTTTTTT-3′). The PCR products were purified and cloned into the vector pHW2000, as described previously First, the hemagglutinin (HA) and neuraminidase (NA) genes of A/Anhui/1/2013 were synthesized by Sangon Biotech (Shanghai) Co. Ltd. The hemagglutinin gene was amplified by PCR using the primer pair (1): Bm-HA-1 (6 cells in MEM with 10% (v/v) FBS. The following day, the cells were trypsinized and resuspended in 50 ml of phosphate-buffered saline in a T25 flask. The cells were next pelleted and resuspended in 0.5 ml of OptiMEM 1 (Hyclone). A total of 5×106 cells was added to a 0.4-cm  electroporation cuvette in a final volume of 400 µl of OptiMEM 1. Twenty micrograms of DNA, containing an equimolar mixture of the eight plasmids in a volume of no more than 25 µl, were next added to the cells in the cuvette. The DNA and cells were mixed gently by tapping and electroporated at 300 V and 950 µF in a Bio-Rad Gene Pulser II. The time constant was in the range 28–35 sec. The contents of the cuvette were again gently mixed by tapping and, 2–3 min after electroporation, 0.7 ml of MEM with 10% (v/v) FBS was added. The cells were again gently mixed by serial pipetting and divided between two wells of a six-well dish containing 2-ml MEM with 10% (v/v) FBS per well. Culture at 33°C followed. The medium was removed on each following day and stored. At 72 h post-electroporation, the final culture supernatants were harvested. Recovered virus was amplified in embryonated chicken eggs and stored at –80°C. Clarified supernatant (0.2-ml amounts) was injected into the allantoic cavities of 10 day-old embryonated chicken eggs. After 48 h of incubation at 35°C, allantoic fluids were harvested and tested for hemagglutination activity. The desired reassortant H7N9/PR8 virus was identified in allantoic fluid from the second passage in eggs.To obtain the reassortant H7N9/PR8 virus, six plasmid DNAs encoding internal proteins of PR8 were combined with the two plasmids encoding the surface antigens HA and NA of A/AnHui/1/2013. One day prior to electroporation, Vero cells at 90–100% confluence were split and seeded at a density of 9×10Eight 4-week-old White Rock chickens received intravenous injections (i.v) of 0.2 ml of 1∶10 dilution of stock virus. Oropharyngeal and cloacal swabs were collected at 3 days post-infection, and we did assays for the presence of virus by injection of 0.1 ml into all of four 10-day-old embryonated chicken eggs. Haemagglutination activity in the allantoic fluid of these eggs was evaluated after incubation at 35°C for 48 hours. On day 14 p.i., all surviving chickens were bled and sera were tested for evidence of sero-conversion by agar gel precipitin (AGP) and HAI assay.50 values of the H7N9/PR8 and PR8 transfectant viruses were determined by intranasal inoculation of groups of wild-type 6-week-old female BALB/c mice . Serial 10-fold dilutions of the viruses were administered to mice anesthetized with pentobarbital sodium. The mice were monitored daily for signs of disease to 14 days post infection. To measure viral replication in various tissues, groups of mice were intranasally inoculated with 106 EID50 of the reassortant H7N9/PR8 vaccine, or wt virus, in volumes of 20 µl, respectively. On day 3 post-infection, animals were sacrificed and various tissues harvested and homogenized in MEM medium to 10% (w/v). Virus in these homogenates was titrated in Madin-Darby canine kidney (MDCK) cells, as described elsewhere.The MLDThe monovalent influenza A (H7N9) split virus vaccine was developed by the HengYe Biological Company, and the seed virus was prepared from the reassortant vaccine H7N9/PR8 virus. The vaccine was prepared in 10-day-old embryonated chicken eggs using techniques identical to those employed in current production of the trivalent inactivated vaccine against seasonal influenza. In brief, virus was harvested from egg cultures and inactivated with formaldehyde. The virus was concentrated, purified, and further sterilized by chromatography (on a Sepharose 4FF column) in a buffer containing Triton X-100. The monovalent influenza A (H7N9) split vaccine was produced on a pilot scale using Good Manufacturing Practices. The vaccine contained all proteins of the viral particle, and the major component was HA . The four experimental vaccines produced were split virus products containing 7.5-, 15-, 30-, and 45-µg HA, and were prepared using WHO-approved protocols Groups of six-week-old BALB/c mice (20 per group) and ten-week-old ferrets  (8 per group) were immunized intramuscularly with two doses  of vaccine containing 7.5, 15, 30, or 45 µg of HA, at 14-d intervals, and sera were collected 2 weeks after priming and 2, 4, and 8 weeks after boosting. The serum titers of hemagglutination-inhibition (HI) antibodies were measured using standard methods; 4 HA units of the two viruses were mixed, in the wells of 96-well plates, with 0.5% (v/v) turkey erythrocytes. Plates used to detect H7N9 influenza-specific IgG, IgG1, and IgG2a in sera were coated using a solution of 5 µg/ml inactivated influenza virions. Samples were serially diluted in fivefold increase in PBS containing 1% (w/v) bovine serum albumin (Serva) and added to coated plates. Bound antibodies were detected using 1∶10,000 dilutions of goat anti-mouse IgG, IgG1, and IgG2a (Sigma), conjugated to horseradish peroxidase (HRP). Plates were stained with the TMB substrate (Sigma) and OD values read at 450 nm. Each sample was assayed in duplicate.3 TCID50 pfu; equivalent to 4.0×LD50). Prior to virus instillation, mice were anesthetized with pentobarbital sodium. After challenge, all mice were observed for signs of illness and weighed daily over the next 14 days. Mice identified as moribund or who lost >25% of body weight were considered to have attained an experimental endpoint and were humanely euthanized. On day 3 post-challenge, mice (five per group) were euthanized and lung tissue collected for virus titration . Mice were humanely euthanized via cervical dislocation and carefully observed and palpated to verify the absence of respiration and heartbeat.Two weeks after the second dose of the H7N9 split vaccine, 15 mice in each group were challenged intranasally with (wt) AnHui H7N9 virus . After two passages, the rescued virus was fully sequenced and was identical as expected.10EID50/ml in eggs. The H7N9/PR8 and PR8 transfectant viruses could form plaques on MDCK cells in the presence of 1 µg/ml TPCK trypsin (Sigma), whereas the wt H7N9 virus formed plaques with similar efficiency in either the presence or absence of TPCK-trypsin (data not shown). Recombinant H7N9/PR8 viral particles were examined by electron microscopy, which revealed spherical structures approximately 80–120 nm in diameter, with the characteristic lipid membrane bilayer on the outer surface  but the H7N9/PR8 transfectant virus was not lethal (MLD50>6.0 log10 EID50). The H7N9/PR8 virus replicated to lower titers than did the wt H7N9 or PR8 transfectant virus in lung tissues of mice. Virus replication was undetectable in the brains, spleens, liver and kidneys of mice when administrated with H7N9/PR8, wt H7N9 Anhui01 virus or PR8-transfectant viruses. These data indicated that the reassortant H7N9/PR8 vaccine virus was attenuated for mice.To explore the pathogenicity of the viral vaccine, we compared the properties of the H7N9/PR8 vaccine and (wt) AnHui virus in mice. The influenza PR8 virus is a highly passaged laboratory strain that is known to be lethal for mice. As shown in H2-type cellular immune response in BALB/c mice.The immunogenicity of the reassortant H7N9/PR8 virus was assessed in mice 2 weeks after the first immunization with the H7N9/PR8 split vaccine. Five groups of BALB/c mice were intramuscularly immunized at weeks 0 and 2, with vaccine containing 7.5, 15, 30, or 45 µg of the HA of the H7N9/PR8 split vaccine. By 14 days after the second vaccination, the titers of HI antibodies had increased sharply in each group. The HI titers against Anhui virus increased in a dose-dependent manner on 2, 4, and 8 weeks after administration of the second of two doses of vaccine . SimilarAs was also observed in mice, HI antibody responses were evident in all ferrets at 2, 4, and 8 weeks after two vaccinations. As was seen in 3 TCID50  of live AnHui H7N9 virus. Mice receiving 15, 30, and 45 µg of vaccine HA were highly resistant to challenge with live AnHui virus; all mice survived for more than 14 days after  lethal challenge and body weight recovered quickly . In contrast, the survival rate of the 7.5-µg HA vaccinated group was only 70%  were euthanized and tissues were collected for virus titration; the remaining mice of each group were observed for a further 2 weeks. As shown in The rapid development of potential candidate vaccines against the 2013 influenza A (H7N9) outbreak began when the potential for disaster was apparent. It remains possible that a pandemic may arise this winter and rapid production of an appropriate vaccine may be needed to protect against H7N9 outbreaks even in 2014 8.5 EID50/ml). The vaccine is not pathogenic for chickens, mice, or ferrets, and failed to replicate in the brain, spleen, liver, or kidney of infected animals. The level of replication of the reassortant H7N9/PR8 virus in the lungs of mice was lower than those of the PR8 or (wt) Anhui virus. A monovalent inactivated split vaccine prepared from the H7N9/PR8 virus was immunogenic and protected mice from subsequent  lethal live AnHui virus challenge. Taken together, our findings demonstrate that reverse genetics is compatible with putting H7N9 HA and NA genes on a PR8 background for vaccine production and warrant human testing of the H7N9/PR8 virus as a potential candidate vaccine in a clinical context. Of course, additional research into the immunogenicity and protective efficacy of the H7N9/PR8 virus is required in ferrets, monkeys, and humans.In this paper, we report our successful efforts to develop an H7N9 vaccine that resembles the currently licensed seasonal influenza A vaccines. The H7N9/PR8 virus has many characteristics that are desirable in an H7N9 vaccine candidate. The absence of pathogenicity and infectivity for chickens should allow manufacture of an egg-based vaccine. Herein, we investigated the properties of the H7N9/PR8 candidate vaccine in chickens, mice and ferrets. Our candidate vaccine virus grows efficiently in eggs (to 10The influenza H7N9 virus-like particle (VLP) vaccine, consisting of full-length, unmodified hemagglutinin (HA) and neuraminidase (NA) from A/AnHui/1/2013, and the matrix 1 (M1) protein from A/Indonesia/05/2005 (H5N1), was immunogenic and protected mice from lethal challenge with wt virus Influenza infection in ferrets has several features resembling human infection in terms of clinical signs and immunity, rendering the ferret an appropriate model in which to assess the efficacy of influenza vaccines It is difficult to predict the influenza subtype that may trigger a pandemic. Thus, it is important to be able to prepare influenza virus vaccines in a short period of time and to evaluate safety and immunogenicity in clinical studies prior to development of a pandemic. Overall, our results indicate that we have the technical expertise and virus stockpiles enabling us to respond rapidly to outbreaks via production of a safe candidate influenza vaccine virus. Of course, much remains to be done before such viruses can be used to protect humans against pandemic influenza. We believe that our work on the research and development of the 2013 influenza A (H7N9) candidate vaccine virus shows that is both challenging and possible to respond to the threat of an influenza pandemic when major efforts are made by vaccine manufacturers and clinical trial organizations.