Human immunodeficiency virus type 1 (HIV-1) genetic diversity poses a challenge to reliable viral load monitoring. Discrepancies between different testing platforms have been observed, especially for non-clade-B virus. Therefore we compare, in antiretroviral therapy (ART)-naïve South African subjects predominantly infected with HIV-1 clade-C, three commercially available assays: the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test version 2.0 by Roche (CAP/CTM v2.0), the BioMérieux NucliSens Version 2.0 Easy Q/Easy Mag (NucliSens v2.0) and the Roche COBAS Amplicor HIV-1 Monitor Test Version 1.5 (Amplicor v1.5). Strong linear correlation was observed and Bland-Altman analyses showed overall good agreement between the assays with mean viral load differences of 0.078 log cp/ml (NucliSens v2.0 – Amplicor v1.5), 0.260 log cp/ml (CAP/CTM v2.0 – Amplicor v1.5) and 0.164 log cp/ml (CAP/CTM v2.0 – NucliSens v2.0), indicating lower mean viral load results for the Amplicor v1.5 and higher mean readings for the CAP/CTM v2.0. Consistent with observations following previous comparisons of CAP/CTM v2.0 versus Amplicor v1.5, the CAP/CTM v2.0 assay detected low-level viremia (median 65 cp/ml) in more than one-third of those in whom viremia had been undetectable (<20 cp/ml) in assays using the NucliSens platform. These levels of viremia are of uncertain clinical significance but may be of importance in early detection of ART resistance in those on treatment. Overall the three assays showed good comparability of results but with consistent, albeit relatively small, discrepancies for HIV-1 clade-C samples, especially in the low-viremic range that should be taken into account when interpreting viral load data. In clinical care the plasma HIV RNA viral load is widely used as a marker of disease progression Approximately 50% of all HIV-1-infections are caused by HIV-1 group M clade-C globally, whereas clade-A and clade-B HIV-infections as the second and third most prevalent subtypes account for only about 10% each To address this gap, we performed a comparison between three different commercial testing platforms for HIV-1 viral load monitoring using predominantly clade-C samples from South Africa, the country with more HIV infections than any other. We conducted a direct comparison between the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test version 2.0 by Roche (CAP/CTM v2.0), the BioMérieux NucliSens Version 2.0 Easy Q/Easy Mag (NucliSens v2.0) and the Roche COBAS Amplicor HIV-1 Monitor Test Version 1.5 (Amplicor v1.5) for HIV-1 RNA quantification in blood plasma from a cohort of HIV-1 clade-C-infected patients.3, median VL 6,100 cp/ml), we had noted an unexpectedly high proportion with low viral loads using the NucliSens v2.0. We therefore selected a subset (n = 44), including samples selected to represent the range of viral loads observed, in order to determine whether, and if so to what degree, the NucliSens v2.0 was reading lower than the CAP/CTM v2.0. In a selected subset of 13 of these subjects, whose viral loads were low (<1000 cp/ml) using the NucliSens v2.0, samples were studied from two separate visits to the clinic in order to increase the power of the study for comparison in the low viremic range. Plasma was separated from EDTA whole blood and stored at −80°C until testing. Viral load measurements were performed on frozen samples without previous freeze-thaw cycles to ensure consistent sample viability for these two assays.In a cohort of 220 asymptomatic, HAART-naïve post-natal women attending a pediatric immunization clinic at the Prince Mshiyeni Memorial Hospital in Umlazi, Durban, South Africa between 2012–2013  recruited from Prince Mshiyeni Memorial Hospital and St Mary's Hospital in Durban, South Africa. In this cohort, viral loads had originally been determined in 2002–2005 from fresh EDTA plasma using the Amplicor v1.5 assay.To compare these two currently available assays with the Amplicor v1.5 assay previously widely used, we selected a representative subset of samples (n = 38) that were collected between 2002–2005 from a similar cohort of antenatal women  compliant laboratory accredited through the South African National Accreditation System (SANAS).The CAP/CTM v2.0 uses the fully automated COBAS AmpliPrep Instrument for specimen processing and the COBAS TaqMan Analyzer for amplification and detection with a reported linear range of 20–10,000,000 cp/ml. The sample input was 850 µl EDTA-plasma for this study.The NucliSens v2.0 uses a p24 Gag targeted nucleic acid sequence-based amplification (NASBA) associated with molecular beacon probes for detection of amplified nucleic acid products. RNA was purified from 100 ul patient EDTA plasma using the fully automated EasyMag extractor. Subsequently isothermic cDNA amplification and detection was performed on the automated EasyQ instrument with a dynamic range of 100–10,000,000 cp/ml with a detection limit of 20 cp/ml.gag gene. It was used as the standard (not the ultra-sensitive) assay with a linear range of 400–750,000 cp/ml using 200 ul of EDTA-plasma.The Amplicor v1.5 is an end-point RT-PCR assay targeting a consensus region in the 1–a2) was plotted against the average of the two measurements ((a1–a2)/2). The bias between the assays was calculated as the mean m of the difference between the two measurements (a1–a2) and its standard deviation (s) was calculated. The 95% limits of agreement between the assays were determined as m±1.96 s.All results were converted to cp/ml and transformed to log cp/ml for further statistical analysis using GraphPad Prism v6.0. Samples with viral loads below the detection limit of the assay were assigned the value of the lower limit of detection, i.e. 20 cp/ml (log cp/ml = 1.30) for NucliSens v2.0 and CAP/CTM v2.0. To determine the linear relationship between the assays the Spearman correlation coefficient (r) was calculated. Bland-Altman analysis was used to assess the agreement between the different methods of viral load measurement For some samples viral load measurements were only available for 2 out of the 3 platforms being compared, hence the number of analyzed samples varies between the different comparisons. Data were available for n = 35 samples for the comparison between Amplicor v1.5/NucliSens v2.0, n = 34 for Amplicor v1.5/CAP/CTM v2.0 and n = 88 for the CAP/CTM v2.0/NucliSens v2.0 comparison as shown in The mean log viral load result for the n = 87 samples used for this comparison was 3.420 log cp/ml (3.107–3.733 log cp/ml) for the NucliSens v2.0 and 3.584 log cp/ml (3.283–3.886 cp/ml) for the CAP/CTM v2.0. In this dataset NucliSens v2.0 and CAP/CTM v2.0 showed a significant linear correlation  for the NucliSens v2.0 and 4.509 cp/ml (4.201–4.817 log cp/ml) for the Amplicor v1.5. The assays showed significant linear correlation within the range of tested samples  indicating slightly higher measurements for the NucliSens v2.0 compared to the Amplicor v1.5 . The 95%Mean viral load results for the n = 34 samples analyzed for this comparison are 4.702 log cp/ml (4.451–4953 log cp/ml) for the CAP/CTM v2.0 and 4.441 log cp/ml (4.120–4.763 log cp/ml) for the Amplicor v1.5. Linear correlation across the analyzed log VL range that was within the quantitative limits of the two assays was strong .In the Bland-Altman model the mean difference between the two assays was m = 0.2603 log cp/ml (s = 0.4783), reading higher on the CAP/CTM v2.0 with 95% limits of agreement of -0.6771 log cp/ml and 1.198 log cp/ml. Three of the 4 outliers (12% of samples) are in the lower log VL range and read higher on the CAP/CTM v2.0.The reliable quantification of HIV-1 RNA viral load is a critical tool in HIV clinical care and research Earlier studies evaluating the previous versions of the NucliSens assays, namely the NucliSens QT and its successors the NucliSens EasyQ v1.1 and v1.2, have shown underestimation of viral loads in non-clade-B samples More recent studies evaluated the new version of the BioMerieux assay, the NucliSens v2.0. A study in China found improved performance in measuring non-B-clade samples gag and LTR regions to improve coverage of HIV-1 sequence polymorphisms The need to adequately detect diverse HIV-subtypes has been appreciated and manufacturers are aiming to improve subtype inclusivity of their assays constantly. The latest version of the Roche platform, the CAP/CTM v2.0, therefore uses two dual-labeled hybridization probes that target both the We therefore performed this study in a South African cohort of HIV-1 clade-C infected subjects to compare recent versions of three widely used testing platforms, the CAP/CTM v2.0, NucliSens v2.0 and the Amplicor v1.5.Our direct comparison between the NucliSens v2.0 and the Amplicor v1.5 has shown overall strong linear correlation  and agreement  with slightly higher readings on the NucliSens platform especially for samples in the range of 3–5 log cp/ml. The CAP/CTM v2.0 measured consistently higher than the Amplicor v1.5 with a bias of m = 0.2603 log cp/ml which is consistent with other studies We also observed higher VL measurements for the CAP/CTM v2.0 compared with the NucliSens v2.0 with a mean difference of m = 0.1642 log cp/ml with strong linear correlation . This bias for clade-C virus between these two assays is lower than the difference that was observed in a study testing clade-B′, BC and AE samples from China measuring a mean difference of 0.588 log IU/ml Further factors to consider include the volume of sample required for the respective assays and costs. For this study we have used the minimum required sample input volume of 100 µl plasma for the NucliSens assay whereas 850 µl were used for the CAP/CTM v2.0. Lower sample volume requirements may be of particular interest in relation to pediatric studies. However, using the minimum volume for the NucliSens assay might compromise the sensitivity of the assay in the low-viremic range as stated by the manufacturer. At current prices the cost of the NucliSens assay is substantially lower than that of the CAP/CTM v2.0, a factor which is always of considerable relevance, but especially so in resource limited settings. It remains open to discussion whether the failure to detect low level viremia in a minority of those samples measuring <20 cp/ml on the NucliSens is of clinical significance and justifies increased costs of using the CAP/CTM v2.0 assay. Indeed, the introduction of novel more sensitive assays has led to an increase in low-positive viral load results of patients on antiretroviral therapy that has raised the question of which clinically relevant threshold should be used as an end-point for treatment efficacy It is important to note the limitations of this study. First, for the comparisons between the Amplicor v1.5 versus the CAP/CTM v2.0 and the NucliSens v2.0, the numbers studied in this dataset were restricted by sample availability, and thus those analyses were underpowered to draw strong conclusions. Second, none of the samples included in the comparisons with the Amplicor v1.5 had a viral load below 3 log cp/ml, and therefore this did not allow the performance of the Amplicor v1.5 to be assessed in the low viremic range. Third, plasma samples for the comparison with the Amplicor v1.5 were collected between 2002–2005 and run on the Amplicor v1.5 immediately but were stored at −80°C until run on the NucliSens v2.0 and CAP/CTM v2.0. The higher measurements on both of the two recent platforms compared to the Amplicor v1.5 might therefore be an underestimate. Finally, the low sample input volume of 100 µl for the NucliSens v2.0 assay may reduce the sensitivity of that assay, as mentioned above.In conclusion, despite overall good agreement, there are discrepancies between the different testing platforms compared in this study using predominantly HIV-1 clade-C samples, especially in the low-viremic range. These differences have to be taken into account in clinical care, but also when comparing viral load results in clinical trials and other scientific studies. The decision to use one assay or the other, will certainly depend on the performance of the assay. But overall costs and throughput rates should also be considered, especially in resource limited settings.Table S1(DOCX)Click here for additional data file.


In this study, the anti-melanogenesis efficacy of clinically used herbal prescription LASAP-C, which consists of four herbal medicines—Rehmanniae Radix Crudus, Lycii Fructus, Scutellariae Radix, and Angelicae Dahuricae Radix, was investigated.The chemical profile of LASAP-C was established by conducting ultra-performance liquid chromatography-electrospray ionization-mass spectrometry. Anti-melanogenic efficacy was evaluated by tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 expression in B16F10 melanoma cells. In vivo evaluation was performed by using zebrafish model.Molecular evidences suggested that melanin synthesis was inhibited via the down-regulation of tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 expression in B16F10 melanoma cells treated with LASAP-C. The anti-melanogenesis efficacy was also confirmed in vivo by using the zebrafish model.The results of this study provide strong evidences that LASAP-C can be used as an active component in cosmeceutical products for reducing excess pigmentation in the human skin. Melanin, the major pigment of human skin, is secreted by melanocytes in the basal layer of the epidermis. It is known to be overproduced in the cases of chronic sun exposure, melasma, or other hyperpigmentation diseases , 2. Melal-dopa—of melanin synthesis by melanocytes -2, 5 diphenyl tetrazolium bromide (MTT) colorimetric assay. This assay measures the metabolic reduction of MTT to formazan (blue) by mitochondrial dehydrogenase, which is active only in living cells . B16F10 4 cells/well) were seeded in a 6-well culture plate and treated with various concentrations  of LASAP-C for 48 h. They were washed with ice-cold PBS and lysed with phosphate buffer (pH 6.8) containing 1 % Triton X-100. They were then disrupted by freezing and thawing, and whole cell lysates were clarified by centrifugation at 10,000 g for 5 min. After protein levels were quantified and protein concentrations were adjusted using lysis buffer, 90 μL of each lysate, containing the same amounts of protein, was placed in each well of a 96-well plate, and 10 μL of 10 mM l-DOPA was added. Control wells contained 90 μL of lysis buffer and 10 μL of 10 mM l-DOPA. After incubation at 37 °C for 15 min, the dopachrome was observed by measuring the absorbance at 475 nm by using an ELISA reader.B16F10 melanoma cells  of LASAP-C for 48 h. The cells were dissolved in 0.2 mL of 1 N NaOH and boiled at 100 °C for 30 min. The amount of melanin was determined by measuring absorbance at 460 nm by using the ELISA microplate reader and normalizing with protein level of cell lysates.β-mercaptoethanol, protease inhibitor , 2 mM phenylmethylsulfonyl fluoride, 10 mM EDTA, 50 mM NaF, and 1 mM Na3VO4). Next, 10 μg of protein per lane was separated by 8 % SDS-polyacrylamide gel electrophoresis and blotted onto polyvinylidene fluoride membranes, which were then saturated with 5 % non-fat dried milk in Tris-buffered saline containing 0.1 % Tween 20. Blots were exposed to specific primary antibodies at a dilution of 1:1000 overnight at 4 °C, followed by incubation with a horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. The blots were developed using an enhanced chemiluminescence detection system .The effects of LASAP-C on the expression of tyrosinase, TRP-1, and TRP-2 were investigated using western blot analysis. The total amount of protein content of each supernatant was measured using Bradford assay . B16F10 melanoma cells were lysed in cell lysis buffer (62.5 mM Tris–HCl (pH 6.8), 2 % sodium dodecyl sulfate (SDS), 5 % Artemia salina). Embryos were obtained from natural spawning, which was induced in the morning by turning on the light. The embryos were collected within 30 min. Synchronized embryos were placed in 96-well plates containing 200 μL embryo medium . LASAP-C (10 and 100 μg/mL in 0.1 % DMSO) was added to the embryo medium from 9 h post fertilization (hpf) to 72 hpf (63 h exposure). Three embryos were assigned for each well. Further, 0.2 mM 1-phenyl-2-thiourea (PTU) was used to generate transparent zebrafish without interfering the developmental process [The effect of LASAP-C on the pigmentation of zebrafish embryos was determined according to the method described previously . Adult z process ; these zt-test. Student’s t-test and paired t-test were performed to assess the differences between the means. P values less than 0.05 were considered statistically significant.Results are presented as means ± standard error of the mean of at least three independent experiments. Statistical analysis was performed using The standard chromatogram for LASAP-C was established by using the UPLC system. The representative chromatogram of LASAP-C is shown in Fig. C-arabinose-8-C-glucose, chrysin-6-C-glucose-8-C-arabinose, baicalin, wogonin-7-O-glucuronide, oroxylin A-7-O-glucuronide, and baicalenin from Scutellariae Radix. These ten compounds were identified based on the UV and MS spectra of previously reproted. Although Rehmanniae Radix Crudus was the major component of LASAP-C in terms of quantity, phytochemicals from Scutellariae Radix were prominent as major peaks in the UPLC-MS study. The raw material of Rehmanniae Radix Crudus contained upto 77 % of water contents depends on cultivars [The retention time, observed mass, mass difference with theoretical mass, and proposed compounds of ten peaks are listed in Table ultivars  and manyultivars , 18. Theultivars . The higActeoside extracted from Rehmanniae Radix Crudus was reported to inhibit melanogenesis in B16F10 cells by the extracellular signal-regulated kinase (ERK) pathway  even thoThe cytotoxicity of LASAP-C was determined by treating B16F10 cells with LASAP-C  for 2 days and measuring the cell viability (data not shown). The LASAP-C extract did not induce any cytotoxicity up to 100 μg/mL concentration, but dose-dependently reduced viability at higher doses . According to the cell viability results, cells were treated with LASAP-C at a maximum concentration of 100 μg/mL.Tyrosinase is a rate-limiting enzyme in melanin synthesis. The biosynthesis of melanin is initiated by the catalytic oxidation of tyrosine to dopa by tyrosinase in the presence of dopa as a cofactor . TyrosinMelanin is synthesized within melanosomes that contain three major pigment enzymes: tyrosinase, TRP-1, and TRP-2 –26. The Recently, the zebrafish model is being actively used in various in vivo assays as a good alternative system to rodents –29. The In this study, the anti-melanogenic effect of LASAP-C was confirmed, and five major components of this traditional prescription were identified by UPLC-ESI-MS for quality control for further research and development.The anti-melanogenic effect of LASAP-C extract was thought to occur via the inhibition of three key enzymes involved in melanin synthesis. The dose-dependent inhibitory effect of LASAP-C on tyrosinase, TRP-1, and TRP-2 expression indicated that this herbal prescription was directly related with the inactivation of genes encoding these proteins.The whitening effect of LASAP-C was also validated using zebrafish as a whole-animal model for phenotype-based screening of LASAP-C extract. As expected, LASAP-C extract remarkably inhibited body pigmentation in zebrafish embryos, reconfirming its anti-melanogenic efficacy. Taken together, these results suggest that LASAP-C has a strong potential to be used as an active ingredient in various cosmeceutical products for skin whitening.l-DOPA, l-3,4-dihydroxyphenylalanine; PTU, 1-phenyl-2-thiourea; ROS, reactive oxygen species; TRP-1, tyrosinase-related protein-1; TRP-2, tyrosinase-related protein-2; UV, ultravioletDMSO, dimethyl sulfoxide;


Visual processing impairments are now well established in schizophrenia, including abnormalities in: contrast sensitivity  visual processing impairments in schizophrenia; (b) visual processing impairments in at-risk states, and the implications of data on an inverse relationship between congenital blindness and incidence of schizophrenia; and (c) broader theoretical papers. The first section begins with three papers on low-level visual impairments in schizophrenia, including findings on: (1) the interaction of color and contrast sensitivity effects  calTaken together, these papers provide a representative example of current work in the vision science of schizophrenia. Our hope is that this set of papers will be of use to those currently working in this field, and will stimulate others to investigate these issues. Findings addressing these questions would be of major benefit to the field of schizophrenia research, and would also inform the study of normal visual perception.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Neutrophils are highly abundant innate immune cells that contribute to asphyxic episodes of acute asthma exacerbations, and secrete the proinflammatory cytokine tumour necrosis factor-α (TNF). Recycling endosomes (REs) are specialized secretory compartments that perform multiple functions including trafficking of cytokines to cell surfaces, although these are not characterized in neutrophils. Our objective is to identify trafficking components in neutrophils that may contribute to cytokine secretion.The effect of bacterial lipopolysaccharide (LPS) stimulation on the trafficking of stored and newly synthesized TNF was determined by treatment of human peripheral blood neutrophils with or without cycloheximide. To visualize intracellular TNF, neutrophils were adhered to glass slides and treated with LPS for 1 h (10 ng/ml). Colocalization of TNFα in neutrophils was performed with transferrin-Alexa 488 and anti-VAMP3 (markers for RE), anti-CD63 and anti-CD66b a membrane markers for the primary and secondary granules in neutrophils, respectively. We also determined Rab5 and Rab7 colocalization with TNF . Imaging was carried out by Deltavision OMX super resolution microscopy.LPS induced 30-40% TNF secretion from stored sources, with the remainder newly synthesized. We found that neutrophils possess REs as determined by transferrin uptake and VAMP3 labeling. TNF also colocalized with REs, primary and secondary granules as well as early and late endosomes, suggesting multiple sites of TNF storage and trafficking in neutrophils. However, TNF only colocalized with VAMP3 around periphery of cells after 1 h stimulation with LPS, suggesting TLR4-induced TNF trafficking via REs.+VAMP-3+ vesicles towards the cell periphery in response to LPS. This suggests that neutrophils utilize REs for trafficking of TNF to the cell surface in response to TLR4 signalling. These findings contribute to our understanding of how neutrophils package, transport, and release cytokines.The present study provides evidence that movement of TNF


In this study, it was found that Nep1Mo-induced cell death could be inhibited by the virus-induced gene silencing of NbALY916 in N. benthamiana. NbALY916-silenced plants showed impaired Nep1Mo-induced stomatal closure, decreased Nep1Mo-induced production of hydrogen peroxide (H2O2) and nitric oxide (NO) in guard cells, and reduced Nep1Mo-induced resistance against Phytophthora nicotianae. It also found that the deletion of AtALY4, an orthologue of NbALY916 in Arabidopsis thaliana, impaired Nep1Mo-triggered stomatal closure, HCD, and defence-related gene expression. The compromised stomatal closure observed in the NbALY916-silenced plants and AtALY4 mutants was inhibited by the application of H2O2 and sodium nitroprusside (an NO donor), and both Nep1Mo and H2O2 stimulated guard cell NO synthesis. Conversely, NO-induced stomatal closure was found not to require H2O2 synthesis; and NO treatment did not induce H2O2 production in guard cells. Taken together, these results demonstrate that the NbAlY916/AtAlY4–H2O2–NO pathway mediates multiple Nep1Mo-triggered responses, including stomatal closure, HCD, and defence-related gene expression.Previously, it was found that Nep1 Plants have evolved multiple defence mechanisms against phytopathogens, including the hypersensitive response (HR) and stomatal closure . HCD is Necrosis and the secretion of ethylene-inducing peptide 1 (Nep1) occurs in taxonomically diverse organisms, including bacteria, fungi, and oomycetes (Mo (Nep1 from Magnaporthe oryzae)-triggered plant responses, and the MAPK signalling associated with HCD exhibits shared and distinct components with that of stomatal closure (omycetes . In someomycetes . It is bNicotiana benthamiana leaves upon elicitor treatment were identified. The involvement of respiratory burst oxidase homologue (RBOH), vacuolar processing enzyme (VPE), G proteins, and MAPKs in elicitor-triggered plant immunity was demonstrated by Potato virus X (PVX)-based VIGS  and yeast ALY proteins contribute to the export of mRNAs from the nucleus before translation, and join with the exon junction complex of proteins marking mRNAs in the course of splicing (High-throughput down-regulated expression screening of a plant cDNA library was previously performed using virus-induced gene silencing (VIGS), and several genes that suppress cell death in sed VIGS b. Here,NbALY916 is involved in the regulation of Nep1Mo-induced stomatal closure, HCD, and pathogen resistance in N. benthamiana. It is also demonstrated that AtALY4, an orthologue of NbALY916 in A. thaliana, plays the same role in Nep1Mo-triggered stomatal closure, HCD, and defence-related gene expression.Here, by employing VIGS, it was found that Nicotiana benthamiana and Arabidopsis were grown in a growth chamber under a 16/8h light/dark cycle at 25 °C. The T-DNA insertion line for AtALY4 used in this study was AtALY4 (CS331800) supplied by the Arabidopsis Resource Center (http://www.arabidopsis.org). The PCR primers  were used to check the T-DNA insertions. To prepare Nep1Mo, overnight cultures of Escherichia coli BL21 cells carrying pET32b harbouring the Nep1Mo gene (GenBank accession no. MGG_08454) were diluted (1:100) in Luria–Bertani medium containing ampicillin (50mg ml–1) and incubated at 37 °C for ~3h. When the OD600 of the culture reached 0.6, Nep1Mo secretion into the culture medium was induced via the addition of 0.4mM isopropyl-β-d-thiogalactopyranoside for 6h. Nep1Mo was expressed as a His-tag fusion protein. Protein purification was performed with Ni-nitrilotriacetic acid resin , and the purified proteins were dialysed against a phosphate-buffered saline (PBS) buffer (pH 7.4) and stored at 20 °C prior to use. Protein concentration was determined using the Bradford reagent (NbALY916 was identified with RACE (rapid amplification of cDNA ends) using a SMART RACE amplification kit . VIGS for the NbALY916 gene (GenBank accession no. AM167906) in N. benthamiana was performed using PVX, as previously described by NbALY916 insert was 450bp, which was derived from the 3′ termini of its open reading frame and inserted into the PVX vector in the antisense direction to generate PVX.NbALY916. The construct containing the insert was transformed into Agrobacterium tumefaciens strain GV3101. Bacterial suspensions were applied to the undersides of N. benthamiana leaves using a 1ml needleless syringe. Plants exhibited mild mosaic symptoms 3 weeks after inoculation. The third or fourth leaf above the inoculated leaf, where silencing was most consistently established, was used for further analyses.To amplify a cDNA encompassing the entire open reading frame of NbALY916, the full-length cDNA for Mo treatment and immediately vacuum-infiltrated for 20min with PBS (pH 7.4) containing 0.5% (w/v) DAB. The leaves were placed in light for 10h and then boiled for 20min in 80% ethanol. Quantitative scoring of hydrogen peroxide (H2O2) staining in leaves was analysed using Quantity One software .Following the methods of Mo inoculation were used for total RNA extraction using the Trizol reagent  following the manufacturer’s protocol and then treated with RNase-free DNase (TaKaRa). qRT–PCR was performed on the ABI 7300 Real-Time PCR System  and SYBR Premix Ex ™Taq according to the manufacturer’s instructions. The N. benthamiana genes (NbEF1α and NbActin) and Arabidopsis genes (AtEF1α and AtS16) were used as the internal reference genes to standardize the RNA sample for evaluating relative expression levels. For qRT–PCR assays, three independent biological samples were used, with each repetition having three technical replicates with a gene-specific primer (Supplementary Table S1 available at JXB online)The leaf fragments after Nep1NbALY916-silenced plants, Col-0, and AtALY4. Abaxial (lower) epidermises were peeled off and floated in 5mM KCl, 50mM CaCl2, and 10mM MES-TRIS (pH 6.15) in light for 3h to open the stomata fully before experimentation to minimize the effects of other factors in stomatal response, because the mesophyll signals can also significantly influence stomatal behaviour. The epidermal strips then were followed by Nep1Mo (50nM), sodium nitroprusside , and H2O2 (800 μM), respectively, to induce a stomatal response. For cPTIO  treatment, cPTIO (400 μM) was applied for 1h prior to SNP treatment. The maximum diameter of stomata was measured under an optical microscope. At least 50 apertures in each treatment were obtained and the experiments were repeated three times.Stomatal apertures were measured as described by 2, and 10mM MES-TRIS (pH 6.15) in the light for 2h, followed by incubation in 20 μM DAF-2DA for 1h in the dark at 30 °C, 65rpm, and finally rinsed three times with 10mM TRIS-HCl (pH 7.4) to wash off excess fluorophore. The dye-loaded tissues were treated with Nep1Mo (50nM), SNP (25mM), and H2O2 (800 μM) for 1h, respectively. And then the fluorescence of guard cells was imaged  and analysed using Quantity One software.NO accumulation was determined using the fluorophore 4,5-diaminofluorescein diacetate  according to 2DCFDA). Epidermal peels were ﬂoated in 5mM KCl, 50mM CaCl2, and 10mM MES-TRIS (pH 6.15) for 2h under light to induce stomatal opening, followed by incubation with 50 μM H2DCFDA for 10min and washing for 20min with incubation buffer. Images of guard cells were obtained 1h after Nep1Mo treatment under a fluorescence microscope . Fluorescence emission from guard cells was analysed using Quantity One software.According to the method described by NbALY916-silenced N. benthamiana was infiltrated with either PBS (10nM) orNep1Mo (50nM). Three hours later the leaves were collected and transferred to Petri dishes containing sterile water-saturated filter paper. A 9 mm×9mm hyphal plug of Phytophthora nicotianae was then placed on the surface of the left side of each leaf, which had not been infiltrated with Nep1Mo or PBS. Samples were kept in the dark at 25 °C. Pictures of the lesions were taken at 48h post-inoculation, Leaves then were fixed with 100% ethanol. Resistance evaluation was based on the measurement of the diameter of P. nicotianae lesion size: inhibition=(diameter of control–diameter of elicitor)/diameter of control 100%. Data are means ±SE from three experiments.One half (right side) of a leaf from PVX Nb and ALY4 mutant leaves collected 3h after Nep1Mo treatment (25 μl) were infiltrated with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) suspension (108 cfu ml–1). Necrotic lesions caused by Pst DC3000 on leaves were observed. Leaves of inoculated plants were harvested and sterilized in 70% ethanol, and homogenized in sterile water. Bacteria were recovered on L-agar medium and induced resistance was deﬁned based on decreases in symptom severity and in planta bacterial numbers . Each clone was used to infect two N. benthamiana plants. A common positive control is silencing of the phytoene desaturase (PDS) gene, which results in photobleaching of the silenced regions and is a readily visible phenotype. When photobleaching was apparent, two leaves of candidate plants were infiltrated with Nep1Mo solution. Several cDNAs, including PVX clone #14-4-4, were found to compromise Nep1Mo-triggered cell death upon silencing. Since the silencing of #14-4-4 produced the strongest and most reproducible inhibition of cell death triggered by Nep1Mo, the #14-4-4-silenced line was therefore chosen for further study. The DNA sequence of #14-4-4 showed strong similarity to the C-terminal domain of ALY916 (GenBank accession no. AM167906) from N. benthamiana. To facilitate a more comprehensive comparative analysis of NbALY916, the full-length cDNA for NbALY916 was isolated from N. benthamiana. Three clones of the full-length cDNA, named NbALY916, were sequenced to confirm that the correct gene had been cloned. The predicted protein for NbALY916 is composed of 275 amino acids. The most closely related protein is AtALY4 from A. thaliana . N. benthamiana database (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi), and that it shared 100% identity with stretches of only 10 or fewer nucleotides in NbALY617, NbALY1693, and NbALY615 (three homologues of NbALY916 in N. benthamiana). To test whether the VIGS of NbALY916 could affect the expression of the other NbALY genes, their transcriptional level was examined by qRT–PCR using primers that specifically annealed to these gene sequences. The results revealed that the expression of these genes was unaffected in the NbALY916-silenced plants . These results indicate a high degree of target specificity using VIGS. It was also observed that the Nep1Mo-triggered HR was not suppressed in NbALY617-, NbALY1693-, and NbALY615-silenced plants. These data demonstrate that the observed inhibition of cell death was due to the specific silencing of NbALY916.Reduced expression of h Nep1Mo . To confNbALY916 is closely related to an A. thaliana gene, AtALY4. To determine whether mutations in the AtALY4 gene affect Nep1Mo-induced cell death, homozygous lines containing T-DNA insertions in this gene were obtained from the Salk Institute Genomic Analysis Laboratory collection by PCR screen. The T-DNA in the AtALY4 mutant is inserted into the fifth exon of AtALY4. In the allele, wild-type AtALY4 transcripts were did not detected by qRT–PCR (Supplementary S2 at JXB online). As expected, Col-0 seedlings showed obvious cell death after treatment with Nep1Mo. Conversely, the AtALY4 mutant showed hyposensitivity to Nep1Mo by displaying no cell death  was decreased significantly in NbALY916-silenced and AtALY4 mutant lines compared with control plants . These data demonstrate that ALYs regulate H2O2 accumulation in response to Nep1Mo. These results suggest that the compromised cell death observed in NbALY916-silenced plants and the AtALY4 mutant may be associated with a Nep1Mo-induced decrease in H2O2.To test whether the alteration in Nep1reatment . N. bentsistance . The expRBOH-, VPE-, and G protein-dependent stomatal closure in N. benthamiana leaves  treatment. An ~90% increase in NO occurred in the guard cells of control plants following Nep1Mo treatment. However, no increase in NO was observed in the guard cells of NbALY916-silenced plants after Nep1Mo treatment  also decreased sharply in the NbALY916-silenced and AtALY4 mutant lines compared with control plants . This suggests that NR may be the main resource of NO production in Nep1Mo signalling. Taken together, these results indicate that ALY proteins are required for Nep1Mo-mediated NO production in guard cells.NO functions as a signal in plant disease resistance  on Nep1Mo-, H2O2-, and NO-induced stomatal closure were assessed in NbALY916-silenced plants or AtALY4 mutant lines. Nep1Mo-induced stomatal closure was greatly reduced in the presence of cPTIO, in agreement with previous reports , indicating a requirement for NO in Nep1Mo-induced stomatal closure and that NO does not require H2O2 generation to initiate stomatal closure.In addition to NO, H2O2-induced stomatal closure was further examined by monitoring NO synthesis in response to applied H2O2. Epidermal fragments were loaded with DAF-2DA to test for changes in NO-induced fluorescence. A significant increase in NO-induced guard cell fluorescence was observed in H2O2-treated epidermal fragments compared with control tissue (P<0.001), demonstrating H2O2-mediated NO production in guard cells .The role of NO in Hrd cells . ImportaP<0.001; . Moreove2O2 is not required for NO-induced stomatal closure. However, Vicia faba guard cells, exogenous NO, applied in the form of the NO donor SNP, did induce H2O2 production. Consequently, the effects of SNP on H2O2 generation in A. thaliana guard cells were analysed using the fluorescent probe H2DCFDA . Expanding disease lesions around the inoculated spots were observed in the silenced plants at 48 hpi. However, Nep1Mo treatment significantly inhibited the expansion of lesions in PVX control-inoculated leaves of N. benthamiana  with Pst DC3000 using a syringe. As expected, Col-0 showed water-soaked necrotic lesions accompanied by chlorosis, while the AtALY4 mutant plants exhibited accelerated necrotic lesions without visible chlorosis upon PBS treatment. Interestingly, when the growth of Pst DC3000 was monitored at 0, 1, 2, and 4 d post-infiltration, the bacterial population on the AtALY4 mutant was significantly increased compared with that on the inoculated control plants in response to Nep1Mo . The jasmonic acid signalling gene LOX encodes a lipoxygenase  in A. thaliana resulted in the inhibition of Nep1Mo-mediated cell death and stomatal closure. The inoculation of NbALY916-silenced N. benthamiana with P. nicotianae induced accelerated necrotic lesions upon Nep1Mo treatment. Similar to NbALY916-silenced N. benthamiana, inoculation with Pst DC3000 of the AtALY4 mutant line induced accelerated necrotic lesions in response to Nep1Mo. Moreover, it was found that NO acts downstream of ALY and H2O2 to participate in Nep1Mo signalling. Based on the phenotypes observed, a model was developed that involves ALY, H2O2, and NO in Nep1Mo-mediated immunity -induced stomatal closure in peas -responsive (LOX) and ethylene-responsive (ERF) gene expression in N. benthamiana and A. thaliana supports the notion that ALYs are involved in SA and ethylene-mediated signalling pathways during pathogen infection. Analyses of NbALY916-silenced and AtALY4 mutant plants revealed that plant disease resistance is conferred by ALY gene expression during pathogen infection.Mo signalling. Although the original intention of the study was to identify genes involved in Nep1Mo-induced HR, a gene, NbALY916, that when silenced inhibited cell death and stomatal closure upon Nep1Mo application was identified. Although the precise role of NbALY916 in the Nep1Mo signalling pathway is debatable and requires confirmation, the results present a new role for ALYs in P. nicotianae and bacterial disease development.In conclusion, a VIGS-based forward genetic screen was developed for the identification of new targets involved in Nep1JXB online.Supplementary data are available at Fig. S1 Screening of Nicotiana benthamiana gene coding hypersensitive cell death induced by Nep1Mo by virus-induced gene silencing.Fig. S2 Expression analysis of NbALY916 and AtALY4 in NbALY916-silenced plants and the AtALY4 mutant by qRT–PCR.Fig. S3 Local induction of hypersensitivity responses on PVX-, PVX.NbALY615-, PVX.NbALY617-, and PVX.NbALY1693-infected N. benthamiana leaves in response to Nep1Mo.Fig. S4 Expression analysis of genes associated with redox control in NbALY916-silenced plants and the AtALY4 mutant by qRT–PCR.Table S1. Gene-specific primers for qRT–PCR.


Pichia pastoris) and its evaluation as vaccine candidate. The gene encoding structural polyprotein of CHIKV from a recent epidemic strain was cloned into yeast expression system. The multicopy integrants were processed for expression of CHIK-VLPs. The VLPs were purified and confirmed through electron microscopic analysis for their morphological identity with CHIKV. The in vitro and in vivo evaluation of CHIK-VLPs as vaccine candidate was determined in Balb/c mice. Induction of both humoral and cellular immune response was observed with different doses of CHIK-VLPs. The humoral immune response was studied through different techniques like enzyme linked immunosorbent assay, IgG Isotyping and plaque reduction neutralization test. CHIK-VLPs were found to elicit high titer of antibodies that are able to recognize native CHIKV. Higher level of IgG2a and IgG1 subtypes was identified suggestive of balanced Th1/Th2 response. Both in vitro and in vivo neutralization activity of CHIK-VLPs antibodies was observed even with low concentration, which shows its high specificity and neutralizing activity against two different CHIKV strains. Neonatal mice receiving anti-CHIK-VLPs antibodies were protected from CHIKV challenge. Induction of cellular immune response was confirmed through higher level of TNF-α, IL-10 and substantial level of IL-2, IL-4 and IFN-γ indicating a balanced response. This is the first report, where CHIK-VLPs has been expressed by Pichia pastoris and evaluated for neutralizing activity against CHIKV. These promising results indicate the utility of CHIK-VLPs as a promising vaccine candidate against emerging CHIKV.Chikungunya virus (CHIKV) has emerged as a global health concern due to its recent spread in both old and new world. So far, no CHIKV specific drug or vaccine is licensed for human use. In this study, we report production of Chikungunya virus like particles (CHIK-VLPs) using novel yeast expression system ( Pichia pastoris) and its evaluation as vaccine candidate. These CHIK-VLPs share morphological identity to native CHIKV. The results indicate that CHIK-VLPs induced both cell mediated as well as humoral response in a balanced manner, which fulfils its criteria as a potent immunogen. Further, antibodies generated against CHIK-VLPs demonstrated efficient in vitro and in vivo neutralization activity, as evaluated through plaque reduction in Vero cells and protection in CHIKV infected neonatal mice respectively using two different CHIKV strains, which makes it a promising vaccine candidate. The yeast expressed CHIK-VLPs has high potential for development of an effective vaccine candidate against CHIKV during epidemic situations.Chikungunya virus (CHIKV) has emerged in many parts of tropics in last decade. The absence of an approved vaccine or antiviral drug for CHIKV makes it one of the important public health challenges. Though attempt to develop a CHIKV vaccine was initiated in 1980s, however it has not succeeded so far. The Virus like particles (VLPs) are now explored as promising vaccine candidate against many viruses viz. HBV, HPV etc. In this study, we report the production of CHIK-VLPs using novel yeast expression system ( Togaviridae and genus Alphavirus. The word Chikungunya comes from Makonde language, meaning “which bends up”, relating to the patients having contoured posture [Chikungunya virus (CHIKV) is an emerging mosquito transmitted arbovirus of immense public health significance. It belongs to family  posture . After i posture , 3.CHIKV is a spherical, enveloped, positive-strand RNA virus of about 60–70 nm. The RNA is enclosed in a ~ 40 nm nucleocapsid that is enveloped by a host derived lipid bilayer supporting viral trimeric glycoprotein-spikes, CHIKV genome is 11,805 nucleotides long. It contains two open reading frames (ORF) encoding the non-structural polyprotein (nsP1-nsP4) and the structural proteins  [Aedes aegypti and Ae. albopictus mosquitoes. The vector control measures that can play important role in control of infection, have so far proved unsuccessful [CHIK-fever caused by CHIKV is a febrile illness associated with rash and severe polyarthralgia that may persist for years. Polyarthralgia, particularly of small joints are the typical symptoms associated with CHIKV , 7. The ccessful .Vaccination has the potential of protecting humans and limiting transmission of CHIKV. Currently there are no licensed antiviral or vaccine available commercially. Since 1960 various efforts has been made to develop a vaccine against CHIKV. These include inactivated vaccine, live-attenuated virus vaccines, DNA vaccines, chimeric virus vaccines, subunit protein vaccines and a virus-like particle (VLP) based vaccines. Among these, majority of vaccine candidates are at preclinical stage and/or phase I trial [Conventional inactivated and live attenuated vaccines have been demonstrated to be immunogenic in humans. However requirement of large quantity of virus and BSL-3 containment for vaccine manufacturing are the main hurdles. Though live-attenuated vaccine elicits effective balanced immune response, however, there is a concern of reversion to virulence . A live-The virus like particles (VLPs) are non-infectious, nano sized caged architecture composed of viral structural proteins. The presence of complete structural proteins makes it an excellent antigen that strongly mimics the native virus but lack the viral genome making it a safer vaccine candidate. VLPs based CHIKV vaccine candidate produced in mammalian and insect expression system induced an effective immune response even at lower antigen doses , 19. TheP. pastoris cells readily grow in suspension cultures, which is a benefit of the yeast expression system. Pichia expression system is an attractive alternate platform for production of VLPs due to its several advantages including production of protein in native conformation, cheaper operating costs, simple chemical media and free from virus contamination [P. Pastoris derived VLP-based vaccines have been approved by US FDA and commercially available worldwide against hepatitis B virus. In India, VLPs based vaccines against HBSAg, derived from P. pastoris are commercially available [P. pastoris derived Dengue VLPs [mination  Further,mination . The firvailable . Other Pgue VLPs , HPV-16 gue VLPs  and Norogue VLPs  are showP. pastoris expression system to develop CHIK-VLPs based vaccine candidate and evaluated its immunogenicity in a mice model.With these advantages, in the present study, we first time exploited 2 humidified incubator, at 37°C.Chikungunya virus (DRDE07) (GenBank Accession number EU372006) having E1:226V belonging to East Central South African (ECSA) genotype isolated from an infected human during 2007 outbreak in Kerala, India and maintained in Division of Virology, DRDE, Gwalior was used as reference standard in this study. Another CHIKV isolate (DRDE06) (GenBank Acc No. EF210157) having E1:226A was used for comparative neutralization study along with DRDE07. All the live virus experiments were performed at BSL-3 facility, DRDE, Gwalior. Vero cell lines were obtained from National Centre for Cell Science (NCCS) Pune, India and were used for virus maintenance, propagation and titration. Vero cells were maintained in Eagles Minimum essential medium (EMEM), supplemented with 10% (v/v) fetal bovine serum (FBS), in a 5% COin vivo neutralization studies. All the animals were obtained from Animal facility, DRDE, Gwalior. The animal experiments had approval from the Institutional Animal Ethics Committee (IAEC) approved by Defence Research Development & Establishment (DRDE), India vide registration number 37/1999/CPCSEA , Government of India and adopted by the Institutional Biosafety Committee (IBSC). Animals were maintained in accordance with CPCSEA, Govt. of India. The food and water was provided ad libitum. After performing the experiment, all animals were euthanized by anesthesia with CO2. The animal studies were conducted in a BSL-3 containment facility, following the standard operating procedures for the facility.Four weeks old Balb/c mice were used for immunization purpose and neonatal Balb/c mice (2 day old) were used for P. pastoris GS115  was used as the host strain for expression of the Chikungunya VLPs. P. pastoris GS115  was grown at 28°C in Yeast Extract Peptone Dextrose (YPD) Medium. 2% agar was added to the media for plate culture. Transformants were screened in media supplemented with 500 μg/ml Geneticin . E. coli DH5α were used in cloning experiments and were grown at 37°C in LB medium supplemented with 100 μg/ml kanamycin.The DNA polymerase, restriction enzymes, and T4 DNA ligase were procured from Fermentas (USA). Synthetic oligonucleotides and chemicals were procured from Sigma-Aldrich (USA). The yeast transfer vector pPIC9K was from Invitrogen (USA). The TACGTAATG GAG TTC ATC CCA ACC C 3’ (SnaBI) and CHIKVLPMRev: 5’ CAC GTG ACC TCG AGC CCT TCA 3’  CHIKVLPMFwd: 5’ TGA AGG GCT CGA GGT CAC GTG 3’  and CHIKYVLPRev: 5’ TA CCTAGGTTA TTA TTC TTA GTG CCT GCT G 3’ (AvrII) were used respectively. The amplification was carried by using Enhanced avian HS RT-PCR kit . The PCR reaction consisted of 5μl of 10X PCR buffer, 3 μl of 25 mM MgCl2, 1 μl each of RNase inhibitor, dNTP mix, eAMV-RT, Fwd and Rev Primers (20 pmol) and AccuTaq LA DNA polymerase, 31 μl Nuclease free water and 5 μl of CHIKV RNA. The PCR thermal conditions used were: 48°C for 45 min, 95°C for 5 min, 94°C for 45 sec, 63°C for 45 sec, 72°C for 2 min, for 40 cycles, and finally 72°C for 10 min. The amplified left and right fragment of Chikungunya polyprotein gene corresponded to 1952 bp and 1795 bp respectively. Since both amplified products naming left and right fragment having XhoI natural restriction site, so for constructing full length of CHIKV polyprotein gene, both amplified products were digested by using AvrII and XhoI & SnaBI and XhoI restriction enzymes. After restriction digestion, both the digested products were ligated at XhoI natural site. Full CHIKV structural polyprotein gene, encoding 3747 bp was cloned into pPIC9K yeast transfer vector  at AvrII and SnaBI restriction sites. This full length structural polyprotein gene of CHIKV was fused with α-mating factor secretion signal of Saccharomyces cerevisiae under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter in pPIC9K yeast transfer vector. The resulting pPIC9K-CHIKV-C-E3-E2-6K-E1 DNA was transformed into E. coli DH5α strain . The recombinant transformants were selected on Luria-Bertani agar  supplemented with 100 ug/ml of Kanamycin. Positive recombinant transformants were confirmed by restriction analysis of plasmid DNA using AvrII and SnaBI and PCR using CHIKYVLP Fwd and CHIKYVLP Rev primers. This was then further confirmed through nucleotide sequencing using ABI 3130 automated DNA sequencer .RNA from Chikungunya virus (DRDE07) was isolated from infected Vero cell supernatant employing a QIAamp viral RNA kit  according to manufacturer’s protocols. The full length gene encoding CHIKV structural polyprotein was amplified as two fragments known as left and right fragments from CHIKV RNA through conventional RT-PCR. For amplification of left and right fragment, primer set having CHIKYVLPFwd: 5’ TAP. pastoris by electroporation as described by manufacture. Briefly, the recombinant plasmid pPIC9K-CHIKV-C-E3-E2-6K-E1 DNA was linearized by SacI and 10 μg of the linear DNA has been transformed in to freshly prepared P. pastoris cells via electroporation using Gene Pulser XCell electroporator  at 1800V, 20μF capacitance and 200Ω resistance. The transformed cells were plated on RD-His plates , and incubated at 28°C for 48 hrs. The colonies obtained were streaked on fresh YPD plates having different concentration of Geneticin  viz 500 μg/ml, 750 μg/ml and 1000 μg/ ml, Integration of CHIKV structural polyprotein gene at His4 locus of Pichia chromosome was verified by amplification with AOX forward and CHIKYVLP Rev primers from genomic DNA of transformants.The yeast expression cassette (pPIC9K-CHIKV-C-E3-E2-6K-E1) plasmid was integrated at His4 locus in GS115 strain of + Mut+Pichia clone was selected for methanol induction. The glycerol stock of recombinant Pichia clone was inoculated into 10 ml YPD medium containing 500 μg/ml Geneticin and was incubated at 28°C in a shaker incubator at 200 rpm until the culture reached an A600 of 2–3. Before induction of protein expression, the cells were centrifuged at 2500 × g for 10 min at room temperature. The dextrose containing YPD medium was then replenished with fresh YPM induction media  as to get an A600 of 3. Incubation was continued at 28°C on shaker incubator (200 rpm) for 48 hrs. Required volume of methanol was added to flask at every 24 hours interval to sustain induction. Supernatant collected both from un-induced and induced cultures were concentrated using cellulose membrane 10 KD pore diameter  by centrifuging at 5000 rpm for 30 min at 4°C. The samples were stored at -80°C until the expression of CHIK-VLP was analyzed on 10% SDS-PAGE as described by Laemmli [2O2/DAB substrate/chromogen .Protein expression was done as described previously with some modifications . Verifie Laemmli . The expThe CHIK-VLPs in the supernatant were purified by ultracentrifugation through a discontinuous sucrose gradient as described previously . BrieflyThe purified CHIK-VLPs and inactivated CHIKV were fixed in 4% formaldehyde and negatively stained with 1.5% phosphotungstic acid (PTA), pH 7.2. Briefly, 1.0 μl of the VLPs and inactivated CHIKV were placed onto a carbon coated Formvar-filmed copper grid  and CHIK-VLPs and inactivated CHIKV were allowed to attach to the surface for 1 min. The grid was washed thrice in sterile triple distilled water by floating the grid on water droplets for 45 seconds to remove excess sample and sucrose. Finally the samples were negatively stained by 1.5% PTA solution as described previously . The airThe immunogenicity of CHIK-VLPs as vaccine candidate against CHIKV was evaluated in 4 week old female Balb/c mice. For the preparation of immunogen, the purified CHIK-VLPs were mixed with equal volume of Freund’s adjuvant . Four groups (n = 10 each) were immunized subcutaneously with 10 μg, 20 μg and 40 μg of yeast derived CHIK-VLPs in Freund’s adjuvant and a control group was immunized with PBS. To determine humoral and cell mediated response all groups were boosted with same formulation on 14, and 28 day after first immunization. Mice immunization was also done with inactivated CHIKV to compare humoral response.2O2  as substrate chromogen. Finally colour development was stopped using 1N H2SO4 and plate was read at 450 nm by microplate reader . Cut-off value was calculated as the mean absorbance (+2 SD) from control sera assayed at 1:250 dilutions. The endpoint IgG titers were then calculated as reciprocal of the highest serum dilution giving an absorbance more than the cut-off.The anti-CHIK-VLPs IgG response against CHIK-VLPs was determined through indirect ELISA at 14, 28, 42, 56 and 140 days of post-vaccination. Briefly, 96-well ELISA plate  was coated with CHIK-VLPs (300 ng/well) followed by blocking with 3% BSA  on next day. The plate was then washed five times with PBST, followed by incubation with two fold serially diluted post-vaccinated sera starting from 1:250 to 1:51200 dilutions in triplicate wells (100 μl/well) including healthy non-vaccinated sera for 1 h at 37°C followed by five washing with PBST. HRP-labeled goat anti-mouse IgG   was added. This was followed by washing (as above) and development with TMB/HSimilar indirect ELISA was performed for evaluating the potential of CHIK-VLPs in recognizing native CHIKV. In this, method 300 ng/well of purified native CHIKV was coated as antigen and the rest procedure remain same.2O2 chromogen and absorbance was measured at 450 nm.The antibody isotyping of CHIK-VLP immunized sera and inactivated CHIKV sera was determined using mouse antibody subtyping kit . Briefly, CHIKV-VLP or inactivated CHIKV coated ELISA plate was incubated with 100μl (1:1000) of immunized sera and incubated for 1 h at 37°C followed by three washing with PBST. Wells were incubated with 100μl (1:1000) of goat anti-mouse IgG specific for each subtype  , at 37°C for 1 h. Following washing, 1:5000 dilution of rabbit anti-goat IgG HRP conjugate  was added and incubated at 37°C for 30 min. Following washing, plate was developed using TMB/H3 PFU of CHIKV was added to the respective wells. The plate was incubated at 37°C for 1 h, with shaking at 20 min intervals. After adsorption, plate was rinsed once with PBS and then replenished with MEM containing 2% FBS and incubated further at 37°C for 24 hrs. The cells were rinsed thrice with PBS, fixed with chilled methanol for 30 min, followed by blocking with 5% BSA for 2 h at 37°C. The cells were then washed thrice with PBS and permeabilized with 0.1% Triton X-100 for 15 min. The cells were incubated for 1h at 37°C with 1:1000 dilution of hyperimmune sera raised against CHIK-VLP. Similarly dilution of pre-immune sera was used as control. The cells were then washed and incubated with Fluorescein isothiocyanate (FITC)—conjugated goat anti mouse antibody (1:100)  for 45 min at room temperature in dark. Cells were then washed and fluorescence was visualized using a Carl-Zeiss Aximot 2  microscope.Sera samples were tested for their ability to recognize the Chikungunya virus by immunofluorescence assay. Vero cells were seeded in a 6 well plate with a cover slip. At 90% confluency, cells were rinsed with PBS and 10In vitro neutralization activity of mice sera immunized with CHIK–VLPs was carried out using two different techniques viz plaque reduction neutralization test (PRNT) and immunofluorescence assay (IFA). Two-fold serial dilutions of heat inactivated sera were prepared in MEM and added to equal volume of the CHIKV containing 102 pfu. The virus control was included without serum. The diluted serum-virus mixtures were incubated at 37°C for one hour. 200 μl/well of serum-virus mixture was then used to infect Vero cells for both PRNT and IFA.2 for 3 days. Cells were washed with PBS and fixed with chilled methanol for 1 hr. The fixed cells were stained with 0.25% crystal violet. The plaques formed were counted. The neutralizing titre is considered as the highest dilution of the immunized sera that showed more than 50% plaque reduction, compared to virus control (serum negative control).Briefly in PRNT, plate was incubated at 37°C for one hour. The serum-virus mixtures were completely removed and the wells were overlaid with 1ml/well of MEM containing 2% FBS and 1.25% methyl cellulose . Plate was incubated at 37°C in 5% COBriefly in IFA, following 24 hrs infection, immunofluorescence was developed by using anti-CHIK-VLP and FITC conjugated goat anti mouse antibody (1:100)  as primary and secondary antibody respectively. Cells were than washed and fluorescence was visualized using a Carl-Zeiss Aximot 2  microscope. The highest dilution of immunized sera that showed reduction in fluorescence as compared to virus control was considered as neutralization titer.In vivo neutralization activity of immunized CHIK-VLP mice sera, was determined via passive immunization of purified IgG intraperitoneally as descried previously [6 PFU of CHIKV. Symptoms were recorded at regular interval of 24 h up to 10 days of post-infection. Gain in body weight, percentage survival and serum viremia were determined. On day 3 and 6 after CHIKV inoculation, 3 mice pups in each experimental and control group were bled to determine serum viremia via qRT-PCR as described earlier [eviously , 32. Tot earlier .Cellular immune response against CHIK-VLPs was determined by cytokine profiling. The expression level of different Th1 and Th2 cytokines i.e. TNF-α, IFN-γ, IL-2, IL-4, IL-6 and IL-10 from the culture supernatants of splenocytes of CHIK-VLPs immunized mice and mock immunized mice were measured by sandwich ELISA using BD OptEIA Kit, .6 cells per well) were seeded in a 24-well plate in triplicate. To measure CHIKV specific responses, cells were stimulated with different doses of inactivated CHIKV  corresponding to respective mice groups. Concanavalin A  as positive control and negative control (without antigen) were also included for each group. The plates were incubated at 37°C in 5% CO2 humidified incubator. After in vitro stimulation, cell supernatants were collected from the wells at 48 and 72 hrs and stored at -80°C prior to use. The cell supernatant was then centrifuged at 15,000g for 30 minutes at 4°C and assayed for cytokines by BD mouse sandwich ELISA Kit . Cytokine expression was determined using standard curve and presented as picograms per millilitre (pg/ml).Briefly, after 7 days of post-booster vaccination, three mice from all the groups were sacrificed and their spleens were retrieved. Splenocyte cell suspension prepared from each mouse , by one way ANOVA using Multiple Comparison Procedure (Fisher LSD Method). Statistical analyses of serum viremia and body weight gain were performed using an unpaired XhoI natural restriction site they were ligated by T4 DNA ligase resulting 3.747 Kbp product. The full CHIKV structural polyprotein gene, encoding 3.747 Kbp was transformed into pPIC9K yeast transfer vector  at AvrII and SnaBI sites resulting pPIC9K-CHIKV-C-E3-E2-6K-E1 construct. Positive transformants having full CHIKV structural polyprotein gene was confirmed by restriction digestion with AvrII and SnaBI and PCR amplification with CHIKYVLP forward and CHIKYVLP reverse primers. Correct integration resulting 3.747 Kbp DNA band . The concentrated Pichia yeast supernatant was purified through 20–60% discontinuous sucrose density gradient centrifugation. SDS-PAGE analysis of various purified fractions revealed localization of majority of CHIK-VLPs at 20–60% sucrose interface  was 1.6 × 104 in 40 μg CHIK-VLPs group whereas 4 × 103 in 20 μg CHIK-VLPs and only 2 × 103 in 10 μg CHIK-VLPs. The peak ELISA antibody titer was obtained on 42 days post-vaccination After second booster (42 days) antibody titer were 3.2 × 104, 2.56 × 104 and 8 × 103 in mice immunized with 40 μg, 20 μg and 10 μg CHIK-VLPs respectively. Serum anti-CHIK-VLPs antibody levels in immunized group was found significantly higher in all of three concentrations compared to controls on 42 day (#P < 0.0001), whereas on 56 and 140 days was significantly higher in 40 μg and 20 μg concentration with respect to control mice sera (#P < 0.0001) and the same pattern persisted up to 140 days post vaccination. Titre of serum antibody increased in dose dependent manner. ELISA titers were also substantially increased following booster doses. The antibody titer in 40 μg CHIK-VLPs was found to significantly higher than 20 μg CHIK-VLPs (****P < 0.0001) and 10 μg CHIK-VLPs ($P < 0.0001) (The assay antibody response in vaccinated mice was examined through an indirect ELISA using CHIK-VLPs. The highest IgG endpoint titer to CHIK-VLPs was 4 × 10 0.0001) .#P < 0.0001) high level of all four isotypes IgG1, IgG2a, IgG2b and IgG3 were observed in all groups  compared to control group. Among the IgG subclasses, IgG1 and IgG2a appeared to have the highest and approximately same levels, although IgG3 levels were also high but IgG2b levels were the lowest. Immunization of different doses of CHIK-VLPs with FCA showed the balanced immune response with approximately high and same amount of IgG1 and IgG2a. There is no significant difference with the level of IgG1 and IgG2a, whereas with respect to IgG2b, the level of IgG1 and IgG2a were found to significantly ($P < 0.0001) higher. The level of IgG3 was found to be significantly lower (¤P < 0.0001) than IgG1 and IgG2a in all doses of CHIK-VLPs  were 1:2048, 1:512 and 1:128 for 40 μg CHIK-VLPs, 20 μg CHIK-VLPs and 10 μg CHIK-VLPs respectively  significantly (**P < 0.001). Major clinical symptoms associated with Chikungunya in mice like hind limb paralysis and retarded growth was not discernable in CHIK-VLPs IgG treated mice group. Whereas, mice group that received control IgG and infected with CHIKV exhibited the typical clinical symptoms at 4–7 days of post infection. All the mice from CHIKV infected group died up to 7 days of post infection, whereas all mice from treated group survived and were healthy . Expression level of IL-2, TNF-α, IFN-γ, IL-4 and IL-10 was significantly higher at 72 hrs, compared to 48 hrs among all the groups (****P < 0.001). A dose dependent pattern has also been seen in all cytokines. The expression of TNF-α, IFN-γ, IL-4 and IL-10 in 40 μg CHIK-VLPs was significantly higher compared with 20 μg  and 10 μg CHIK-VLPs (¤P < 0.001) at 72 hrs  and insect expression (40 mg/l) [Further the optimization of expression parameters of CHIK-VLP revealed YPD with 2% methanol as induction medium and yield of expressed protein was highest at 48 hrs. Induction of CHIK-VLPs expression was carried out using methanol which is very cheap and economical carbon source. In denaturing PAGE, approximately 52 KD, 35 KD and 13 KD bands have been observed which corresponds to the size of CHIKV E1/E2, capsid and E3 protein respectively. So, these observations indicated the presence of all structural proteins in the VLPs. The yield of CHIK-VLP was found to be 60 mg/l in this study. This further indicates the higher productivity of VLP in 40 mg/l) . These V40 mg/l) , 19.Pichia system proved economical for large scale production of recombinant proteins with high safety [P. pastoris can serve as an alternate host system for large scale production of Chikungunya VLPs.VLPs have been known to stimulate similar immune response as native virus. Polyvalence of VLPs leads to high frequency of cross linking between B-cell receptors and VLPs epitopes that resulted in stronger humoral response as well as broader protection . VLPs vah safety . This stVLPs stimulate innate as well as adaptive immune response and act as safer effective vaccine candidate. Recently, phase I clinical trial on HEK cell based CHIK-VLPs has been successfully conducted by National Institute of Health (NIH) and it has been shown to be safe, well tolerated and immunogenic in healthy human volunteers .4, 2.56 × 104 and 8 × 103 with 40 μg, 20 μg and 10 μg CHIK-VLPs respectively. Humoral response plays a crucial role in neutralization of viruses and their clearance. In the present study, successful recognition of native CHIKV by antibodies raised against different doses of CHIK-VLPs was demonstrated in infected Vero cells. Virus neutralizing titre was determined for sera raised against different doses of CHIK-VLPs with two different viral strains belonging to ECSA genotype. The PRNT50 titer of mice sera was found to be dose dependent. This result support that antibodies raised against CHIK-VLPs not only effectively recognizes native CHIKV but also neutralize different strains of CHIKV. In vivo neutralization of different strains of CHIKV by anti CHIK-VLP sera was demonstrated in suckling mice through passive immunization. It was observed that CHIKV infection in suckling mice led to appearance of classic symptoms including increased viremia, retarded growth and hind limb paralysis. Immunized suckling mice were found to be protected as there was a 10 fold lower CHKV RNA copies at day 3 dpi and further 2500 fold lower CHIKV RNA copies were detected at day 6 dpi in IgG treated groups compared to virus control group. This passive immunization study clearly confirmed the ability of anti CHIK-VLP in both in vivo recognition as well as binding to antigenic sites of CHIKV leading to efficient neutralization.Our evaluation indicated that the CHIK-VLPs elicited both humoral as well as cell mediated immune response in mice. The humoral immune response was characterized by high titers of ELISA antibodies. Further, high titer of neutralizing antibodies was demonstrated by plaque reduction neutralization assay. All doses exhibited promising antibody titre. The peak ELISA antibody titer was obtained on 42 days of post-vaccination; antibody titers were 3.2 × 10Earlier studies of CHIK-VLPs in non human primates and mice revealed the important role of neutralizing antibody in the protection , 42. In Titre of IgG1 and IgG2a was comparable in CHIK-VLPs and inactivated CHIKV immunized mice. This demonstrates the effectiveness of CHIK-VLPs to induce a strong immune response comparable to inactivated CHIKV. In previous studies CHIK-VLPs have been shown to protect mice against CHIKV . In thesCHIKV specific cell mediated immune response against different formulations of CHIK-VLPs were evaluated through stimulation of splenocytes by inactivated CHIKV. Efficient stimulation of both humoral and cell mediated immune response is considered crucial for an effective vaccine. Hence the present study demonstrating induction of efficient cell mediated immune response by CHIK-VLPs is an important finding. Results of cell mediated response against CHIK-VLPs demonstrated the balanced Th1 and Th2 response. IL-2, TNF-α and IFN-γ mediate Th1 response and IL-4 and IL-10 mediate Th2 response. Higher expression of TNF-α, IL-10 and moderate expression of IL-2, IL-4 and IFN-γ indicated a balanced Th1/Th2 response. Significant up regulation of TNF-α, IL-2 and IgG2a favored the Th1 response whereas production of IL-4, IL-10 and IgG1 favored the Th2 response.Role of cell mediated immune response in protection against CHIKV has been studied earlier in wild-type mice , where eP. pastoris to express CHIK-VLPs has been demonstrated. CHIK-VLPs are also found immunogenic in mice and found efficient in inducing virus neutralizing antibodies and balance Th1/Th2 response. Purified IgG from CHIK-VLPs immunized mice sera showed protection against CHIKV viremia and hind limb paralysis in neonatal mice. These results confirm the protective efficacy of CHIK-VLPs against emerging Chikungunya virus.In conclusion, this is the first report where, the potential of S1 FigSnaBI & AvrII digest, 2- pPICK9K plasmid control SnaBI & AvrII digest, 3- pPICK9K plasmid uncut; (D) Schematic diagram showing the CHIKV C-E3-E2-6K-E1expression within pPIC9K yeast transfer vector. The structural polyprotein gene of CHIKV is inserted at Avr II-SnaB I sites under the control of AOX1 promoter in fusion with the secretory signal (SS) at 5’ end. The transgene is integrated within the Pichia genome is at HIS 4 locus. Kan R gene present within the expression cassette confers resistance to Geneticin (in Yeast) and Kanamycin (in bacteria).(A) RT-PCR of PCR CHIKV structural polyprotein gene as Right and Left fragment; Lane M: GeneRuler 1 kb DNA Ladder ; Lane 1–4: Amplified Right fragment of CHIKV structural polyprotein gene; Lane 5–8: Amplified Left fragment of CHIKV structural polyprotein gene; (B) PCR analysis of pPIC9K-CHIKV-C-E3-E2-6K-E1 clone; M- DNA ladder, 1- PCR amplification from pPIC9K plasmid (-ve control), 2- PCR amplification from pPIC9K-CHIKV-C-E3-E2-6K-E1 clone, 3- PCR amplification from pTZ57-C/E3/E2/6K/E1(+ve control); (C) Restriction analysis of recombinant clone; M- DNA ladder, 1- pPIC9K-CHIKV-C-E3-E2-6K-E1 clone (TIF)Click here for additional data file.S2 FigP. pastoris having structural polyprotein gene of Chikungunya virus integrated in genomic DNA.; (B) Genomic DNA PCR confirmation of transgene integration in CHIK-VLP-Pichia transformants; Lane M- DNA ladder (1 Kb), Lane 1–10 PCR amplification from Genomic DNA, Lane 11- PCR amplification from pPIC9K-CHIKV-C-E3-E2-6K-E1 plasmid DNA (Positive control), Lane 12- NTC.(A) Geneticin sensitivity assay for recombinant (TIF)Click here for additional data file.S3 Fig#P < 0.0001(significance with respect to control); ****P < 0.0001(significance with respect to 20 μg inactivated CHIKV); °P < 0.0001(significance with respect to 10 μg inactivated CHIKV); $P < 0.0001(significance with respect to IgG2b); §P < 0.001(significance with respect to IgG2b); ¤P < 0.0001(significance with respect to IgG3).Profile of IgG isotypes in sera after immunization with inactivated CHIKV . Data represented in mean antibody titers with S.D. of ten Balb/c mice in each group. Analysis was done by one way ANOVA, (Fisher LSD) (TIF)Click here for additional data file.


Thus TNF-α appears to induce neuronal loss via microglial activation and phagocytosis of neurons, causing neuronal death by phagoptosis.Tumour necrosis factor-α (TNF-α) is a pro-inflammatory cytokine, expressed in many brain pathologies and associated with neuronal loss. We show here that addition of TNF-α to neuronal–glial co-cultures increases microglial proliferation and phagocytosis, and results in neuronal loss that is prevented by eliminating microglia. Blocking microglial phagocytosis by inhibiting phagocytic vitronectin and P2Y In the healthy brain, it is expressed at very low levels by a variety of brain cells including neurons In culture, we have found that activation of microglial TLRs results in high levels of TNF-α production As activation of microglia can cause both TNF-α production and neuronal phagoptosis, we tested here whether soluble, extracellular TNF-α was able and sufficient to induce microglia-dependent neuronal loss by phagoptosis.22.1Salmonella typhimurium (Sigma), Alexa Fluor 488 conjugate of isolectin B4 from Griffonia simplicifolia (Invitrogen), carboxylate-modified fluorescent microspheres (Invitrogen), cyclo(RGDfV) peptide (Bachem), recombinant MFG-E8 (R&D Systems), human soluble TNF receptor inhibitor/Fc chimera (GenScrip Corporation), and MRS2578 compound (Tocris). Cell culture reagents were from PAA. Other reagents were from Sigma.Rat recombinant TNF-α (Sigma), lipopolysaccharide from 2.2Mfge8−/− mice 5 cells/well on poly-l-lysine coated 24-well plates. Glial cultures and pure microglial cultures were prepared as described previously l-leucine methyl ester as described previously All experiments were performed in accordance with the UK Animals (Scientific Procedures) Act (1986) and approved by the Cambridge University local ethical committee. Primary mixed neuronal/glial cultures from postnatal days 5–7 Wistar rat or 2.32. The medium was removed, and the culture was washed several times to remove excess beads. Microglia cells were then labelled with Alexa Fluor 488-tagged isolectin B4 (2 μg/ml) and bead number per cell was evaluated in >50 cells per condition.Phagocytic capacity of microglial cells was evaluated as described previously 2.45 microglia (untreated and treated) were added to each well (in a 24 well plate) of a mixed neuronal/glial culture, which had previously been stained for 15 min with TAMRA (red fluorescence) and washed. Phagocytosis was assayed in a medium half from a glial culture and half from a mixed neuronal/glial culture. Phagocytosis of neurons by microglia was evaluated by microscopy at 6 h after adding microglia as the number of microglia per field containing red fluorescent debris. Cells were also stained with Hoechst 33342 (for nuclei) and green fluorescent isolectin B4 (for microglia).Glial cultures were treated ± 50 ng/ml of TNF-α for 24 h. Microglia from untreated and treated flasks were detached from other glia by shacking the flask. 102.5post-hoc Bonferroni tests. All such significant changes are reported in the Figures – those not reported as significant are not significant. P values < 0.05 were considered as significant. Numbers of alive, apoptotic and necrotic neurons were compared separately. All data presented are expressed as mean ± standard error of the mean (S.E.M.).For all experiments, each condition/treatment was repeated at least in duplicate, and each experiment was replicated in at least three independent cultures – except the experiment on microglial phagocytosis of neurons, which was repeated in quadruplicate but on one culture. Statistical analysis was performed using IBM SPSS Statistics v20 software. Normality of data was verified by Shapiro–Wilk test. Means were compared by one-way ANOVA, and the significance of the difference between each treatment mean and the control or TNF-α treatment mean was quantified by 3TNF-α  caused microglial proliferation Figs. A and 2A,l-leucine methyl ester Treatment with TNF-α did not increase the number of apoptotic or necrotic neurons in the neuronal glial cultures A and B, vβ3 or αvβ5 integrin) Microglia-induced loss of neurons may be mediated via variety of mechanisms: microglial release of glutamate to induce excitotoxicity Mfge8−/− mice Mfge8−/− culture was reconstituted by adding recombinant MFG-E8 protein together with TNF-α, whereas adding MFG-E8 alone had no effect (In order to test whether the VNR-specific opsonin MFG-E8 was required for TNF-α-induced neuronal loss, we isolated neuronal–glial cultures from the cerebella of o effect B. Thus M6 receptor is required for microglial phagocytosis of neurons, as UDP released from damaged neurons induces formation of the phagocytic cup via activating microglial P2Y66 receptors is required for the microglial phagocytosis of neurons induced by LPS or amyloid β 6 with 1 μM MRS2578 prevented TNF-α-induced neuronal loss, and found that it did (6 phagocytic receptor is required for this neuronal loss.The microglial P2Yt it did C, indica6, inhibition of VNR or lack of MFG-E8 prevented neuronal loss without statistically significant increases of the number of apoptotic or necrotic neurons in the cultures treated with TNF-α (Importantly, inhibition of P2Yth TNF-α . If micrIt has been previously established that TNF-α activates microglia and triggers NADPH oxidase activation Mfge8−/− cultures, indicating that neuronal loss was mediated by the MFG-E8/VNR phagocytic pathway of microglial cells (6 also prevented neuronal loss indicating that this neuronal loss also required the UDP/P2Y6 phagocytic pathway (Treatment with TNF-α alone was sufficient to induce microglia-dependent neuronal loss A and B. al cells A and B.  pathway C. While 6 receptor.Altogether, the data presented here revealed that TNF-α induces neuronal death by microglial phagocytosis. Consequently the delayed neuronal loss that occurs in many pathologies accompanied by inflammation may be due to TNF-α-induced phagoptosis, and might be prevented by blocking TNF-α production or function, its receptor, microglial activation, MFG-E8, the vitronectin receptor or the P2Y


FWA:CYCD7;1) triggered nuclear divisions and partial ovule abortion, reducing seed number in each silique and leading to increased seed size. A similar effect on seed size was observed with other segregating embryo lethal mutations, suggesting caution is needed in interpreting apparent seed size phenotypes. Here, we show that the positive effect of FWA:CYCD7;1 on Arabidopsis seed size is modulated by the architecture of the mother plant. Larger seeds were produced in FWA:CYCD7;1 lines with unmodified inflorescences, and also upon removal of side branches and axillary stems. This phenotype was absent from inflorescences with increased axillary floral stems produced by pruning of the main stem. Given this apparent confounding influence of resource allocation on transgenes effect, we conclude that plant architecture is a further important factor to consider in appraising seed phenotypes.The size of seeds is the result of cell proliferation and growth in the three seed compartments: the embryo, endosperm and integuments. Targeting expression of the D-type cyclin CYCD7;1 to the central cell and early endosperm ( Plants have a sessile lifestyle and their development and growth respond to the environment.The mitotic cell cycle of eukaryotes is composed of two alternating phases, during which DNA is first replicated (S phase) and then chromosomes are partitioned , interrupted by two gaps G1 (between M and S) and G2 (between S and M). In eukaryotes, progression through the cell cycle requires the modulation of the activity of kinase complex composed of the cyclin-dependent kinase (CDK) and the regulatory subunit, cyclin (CYC).Arabidopsis, CDKA1;1/CYCD kinasesCell cycle progression requires tight regulation that occurs at two main checkpoints: G1-to-S and G2-to-M. In CYCD7;1 using the highly specific FWA promoter to address the consequences of altering the normal pattern of divisions early in Arabidopsis seed development.FWA promoter is active pre- and post-fertilization in the central cell of the female gametophyte and developing endosperm of the seed. Interestingly, targeted expression of CYCD7;1 using the FWA promoter overcomes cell cycle arrest in the central cell of the female gametophyte before fertilization, resulting in the central cell becoming multinucleate with high frequency. Post-fertilization there is an acceleration of early endosperm and embryo development, although this slows after heart phase of embryo development, by which time the FWA promoter is no longer active. We also observed a high degree of lethality of embryos, leading to abortion.We have used targeted expression of CYCD7;1 expression, we recently reported that FWA:CYCD7;1 transgenic lines produce seeds with increased final size.Associated with these phenotypes provoked by CYCD7;1 expression in the seed under the control of the FWA promoter was also tested using the UAS/GAL4 system,FWA>>CYCD7;1 transgenic lines, the trade-off between seed size and number was further explored within the context of resource allocation. The number of seed pods was artificially decreased by removal of the branches of the floral stalk as well as the axillary shoots . However on SUB shoots where the main stem was removed to increase side branching, mean seed size was decreased by 4% (p = 0.76). In each case, overall mean seed size from the whole plant was determined as described.Hence in this study, we showed evidence for the trade-off between seed size and seed number in the siliques.FWA:CYCD7;1 seeds,FWA>>CYCD7;1 NORMAL and SUPRA plants produce larger seeds compared to WT controls  and in SUPRA conditions FWA>>CYCD7;1 seeds showed a 9% increase in overall area. However, on the SUB plants with increased branching FWA>>CYCD7;1 mean seed size was 4% smaller than the WT control with increased branching, indicating that the beneficial effect of targeted CYCD7;1 expression is lost when shoot growth is stimulated. The length and width showed results similar to those observed for the overall area, suggesting that these trends are the same in both dimensions (FWA driven transgene, but measuring seeds from developmentally comparable shoots suggests that the effect of the transgene depends rather on the architecture of the mother's shoot.In line with our analysis of the mensions . At thisCYCD7;1 ectopic expression appears to be dependent on the availability of sufficient resources. This confounding influence of plant architecture on the effects of ectopic expression CYCD conferred phenotypes might have implications in our evaluation of phenotypes.These observations suggest that not only is seed size affected by sibling lethality but this effect can also be dependent on other aspects of growth. In this case, the manifestation of the enlarged seed phenotype conferred by


IFN-α has been used for decades to treat chronic hepatitis B and C, and as an off-label treatment for some cases of hepatitis E virus (HEV) infection. TNF-α is another important cytokine involved in inflammatory disease, which can interact with interferon signaling. Because interferon-stimulated genes (ISGs) are the ultimate antiviral effectors of the interferon signaling, this study aimed to understand the regulation of ISG transcription and the antiviral activity by IFN-α and TNF-α. In this study, treatment of TNF-α inhibited replication of HCV by 71 ± 2.4% and HEV by 41 ± 4.9%. Interestingly, TNF-α induced the expression of a panel of antiviral ISGs (2-11 fold). Blocking the TNF-α signaling by Humira abrogated ISG induction and its antiviral activity. Chip-seq data analysis and mutagenesis assay further revealed that the NF-κB protein complex, a key downstream element of TNF-α signaling, directly binds to the ISRE motif in the ISG promoters and thereby drives their transcription. This process is independent of interferons and JAK-STAT cascade. Importantly, when combined with IFN-α, TNF-α works cooperatively on ISG induction, explaining their additive antiviral effects. Thus, our study reveals a novel mechanism of convergent transcription of ISGs by TNF-α and IFN-α, which augments their antiviral activity against HCV and HEV. Cytokines orchestrate cellular communication in an autocrine, juxtacrine, or paracrine fashion through binding to distinct families of receptors, triggering specific immune responses against invading pathogens. The interferon (IFN)-mediated innate immune response is probably the most prominent response and provides a robust first defense line. Among different types of interferons, IFN-α (a type I member) has been used for decades to treat chronic hepatitis B or C infection in the clinicIt is generally believed that ISGs are predominantly induced by interferons. However, ISGs are still up-regulated in embryonic fibroblasts from IFN alpha/beta receptor knockout mouse upon infection of West Nile virusTumor necrosis factor alpha . This is through the activation of the NF-κ B signaling but independent of the canonical interferon pathway. Surprisingly, we found a consensus DNA binding sequence between the NF-κ B and ISRE motif with bioinformatics analysis. Functional assays revealed that the NF-κ B complex is able to bind to the ISRE motif and directly activates the transcription of antiviral ISGs. Combination of TNF-α with IFN-α further boosts the induction of ISGs and results in augmented antiviral activity against HCV and HEV. Thus, this study identified a non-canonical mechanism of driving antiviral ISG transcription, which provides the molecular basis for the antiviral action of TNF-α and its additive antiviral effect with interferon.P <  0.001), HEV to 59 ±  4.9%  at 72 hrs.TNF-α is involved in host responses to a variety of pathogen invasions, including HCV and HEV infections9Since TNF-α has been reported to interacts with interferon signaling and ISGs are the ultimate antiviral effectors of the interferon cascade, we thus attempted to investigate whether TNF-α alone has any effect on ISG transcription. Based on the knowledge that interferon induces ISG expression via the activation of the ISRE motifs within the promoters of ISGs, a Huh7 cell line stably harboring a ISRE-driven luciferase reporter was usedThe fact that TNF-α can induce ISGs inspired us to investigate the straightforward possibility that TNF-α merely triggers the production of interferons. Interferon regulatory factor 1 (IRF1) was demonstrated to be important in a TNF-α triggered IFN-β autocrine loop in primary macrophage cellsWe next investigated the effects of TNF-α on gene expression of type I interferons. As determined by qRT-PCR, the constitutive expression levels of IFN-α and β 1 in Huh7 cells are rather low, compared to the reference genes GAPDH and RP2 . MoreoveClassically, ISGs are induced by interferons via the JAK-STAT signaling. Following receptor activation by interferons, JAK1 phosphorylates STAT1 and Tyrosine kinase 2 (TYK2) phosphorylates STAT2. This provokes STAT1 and STAT2 dimerization and subsequent binding to IRF9 to form the ISGF3 complex. The ISGF3 complex translocates into the nucleus, binds to the ISRE motif [5′ -CAGTTTCACTTTCC-3′ ] and drives the transcription of ISGs . To testFurthermore, to see if TNF-α treatment has any effect on STATs activation and translocation, we examined the phosphorylation status of STAT1 at amino acid 701 (Y701P) and STAT2 at amino acid 690 (Y690), which are indispensable signature of STAT1 and STAT2 activation, respectively. WB results showed TNF-α treatment had no effects on the phosphorylation of both STAT1 and STAT2 at indicated sites . ConfocaIn addition, the role of IRF9 was also verified, which is a key downstream element of interferon pathway. IRF9 was up-regulated and translocated into cell nucleus upon IFN-α stimulation, whereas TNF-α stimulation did not induce the translocation of IRF9 into cell nucleus . These rTNF receptor (TNFR) is the important upstream component in TNF-α induced signaling transduction. TNF acts through two receptors, TNFR1 and TNFR2. TNFR1 is the major signaling receptor for TNF-α and is expressed by all human tissues, while TNFR2 is mostly expressed in immune cells and mediates limited biological responsesTo further confirm these results, the clinically widely used drug for rheumatoid arthritis patients and Crohn′ s disease, Humira , was used. Humira binds specifically to TNF-α and blocks its interaction with TNF receptors. As expected, Humira effectively blocks TNF-α induced activation of NF-κ B luciferase activity , NF-κ B Activation of NF-κ B signaling is one of the most important canonical responses to the stimulation of TNF-α . Following TNF receptor activation by TNF-α , inhibitor of kappa B (IkB) proteins undergo phosphorylation dependent ubiquitination and degradation, resulting in the activation and translocation of NF-κ B dimers into the cell nucleus. In the cell nucleus, NF-κ B dimers bind to the specific NF-κ B motifs, [5′ -GGGAA/CTTTCC-3′ ], within the promoter regions driving the expression of NF-κ B target genes . Because18The NF-κ B complex is the endpoint of its signal transduction, which comprises the heterodimeric RelA (P65)-P50 complex. Indeed, unstimulated cells display little nuclear RelA, but the RelA protein level in the cell nucleus was substantially elevated following TNF-α stimulation . Thus, tin silico analysis comparing the ISRE motif and the NF-κ B DNA binding site. Surprisingly, we identified a partial consensus sequence region in common within these two motifs  can directly bind to the promoter region of a large cohort of ISGs. Our loss-of-function and mutagenesis assay further confirmed that NF-κ B could directly drive ISRE-controlled gene transcription. Since NF-κ B is also the key downstream effector of most Toll-like receptors (TLR), this novel mechanism may also partially explain the antiviral activities of TLR agonists in clinic, such as the TLR7 agonists, which are being therapeutically targeted and explored for HCV treatment in clinic trialAs a first line defense, TNF-α and type I interferons are induced by microbial stimuli and mediate innate immune responses. Despite the fact that cells at sites of infection are continuously exposed to both cytokines, the interactions between TNF-α and interferons remain under investigated89More excitingly, TNF-α not only activates antiviral ISGs transcription, but also cooperates with IFN-α , explaining the additive antiviral outcome of their combination. This highlights the important facts that different cytokines orchestrate innate immune responses by activating signaling cascades to protect against infection efficiently.In conclusion, we revealed a novel antiviral mechanism of TNF-α . TNF-α ,via the activation of NF-κ B cascade, can drive the transcription of antiviral ISGs through direct binding of ISREs. This antiviral mechanism may provide clues for tackling the high rise of infections caused by TNF-α inhibitor treatment in patients. More interestingly, TNF-α also acts cooperatively with IFN-α in antiviral ISGs induction to exert additive antiviral effects. These findings not only provide new clues for understanding virus-host interactions but also assign a novel function of the canonical NF-κ B pathway.The HCV subgenomic replicon comprised Huh7 cells containing a subgenomic HCV bicistronic replicon (1389/NS3-3 V/LucUbiNeo-ET) linked to the firefly luciferase reporter gene were maintained with 250 μ g/ml G418 . The HEV subgenomic model was based on Huh7 cells containing the subgenomic HEV sequence (Kernow-C1 p6/luc) coupled to a Gaussia luciferase reporter gene. Lentiviral pLKO knockdown vectors (Sigma–Aldrich) targeting IRF1, TNFR1, RelA were obtained from the Erasmus Biomics Center and produced in HEK293T cells as previously describedHow to cite this article: Wang, W. et al. Convergent Transcription of Interferon-stimulated Genes by TNF-α and IFN-α Augments Antiviral Activity against HCV and HEV. Sci. Rep.6, 25482; doi: 10.1038/srep25482 (2016).


Additional National Eye Institute/National Institute Health grants to the eighth author were incorrectly omitted from the Funding Statement. The Funding Statement should read: "This work was supported by a Burroughs-Wellcome Career development award to GKA, grants from the Hope for Vision foundation to GDA and GKA, a grant from the Pennsylvania Lion’s Foundation to GKA, National Eye Institute (NEI) / National Institutes of Health (NIH) grants EY020516 (GKA), -06855 (GDA), and -14579 (GDA), Foundation Fighting Blindness (GDA), and the ONCE International Price for R&D in Biomedicine and New Technologies for the Blind . GDA also supported by National Eye Institute (NEI) / National Institutes of Health (NIH) grants EY019304 and EY018241. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."