Tumor necrosis factor (TNF) blockers have a high efficacy in treating Ankylosing Spondylitis (AS), yet up to 40% of AS patients show poor or even no response to this treatment. In this paper, we aim to build an approach to predict the response prior to clinical treatment.AS patients during the active progression were included and treated with TNF blocker for 3 months. Patients who do not fulfill ASASAS40 were considered as poor responders. The Immunoglobulin G galactosylation  ratio representing the quantity of IgG galactosylation was calculated and candidate single nucleotide polymorphisms (SNPs) in patients treated with etanercept was obtained. Machine-learning models and cross-validation were conducted to predict responsiveness.MYOM2-rs2294066 was found to be significantly associated with responsiveness of etanercept treatment. A three-stage approach consisting of baseline IgG-Gal ratio, differential IgG-Gal ratio in 2 weeks, and rs2294066 genotype demonstrated the ability to precisely predict the response of anti-TNF therapy .Both IgG-Gal ratio at each time point and differential IgG-Gal ratios between week 0 and weeks 2, 4, 8, 12 showed significant difference between responders and poor-responders. Area under curve (AUC) of the IgG-Gal ratio prediction model was 0.8 after cross-validation, significantly higher than current clinical indexes (C-reactive protein (CRP) = 0.65, erythrocyte sedimentation rate (ESR) = 0.59). The SNP Combination of different omics can more precisely to predict the response of TNF blocker and it is potential to be applied clinically in the future.The online version of this article (10.1186/s10020-019-0093-2) contains supplementary material, which is available to authorized users. Ankylosing spondylitis (AS) is an immune-mediated inflammatory disorder of the spine and sacroiliac joints, which could lead to vertebral fusion  plays an important role in humoral immune response by binding to antigen and Fcγ receptors (FcγR). Immune cells activity can be regulated by the glycan attached to asparagine-297 in the Fc part of IgG  with incubation for 12 h at 37 °C. The released oligosaccharides were subsequently purified using PGC (porous graphitic carbon) and analyzed by AXIMA Resonance MALDI MS (Shimadzu Corp. JP) equipped with a 337 nm nitrogen laser in reflector positive ionization mode. Tandem mass spectrometry (MS/MS) was utilized to validate the component of the detected glycans. The GlycoWorkbench software was used for the annotation of MS spectra. Progenesis MALDI was used for MS data process. The biantennary, core-fucosylated structures carrying two, one, and no galactose residues are the three most significant glycans identified from IgG, named G2, G1, and G0. The distribution of IgG galactosylation  was calculated using the relative intensities of G0, G1 and G2 according to the formula G0/ (G1 + G2 × 2).Whole-exome sequencing was performed on 33 (24 responders and 9 poor-responders) samples with an average coverage of 100× using Illumina HiSeq 2000. Burrows Wheeler Alignment V.0.7.10, and the genome analysis toolkit (GATK) V.1.1.28 were performed to map reads and call variants respectively against the reference sequences and Annovar was used for variant annotation.Genotyping of all samples was performed by Sanger sequencing and candidate SNP was used to predict the response to the TNF blocker.The response to Etanercept of each patient was used as a dependent variable and several candidate indexes including Bath Ankylosing Spondylitis Di (BASDAI), Bath Ankylosing Spondylitis Fu (BASFI), Erythrocyte sedimentation rate (ESR), C reactive protein (CRP), Ankylosing Spondylitis Disease (ASDAS) and IgG-Gal ratio were used as independent variables to build the regression model. Cross-validation was applied as follows. The data of 78 patients were used to build the model and determine the best cutoff value; the value was used to predict the response of the 79th patient. The process was duplicated by 79 times.As for the combination model of IgG-Gal ratio and candidate SNP, the methods logistic regression (LG), randomforest (RF), support vector machine (SVM), naviebayes (NB), neural network (NN), linear discriminant analysis (LDA), mixture discriminant analysis (MDA) and flexible discriminant analysis (FDA) were performed. Cross-validation was applied as follows: we randomly divided the responders and poor-responders into 10 groups; groups 1 to 8 were used to build model (training), and groups 9 and 10 were used to test the model  and then rotate 10 times. All models were performed by R software with “randomForest”, “e1071”, “nnet”, “MASS” and “mda” packages.ROC (receiver operating characteristic) curve was plotted and sensitivity as well as specificity were calculated to test the power of the index to predict patient response to Etanercept.ROC curve was performed by “ggplot2” package in R.Etanercept is the most widely used type of TNF blocker for AS patients in China.. Plasma from 79 patients were collected at weeks 0, 2, 4, 8, and 12 during treatment for measuring IgG galactosylation level   was measured by the method reported in our previous study . After treatment, the average IgG–Gal ratio of responders decreased slowly from 1.1 to 0.8 (Ptrend = 2.9 × 10− 5), while that of poor-responders persisted (Ptrend = 0.82)  and poor-responders (n = 10) , Vacuolar Protein Sorting 13 Homolog B (VPS13B), Dispatched RND Transporter Family Member 1 (DISP1) and Interleukin (IL27) genes were most significantly different between responders and poor-responders. After the validation stage , only MYOM2 SNP rs2294066 remained significant   with 8 machine-learning models to achieve a more precise prediction of AS patient response to etanercept treatment.We combined IgG-Gal ratio and MYOM2 rs2294066 to be a reliable predictor.After 10-times cross-validation, as depicted in Fig. week2 – IgG-Gal ratioweek0) and MYOM2 rs2294066 were used to differentiate responders from non-responders in each stage respectively in clinical practice.It is of great importance to screen patients and separate responders from non-responders. IgG-Gal ratio prior to treatment, ΔIgG-Gal ratio  to predict the response of TNF blocker.Genetics and environment are two important factors to the pathogenesis of AS  served as a biomarker for dilated cardiomyopathy and TNF-α was up-regulated during brief myocardial ischemia, or coronary occlusion . If he carries CT/TT genotype he will be advised to change to another treatment. If he carries CC genotype, we suggest him to continue the treatment with TNF blocker.Therefore, we proposed a novel three-stage approach to combine genetic markers and post-translational modifications to predict precisely the response of the TNF blocker etanercept in AS patients. Briefly, when an AS patient is considered to be treated with TNF blocker, first we detect IgG-Gal ratio and if it is over 0.8 we advise him to be treated with TNF blocker for long-term. If it is below 0.8, we suggest him to be treated with TNF blocker for one time and IgG-Gal ratio will be detected again after two weeks. If ΔIgG-Gal ratio is below − 0.1, he can be treated with TNF blocker for long-term, or he should further be detected the genotype of The limitation of this study was that it only included the result from single medical center and the sample number is not very large. Therefore, in the future the result will be further validated in another medical center.In this study, in order to predict the response of TNF blocker more precisely, we combined genetic data with protein modification data. This was a pilot study, but it largely increased the power of prediction. We propose that it is a potential biomarker to combine different omics data in the future.Additional file 1:Figure S1. A: The variance of BASFI with time course. Figure S2. A: ΔIgG-Gal ratio of responders and poor-responders after IgG-Gal ratio screening. Figure S3.MYOM2 gene expression of different alleles (Wilcoxon test). Table S1. Inclusion criteria. Table S2. Mean value of sensitivity, specificity and AUC of different indexes after cross-validation. Table S3. Validation results of the other 5 SNPs that were associated with the response to TNF blocker in the first stage. (DOCX 886 kb)


In multiple regression analysis for N1/P2 sLORETA-LDAEP, lower N1/P2 sLORETA-LDAEP was significantly related to higher TNF-α  when all subjects were dichotomized based on the median TNF-α level (7.16 pg/mL) into pretreatment low- and high-TNF-α groups. In addition, the pretreatment Beck Depression Inventory, P2 LDAEP, and N1/P2 sLORETA-LDAEP were greater in the high-TNF-α groups than in the low-TNF-α groups (p < 0.05). Moreover, the posttreatment TNF-α level was significantly decreased compared to the pretreatment TNF-α level . However, the posttreatment TNF-α levels were not associated with posttreatment LDAEP. Conclusions: Higher TNF-α level is associated with decreased LDAEP, which could indicate compensatory elevation of central serotonin activity in outpatients with MDD, although this effect disappeared and TNF-α level was reduced after three months of antidepressant treatment.Background: Proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), are associated with the pathophysiology of major depressive disorder (MDD). Several studies have reported that increased TNF-α might be associated with tryptophan depletion, which eventually could result in MDD. However, other studies revealed that TNF-α increased serotonin firing in raphe. Therefore, whether TNF-α increases or decreases serotonin activity remains unclear. Here, we aimed to determine the relationship between serum TNF-α level and central serotonergic activity using the loudness dependence of auditory evoked potentials (LDAEP) and standardized low-resolution brain electromagnetic tomography (sLORETA), as well as to evaluate the effects of antidepressants on TNF-α levels. Methods: LDAEP, serum TNF-α level, and depression severity were measured in 64 MDD outpatients pre and post 3 months of treatment. Results: Pretreatment TNF-α levels were negatively correlated with the pretreatment N1 sLORETA-LDAEP, P2 sLORETA-LDAEP, and N1/P2 sLORETA-LDAEP ( The monoamine hypothesis is considered as the main pathophysiology underlying a major depressive disorder (MDD). However, various hypotheses have emerged recently, of which one states that proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) play a significant role in MDD. Recent meta-analysis revealed higher levels of TNF-α in patients with MDD than those in healthy subjects . AnotherStress activates the hypothalamic–pituitary–adrenal (HPA)-axis and leads to secretion of cortisol, which eventually is responsible for secretion of proinflammatory cytokines such as TNF-α from the microglial cells. In addition, TNF-α can be produced from a variety of cells such as fibroblasts, endothelial cells, neurons, mast cells, and adipose tissue . The infNevertheless, in contrast, few previous studies suggested that TNF-α elevation increases the serotonin activity or levels ,7. That The loudness dependence of auditory evoked potentials (LDAEP), which is calculated using the amplitude of event-related potentials such as N100 and P200 induced by auditory stimuli, has recently been used to measure the central serotonergic activity . Some clIn total, 64 outpatients aged between 18 and 65 years, who met the DSM-IV Text Revision criteria for MDD, have been recruited at Ilsan Paik Hospital, Korea. The inclusion criteria for participation of these patients were: (1) Having total Beck Depression Inventory (BDI) and Hamilton Depression Rating Scale (HAMD) scores exceeding 19 or 17, respectively, prior to the treatment, and (2) no history of antidepressant administration during the 12 weeks before their first visit to our hospital. The exclusion criteria were: The presence of a (1) previous hypomanic episode, (2) high suicide risk, (3) history of brain trauma or organic brain disease, or (4) neurological disease. Depression severity was assessed using the HAMD and BDI scores at baseline and at 3 months. The serum TNF-α level and LDAEP were also measured at baseline. In addition, the follow-up serum TNF-α and LDAEP were remeasured in 22 subjects after the 3-month antidepressant monotherapy , as the remaining patients did not wish to undergo follow-up blood sampling. These data were included in the Korean MDD cohort for suicide prevention (KOMDD study) .The study protocol was approved by the Ethics Committee of Ilsan Paik Hospital, and written informed consent to participate was obtained from all patients before commencing the investigations.Blood samples for measuring serum TNF-α level were collected between 8:00 a.m. and 9:00 a.m., following an overnight fast by venipuncture, and were added into the anticoagulant-free vacuum tubes. The samples were allowed to clot for 30 min at room temperature, and were then centrifuged at 2000 rpm for 15 min. Serum was separated and aliquots were stored at −70 °C until further analysis. The serum TNF-α level was evaluated at baseline and after 3 months of treatment.Serum TNF-α level was analyzed using enzyme-linked immunosorbent assay (ELISA) kits . Each assay was performed in duplicate. The actual concentration of each sample was calculated using the five-parameter-fit logistic-curve equation. The ELISA plate readings were recorded using a microplate reader .All subjects were seated in a comfortable chair in a sound-attenuated room. They were asked to keep their eyes open throughout the testing, fixated on a pointer displayed on the monitor. The auditory processing comprised 1000 stimuli with an interstimulus interval of 500–900 ms. Tones at 1000 Hz and with a duration of 80 ms  were generated by E-Prime software , and presented in a randomized order at 5 intensities  via headphones . Electroencephalography data were recorded from 64 scalp sites using silver/silver chloride electrodes according to the International 10–20 system (impedance <10 K) using an Auditory Neuroscan NuAmp amplifier . Data were collected at a sampling rate of 1000 Hz after passing the signals through a bandpass filter from 0.5 to 100 Hz. In addition, four electrodes were used to measure the horizontal and vertical electrooculograms.Data were reanalyzed using Scan 4.3 software with a bandpass filter from 1 to 30 Hz, and the ocular contamination was removed using standard blink correction algorithms . Event-rOn the basis of the averaged scalp-recorded electric potential, standardized low-resolution brain electromagnetic tomography (sLORETA) was used to estimate the current densities . The sLOp < 0.05. The statistical analysis was performed using the SALT 2.5 software packages.The Kolmogorov–Smirnov test was used to check whether the clinical variables were normally distributed. Correlation analysis using Pearson’s and Spearman’s correlations was carried to identify the significant correlations between clinical variables and the LDAEP and TNF-α level. All subjects were dichotomized according to the median TNF-α level (7.16 pg/mL) into baseline low- and high-TNF-α groups. Student’s t-test, Mann–Whitney U-test, and the Chi-squared test were used to compare the clinical variables between the two groups. For multiple regression analysis of the relationship between TNF-α and LDAEP, all subjects were dichotomized according to the median TNF-α level (7.16 pg/mL) into baseline low- and high-TNF-α groups. The pre- and posttreatment clinical variables, LDAEP and TNF-α, in the MDD patients were compared by paired t-tests or Wilcoxon’s signed ranks test. All tests were two-tailed, and the cutoff for significant group differences was In total, 64 subjects  aged 40.47 ± 13.71 years (mean ± SD) were evaluated in this study. The age, sex distribution, and baseline scores on the BDI, HAMD, LDAEP values, and TNF-α levels are presented in p < 0.05). In addition, the average P2 LDAEP, left N1 sLORETA-LDAEP, left P2 sLORETA-LDAEP, average P2 sLORETA-LDAEP, left N1/P2 sLORETA-LDAEP, and average N1/P2 sLORETA-LDAEP in the high- TNF-α groups were significantly lower than those in the low-TNF-α groups (p < 0.05). In multiple regression analysis for pretreatment N1/P2 sLORETA-LDAEP, lower pretreatment N1/P2 sLORETA-LDAEP was significantly related with pretreatment of high-TNF-α groups (CE(Coefficient) = −0.047, p = 0.017, When comparing the clinical variables and LDAEP between pretreatment low- and high-TNF-α groups according to the median TNF-α level , left P2 sLORETA-LDAEP , left N1/P2 sLORETA-LDAEP , and average N1/P2 sLORETA-LDAEP .Pretreatment TNF-α was negatively correlated with the pretreatment average N1 sLORETA-LDAEP (n = 22) were compared with each other. Each mean TNF-α level was, respectively, 11.14 ± 3.89 and 9.41 ± 3.54 pg/mL, and significantly differed from each other  under an activated HPA axis . This IDFew investigators have hypothesized that TNF-α is able to increase the serotonin levels in the synaptic cleft as long as the uptake activity via SERT is intact . They alSeveral studies have investigated the effects of antidepressants on TNF-α; however, the results still revealed contradictory findings like decreased, increased, or unchanged TNF-α levels after antidepressant treatment ,28,29. IThe LDAEP has been widely studied in the field of psychopathology, for its relationships with suicide and treatment responses, and as a biological marker associated with serotonin, since Hegerl and Juckel first discovered this noninvasive method . HoweverThis study was subject to several limitations. The sample was relatively small and it did not include healthy controls. In addition, other cytokines were not analyzed in this study. Nevertheless, this study supports that pretreatment TNF-α levels are associated with central serotonergic activity, and antidepressant treatment can reduce the pretreatment TNF-α levels in patients with MDD. Moreover, despite TNF-α being produced in a circadian rhythm-manner and different sleep patterns being observed among the patients, these factors were not considered while designing the experiments. However, blood samples for measuring serum TNF-α level were collected between 8:00 a.m. and 9:00 a.m., following an overnight fast to reduce a bias.


Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia. Accumulating experimental evidence shows the important linkage between tumor necrosis factor-α (TNF) and AD, but the exact role of TNF in AD is still not completely understood. Although TNF-inhibitors are successfully used for treating several diseases, total inhibition of TNF can cause side effects, particularly in neurological diseases. This is attributed to the opposing roles of the two TNF receptors. TNF receptor 1 (TNFR1) predominantly mediates inflammatory and pro-apoptotic signaling pathways, whereas TNF receptor 2 (TNFR2) is neuroprotective and promotes tissue regeneration. Therefore, the specific activation of TNFR2 signaling, either by directly targeting TNFR2 via TNFR2 agonists or by blocking TNFR1 signaling with TNFR1-selective antagonists, seems a promising strategy for AD therapy. This mini-review discusses the involvement of TNFR2 and its signaling pathway in AD and outlines its potential application as therapeutic target. A better understanding of the function of TNFR2 may lead to the development of a treatment for AD. The pleiotropic pro-inflammatory cytokine tumor necrosis factor-α (TNF), known as a member of the TNF superfamily of ligands, is a master regulator of the innate and adaptive immune system. TNF plays a vital role in the initiation and orchestration of inflammation and immunity , and is Alzheimer’s disease is an age-related neurodegenerative disorder associated with severe cognitive impairment such as memory deficits, deterioration of visuospatial skills and executive dysfunction. Its neuropathological hallmarks include both positive and negative features. Positive lesions consist of amyloid plaques, neurofibrillary tangles, neuropil threads, tau protein hyperphosphorylation, and glial activation. Negative lesions include losses of neurons, neuropil, and synaptic elements. Besides the well-known pathological characteristics mentioned above, neuroinflammation has been considered a major contributor to AD, in which TNF is an important mediator .Tumor necrosis factor is expressed as a 26-kDa monomeric type II transmembrane protein (tmTNF) that can be cleaved to release a 17-kDa soluble monomeric protein (solTNF) by the matrix metalloprotease TNF converting enzyme (TACE/ADAM17) . TNF binreg) cells, and by endothelial cells, and preferentially binds tmTNF , p38, c-jun N-terminal kinase (JNK), and the ceramide/sphingomyelinase signaling pathway, result in multifarious processes including inflammation, proliferation, cell migration, apoptosis, and necrosis . TNFR2-mMounting evidence convincingly shows that TNF expression is increased in AD by various methods and means, including genetic , proteinAltogether, these results support the hypothesis that increased expression of TNFR1 aggravates AD pathology, whereas TNFR2 is able to prevent it. Therefore, TNFR1 and TNFR2 represent promising therapeutic targets for the treatment or prevention of AD.Existing therapies that target TNF for clinical use are based on monoclonal antibodies that inhibit its signaling via its receptors. These therapies have been approved for treatment of several inflammatory and autoimmune diseases, including rheumatoid arthritis, psoriatic arthritis or plaque psoriasis. The therapeutic potential of these anti-TNF antibodies has more recently been studied in neurodegenerative diseases. Infliximab is a chimeric IgG1 monoclonal antibody that has a high binding affinity for human TNF. Two similar studies that were performed in AD mouse models showed that intracerebroventricular injection of infliximab reduced tau phosphorylation, TNF levels and Aβ plaques , and impAnother biologic anti-TNF drug is etanercept, a combination of the extracellular domain of TNFR2 linked to the Fc portion of a human IgG1. A pilot study conducted in 15 AD patients that received perispinal administration of etanercept resulted in a reduction of cognitive deficits based on the AD Assessment Scale-Cognitive Subscale, the Severe Impairment Battery, and the Mini-Mental State Examination . A folloAnother limitation of the abovementioned etanercept studies in AD patients is that they did not include a control group receiving placebo and the long-term effects of etanercept were not stated. In fact, The use of a similar biological anti-TNF drug, lenercept, was unsuccessful as treatment for relapsing-remitting multiple sclerosis in a placebo-controlled, double-blind phase 2 study. Indeed, patients receiving lenercept experienced an earlier and significant aggravation of the disease compared with patients receiving placebo . Other sThese studies collectively show that anti-TNF therapies may be disadvantageous, especially in the treatment of neurological diseases. In this respect, total inhibition of TNF might nullify its positive effects, particularly via its TNFR2. Given the evidence described, we will focus on new experimental therapies that selectively interfere with the TNF receptors signaling for the treatment of neurodegenerative diseases:As concluded in the previous section, TNFR1 plays a key role in neuroinflammation and apoptosis occurring AD. Since solTNF primarily activates TNFR1, a specific inhibition of solTNF would, in principle, block the functions of TNFR1 without preventing the neuroprotective functions of TNFR2.XPro-1595 is a selective solTNF inhibitor . ResultsFurthermore, in a recent study using a rat model of Parkinson’s disease, peripheral injection of XPro-1595 significantly reduced apoptosis, inflammation, and loss of dopaminergic neurons . This stin vivo model is generated by an exposure to glutamate, which causes neuronal cell death and, mimics acute neurodegenerative diseases. The NBM lesion model provokes an activation of macrophages and microglia (inflammation) and a loss of cholinergic fibers similar to that in AD  that ameliorate inflammation and apoptosis, and TNFR2 agonists that enhance neuro-regeneration and tissue homeostasis, are promising strategies to treat neurodegeneration. As discussed in this mini-review, a considerable number of studies have shown the efficacy of targeting TNF receptors in several neurodegenerative diseases, suggesting that these drugs might have potential in the therapy of AD. In the future, a deeper understanding of the diverse molecular pathways of TNF signaling can contribute to the discovery of more specific and refined strategies to treat AD and other neurodegenerative diseases.NO-C and YW wrote the manuscript. PN, PDD, IZ, and UE reviewed and edited it, and provided key guidance.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.


Norovirus (NoV) genotype GII.4 is responsible for the majority of NoV infections causing pandemics every few years. A NoV virus-like particle (VLP)-based vaccine should optimally cover the high antigenic variation within the GII.4 genotype. We compared the immune responses generated by VLPs of the ancestral GII.4 1999 strain (GII.4 1995/96 US variant) and the most recent GII.4 Sydney 2012 pandemic strains in mice. No significant differences were observed in the type-specific responses but GII.4 1999 VLPs were more potent in inducing high-avidity antibodies with better cross-reactivity. GII.4 1999 immune sera blocked binding of GII.4 2006 and GII.4 2012 VLPs to the putative receptors in a surrogate neutralization assay, whereas GII.4 2012 immune sera only had low blocking activity against GII.4 2006 VLPs. Amino acid substitution in the NERK motif , altering the access to conserved blocking epitope F, moderately improved the cross-blocking responses against mutated GII.4 2012 VLPs (D310N). NoV GII.4 1999 VLPs, uptaken and processed by antigen-presenting cells, induced stronger interferon gamma (IFN-γ) production from mice splenocytes than GII.4 2012 VLPs. These results support the use of GII.4 1999 VLPs as a major component of a NoV vaccine. Norovirus (NoV) GII.4 is the predominating NoV genotype, causing up to 85% of acute gastroenteritis outbreaks of NoV and sporadic infections worldwide . It is aThe NoV particle consists of 90 dimers of capsid VP1 protein organized in T = 3 icosahedral symmetry . VP1 is GII.4 NoV undergoes epochal evolution similar to influenza virus; periods of stasis lead into rapid antigenic drift in common structural epitopes that are under immune pressure ,17. NoV-Virions are dynamic structures reacting to environment with conformational changes, which enable biologically relevant functions such as receptor/ligand binding . MutatioIn this study we investigated type-specific and cross-reactive humoral and cellular immune responses induced in mice with the first  and the latest  pandemic GII.4 variant VLPs. In addition, we studied the effect of an amino acid mutation in the NERK motif on cross-blocking antibody responses. The results presented here add to the current knowledge and understanding of cross-reactive immune responses induced in vivo by different variant GII.4 VLPs.Three different NoV GII.4 variant VLPs were used as immunogens and/or in vitro antigens in this study. GII.4 1999  to the VP1 sequence of the reference strain GII.4 1995/96 US, Genbank accession number AF080551) and GII.4 2012 (accession number AFV08795.1) VLPs were produced in baculovirus-insect cell system and purified as previously described by our laboratory ,33,34. G3 to the Seven-week-old female BALB/c OlaHsd mice obtained from Envigo RMS BV  were immunized intramuscularly (IM) twice (weeks 0 and 3) with 10 µg of GII.4 1999 (5 mice) or GII.4 2012 VLPs (4 mice). Mice receiving carrier buffer only  were used as negative controls (5 mice). Whole blood and lymphoid tissue were collected at the time of euthanization on study week 5. For bone marrow-derived dendritic cell (BMDC) generation, femurs and tibia were collected from naive control mice and the exterior tissues were sterilized with 70% ethanol and kept on ice until bone marrow extraction (described in o-phenylenediamine dihydrochloride (OPD)-substrate (30 min RT). All incubations were performed at 37 °C for one hour unless otherwise stated. After stopping the substrate reaction with 2M H2SO4 the optical density (OD) was measured at 490 nm . Each sample/dilution was assayed in duplicate wells and sample volumes were 50 µl/well. The background signal from wells lacking serum (blank wells) was subtracted from all of the OD readings at a plate. The cut-off value was calculated as mean OD + 3 × SD of negative control mice sera at a given dilution. A sample resulting in an OD value above the set cut-off OD and at least 0.100 OD was considered positive. End-point antibody titers were defined as the highest dilution of serum giving an OD above the set cut-off value. Geometric mean titers (GMTs) with 95% confidence intervals (CIs) for each immunization group were counted from individual mice end-point titers.Antigen-specific and cross-reactive immunoglobulin G (IgG) responses in mice sera were measured by enzyme-linked immunosorbent assay (ELISA) as described in detail elsewhere ,38. IndiSerum IgG avidity was measured by ELISA as described above, but after serum incubation the plates were incubated twice (for 5 min for each treatment) with 8 M urea (Sigma-Aldrich) to elute low-avidity antibodies . The avia, Lewisb, H-type-1, H-type-3, A-trimer, and B-trimer, Glycotech, Gaithersburg, MD, USA) [HBGA binding of mutated GII.4 2012 D310N VLPs in comparison to wild-type GII.4 2012 VLPs was analyzed by using three different sources of HBGAs: human type A saliva ,40, pig MD, USA) ,18. BrieMD, USA) . The plaBlocking assay was used to measure antibodies that block binding of NoV VLPs to HBGAs present in human saliva (type A) or PGM, or to synthetic biotinylated H-type-1 carbohydrate as previously described ,42. NoV g for 10 min and suspended in CM containing 20 ng/mL recombinant mouse granulocyte-macrophage colony-stimulating factor . BM-cells were seeded at 1×106 cells/mL (10 mL per plate) in non-treated 90 × 14.2-mm sterile petri dishes  and cultured at 37 °C, 5% CO2 for 8 days. Fresh CM with GM-CSF (5 mL/plate) was added on the dishes on days 4 and 7 and the cells were harvested on day 8. The generated cells were surface stained with phycoerythrin (PE)-conjugated anti-mouse CD11c and Horizon Viability Stain 780 (both from BD) and acquired using BD FACSCanto II flow cytometer as described earlier [The method for generating mouse BMDCs was adapted from a published procedure  with som earlier  which co earlier  in ice-cg, 10 min) and seeded 2×106 cells/mL in CM in non-treated cell-culture 24-well plates (Corning Costar) for antigen pulsing. GII.4 1999, GII.4 2012 and GII.4 2006 VLPs were added to the cells at 100 µg/mL. Cells lacking pulsing antigen (un-pulsed BMDCs) were used as a negative control. The cells were incubated  for 20–22 hours, washed twice, and were used as antigen-presenting cells (APCs) in enzyme-linked immunospot (ELISPOT) assay.The BMDCs were thawed, washed twice (300× 6 cells/well) on top of BMDCs and the plates were incubated for 20 h at 37 °C and 5% CO2. Thereafter, the cells were discarded and the plates were developed with biotinylated anti-mouse IFN-γ monoclonal antibody  and alkaline-phosphatase (ALP) conjugated streptavidin  reacting with BCIP/NBT substrate . After 12 minutes the color reaction was stopped with tap water. The spots were counted by an ImmunoSpot® automatic CTL analyzer  and the results are expressed as mean spot forming cells (SFCs) per 106 live splenocytes of replicate wells.Homologous and cross-reactive T-cell responses were analyzed by quantification of IFN-γ production from group-wise pooled mice splenocytes in response to VLP pulsed BMDCs by an ELISPOT-IFN-γ assay ,38. Briep-value of <0.05. Data were analyzed with IBM SPSS Statistics version 25.0 .The Kruskal–Wallis’ test was used to assess the statistical differences in antibody titers and avidity indices between individual immunization groups. A statistically significant difference was defined as a p = 0.264) of type-specific IgG response, with GMTs of 102,400 and 86,100 , respectively. IgG responses against homologous VLPs were significantly higher (p < 0.05) than cross-reactive responses induced by heterologous antigen. GII.4 1999 VLP immunization induced significantly higher (p = 0.018) cross-reactive IgG response against GII.4 2006 VLPs  than GII.4 2012 VLP immunization . When GII.4 1999 and GII.4 2012 cross-reactive responses were compared against each other, GII.4 1999 immunization resulted in 2-fold higher GII.4 2012-specific titer  than GII.4 2012 immunization against GII.4 99  but the difference was not statistically significant (p = 0.15). Control mice did not develop specific IgG response to any of the VLPs tested.Immunized mice sera were tested using ELISA to quantify type-specific and cross-reactive IgG antibodies against GII.4 1999, GII.4 2006, and GII.4 2012 VLPs a. GII.4 p = 0.027) higher avidity than GII.4 2012 VLP immunization.Comparison of the avidity of type-specific and cross-reactive antibodies are shown in p = 0.079, p = 0.036) and completely failed to block GII.4 1999 binding. Control mice sera blocked all VLP binding <15% at the lowest serum dilution (1:20).The ability of mouse immune sera to block homologous and heterologous VLPs binding to HBGAs present in human saliva (type A) was tested in a blocking assay ,40. BothThe NERK motif  is suggested to control antibody access to epitope F, a putative universal blocking epitope in the GII.4 lineage [To study the effect of amino acid 310 mutation on cross-blocking activity we used GII.4 1999 pooled immune sera to block wild-type GII.4 2012 and mutated GII.4 2012 D310N VLP binding to type A saliva a, PGM Fb, and sy6 cells.ELISPOT IFN-γ was used to investigate whether there are differences in the development of homotypic or heterotypic T-cell immunity after GII.4 1999 and GII.4 2012 VLP immunization. IFN-γ-producing cells were quantified from mice splenocytes in response to autologous BMDCs pulsed with different NoV GII.4 VLPs. GII.4 1999 or GII.4 2012 VLP-immunized mice splenocytes responded with similar intensity to GII.4 1999 a, GII.4 An important issue in NoV VLP vaccine development is the antigenic diversity of NoV genotypes and the evolution of the predominant GII.4 strain resulting in variants able to escape herd immunity . A similAvidity of antibodies is considered to be an important surrogate of protective efficacy of several vaccines ,52 and hWe observed that GII.4 1999 immune serum had blocking activity against GII.4 2012 but not vice versa. The finding is in concordance with our earlier results where GII.4 1999 immune serum was able to block the VLP binding of another contemporary variant (GII.4 NO) to HBGAs whereas GII.4 NO immune serum completely lacked blocking activity against GII.4 1999 . There mGII.4 1999 immune serum cross-blocked GII.4 2006 VLP binding similarly to homologous VLP, whereas GII.4 2012 serum cross-blocking activity against GII.4 2006 VLPs was significantly lower. Other investigators have bioinformatically identified blocking epitopes A-F and shown that evolution in these epitopes, especially in evolving epitopes A and D, might have resulted GII.4 variants to escape from herd immunity ,26,31. ABroadly conserved epitopes are often occluded in the capsid and can be shielded by the virion via selected motifs regulating the exposition of these epitopes . EpitopeThe role of T-cell immunity in protection against NoV infection and disease is not well known but it might have a role in the generation of heterologous immunity ,57. We aWe have previously proposed a candidate NoV VLP vaccine including GII.4 1999 and GI.3 VLPs . The res


As is a prerequisite of belonging to the scavenger receptor super family, SCARF1  is known to play a key role in the binding and endocytosis of a wide range of endogenous and exogenous ligands.Unlike most scavenger receptors, SCARF1 is an essential protein, as SCARF1-deficient mice exhibit a severe resting phenotype in which they develop systemic lupus erythematosus (SLE)-like disease, thus highlighting the importance of SCARF1-mediated clearance of apoptotic host cells in homeostasis. In addition, a number of other roles in homeostasis and disease pathology have also been suggested, including roles in both innate and adaptive immunity; however, the majority of these studies have utilised transfected cell lines engineered to ectopically express SCARF1 and very few have utilised in vivo or ex vivo approaches.This review summarises our current knowledge on SCARF1 biology and reflects on future directions for research on this multifaceted, yet largely understudied, scavenger receptor. Caenorhabditis elegans scavenger receptor, CED-1 [C. elegans [C. elegans is highly suggestive of an important role for SCARF1 in mammalian biology and, unlike the majority of scavenger receptor-deficient mouse lines which do not exhibit a resting state phenotype, the importance of SCARF1 becomes apparent in SCARF1-deficient (SCARF1−/−) mice which spontaneously develop systemic lupus erythematosus (SLE)-like autoimmune disease from 20 weeks of age. This autoimmune disease phenotype results from a significantly impaired clearance of apoptotic cells from key immunological organs, such as the spleen, and manifests in SLE-like symptoms, such as increased production of autoantibodies, splenomegaly and larger germinal centres with increased CD4+ T cells and B cells, severe dermatitis, and nephritis [Scavenger receptors are a large super family of proteins which are defined by their ability to bind and endocytose a vast range of ligands, eliciting their ‘scavenging’ of unsolicited endogenous and exogenous products . Surprisr, CED-1 , a recep elegans , 7. Its ephritis .SCARF1 was first cloned from human umbilical vein endothelial cells (HUVEC) , but hasMany scavenger receptors are known to exist in general circulation in a soluble form, which is often released via cell surface cleavage by exofacial proteases , and SCAAs is a prerequisite of scavenger receptor classification, SCARF1 is a highly promiscuous receptor and has been shown to bind a wide range of endogenous and exogenous ligands  , 29; the9 cells undergo apoptosis in the human body per day, a rapid and immunologically ‘clean’ removal of apoptotic cells by neighbouring phagocytic cells is essential for the maintenance of homeostasis and avoidance of inflammation [−/− mice spontaneously develop SLE-like autoimmune disease at 20 weeks of age as a consequence of impaired apoptotic cell clearance, thus highlighting the prominence of SCARF1 in this homeostatic process [Given that more than 10ammation . The firammation , an impoammation . Followi process . Their s process , which,  process . NeverthNeisseria gonorrhoeae porin protein, PorBIA, in in vitro bacterial adhesion assays [Staphylococcus aureus is able to avidly bind SCARF1 expressed on nasal epithelial cells via its wall teichoic acid (WTA) [SCARF1 has been shown to bind a diverse range of bacterial (both Gram-positive and Gram-negative), viral, and fungal antigens  . Howeverid (WTA) , 38, yetIt has also been suggested that SCARF1 could play an indirect role in the capture and uptake of bacteria via two homologous endogenous ligands, the Tamm–Horsfall protein (THP)  and pancet al. demonstrated that SCARF1 was able to directly bind an outer membrane protein (OmpA) isolated from Klebsiella pneumonia, but required the presence of TLR2 to elicit the release of the potent neutrophil chemoattractant and proinflammatory cytokine, IL-8, from transfected Chinese hamster ovarian (CHO) cells [Cryptococcus neoformans and Candida albicans, via β-glucan, but, again, the presence of TLR2 was required to elicit macrophage activation and the initiation of a proinflammatory immune response [In addition, a number of studies have shown evidence to suggest that SCARF1 acts in cooperation with a family of essential pattern recognition receptors (PRRs), called the Toll-like receptors (TLRs), to bind a range of pathogen-associated molecular patterns (PAMPs) and elicit a proinflammatory immunological response. A study by Jeannin O) cells . A seminresponse . Furtherresponse . More reresponse , or the response .As mentioned previously, much of this work has utilised in vitro experimentation in cell lines ectopically expressing SCARF1; and, although these results are promising, their validation in primary cells which naturally express SCARF1, in more sophisticated multi-cellular in vitro models , or in murine models of infection is required to fully appreciate the role of SCARF1 in the innate immunity.+ or CD4+ T cells [In addition to being implicated in the innate immunity, there is evidence to suggest that SCARF1 is also involved in the adaptive immunity and facilitates the uptake of chaperone-bound antigens and their subsequent antigen presentation to T cells , 17–19.  T cells , 17, 19,+ T cells to the sinusoidal endothelium under physiological shear stress conditions [+ adhesive cups form on the surface of the HSEC [+ T cells do not express SCARF1 [−/− mice.The leukocyte adhesion cascade is a multi-step process which facilitates the extravasation of leukocytes from circulation into inflamed tissues during injury or infection, with the primary aim of eliminating the inflammatory trigger and/or contributing to tissue repair. The adhesion cascade is mediated by a large number of chemoattractant cytokines (chemokines)  and adhenditions . In thisthe HSEC ; howevers SCARF1  or SCARFSCARF1 is known to bind a wide range of ligands both endogenous and exogenous in nature; consequently, a number of biological roles in homeostasis, immunity, and disease pathology have previously been suggested. However, the majority of studies undertaken thus far have relied heavily on in vitro experiments involving the ectopic expression of SCARF1 in cell lines and, whilst the validity and value of these studies is not being called into question here, this review highlights the distinct lack of research with regards to SCARF1 which utilises naturally expressing primary cells and ex vivo or in vivo approaches. Consequently, the cellular and tissue expression and function of this highly promiscuous scavenger receptor still remain a mystery and future studies should aim to reduce this significant deficit.


We used the ARPE-19 human retinal pigment epithelial cell line as ocular cells considered to play an important role in the blood-ocular barrier, and the MT2 HTLV-1-infected cell line. Jurkat cells were used as controls. Infliximab (IFX) was used as an anti-TNF-α antibody to achieve TNF-α inhibition. We evaluated the production of inflammatory cytokines and intercellular adhesion molecule (ICAM)-1, proliferation of ARPE-19, expression of TNF-α receptor (TNF-R) and HTLV-1 proviral DNA, and the percentage of apoptotic ARPE-19. Inflammatory cytokines such as interleukin (IL)-6, IL-8, TNF, and ICAM-1 were significantly elevated through contact between ARPE-19 and MT2. Treatment with IFX tented to inhibit TNF production, although the level of production was low, but changes in IL-6, IL-8, and ICAM-1 remained unaffected. Expression of TNFR was unaltered by IFX treatment. HTLV-1 proviral DNA was not significantly changed with treatment. No change in cell growth rate or apoptotic rate of ARPE-19 was seen with the addition of IFX. In conclusion, IFX did not exacerbate production of inflammatory cytokines, and did not affect expression of TNFR, proliferation of ARPE-19, HTLV-1 proviral load, or apoptosis of ARPE-19. These results suggest that IFX does not exacerbate HTLV-1-related inflammation in the eye and represents an acceptable treatment option under HTLV-1 infectious conditions.Use of biologics has been widely advocated for inflammatory diseases recently. Anti-tumor necrosis factor (TNF)-α antibody therapy is reportedly effective against ocular inflammation. However, side effects of TNF-α inhibition have been reported, particularly in the form of exacerbation of infections such as tuberculosis. Paradoxical reactions such as exacerbated inflammation are also well known. Around 20 million humans are infected with human T-cell leukemia virus type 1 (HTLV-1) globally, and this virus can cause adult T-cell leukemia, HTLV-1-associated myelopathy and HTLV-1 uveitis. As for ophthalmic concerns, it has not been identified whether anti-TNF-α antibody stimulates HTLV-1-infected cells and ocular cells to induce HTLV-1 uveitis in HTLV-1 carriers. Here we investigated the effects of anti-TNF-α antibody on ocular status under HTLV-1 infectious conditions using ocular cells and HTLV-1-infected cells  Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that infects an estimated 20 million people globally . VariousBiologics have been widely advocated for use against inflammatory diseases recently. In particular, anti-tumor necrosis factor (TNF)-α antibody therapy has seen wide use for rheumatoid arthritis (RA) , psoriasAlthough significant efficacy of anti-TNF has been reported, side effects of TNF-α inhibition such as infection , malignain vitro , after providing anti-TNF-α antibody therapy . Basicalin vitro .As mentioned above, several reports have examined the safety of anti-TNF-α antibody therapy in internal medicine fields, but no report from the ophthalmological field has clarified whether TNF-α inhibition can affect ocular conditions in HTLV-1 carriers. HU is one of the most important clinical entities among HTLV-1-infected individuals. As a result, the risk of induction of HTLV-1-related ocular inflammation  following administration of anti-TNF-α antibody into HTLV-1 carriers is a concern warranting investigation. In addition, guidelines for the use of anti-TNF-α antibody have not mentioned screening for HTLV-1 infection prior to initiation of treatment.in vitro. The present investigation chose a retinal pigment epithelium (RPE) cell line as a representative ocular cell line, as the RPE plays a major role in the blood-ocular barrier and the maintenance of immunological homeostasis in the eye (The present study investigated the effects of anti-TNF-α antibody on ocular status among HTLV-1 carriers using an ocular cell line and an HTLV-1-infected cell line  the eye .2. In vitro infection by HTLV-1 was performed using the standard co-culture method (5 ARPE-19 cells were used in cytometric bead assay (CBA) and annexin V assay. 2 × 104 ARPE-19 cells were used in cell counts, TNF receptor analysis, and the measurement of HTLV-1 proviral load.We used the ARPE-19 human retinal pigment epithelial cell line  as ocular cells, and the MT-2 cell line as an HTLV-1-infected T-cell line. We used Jurkat cells as a control T-cell line. MT2 and Jurkat cells were cultured in RPMI 1640  supplemented with 10% fetal bovine serum (FBS)  and 1% penicillin/streptomycin. ARPE-19 cells were cultured in minimum essential medium  with the same supplements. All cell lines were incubated in a humidified incubator at 37°C under an atmosphere of 5% COe method . Brieflyin vitro method for HTLV-1-associated myelopathy experiments  was used as an anti-TNF-α antibody, with 10 μg/ml/well, in line with a previously established eriments .Cultured supernatants were examined using CBA human inflammation cytokine kits . Results were analyzed with FCAP Array version 3.0 software (BD Biosciences) according to the instructions from the manufacturer. Cytokines measured by the kits included interleukin (IL)-6, IL-8, IL-1β, IL-12p70, IL-10, and TNF.To measure levels of soluble intercellular adhesion molecule (ICAM)-1 in supernatants, ELISA kits  were used in accordance with the instructions from the manufacturer.4) were co-cultured with three times the number of MT2 or Jurkat cells with or without IFX. After 0, 24, 48, or 72 h of co-culture, we removed the supernatants, trypsinized ARPE-19, and counted the number of ARPE-19 cells under light microscopy.ARPE-19 cells (2 × 10Fluorescence-activated cell sorting (FACS) analysis was performed to examine the cell surface expression of TNF-R1 and TNF-R2, using fluorescein isothiocyanate (FITC)-conjugated anti-CD120a (TNF-R1) and anti-CD120b (TNF-R2) human monoclonal antibodies . ARPE-19 cells were washed and trypsinized, then incubated with TNF-R1 and TNF-R2 antibodies according to the instructions from the manufacturer. Analysis was performed using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences).ARPE-19 cells were cultured on glass bottom plates  for 24 h, then co-cultured with MT2 and Jurkat cells using cell culture inserts (Thermo Fisher Scientific) for 48 h. After three washes with phosphate-buffered saline (PBS) , ARPE-19 cells were fixed by cold fixing buffer  at −20°C for 20 min and blocked with 10% FBS in PBS for 15 min. Cells were then incubated in the diluted primary antibodies for 1 h at room temperature, followed by incubation with Alexa fluor488-labeled anti-rabbit secondary antibody  along with 4′,6-diamidino-2-phenylindole dihydrochloride  incubation for 1 h at room temperature. The following antibodies were used as primary antibodies: TNF receptor 1 polyclonal antibody  and TNF receptor 2 polyclonal antibody . We scanned using a TCS-SP8 microscope .DNA was prepared from each sample using EZ1 Virus Mini Kits v2.0  according to the instructions from the manufacturer. Quantitative real-time polymerase chain reaction (PCR) assay was used to measure the PVL of HTLV-1 in cells, as described previously . PVL wasFACS analysis to evaluate of apoptosis and cell death using Annexin V-FITC was performed using assay kits  according to the instructions from the manufacturer. Apoptotic cells were defined by detecting propidium iodide-negative, annexin V-positive cells. The rate of apoptotic cells among all cells was calculated by FACSCalibur flow cytometer and CellQuest software (BD Biosciences).t-test or Welch’s unpaired t-test after Bonferroni correction for multiple testing, which adjusts the values of p by multiplying them by the number of tests . More precisely, EZR is a modified version of R Commander (version 1.6-3) designed to add statistical functions used frequently in biostatistics.Student’s unpaired of tests , was useLevels of IL-6, IL-8, IL-1β, IL-12p70, IL-10, TNF, and ICAM-1 secreted by each of ARPE-19, MT2, Jurkat, ARPE-19 co-cultured with MT2, and ARPE-19 co-cultured with Jurkat were measured . MT2 spop = 0.14). Levels of IL-12p70, IL-1β, and IL-10 were below measurable limits under all conditions . Likewise, there were no differences in the histograms of TNF-R1 and TNF-R2 on ARPE-19 by treatment with IFX . FurtherPVL was measured by real-time PCR to assess whether IFX affected HTLV-1 related disease progression in ARPE-19 cells. PVL in ARPE-19 cells co-cultured with MT2 cells was significantly increased compared to that in co-culture with Jurkat cells. As for controls, undetectable PVL was confirmed in ARPE-19 cells co-cultured with Jurkat cells. In HTLV-1-infected ARPE-19, PVL did not increase with IFX treatment .p = 0.10). No change in the apoptotic rate of ARPE-19 co-cultured with MT2 was seen with the addition of IFX (Annexin V assay was performed to assess the apoptosis in ARPE-19 co-cultured with MT2 and the effect of IFX on ARPE-19 was examined. The apoptotic rate of ARPE-19 co-cultured with MT2 tended to be increased compared to that in co-culture with Jurkat, but the difference was not significant (n of IFX .Biologics are reportedly effective for ocular inflammation and have been used worldwide. Although HTLV-1 carriers are common all over the world, no basic studies have been conducted to clarify the safety of biologics for HTLV-1-infected patients in the ophthalmological field. In Japan, several decades have passed since the first biologic was applied to inflammatory diseases, and Japan is now one of the countries with the greatest use of biologics by physicians. A case has already been reported in which an RA patient with HTLV-1 infection experienced exacerbation of HTLV-1-associated conditions such as myopathy and uveitis after using a biologic agent . ClarifiThe intraocular concentrations of cytokines and chemokines greatly influence the pathophysiology of ocular inflammation . The RPEAs for TNF-α, our experiments showed that levels of this cytokine were low, but decreased further when HTLV-1-infected T-cells came into contact with RPE, compared to HTLV-1-infected T-cells alone . This phin vitro investigation of HTLV-1-associated myelopathy (We next focused on another important molecule, ICAM-1. ICAM-1 acts to mediate cell-to-cell interactions,  and ICAMelopathy .via interference of TNF binding to two known receptors, namely TNF-R1, which binds to soluble TNF, and TNF-R2, which binds to membrane-bound TNF. As shown in previous studies, TNF-R1 and TNF-R2 are expressed on the RPE (Inhibition of IFX is mediated  the RPE , and cha the RPE ,B. This The PVL in peripheral blood is related to the development of HTLV-1-associated diseases , and PVLAn increase in apoptotic ARPE cells represents critical damage to the maintenance of immunological homeostasis in the eye . In the in vitro investigation using cell lines might not accurately reflect all aspects of clinical events. As we placed particular focus on changes to ocular tissue in this investigation, we did not examine the risk of ATL induction using IFX. Although previous basic and clinical papers have not indicated any increased PVL in peripheral blood mononuclear cells with IFX treatment (Some limitations to this study merit attention when considering the potential effects of IFX therapy in patients with ocular inflammation complicated by HTLV-1 infection. For instance, in our experiments involving inflammatory cytokines, we did not observe the increases in IL-1 and IL-10 in co-cultures of ARPE-19 and MT2 that were described in previous clinical reports . This inreatment , we woulin vitro.IFX did not exacerbate the production of inflammatory cytokines, and did not affect the expression of TNFR, cell proliferation, HTLV-1 PVL, or apoptosis on RPE. These results suggest that IFX did not exacerbate HTLV-1-related inflammation in the eye and represents an acceptable treatment option, at least according to HTLV-1 infectious conditions MU performed the experiments and wrote the draft of the manuscript. KK designed the experiments, analyzed the data, and wrote the manuscript. NA, CW, and HK performed the experiments. KO-M contributed to analysis and interpretation of data, and assisted in the preparation of the manuscript. All authors critically reviewed and approved the final manuscript.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.


The interaction of intracellular TNF-α signal with the insulin signaling pathway is not well identified. Hence, we aimed to investigate the effect of TNF-α elimination on the diabetic model of palmitate-induced insulin-resistant hepatocytes (HepG2). The changes of phosphorylation rate in IRS-1 protein are determined to know the effect of TNF-α on this key protein of the insulin signaling pathway. HepG2 cells were treated with 0.5 Mm palmitate, and TNF-α gene knockdown was performed by shRNA-mediated technique. Western blot analysis was used to evaluate the phosphorylated activity of the insulin signaling pathway. Palmitate-induced IR could increase TNF-α protein expression 1.2-, 2.78-, and 2.25-fold compared to the control cells at times of 8 h, 16 h, and 24 h, respectively. TNF-α expression in downregulated cells transfected with shRNA-TNF-α is approximately 47.0% of normal cells and 49.0% in the case of scrambled cells. IRS-1 phosphorylation in TNF-α-downregulated and stimulated cells with 100 nM insulin, after treatment and in the absence of palmitate, was 45% and 29% higher than the normal cells. These data support the evidence that TNF-α downregulation strategy contributes to the improvement of IRS-1 phosphorylation after insulin stimulation and insulin response in HepG2 liver cells.One of the major mechanisms of hyperglycemia in type 2 diabetes is insulin resistance (IR) which can induce free fatty acids like palmitate. In hepatic cell, as an insulin target tissue, insulin resistance can be stimulated by inflammatory cytokine TNF- It has been supported by the fact that in obesity and high-fat diet, removal of TNF-α function improves insulin sensitivity and glucose homeostasis in obese mice [α infusion in healthy humans leads to insulin resistance through impaired insulin signaling and decreased glucose uptake [α to the cell surface receptor leads to the activation of two major transcription factors: c-Jun and nuclear factor-κβ (NF-κβ), which subsequently results in the activation of genes involved in the inflammatory process, stress responses, and cell growth and development [α expression in insulin-resistant obese muscle cells [α expression in pathogenesis of palmitate-induced insulin resistance and inflammation in liver cells is poorly described. The current study is aimed at investigating the effect of TNF-α elimination on the palmitate-induced insulin resistance. It is an insight into the regulation of the hepatic insulin signaling pathway and glucose uptake through IRS. We determined the beneficiary phosphorylation of this key protein in TNF-α knockdown and control hepatic (HepG2) cells under the presence and absence of PA. It has been purposed to demonstrate the novel potential background for eliminated expression of the inflammatory factor TNF-α in the improvement of hepatic diabetic cells.Millions of people around the world have been diagnosed with type 2 diabetes, and many more remain undiagnosed. It has been considered as epidemic-like proportion since it is likely to be more than double by 2030  and typenditions . The rolese mice –11. In ae uptake , 11. Theelopment , 9. The le cells –14. Howew/w endotoxin/fatty acid-free BSA to make a 50 mM working stoke and incubated in a water bath. The conjugated PA solution was sterile filtered and kept in -20°C.Palmitate was prepared according to the protein absorption method . To incrv/v heat-inactivated fetal bovine serum (FBS) and 1% v/v penicillin/streptomycin antibiotic solution at 37°C under 5% CO2 atmosphere. For mRNA or protein analysis, HepG2 cells were cultured for 24 h in DMEM supplemented with 1% FBS and the presence or absence of PA (25 mM). For PA treatment, the PA-free BSA samples were incubated at 37°C for 2 hours and used for the treatment of HepG2-cultured cells.Hepatocellular carcinoma cells (HepG2 cells) possess the same bioactivity as normal hepatic cells. These cells are valuable for investigating liver-associated functions, and they are stable during many passages. The HepG2 cell line was purchased and cultured in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) . It has been supplemented with 10% α gene, shRNA-mediated technique was performed [α in liver cells. HepG2 cells were transduced with shRNA lentiviral particles and with noneffective scrambled shRNA sequences as the control (scrambled control). The cells at density of 3 × 105 cells per dish were seeded onto 35 mm dishes. After one day of seeding, 200 ml of lentiviral particles in 2 ml DMEM medium including 10% FBS was added to the cultures and then incubated for 24 h in 5% CO2 at 37°C. The cells successfully infected by lentiviral particles were selected using 3 mg/ml puromycin in the presence of 10% FBS for 48 h. The processed cells were then harvested and applied to Western blot analysis. A sample checking was done to determine the efficiency of the used lentiviral particles for suppressing TNF-α gene expression.In order to knock down TNF-erformed  by usingα protein level, Western blot method was used [4P2O7, 2 mM NaVO4, and protease inhibitor cocktail was used. Protein concentration was determined using Bradford's method [α, p-IRS-1 (Tyr632) , and β-actin . The membrane was then incubated with secondary antibody conjugated to HRP  for 1 hour, and detection was performed using ECL reagents . Films were scanned, and protein bands were quantified using the TotalLab software. The density of different protein bands measured and the density of TNF-α band of each sample were divided to the density of β-actin on the same sample to remove loading-related errors. Each experiment was performed at least three times.To confirm the downregulation of the TNF-was used , 7, 15. s method . 20-30 mp < 0.05 were considered statistically significant.The statistical analyses were performed by SPSS 19.0  software. All normally distributed continuous variables were expressed as mean ± SD. Each experiment was repeated at least three times. The comparison between all continuous variables was performed by using one-way analysis of variance (ANOVA). If there were statistically significant differences, then Tukey's post hoc test was applied. The values of p < 0.05) the expression levels of TNF-α in HepG2 cells  was used to knock down TNF-α gene in hepatocytes. Subsequently, the rate of TNF-α protein expression was analyzed by the Western blot method (α (TNF-α knockdown cells), plasmids lacking shRNA-TNF-α (control cells), and plasmids containing sh-RNA which interferes with none of the intracellular mRNA (scrambled cells). The transfection procedures were done by a related commercial method. The concentration of TNF-α produced by hepatocytes was quantitated [α expression in knockdown cells transfected with shRNA-TNF-α is approximately 47.0% of control cells. In the case of scrambled cells, it is 49.0% compared to the control cells.A gene silencing approach directed to TNF-t method . HepG2 cntitated , and equα-downregulated and normal cells (p < 0.05). The results showed the phosphorylation of tyrosine 632 of IRS-1 after treatment of cells with palmitate (to make the diabetic model of insulin resistance) and stimulation with insulin  in TNF-α-downregulated cells was 45% (p < 0.05) higher than the normal cells. Following the absence of palmitate and stimulation with 100 nM insulin, the phosphorylation of IRS-1 in TNF-α-downregulated cells evaluated was 29% higher (p < 0.05) compared to the normal cells , was analyzed by a Western blot method. In the absence of palmitate and no treatment with insulin, there was no any significant difference in tyrosine 632 phosphorylation of IRS-1 molecules in TNF-al cells .α has taken much concentration recently. Studies have shown that insulin resistance is associated with elevated plasma levels of TNF-α [α as an inflammatory mediator cytokine on insulin signaling inhibition, we designed the current course of TNF-α levels in the simulated obese HepG2 cells under treatment with palmitate. The idea is consistent with the previous studies regarding elevated expression of TNF-α in the liver [α protein levels on 16-hour treatment with palmitate (0.5 mM). This indicates the etiological role of elevated palmitate on TNF-α overexpression in obese insulin resistance hepatocytes. The molecular mechanisms of insulin resistance have been demonstrated extensively [α, can reduce IRS-1 serine phosphorylation and thus inhibits its function [α-induced insulin resistance. The elevated TNF-α levels have been reported in obesity and other insulin-resistant states [α knockdown was carried out by shRNA-TNF-α lentiviral particles containing shRNA specifically for TNF-α. This process resulted in nearly 53.0% reduction in TNF-α protein levels . It is noted that due to t-test, the value rate of gene expression was reduced to p < 0.05 and considered to be significant. It suggests that the particles containing shRNA to silence TNF-α gene cause the efficient rate of TNF-α knockdown. The process of TNF-α downregulation was then followed by palmitate treatment to avoid coincidence reactions between TNF-α level changes and inhibition of insulin signaling. The tyrosine 632 phosphorylation of IRS-1 after treatment of cells with palmitate and stimulation with insulin showed less reduction of 55.0% (p < 0.05) in TNF-α-downregulated cells compared to the normal cells of 71.0% (p < 0.05). Although the treatment with palmitate 0.5 mM generally reduced the tyrosine 632 phosphorylation of the molecule IRS-1 in both TNF-α-downregulated and normal cells, the amount of phosphorylation in TNF-α-downregulated cells is approximately 1.3 times (p < 0.05) higher than the normal cells. The achieved results confirm that the reduction of TNF-α expression in HepG2 cells improves insulin-stimulated Tyr632 IRS-1 phosphorylation. Our results were parallel to the other studies approaching the reduction of TNF-α through different strategies and have demonstrated improved insulin sensitivity in skeletal muscle, liver cells, and animal models [α in hepatocytes.Several genetic and environmental factors have been determined to involve in insulin resistance, of which TNF-of TNF-α  and causof TNF-α , 10, 12 of TNF-α , 14, 18.of TNF-α –23. In ohe liver , 7 and she liver , 24–26. ensively . Multiplfunction , 29. Dect states . The expl models , 31–33. α downregulation contributes to the improvement of insulin sensitivity in hepatic cells, even in the presence of palmitate. Taking these findings together, the inhibition of TNF-α signaling has a potential possibility for treatment target. It could be a strong point in management of metabolic and cardiovascular diseases, lipid-induced insulin resistance, and type 2 diabetes.These data support the evidence that TNF-


Virus-like particle (VLP) platform represents a promising approach for the generation of efficient and immunogenic subunit vaccines. Here, the feasibility of using grapevine fanleaf virus (GFLV) VLPs as a new carrier for the presentation of human papillomavirus (HPV) L2 epitope was studied. To achieve this goal, a model of the HPV L2 epitope secondary structure was predicted and its insertion within 5 external loops in the GFLV capsid protein (CP) was evaluated.” domain C of the GFLV CP, which was then over-expressed in Pichia pastoris and Escherichia coli. The highest expression yield was obtained in E. coli. Using this system, VLP formation requires a denaturation-refolding step, whereas VLPs with lower production yield were directly formed using P. pastoris, as confirmed by electron microscopy and immunostaining electron microscopy. Since the GFLV L2 VLPs were found to interact with the HPV L2 antibody under native conditions in capillary electrophoresis and in ELISA, it can be assumed that the inserted epitope is located at the VLP surface with its proper ternary structure.The epitope sequence was genetically inserted in the αB-αBThe results demonstrate that GFLV VLPs constitute a potential scaffold for surface display of the epitope of interest. Virus-like particles (VLPs) are among the most easily-produced nanomaterials, since they result from the self-assembly of the capsid protein (CP). VLPs can be modified either genetically or chemically for various downstream applications i.e. as biomaterials , drug-dePlant viruses are particularly interesting since they are not infectious for humans and animals. Compared to most animal viruses, the structure of the majority of plant viruses is very simple and often made up of a single CP type. Moreover, most of them are nonenveloped and they present a very high level of accumulation in their host –19. In rNepovirus. Considering the structural features of the GFLV capsid, it is composed of 60 copies of a single coat protein  without any envelope. The ternary structure of the CP subunit is defined by three jelly-roll domains named C, B and A from the N- to C- termini, respectively. The viral structure has been elucidated at 2.7 Å resolution [VLPs based on several plant viruses were tested, including cowpea mosaic virus (CPMV), alfalfa mosaic virus, tobacco mosaic virus, potato virus C, tomato bushy stunt virus, zucchini yellow mosaic virus, plum pox virus, papaya mosaic virus, cucumber mosaic virus and cowpea chlorotic mottle virus (CCMV) –27. In ssolution .Nicotiana benthamiana [Recently, GFLV VLPs have been transiently expressed using pEAQ-HT vector in thamiana . These aEscherichia coli, Pseudomonas fluorescens, Pichia pastoris and Saccharomyces cerevisiae [E. coli. Nevertheless, soluble proteins are obtained without an extra solubilization step. Moreover, this system is easy to manipulate and offers high expression yields as well as ease of scale-up [VLPs from plant viruses have been produced in various microbial expression systems, namely revisiae , 31–34. revisiae . Moreoverevisiae . Concernscale-up .High-risk genotypes of human papillomavirus (HPV) are responsible for cancers in females and males. Around 70 % of the cervical cancers are due to HPV16 and 18 genotypes . The curEscherichia coli and Pichia pastoris systems.Here we have studied the feasibility of using GFLV VLPs as scaffold for HPV L2 epitope presentation albeit maintaining VLP formation. To achieve this goal, GFLV VLPs displaying HPV L2 epitope as well as GFLV CP VLPs were expressed in both For GFLV-L2 VLP modeling, the secondary structures of the GFLV CP and the selected HPV L2 epitope (amino acids 17–31) were needed. The GFLV CP 3D structure was previously solved by  (Protein” domain C), 190–195 (βB-βC domain B), 211–212 (βC-βC” domain B), 375–378 (βC-βC” domain A). These sites were assumed to have at least one advantage for the insertion of foreign epitopes, i.e. the fact that the inserted epitope would be displayed on the surface of the particle since these regions are located in the outer area of the particle , 79–85 . The detection of a distinct band at 56–57 kDa in lanes 3 and 4 indicates that large amounts of the recombinant GFLV CP and GFLV L2 in an insoluble form were produced in E. coli.Restriction enzyme analysis confirmed that the GFLV CP and GFLV L2 genes 1.5 kb) were inserted in the pET26B vector between .5 kb werE. coli, such as different E. coli strains (BL21 and SHuffle T7 cells), induction times , isopropyl β-D-1-thiogalactopyranoside (IPTG) concentrations  and growth temperatures . However, under all tested conditions, the proteins were expressed as inclusion bodies (IBs). The highest expression yield was obtained using the strain BL21 (DE3), 0.25 mM IPTG at 37 °C during 4 h in a shaker.Various expression conditions were examined in order to obtain the GFLV CP and GFLV L2 in a soluble form in To solubilize the proteins of interest, different solutions containing one or several compounds were tested ; 8 M urea, 1% triton X-100, 10 mM DTT and 14 mM mercaptoethanol, pH = 8 for 4 and 6 h at 4 °C; 10 mM SDS and 15 mM DTT; 8 M urea and 10 mM DTT, pH = 7 for 16 h at 4 °C). The IBs were found to be solubilized in solutions containing 10 mM SDS and 15 mM DTT or 8 M urea and 10 mM DTT, pH = 7 for 16 h at 4 °C. The solubilization of the IBs in the solution containing urea during 16 h at 4 °C was found to be the best protocol. Indeed, as shown in Additional file P. pastoris transformed with the empty pPICZα vector in the presence of methanol (lane 5). The cultures of the selected clones were concentrated by sucrose cushion ultracentrifugation  and c (P. pastoris), lane 2), indicating that the GFLV antibody interacts with the GFLV L2 protein, as is the case for the GFLV CP  and d (P. pastoris), lane 1). Considering the absence of a band at the corresponding MW for the GFLV sample  and d (P. pastoris), lane 2), it can be assumed that the GFLV CP does not interact with the HPV L2 antibody, which therefore confirms that this antibody specifically detects the HPV L2 epitope in the GFLV L2 protein.Western blot analysis using an antibody directed against the GFLV CP showed the presence of the protein of interest for the GFLV L2 samples  analysis showed a population of spheroid particles having a diameter of 29 ± 1.5 nm and 31.9 ± 2.5 nm in E. coli  and in vivo (P. pastoris). In agreement with the TEM results, the immunostaining electron microscopy (ISEM) analysis confirmed the presence of GFLV VLPs obtained from E. coli and P. pastoris that clearly immunoreact with anti-GFLV CP polyclonal antibody . VLPs were analyzed by SEC and a major peak was observed at 1.3 mL in both cases  is a very powerful technique that allows the analysis of intact viral particles but also the study of biomolecular interactions under native conditions . It is wE. coli  [Ageratum yellow vein virus [Based on in silico data, loop structures βB-βC and αB-αB” (domain C) were predicted as epitope insertion sites and the latter was selected for expression experiments. Expression of GFLV constructs in ws virus , 44. Thiws virus . To soluCMV VLPs . TEM exafor CCMV . When cosh leaf) . Transiesh leaf) , turnip sh leaf)  and Agerin virus . Unlike in virus .From the GFLV CP 3D structure, it could be deduced that the selected 79–85 loop of all sixty CP subunits is surface accessible. The insertion in this loop did not interfere in the CP- CP interactions necessary for VLP assembly and stability and resulted in the exposure of the HPV L2 epitope at the particle outer surface in its native conformation. In agreement with our results, Schellenberger et al. reported that the replacement in the CP region R1 of GFLV (amino acids 79–85) to CP region R1 of Arabis mosaic virus, which is located at the external surface of the particles, did not interfere with virus encapsidation .Since GFLV L2 VLPs were found to interact with the HPV L2 antibody under native conditions in capillary electrophoresis and in ELISA, it can be assumed that the inserted epitope is located at the VLP surface with its proper ternary structure. This is obviously crucial for the use of GFLV L2 VLPs as a potential vaccine. Previous studies reported that this HPV L2 epitope located at the N-terminal part of MS2 phage has more conformational flexibility than in the AB-loop of PP7 phage, allowing the formation of a disulfide bond in the epitope and leading to a better presentation of the peptide, inducing a more broadly reactive antibody response against the HPV L2 epitope , 51.The absence of GFLV mRNA was confirmed using purified VLPs as input for RT-PCR. However, the encapsulation of non-specific mRNA and DNA within the VLPs cannot be excluded. To avoid any encapsulation of nucleic acids, the subunits might be treated using RNaseA and DNase before VLP formation.E. coli and P. pastoris was demonstrated. VLPs containing an epitope from the HPV minor CP (HPV L2 epitope) were obtained. The application of GFLV L2 VLPs as a promising vaccine antigen will be clear after immunization tests. Besides loop αB-αB”, βB-βC loop might be investigated as another insertion site for possible divalent exposure of candidate peptides at the same time.In this study, the generation of a new icosahedral VLP carrier through the cloning and expression of the GFLV CP and GFLV L2 genes in NH3-QLYKTCKQAGTCPPD-COOH) was constructed by examining small epitope fragments in Protein Design Assistant . To this end, the HPV L2 epitope sequence was divided into three parts, namely QLYKT, CKQAGTC and CPPD, which were used as query sequences in ProDA. Finally, the PDB files of the described structures were used as templates to model HPV L2 epitope using MODELLER software [http://www.yasara.org/).Since the crystal structure of HPV L2 minor protein is not available, the secondary structure model of the HPV L2 epitope . pGEM-GFLV CP [E. coli DG1 . The grown colonies were tested and analysed using colony PCR, digestion analyses and sequencing to confirm GFLV CP and GFLV L2 constructs. Finally, the positive plasmids were transformed into E. coli BL21 (DE3) (New England Biolabs) using the calcium chloride transformation method.To produce pBluescript II SK-GFLV-L2 construct, GFLV L2 gene was synthesized and cloned into the -GFLV CP  and pBluE. coli strains BL21 (DE3) transformed with pET26 GFLV CP and pET26 GFLV L2 were grown overnight in Luria–Bertani (LB) medium containing 50 mg/L kanamycin. One mL of the overnight culture was added to 100 mL of fresh LB culture medium and incubated at 37 °C. When the OD600 reached a value of 0.8–1, 0.1–0.25 mM IPTG was added and the cultures were incubated at 18–37 °C for 1–4 h. Pelleted cells were collected by centrifugation and stored at − 20 °C for use in subsequent steps. The negative control was empty pET26 vector.E. coli were dissolved in 15 mL of disassembly buffer  with slow rotation during 16 h at 4 °C. The dissolved proteins were centrifuged at 16000 rpm for 15 min. The supernatants were filtered and purified by SEC using a Superdex 200 column. SEC runs were performed at a constant flow rate of 2 mL/min using the disassembly buffer  containing 2.5 M NaCl as eluent. Twenty μL of the peak fractions were analysed by SDS-PAGE. The fractions containing the purified GFLV CP and GFLV L2 were dialyzed 6 times against the dialysis buffer  and were refolded using 2 cycles of dialysis against the reassembly buffer . Finally, the GFLV VLPs concentrated using ultracentrifugation were dissolved in the virus buffer  .To solubilize GFLV CP and GFLV L2 proteins, a process of denaturing and refolding was adapted from a previous work  with somP. pastoris X-33 by electroporation as described in the Pichia Expression Kit protocol .The PCR of the GFLV CP and GFLV L2 genes were done using a primer set  and the recombinant plasmid (pPICZα-CP) with α-factor sequence was constructed by Gibson assembly method . Five-teThe PCR-positive colonies on YPDS-Zeocin™ (100 mg/mL) agar plates were cultured in 250 μL YPD medium and incubated for 48 h at 28–30 °C. Ten μL of each culture was then recultured in 240 μL fresh YPD medium and incubated at 30 °C for 24 h. This step was repeated 3 times. Ten μL of the last microplate was cultured in 240 μL fresh YPD medium with three concentrations of Zeocin™, i. e. 0.25 mg/mL, 0.5 mg/mL and 1.0 mg/mL. The microplates were incubated for 3–4 days at 30 °C until the cells grew.Sixty μL of the selected transformants were inoculated into 5 mL YPD medium and shaked at 200 rpm for 16–18 h at 30 °C. Afterwards, 20 mL of BMGY medium were inoculated with 250 μL of YPD culture and incubated as described above. When the OD600 had a value of 2–6, a centrifugation at 4800 rpm for 10 min was performed. Pelleted cells were resuspended in 20 mL of BMMY medium and incubated at 28 °C until the OD600 reached a value of 2–6. Sterile pure methanol  was then added every 24 h to maintain induction. Every 12 h, 1 mL of the induced culture was sampled and 1 mL of BMMY medium was added to keep the original volume of the culture, for the determination of the protein expression level in the supernatant by SDS-PAGE and Western blotting. All materials used in the culture media were purchased from Sigma Aldrich .P. pastoris containing the expressed protein were first centrifuged  for 30 min. The supernatants were centrifuged at 73,360 g  for 2 h to pellet VLPs [E. coli, the sucrose cushion method described above was also applied.The cultures of let VLPs . VLPs weThe proteins expressed in both systems were analyzed in a 12% SDS-PAGE gel followed by Western blot . Western blotting analysis was performed using anti-GFLV CP polyclonal antibody (1:1000)  and mouse anti-L2 polyclonal sera . The development of the signals was carried out using the Super Signal West Pico Chemiluminescent Substrate  (Amersham™ Imager 600).GFLV VLPs were analyzed by TEM . For this purpose, 10 μL of the samples, previously diluted 10–15 times in the virus buffer were placed on 400 mesh copper grids  for 2 min followed by negative staining. The mean particle diameter was determined on the basis of the analysis of 20 particles. The ISEM was performed following the procedure published by . Thirty To confirm VLP formation and to evaluate the exposition of HPV L2 epitope on the VLP surface, ELISA analysis was performed as previously described in , 56. VLPVLPs were purified by size exclusion chromatography on a 2 mL column of Sephacryl 300 using 100 mM HEPES and 0.001 M EDTA (pH 8) buffer.3D CE system . This instrument is equipped with an autosampler and a temperature control system (15–60 °C ± 0.1 °C). The detection was carried out using an on-column diode array detector. UV detection was set at 280 nm. Bare fused-silica capillaries with an internal diameter of 50 μm were purchased from Optronis . Capillaries of 48.5 cm total length (8.5 cm effective length) were dynamically coated with poly (ethylene oxide) (PEO) according to the procedure previously reported by [The experiments were performed on a HPorted by . ExperimRT-PCR test was performed to evaluate the presence or the absence of nucleic acid in GFLV VLPs. Total RNA was purified from VLPs using Trizol according to the manufacturer’s instructions  and specific primers of GFLV CP were used for the detection of viral mRNA in the VLPs.Additional file 1: Figure S1. (A) SDS–PAGE analysis of the protein expression in E. coli. Lane M: unstained PageRuler® molecular weight markers, lane 1: Total fraction of GFLV-L2, lane 2: soluble fraction of GFLV-L2, lane 3: insoluble fraction of GFLV-L2, lane 4: insoluble fraction of GFLV CP. (B) SDS–PAGE analysis after application of the solubilisation protocol. Lane M: unstained PageRuler® molecular weight markers, lane 1: pellet fraction of GFLV CP, lane 2: pellet fraction of GFLV L2, lane 3: supernatant fraction of GFLV CP, lane 4: supernatant fraction of GFLV L2. (C) SDS–PAGE analysis after sucrose cushion ultracentrifugation. Lane M: unstained PageRuler® molecular weight markers, lane 1: GFLV CP VLPs, lane 2: GFLV L2 VLPs. The proteins of interest are indicated by an arrow. Gels were stained with coomassie blue.Additional file 2: Figure S2. (A) SDS-PAGE analysis of the GFLV VLP samples expressed in P. pastoris. Lane M: unstained PageRuler® molecular weight markers, lane 1: clone A1 of GFLV CP, lane 2: clone A2 of GFLV CP, lane 3: clone A3 of GFLV L2, lane 4: clone A4 of GFLV L2, lane 5: negative control (P. pastoris with pPICZα empty vector). (B) SDS–PAGE analysis of the GFLV VLP samples expressed in P. pastoris after sucrose cushion ultracentrifugation. Lane M: unstained PageRuler® molecular weight markers, lane 1: clone A1 of GFLV CP, lane 2: clone A3 of GFLV L2. Gel was stained with coomassie blue.Additional file 3: Figure S3. Size exclusion chromatography (SEC) analysis of GFLV VLPs using a Sephacryl 300 column (absorbance at 280 nm). (A) SEC analysis of the VLPs produced in E. coli, a GFLV CP VLPs, b GFLV L2 VLPs. (B) SEC analysis of the VLPs produced in P. pastoris,a GFLV CP VLPs, b GFLV L2 VLPs.Additional file 4: Figure S4. Analysis of RT-PCR products of GFLV CP and GFLV L2 mRNA by agarose gel electrophoresis. Lanes 1–2: nucleic acids extracted from 200 μL GFLV CP and GFLV L2 VLPs samples from E. coli, lanes 3–4: nucleic acids extracted from 200 μL GFLV CP and GFLV L2 VLPs samples from P. pastoris. Lane 5: positive control (i.e. nucleic acids extracted from 0.1 g leaf tissue infected by GFLV), lane 6: negative control (E. coli with pET26 empty vector used for expression steps), lane M: DNA molecular weight marker (1 kb).


We produced and isolated tobacco mosaic virus-like particles (TMV VLPs) from bacteria, which are devoid of infectious genomes, and found that they have a net negative charge and can bind calcium ions. Moreover, we showed that the TMV VLPs could associate strongly with nanocellulose slurry after a simple mixing step. We sequentially exposed nanocellulose alone or slurries mixed with the TMV VLPs to calcium and phosphate salts and utilized physicochemical approaches to demonstrate that bone mineral (hydroxyapatite) was deposited only in nanocellulose mixed with the TMV VLPs. The TMV VLPs confer mineralization properties to the nanocellulose for the generation of new composite materials. There is much demand for natural renewable biomass derived nanomaterials not only due to their revalorization of waste streams, but also their broad range of customizable physical and chemical properties which can be deployed to different market sectors. One such nanomaterial are nanocelluloses which can be derived from celluloses of waste herbaceous plants, wood and bacteria, using a plethora of different chemical, mechanical, and enzymatic disruption methods. They are typically inexpensive to produce from waste materials, have low toxicity, biodegradability and biocompatibility, and are thus safe and environmentally friendly materials . These nFrom the perspective of medicine and biosciences the tunable chemistries of nanocelluloses have been exploited to incorporate reactive biologics that may be utilized as new nanomaterials for controlled drug release, wound repair, tissue engineering, enzymatic bioreactor formulations, and antimicrobial components. Incorporation of biologics into nanocellulose in some situations may be a simple mixing process which could just rely on factors such as pH, surface charge, and hydrophobicity to facilitate attachment. For example, lysozyme mixed with cationic nanocellulose fibres had increased retention, stability, and enhanced antibacterial activities when compared with lysozyme mixed with unmodified or anionic nanocelluloses . Integra10(PO4)6(OH)2), after exposure to precursors such as CaCl2 at non supersaturating concentrations. Such deposition was not observed with unmodified nanocelluloses which indicates that the hydroxyl groups are not reactive enough to grow HA crystals [2+ ions in the salt, producing nuclei for seeding HA crystal growth [In addition to incorporating proteins into the nanocellulose matrices, there has been a wide interest in recent years in coating these structures with bone minerals such as hydroxyapatite (HA). These nanocellulose-HA materials have potential bone repair applications ,11 and ccrystals ,15. It w2+ ions. We observed that these VLPs could robustly integrate into nanocellulose matrices via a simple mixing step and that they could confer HA mineralizing properties to the nanocellulose upon exposure to calcium and phosphate salts. Chemical analysis confirmed that the crystalline structures on the nanocellulose/VLPs were HA. Thus non infectious virus derived structures can be utilized to alter the properties of nanocellulose to facilitate bone mineral deposition for formation of new composites.In this paper we describe a novel approach for the facilitated deposition of HA onto plant nanocelluloses, a substrate which would not typically support HA nucleation, by incorporating virus-like particles (VLPs) derived from the Tobacco mosaic virus (TMV) coat protein subunit (CP). TMV VLPs can be easily generated by expressing in bacteria a modified CP (E50Q and D77N mutations) which can assemble into 18 nm diameter, 100–1000 nm length rods in the absence of viral RNA . These pBacteria which expressed the TMV CP subunit with the modified E50Q and D77N , which i2+ and subsequent nucleation of Ca2+ and phosphates to form HA [2+; this was subsequently assessed using isothermal titration calorimetry (ITC). The ITC binding curves were produced by monitoring temperature changes in a reaction vessel containing 0.05 mM TMV VLPs in water, to which 1 mM CaCl2 was periodically added over time (Ka = (0.25 ± 0.05) × 104 M−1) is reminiscent to that observed in moderately strong enzyme-substrate and enzyme-inhibitor interactions.It has previously been reported that negative surface charges can promote binding of Ca form HA . We measver time . From th2+ binding functionality could be introduced into nanocellulose, in order to produce a new material which has potential mineralization properties. For this process it is crucial that the VLPs are able to closely interact with the nanocellulose, and this was tested initially by mixing the TMV VLPs with the nanocellulose, washing three times, and then examining the association under TEM. It was found that the unmixed nanocellulose samples comprised long micron-sized filamentous strands of diverse thickness  × 104 M−1). In toto this demonstrates a strong association and integration of the VLPs into the nanocellulose matrix.To more robustly characterize these interactions ITC was performed on nanocellulose mixed with the VLPs  and X-ray diffraction (XRD) approaches. Under TEM it was found that the nanocellulose only sample exposed to the HA precursors had the characteristic filamentous structure  on the XRD data was used to extrapolate the sizes of the hydroxyapatite crystals and it was found the TMV VLPs/nanocellulose had average HA crystal grain sizes of 8 nm, which is of a similar magnitude to the measurements obtained using microscopy. Thus incorporation of non-infectious TMV VLPs into the nanocellulose network promotes its hydroxyapatite mineralization after exposure to precursor salts.The XRD characterization  of these2+), comprising C-terminal carboxyls, aspartic acid and glutamic acid residues . Plasmids were transformed into BL21(DES)pLysS chemically competent cells  by following the manufacturers recommendations.For the TMV VLPs, the TMV E50Q and D77N group modified coat protein sequence  was cod600 of 0.5. Cultures were then incubated overnight at 20 °C with shaking at 200 rpm. After centrifugation at 4000× g for 15 min, the supernatant was removed, and the bacterial cells were lysed by freeze-thawing 3 times using liquid nitrogen and a 37 °C water bath. Crude lysates were mixed with B-PER Complete reagent  at room temperature for 30 min prior to centrifugation at 10,000× g for 20 min. Six parts of this supernatant was mixed with 1 part chloroform for 10 min at 4 °C, and then centrifuged at 10,000× g for 10 min at 4 °C. To the upper phase PEG 8000 and NaCl were added to a final concentration of 2% (w/v) and 1% (w/v) respectively. This was left overnight at 4 °C, and then centrifuged at 10,000× g for 20 min at 4 °C. The VLP pellet was resuspended in 25 mM tris-HCl (pH 7.8), centrifuged at 10,000× g for 10 min at 4 °C to clarify and the supernatants were retained. Supernatants were loaded onto a 20% sucrose 25 mM tris-HCl (pH 7.8) cushion, which was then centrifuged at 175,500× g in a SW41 rotor at 4 °C for 2 h. Virus pellets were resuspended in 0.01 M Tris-HCl (pH 7.8), centrifuged at 175,500× g in a SW41 rotor at 4 °C for 2 h, prior to resuspension in deionized water.Bacteria were grown at 37 °C with shaking at 200 rpm in LB media supplemented with 100 µg/mL ampicillin and 34 µg/mL chloramphenicol. IPTG was added to a final concentration of 0.1 mM, once cultures reached an ODCrude bacterial lysates were mixed 1:4 parts with 4× Laemmli loading buffer and then boiled for 10 min to denature the proteins. Twenty microliters of each of these samples were loaded onto 15% SDS-PAGE gels, along with 15 μL of PageRuler Plus prestained ladder (ThermoFisher Scientific). Gels were run at 200 V for a 45 min and then stained in Quick Blue Coomassie stain  prior to photography.Nanocellulose formulation 300315-M 0.69% was obtained from CelluComp .2 and then to 125 µL of 60 mM Na2HPO4. The reactions were allowed to progress overnight prior to analysis.Two hundred and fifty microliters of nanocellulose (0.69%) was combined with 20 µL of VLPs (20 mg/mL) and this was added to 125 µL of 100 mM CaClw/v) uranyl acetate. After incubation for 1 min, the grids were wicked and left to air dry. VLPs were then observed in a JEOL 1400 transmission electron microscope  at 80 kV.Ten microliter volumes of neat, 1:10 and 1:100 dilutions of VLPs, nanocellulose or VLPs with nanocellulose were pipetted on to 200 mesh carbon coated copper grids and incubated for 30 s. Grids were wicked by using Whatman 3MM blotting paper and washed with 20 µL deionized water prior to application of 20 µL of 2%  operating at 100 kV.Kα = 1.5418 Å) operating at 30 kV and 20 mA (wavelength). The diffraction patterns were collected over a 2θ range from 20° to 60°. The Scherrer equation (Dhkl= 0.9λ/Bcosθhkl) was used to estimate crystal sizes.Dry powders of the samples were examined by X-ray diffraction in a DRON-3M X-ray diffractometer   with sample temperatures maintained at 0.1 °C using a Peltier thermostatting system. Zeta potential measurements were performed on 1 mL of 0.15–0.3 mg/mL concentrations of TMV VLPs in folded capillary cells .2+, VLPs (in deionised water) were placed in a cell in the ITC apparatus at a molarity of 0.05 mM  and 1 mM CaCl2 was used as an injectant. Data was recorded over a 3 h period. VLP and nanocellulose interactions were carried out in a similar manner except that nanocellulose (10 mg/mL) was placed in the cell and 5 mg/mL of VLPs was used as the titrant.ITC assays were conducted with a MicroCal VP-ITC . For assessment of interaction of VLPs with Ca