However, the instability of CPPs and mRNAs has limited their application. Bacteriophage PP7 virus-like particles (VLPs) may protect peptides and RNAs from degradation through displaying foreign peptides on their surface and encapsidating RNA linked with the Escherichia coli BL21 (DE3) with high yield and thermal stability, and were easily purified. The VLPs were also non-replicative, non-infectious, and non-toxic. Moreover, they penetrated the mouse prostate cancer cells RM-1 after 24 h incubation. Last, PP7 VLPs carrying the LMWP could encapsidate the GFP mRNA, which was translated into mature protein in mammalian cells.In this study, the cDNA of the PP7 coat protein single-chain dimer carrying low molecular weight protamine (LMWP) and the cDNA of green fluorescent protein (GFP) were inserted into two multiple cloning sites of pETDuet-1, respectively. PP7 VLPs carrying the LMWP peptide and GFP mRNA were subsequently expressed in Recombinant PP7 VLPs can be used simultaneously as a targeted delivery vector for both peptides and mRNA due to their abilities to package RNA and display peptides.The online version of this article (doi:10.1186/s12896-016-0274-9) contains supplementary material, which is available to authorized users. Cell-penetrating peptides (CPPs) are short positively charged peptides containing less than 30 amino acid residues. They exhibit low toxicity and have the capacity to cross cellular membranes by energy-dependent and/or independent mechanisms , 2. In tSeveral factors currently limit the preclinical and clinical development of CPPs. Fist, CPPs require custom synthesis, are expensive, and can be easily degraded by intracellular or extracellular proteases . Second,Escherichia coli (E. coli) strains such as CSH41F [Bacteriophage PP7 virus-like particles (VLPs) may be a promising tool in meeting the above requirements as a CPP delivery vehicle because PP7 VLPs can be expressed in s CSH41F . Additios CSH41F , 13. Spes CSH41F . These ps CSH41F . The inss CSH41F . Furthers CSH41F .E. coli strain BL21 (DE3). We also evaluated the cell-penetrating ability of the inserted LMWP and analyzed whether PP7 VLPs could package the foreign mRNA.To date, the PP7 VLPs have been used as peptide vaccine carriers against the human papillomavirus  and malaKpnI  with high-efficiency; however, the fusion of the peptide to PP7 coat protein and the inserted GFP gene in the second multiple cloning sites (MCSs) of the plasmid pETDuet-2PP7-Protamine decreased the expression of the PP7 VLPs.The VLPs were then purified by size-exclusion chromatography Fig. . The wesTo confirm that the purified products successfully assembled into PP7 VLPs, western blotting and TEM were performed. The western blotting results showed only one specific band appearing in every lane, which demonstrated that all three recombinant VLPs could be detected by the murine antibody of the anti-coat protein of PP7 phage Fig. . In addiThe chromatogram showed nucleic acid was present in the first peak, indicating that nucleic acids were packaged by the PP7 VLPs Fig. . To veriFollowing the above assay, the DNAs or RNAs packaged by these VLPs were further verified by RT-PCR. The results showed that the RNA and not DNA was the nucleic acid packaged by these VLPs Fig. . MoreoveThe cytotoxicity of 2PP7, 2PP7-Protamine, and 2PP7-Protamine-GFP VLPs was examined in the African green monkey kidney fibroblast cell line Cos-7 using CCK-8 assay. The results showed no dose-dependent cytotoxicity in these three VLPs Fig. . Even atP < 0.01, Fig. At the same time, the thermal stability of these three VLPs was determined by measuring the protein concentration at different temperatures. The results showed that the concentrations of 2PP7, 2PP7-Protamine and 2PP7-Protamine-GFP VLPs decreased significantly at above 60 °C  and incubated with cells from the mouse prostate cancer cell line RM-1. After 12 h, the RM-1 cells incubated with 2PP7-Protamine VLPs exhibited a high level of fluorescence Fig. . In contWe investigated whether the GFP mRNA packaged by the PP7 capsid carrying the LMWP peptide could be translated into mature protein in RM-1 cells. After 24 h of incubation with 2PP7-Protamine-GFP VLPs, most of the RM-1 cells incubated with 2PP7-Protamine-GFP VLPs exhibited green fluorescence Fig. , which wP < 0.001). Moreover, in this group, the level of GFP mRNA at 48 h was significantly higher than that at 24 h. All these results showed that PP7 VLPs carrying the LMWP peptide could penetrate cell membrane and that the mRNA packaged by them was translated into mature protein in mammalian cells.Additionally, the levels of GFP mRNA level in the RM-1 cells were verified by quantitative real-time PCR (qPCR) at 24 h and 48 h Fig. . As showin vivo application of mRNAs, including the low in vivo delivery efficiency of mRNA, their lack of cell-type specificity, and instability. In this study, we introduced a delivery system based on PP7 VLPs and a cell-penetrating peptide as a solution to the above limitations of mRNA-targeted delivery.Molecules such as mRNAs have been used for the prevention and treatment of various diseases –21, inclIn order to improve the delivery efficiency of mRNA, the PP7 VLPs were decorated with a non-toxic cell-penetrating peptide, LMWP. This peptide, similar to the peptide TAT, enhanced the intracellular delivery of the linked particles without any dependence on receptors, temperature conditions, or energy use and without causing any alterations to the cell membrane –24. The pac site [, and our results showed PP7 capsids could also do this. As was expected, we found that PP7 VLPs carrying the cell-penetrating peptide LMWP packaged a specific mRNA linked with the pac site and protected the RNA from rapid extracellular degradation by RNase. Furthermore, the PP7 VLPs carrying the cell-penetrating peptide LMWP and GFP mRNA efficiently penetrated the cell membrane. To date, the greatest length of RNA packaged using one wild-type pac site in MS2 VLPs fused with a peptide was 970 bases [E. coli. The size and characteristics of the foreign RNA that were packaged by the peptide fused to the PP7 coat protein will therefore need to be studied further.Previous studies have shown that MS2 capsids specifically encapsidate specific mRNAs that are linked to the MS2 pac site –27, and 70 bases ; the lenIn addition, the mRNA packaged by the PP7 VLPs showed a high level of protein expression in mammalian cells within 24 h. This result was not in accordance with that observed for the naked mRNA vaccine, which showed maximum expression within 12–18 h , 30. ThiIn 2011, Caldeira and Peabody proved that PP7 VLPs with a peptide insertion were generally stable up to about 70 °C . In thisE. coli with high efficiency, are easily purified, and are nuclease-resistant, non-replicative, nontoxic, and noninfectious.In summary, our current study demonstrates that PP7 VLPs carrying a cell-penetrating peptide and mRNA can be expressed in The current study provides a stable and efficient mRNA delivery system that is based on the inherent abilities of PP7 VLPs to encapsidate mRNA and tolerate foreign peptides. Moreover, the recombinant PP7 VLPs carrying the CPP and mRNA can be easily expressed in a prokaryotic expression system at high yield, are nuclease-resistant, non-replicative, nontoxic, and non-infectious. In addition, the mRNA packaged by these VLPs showed a high level of protein expression in mammalian cells. In conclusion, this system holds great potential in the development of mRNA and peptide-based targeted therapeutics to be used for the prevention and treatment of cancer and other diseases.KpnI site was amplified by the overlap extension PCR, and inserted into restriction enzyme sites NdeI and XhoI of the vector pETDuet-1 . This constructed plasmid was named as pETDuet-2PP7, which was finally verified by sequencing analysis .The single-chain dimer gene of PP7 phage coat protein (accession number: NC_001628.1) with one KpnI and XhoI , and ligated with the plasmid pETDuet-2PP7. After that, the plasmid pETDuet-2PP7-Protamine-GFP was constructed with GFP DNA (accession number: NC_025025.1) inserted between XbaI and BamHI of the plasmid pETDuet-2PP7-Protamine. The correct construct was verified by sequencing analysis. The sequence of all primers are listed in Table The plasmid pETDuet-2PP7-Protamine was constructed based on the plasmid pETDuet-2PP7. Briefly, the gene containing LMWP was amplified by PCR using primers 2PP7-Protamine-for and PP7-r Table , digeste600 of approximately 0.6 at 37 °C, isopropyl thio-β-D-galactoside (IPTG) was added with a final concentration of 1 mM and these cultures were incubated overnight at 37 °C. The cells were harvested using centrifugation at 12,000 rpm for 1 min at 4 °C, resuspended in a final volume of 30 mL TBS , and disrupted by sonication  on ice 200 times . The homogenate was centrifuged at 12,000 rpm, 4 °C for 30 min. The supernatant was collected, and 30 mL of chloroform was added and mixed with the solution. The mixture was centrifuged at 12,000 rpm, 4 °C for 15 min, and the supernatant was collected. The nanoparticles in the supernatant were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) on 12 % gels. Then the supernatant was incubated with DNase I (20 U/mL) and RNase A (10 mg/mL)  at 37 °C for 3 h, and purified by Bio-Gel A-1.5 m gel  filtration chromatography .The plasmids mentioned above were isolated from 2 mL overnight cultures of single colonies using a QIAprep Spin Plasmid Kit (Qiagen) and separately transformed into the competent BL21 (DE3) cells. The transformed cells were cultured in 5 ml of LB medium supplemented with ampicillin (50 mg/L). The precultures were used to inoculate 500 mL of LB medium supplemented with ampicillin. When these cultures were grown to an ODThe purified products were verified by western blotting. The purified VLPs was transferred from 12 % SDS-PAGE gels onto a 0.45 μm polyvinylidene fluoride (PVDF) membrane in the buffer containing 25 mM Tris-HCl (pH 8.3), 192 mM glycine, 20 % methanol, and blocked with 5 % fat-free dry milk in phosphate-buffered saline (PBS) for 1 h. These membranes were incubated with anti-PP7 coat protein (prepared using wild-type PP7 phage coat protein as the immunogen by our lab) overnight at 4 °C and with HRP-labeled secondary antibodies  at 37 °C for 1 h in order. The signal was detected using the Image Lab software .The recombinant PP7 VLPs were adsorbed on carbon-coated glow-discharged copper grids for 2 min, and were negatively stained with 2 % uranyl acetate for 2 min. The VLPs were visualized using a Hitachi H7500 (Japan) transmission electron microscope at a magnification of 100,000 × .The purified 2PP7 VLPs, 2PP7-Protamine VLPs and 2PP7-Protamine-GFP VLPs (800 μg/ml) were separately mixed with DNase I (20 U/mL) and RNase A (10 mg/mL), incubated at 37 °C for 12 h, and verified by 1 % agarose gel electrophoresis.RNA isolation was performed using TRIzol  according to the manufacturer’s instructions. To identify of the nucleic acid packaged by 2PP7-Protamine VLPs and 2PP7-Protamine-GFP VLPs, respectively, ordinary PCR or reverse transcription-polymerase chain reaction (RT-PCR) was performed with the appropriate primers and templates at 94 °C for 5 min, followed by 30 cycles of 10 s at 98 °C, 30 s at 60 °C, and 60 s at 72 °C, and followed by 7 min at 72 °C. 2PP7 gene and GFP gene were amplified using primers PP7-for paired with PP7-r and GFP-for paired with GFP-r, respectively. The PCR products were verified by 1 % agarose gel electrophoresis with ethidium bromide staining, then purified and ligated into the pMD18-T plasmid  for further verification by sequencing.4/well) were incubated with various concentrations of PP7 VLPs  for 24 h or 48 h in triplicate. 10 μL CCK (cell counting kit)-8 solution  was carefully added to each well 2 h before detection. The absorbance of each well was measured at 450 nm with an ELISA plate reader .Cos-7 cells , according to the manufacturer’s instructions. Briefly, 50 μL of 1 mg/mL FITC was mixed with 950 μL of 2 mg/mL 2PP7 or 2PP7-Protamine VLPs in a dialysis bag . The mixture was dialyzed in 0.1 M carbonate-bicarbonate buffer (pH 9.0) at 4 °C in darkness for 12 h. After that, the mixture was dialyzed in 2 L of PBS at 4 °C for 12 h. The FITC-labeled 2PP7-Protamine VLPs were purified using Sephacryl S-200 gel exclusion chromatography. Following that, the purified VLPs were added to 1 × 10The thermal stability of VLPs was determined by measuring the fraction of proteins that remained soluble after 2 min at a given temperature . 25 μL o4 RM-1 cells in 24-well plates in triplicate. After 24 h, the expression of GFP protein was monitored firstly by a fluorescence microscope. Then the cells were stained with red fluorescent reactive dye  and DAPI  in order before detection. The expression of GFP protein was further monitored by confocal laser scanning microscopy .The 2PP7-Protamine-GFP VLPs (100 nM/mL) were added to 3 × 105 cells/well was cultured. The 2PP7-TAT-23b or 2PP7-TAT-con VLPs (100nM) were then added to each well and incubated with the cells for 24 h or 48 h at 37 °C. Total RNA was extracted from the harvested cells with TRIzol. For GFP mRNA detection, the primers were designed on the base of these sequences, as previously described . Data were considered statistically significant when


To identify the share of open access (OA) papers in the total number of journal publications authored by the members of the University of Zagreb School of Medicine (UZSM) in 2014.Bibliographic data on 543 UZSM papers published in 2014 were collected using PubMed advanced search strategies and manual data collection methods. The items that had “free full text” icons were considered as gold OA papers. Their OA availability was checked using the provided link to full-text. The rest of the UZSM papers were analyzed for potential green OA through self-archiving in institutional repository. Papers published by Croatian journals were particularly analyzed.Full texts of approximately 65% of all UZSM papers were freely available. Most of them were published in gold OA journals . In the UZSM repository, there were additional 52 freely available authors’ manuscripts from subscription-based journals .The overall proportion of OA in our study is higher than in similar studies, but only half of gold OA papers are accessible via PubMed directly. The results of our study indicate that increased quality of metadata and linking of the bibliographic records to full texts could assure better visibility. Moreover, only a quarter of papers from subscription-based journals that allow self-archiving are deposited in the UZSM repository. We believe that UZSM should consider mandating all faculty members to deposit their publications in UZSM OA repository to increase visibility and improve access to its scientific output. Open access (OA) availability and searchability of new research results in medicine have a significant and positive impact, from research and education to everyday medical practice . In the Gargouri et al  found thCroatian Medical Journal, partly owned by the UZSM, has been using diamond open access model since 1997   mentionsnce 1997 .The primary aims of this study were to identify the proportion of OA papers in the total number of journal publications authored by faculty members of the UZSM and indexed by PubMed in 2014 and determine the type of OA they belong to. This was done in order to examine if UZSM follows global trends in medical OA publishing and to identify the potential differences. Since all 12 Croatian journals indexed by PubMed comply with the national policy recommending OA to the results of publicly funded research, an additional aim was to analyze their availability and visibility in a broader international context.We searched PubMed, a freely available biomedical bibliographic database that regularly covers more than 5500 medical journals. Our data set included papers from PubMed published between January 1 and December 31, 2014, in which at least one author was affiliated to UZSM. PubMed was searched by combination of the most frequently used variations of affiliation name ) and publication date (2014). We double-checked the author names in each paper retrieved using the official UZSM teaching staff list.Close examination of search results revealed the most important methodological limitation of our research. It showed that many papers published in a number of PubMed-indexed Croatian journals were not retrieved. The main reason was that the authors’ affiliation data in these records were incomplete. We browsed all PubMed records from Croatian journals published in 2014 and manually added papers authored by UZSM teaching staff to the initial search results. Since there could be more records with incomplete affiliation data in PubMed, we can only assume that most PubMed papers published by the UZSM in other indexed journals were included in our study.The final set of UZSM affiliated papers was searched again using the PubMed filter “free full-text.” Each retrieved record that had “free full text” or PubMed Central (PMC) icons was considered as an OA paper. We checked the provided link to determine whether the full text was freely available.In the second stage of our study, we investigated the rest of the UZSM papers. In order to find out how many of them are potentially available via green OA, we investigated copyright and self-archiving policies of the respective journals. We used journals’ web pages and Sherpa-Romeo, a searchable database of publishers ' policies regarding self-archiving of journal papers on the web and in OA repositories . We founThe combination of PubMed search and browsing methods resulted in 543 records of papers published in 2014 with at least one author affiliated to the UZSM. Two thirds of these papers (360 or 66.3%) were published in foreign medical journals and one third (183 or 33.7%) in 10 of 12 Croatian journals .Croatian Medical Journal, Biochemia Medica, and Psychiatria Danubina). The rest were published by international OA journals: 88 papers in gold OA journals (59.1%) and 23 papers (15.4%) in hybrid journals, mainly by prominent publishers, like Elsevier, Oxford University Press, Springer. etc. All of these papers had a direct PubMed link to their full-text version either in the PubMed Central archive or on the journals’ web pages.The repeated search using the “free full-text” filter found 149 OA papers . Out of them, 38 papers (25.5%) were published by Croatian OA biomedical journals . According to Sherpa-Romeo and journal publishers’ web pages, authors of 196 papers were allowed to archive the accepted manuscript of their papers  on their own websites and/or institutional repositories. This represents 80.7% of all UZSM papers published in subscription-based journals. Many of the papers available for green OA in our study were published by major commercial publishers like Elsevier or Springer, and most of them allow public release of full texts after an embargo period. The embargo period for scientific, technical, and medical fields varies from 6 to 36 months, but it is most often 12 months. At the time we started this research, there were 52 authors’ manuscripts from subscription-based journals archived and freely accessible in the UZSM institutional repository.Altogether, 352 OA UZMS papers (64.8%) published in 2014 had full texts available across different types of outlets . KeepingThe overall proportion of OA papers in our study was 64.8%. It is higher than in a study examining OA availability in the members of European Research Area and four additional countries . In the Also, the share of gold OA in our study (55.2%) is greater than in a similar study analyzing real and potential OA papers produced by university medical departments, where gold OA proportion varied between 27.8 and 42.1% .However, when we compare our results with the share of records with a “free full text” link among all PubMed indexed papers published in 2014, the situation is not so satisfactory. The percentage of UZSM papers with a “free full text” link was 27.4%, and the respective share for all PubMed-indexed papers was 37.6%. In fact, only half of UZSM gold OA papers are accessible directly via PubMed. Moreover, although papers published by Croatian journals were 100% OA, only 20.8% had the PubMed “free full text” link.Considering that more than half of the analyzed papers (59.3%) are written in English, it is against the interests of both authors and journals that international PubMed users’ community has no indication of their free and open accessibility. Moreover, it is also unfavorable for the visibility and impact of papers written in the Croatian language, because open access can reduce language barriers by eliminating restrictions on re-publication and re-use (including translations) .One of the reasons why nearly all Croatian scientific journals are open access may be found in their funding model. They are all supported by the governmental funds following criteria set for each scientific domain . In the However, is such a high proportion of OA a reflection of authors’ choice? With the exception of those UZSM authors who are recipients of the international research grants subject to a mandate of OA publishing or archiving, we believe that our authors do not select journals for publishing their papers according to journals’ access model. Though OA to journal papers increases their visibility and impact ,26, authFinally, could the rate of OA at UZSM be even higher? Some of UZSM authors participate in contemporary information sharing practices of academic and research community by posting full texts of their papers in institutional repository (green OA) or on social networking sites. However, most of our authors are still not aware of all OA benefits. This research showed that only 26.5% full texts of papers from subscription-based journals that allow open access are deposited in the UZSM repository. This corresponds to the results of Gargouri et al , who fou


Acting on the glucocorticoid receptor (NR3C1), glucocorticoids are widely used to treat inflammatory diseases. However, glucocorticoid resistance often leads to suboptimal asthma control. Since glucocorticoid-induced gene expression contributes to glucocorticoid activity, the aim of this study was to use a 2×glucocorticoid response element (GRE) reporter and glucocorticoid-induced gene expression to investigate approaches to combat cytokine-induced glucocorticoid resistance. Pre-treatment with tumor necrosis factor-α (TNF) or interleukin-1β inhibited dexamethasone-induced mRNA expression of the putative anti-inflammatory genes RGS2 and TSC22D3, or just TSC22D3, in primary human airway epithelial and smooth muscle cells, respectively. Dexamethasone-induced DUSP1 mRNA was unaffected. In human bronchial epithelial BEAS-2B cells, dexamethasone-induced TSC22D3 and CDKN1C expression (at 6 h) was reduced by TNF pre-treatment, whereas DUSP1 and RGS2 mRNAs were unaffected. TNF pre-treatment also reduced dexamethasone-dependent 2×GRE reporter activation. This was partially reversed by PS-1145 and c-jun N-terminal kinase (JNK) inhibitor VIII, inhibitors of IKK2 and JNK, respectively. However, neither inhibitor affected TNF-dependent loss of dexamethasone-induced CDKN1C or TSC22D3 mRNA. Similarly, inhibitors of the extracellular signal-regulated kinase, p38, phosphoinositide 3-kinase or protein kinase C pathways failed to attenuate TNF-dependent repression of the 2×GRE reporter. Fluticasone furoate, fluticasone propionate and budesonide were full agonists relative to dexamethasone, while GSK9027, RU24858, des-ciclesonide and GW870086X were partial agonists on the 2×GRE reporter. TNF reduced reporter activity in proportion with agonist efficacy. Full and partial agonists showed various degrees of agonism on RGS2 and TSC22D3 expression, but were equally effective at inducing CDKN1C and DUSP1, and did not affect the repression of CDKN1C or TSC22D3 expression by TNF. Finally, formoterol-enhanced 2×GRE reporter activity was also proportional to agonist efficacy and functionally reversed repression by TNF. As similar effects were apparent on glucocorticoid-induced gene expression, the most effective strategy to overcome glucocorticoid resistance in this model was addition of formoterol to high efficacy NR3C1 agonists. Acting via the glucocorticoid receptor GR; NR3C1), glucocorticoids may reduce inflammatory gene expression by directly inhibiting the activity of inflammatory transcription factors (transrepression) and by increasing the transcription of genes (transactivation) with anti-inflammatory activity , glucoco,3. In ad2-adrenoceptor agonists (LABAs) [in vitro model of glucocorticoid resistance [There are a number of potential approaches to overcoming glucocorticoid resistance: 1) increase the glucocorticoid dose; 2) reverse the resistance by inhibiting inflammatory signaling pathways; 3) identify glucocorticoids, or other NR3C1 ligands, that are not subject to resistance; and, 4) potentiate glucocorticoid activity using long-acting β (LABAs) . Alterna (LABAs) . Likewis (LABAs) . Furthersistance . A bettesistance –13. TargIdeally, glucocorticoid resistance could be overcome by identifying, if possible, novel NR3C1 ligands that are not susceptible to the mechanisms of resistance. However, in generating new glucocorticoids, pharmaceutical companies have focused on reducing side effects, producing compounds with differing potency, efficacy and metabolism ,15. ManyA further approach to combating glucocorticoid resistance could be to enhance glucocorticoid activity. Thus, the addition of a LABA potentiates the effects of ICS therapy in asthma, providing greater symptom control than doubling, or even quadrupling, the glucocorticoid dose . Furtherq-linked signaling and is bronchoprotective in vivo [Given that glucocorticoid-inducible gene expression is up-regulated in asthmatics taking ICS , we have in vivo –29. Like in vivo ,30,31, w in vivo . We eval3, 2 mM L-glutamine and 10% fetal calf serum . Primary human ASM and HBE cells were isolated from non-transplanted normal human lungs obtained using the tissue retrieval service at the International Institute for the Advancement of Medicine, Edison, NJ, USA [2/95% air and were incubated in serum- or additive-free medium overnight prior to experiments. TNF and IL1B  were dissolved in phosphate-buffered saline containing 0.1% bovine serum albumin . Dexamethasone (Sigma-Aldrich) was dissolved in Hank’s balanced salt solution (Life Technologies). Budesonide and formoterol , des-ciclesonide , PS-1145 , fluticasone furoate, fluticasone propionate, GW870086X , GF109203X, Gö6976, JNK inhibitor VIII, PD098059, SB203580 , GSK9027, PI103 , LY294002, wortmannin (Sigma-Aldrich), Ro 31–8220  and RU24858  were dissolved in dimethyl sulphoxide (Sigma-Aldrich). Final dimethyl sulphoxide concentrations on cells were ≤ 0.1%.Human bronchial epithelial (BEAS-2B) cells   were cul NJ, USA ,34. IndiBEAS-2B cells stably transfected with a glucocorticoid response element (2×GRE) driven luciferase reporter plasmid, pGL3.neo.TATA.neo , were cuTSC22D3, forward 5′-GGC CAT AGA CAA CAA GAT CG-3′, reverse 5′-ACT TAC ACC GCA GAA CCA CCA-3′; CDKN1C, forward 5′-CGG CGA TCA AGA AGC TGT C-3′, reverse 5′-GGC TCT AAA TTG GCT CAC CG-3′; DUSP1, forward 5′-GCT CAG CCT TCC CCT GAG TA-3′, reverse 5′-GAT ACG CAC TGC CCA GGT ACA-3′; RGS2, forward 5′-CCT CAA AAG CAA GGA AAA TAT ATA CTG A-3′, reverse 5′-AGT TGT AAA GCA GCC ACT TGT AGC T-3′; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward 5′-TTC ACC ACC ATG GAG AAG GC-3′, reverse 5′-AGG AGG CAT TGC TGA TGA TCT-3′.Total RNA was extracted using RNeasy Mini Kits  and 0.5 μg used for cDNA synthesis using the qScript cDNA synthesis kit . Following a 1:4 dilution, real-time PCR was performed on 2.5 μl of cDNA in 10 μl reactions using a 7900HT instrument  with SYBR GreenER chemistry (Life Technologies). Amplification conditions of 50°C, 2 min; 95°C, 10 min; then 40 cycles of 95°C, 15 s; 60°C, 1 min were used, before melt curve analysis to confirm primer specificity. Primer sequences were: Cells were lysed in 1× Laemmli buffer supplemented with phosphatase inhibitors (Sigma-Aldrich) and 1× complete protease inhibitor cocktail , size fractionated on 12% acrylamide gels and subsequently electro-transferred onto Hybond enhanced chemiluminescent membranes . Membranes were blocked in 5% milk in tris-buffered saline containing 1% tween 20 and probed with antibodies against phospho-jun proto-oncogene  , nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha  , phospho-NFKBIA , phospho-AKT1 , AKT  and GAPDH (4699–9555(ST); AbD Serotec; Raleigh, NC). After washing, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit  or anti-mouse immunoglobulin . Detection was by enhanced chemiluminescence  and visualization by autoradiography.All data are plotted as means ± SE. Statistical analyses were performed using Prism version 6.01 . Student’s t test or analysis of variance (ANOVA) with Bonferroni’s correction for multiple comparisons or a Dunnett’s post test were used as indicated.Primary HBE cells were pre-treated for 1 h with maximally effective concentrations of TNF or IL1B , before In primary human ASM cells, pre-treatment with either TNF or IL1B had no effect on dexamethasone-induced DUSP1 mRNA expression . HoweverTo evaluate the BEAS-2B human bronchial epithelial cell line as a model for cytokine-induced glucocorticoid resistance, the effect of TNF was tested on the dexamethasone-induced expression of DUSP1, CDKN1C, RGS2 and TSC22D3 at 6 h. As described for both the HBE and ASM cells, TNF pre-treatment did not affect dexamethasone-induced DUSP1 expression . DexametBEAS-2B cells harboring a 2×GRE luciferase reporter were pre-treated for 30 min with maximally effective concentrations of the inhibitors PS-1145, JNK inhibitor VIII, PD098059 or SB203580, prior to TNF treatment ,28,35,3650 of ~1 μM. Likewise, JNK inhibitor VIII decreased TNF-induced JUN phosphorylation in a concentration-dependent manner (EC50 ~0.3 μM) (50 0.6 μM) the TNF-induced repression from ~60% to 76% of the maximum produced by dexamethasone. Likewise, JNK inhibitor VIII reversed (EC50 1.6 μM) the TNF-induced repression to 87% of the response to dexamethasone  and JUN (c-Jun) . PS-1145~0.3 μM) . The eff~0.3 μM) . PS-1145ethasone . FinallyTo examine whether simultaneous inhibition of IKK2 and JNK may combine to fully reverse the repression of glucocorticoid activity by TNF, various concentrations of JNK inhibitor VIII were tested in the absence and presence of a maximally effective concentration (10 μM) of PS-1145 . However50 values of ~900, 70 and 20 nM for LY294002, PI103 and wortmannin respectively . Fluticasone furoate, fluticasone propionate and budesonide all produced similar EMax values to dexamethasone  (EMax produced by each ligand in the absence of TNF.The experiments in  = 0.71) , i.e. thr2 = 0.72)  . In the  = 0.72)  & B. Thubona fides glucocorticoid inducible genes, BEAS-2B cells were pre-treated with PS-1145 or JNK inhibitor VIII for 30 min prior to TNF addition. After 1 h, dexamethasone and/or formoterol were added and the cells harvested 6 h later for gene expression analysis , before addition of tumor necrosis factor-α (TNF). Cells were harvested after 2 min for western blotting and probed for phospho nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha , NFKBIA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). B. BEAS-2B cells were pre-treated for 30 min with the indicated concentrations of JNK inhibitor VIII (JNK), before addition of 10 ng/ml TNF. Cells were harvested after 15 min for western blotting and probed for phospho jun proto-oncogene (pJUN) and GAPDH.(PDF)Click here for additional data file.S2 FigA. LY294002 (LY), B. PI103 (PI) or C. wortmannin (W), before addition of 10 ng/ml tumor necrosis factor-α (TNF). Cells were harvested after 30 min for western blotting and probed for phospho v-akt murine thymoma viral oncogene homolog 1 (pAKT1), AKT1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH).Human bronchial epithelial, BEAS-2B, cells were pre-treated for 30 min with the indicated concentrations of (PDF)Click here for additional data file.


Such a detection limit was achieved in both linear and subthreshold regimes of MoS2 transistors. In both regimes, all sets of transistors exhibited consistent calibrated responses with respect to TNF-α concentration, and they resulted in a standard curve, from which the equilibrium constant of the antibody-(TNF-α) pair was extracted to be KD = 369 ± 48 fM. Based on this calibrated sensor model, the time-dependent binding kinetics was also measured and the association/dissociation rates of the antibody-(TNF-α) pair were extracted to be (5.03 ± 0.16) × 108 M−1s−1 and (1.97 ± 0.08) × 10−4 s−1, respectively. This work advanced the critical device physics for leveraging the excellent electronic/structural properties of TMDCs in biosensing applications as well as the research capability in analyzing the biomolecule interactions with fM-level sensitivities.Atomically layered transition metal dichalcogenides (TMDCs) exhibit a significant potential to enable next-generation low-cost transistor biosensors that permit single-molecule-level quantification of biomolecules. To realize such potential biosensing capability, device-oriented research is needed for calibrating the sensor responses to enable the quantification of the affinities/kinetics of biomolecule interactions. In this work, we demonstrated MoS The community currently lacks proper top-down planar nanofabrication processes to produce ordered arrays of such nanostructures, which makes it very challenging to realize parallel high-throughput assay using biosensors made from these nanostructures. High-quality Si NW biosensor arrays can be made using top-down lithographic techniquesUsing field-effect transistor (FET)-based biosensors created from nanowires (NWs) and carbon nanotubes (CNTs), researchers have demonstrated detection of cancer biomarkers from nM to fM range in serum123456e.g., Si and III-V compounds), the transistors based on MoS2 and other atomically layered semiconductors are expected to exhibit much more sensitive electrical responses to antigen-antibody binding events. Furthermore, all 2D layers have an extremely low density of dangling bonds on their surfaces, which can result in high-quality FET channels with low densities of scattering centers (and hence low Flicker noise level), and enable highly sensitive, low-noise-level detecting of biomolecules27313233et al. have demonstrated graphene-based FET sensors capable of detecting individual gas molecules absorbed on the graphene channels35Emerging two-dimensional (2D) atomically layered materials, such as graphene, topological insulators (TIs), and TMDCs, recently attracted a great deal of interest because of their attractive electronic/optoelectronic properties, large abundance, and compatibility to planar nanofabrication processes141516171819202122232426272829e.g., MoS2) have sizable bandgaps. Therefore, TMDC-based FETs exhibit high On/Off current ratios up to 108, which, in combination with their atomically thin structures, can potentially enable the higher detection sensitivities for gas, chemical, and biological sensing applications in comparison with graphene FETs3738et al. and Sarkar et al. recently demonstrated that FET biosensors made from microscale few-layer-MoS2 flakes exhibit 100–400 fM LODs for detecting cancer-related biomarkers39e.g., glass, plastic, or rubber) by using cost-efficient processes such as CVD followed with roll-to-roll transfer4344In contrast to zero-bandgap graphene, semiconducting TMDCs  as well as the affinities/kinetics of biomolecule interactions. Although individual MoS2-based transistor biosensors and demonstrated that these devices can be synergistically utilized to measure the concentrations of analyte solutions as well as the affinity and kinetic properties of the analyte-receptor pair. The biomolecule under study, TNF-α, is a pro-inflammatory cytokine and a key biomarker associated with host defense and immunosurveillance464748502 transistors. In both transport regimes, the electrically measured sensor responses were calibrated into signal quantities independent of the transistor performance. All sets of transistor biosensors exhibited consistent relationships between calibrated sensor responses and TNF-α concentrations. They generated a standard curve, from which the equilibrium constant of the antibody-(TNF-α) pair was extracted to be KD = 369 ± 48 fM. Based on this calibrated sensor model, the time-dependent association-dissociation kinetics of the antibody-(TNF-α) pair was further studied and the association/dissociation rates of the antibody-(TNF-α) pair were measured to be (5.03 ± 0.16) × 108 M−1s−1 and (1.97 ± 0.08) × 10−4 s−1, respectively.In this work, we fabricated multiple sets of MoS2 sensing channel. First, a pristine few-layer MoS2 flake is printed onto a p+-doped Si substrate coated with 300 nm thick SiO2 522 printing, metallic drain/source (D/S) contacts (5 nm Ti/50 nm Au) are fabricated using photolithography followed with metal deposition and lift-off, and a back-gated MoS2 transistor is subsequently formed   pair, a large open liquid reservoir made from polydimethylsiloxane (PDMS) is integrated on top of the MoS2 transistor  . This Hfmponents . Before ansistor . Such a ansistor . Such a 2 transistor with channel length (L) and width (W) of 5 and 6 μm, respectively. 2 transistor biosensor integrated with a PDMS liquid reservoir. The reservoir is ~4 mm deep and is ~1 mm in diameter, which is drilled into a PDMS block with length, width, thickness of 2, 1, and 0.4 cm, respectively. i.e., drain-source current (IDS) – back gate voltage (VG) curves acquired under a fixed drain-source voltage (VDS)) were measured at each of the biodetection stages, following the sequence of (1) bare transistor, (2) antibody functionalization, and inputs of TNF-α solutions with concentrations of (3) 60 fM, (4) 300 fM, (5) 600 fM, (6) 3 pM, and (7) 6 pM. To eliminate the effect of the IDS-VG hysteresis, all IDS-VG curves were measured by sweeping VG from -100 V to 100 V with a sweep rate of 10 V/s. Other details about different biodetection stages and transistor characterizations are described in the Method and Material section.First we measured the sensor responses from different TNF-α concentrations under the thermodynamic equilibrium condition. The biosensor setup shown in 2 transistor sensors. Specifically, IDS data are plotted in the linear scale. The transfer characteristics of this sensor exhibit a strong dependence on TNF-α concentrations, and the TNF-α detection limit is estimated to be ~60 fM. We choose a fixed VG within the linear regimes of all IDS-VG curves  and threshold voltage (VT)). Therefore, in the analysis of a given biodetection state, the IDS signals acquired by different MoS2 transistors may exhibit a poor device-to-device consistency due to the nonuniformity of MoS2 transistors. Although such an issue could be mitigated through optimizing the material deposition and device fabrication processes, a calibrated sensor response quantity independent of the device performance is highly desirable.IDS-VG characteristic curve measured from a microscale MoS2 transistor sensor in a specific biodetection state can be expressed as Equation gm values extracted from different IDS-VG curves that correspond to different biodetection states are very close and can be approximated as a constant for this sensor. For example, the gm value of the sensor shown in VDS = 1 V. Based on this observation and Equation et al. and Ishikaw et al.10S) is derived and expressed in Equation IDS(anti) is the IDS value measured in the “antibody functionalization” state of a sensor biased under a set of fixed VDS and VG, and IDS-IDS(anti) indicates the IDS variation induced by the introduction of TNF-α molecules. Such an IDS variation normalized by the gm of this sensor results in a sensor response quantity directly related to the change in the VT of the sensor . It should be noted that although ΔVT is assumed to be completely induced by the charge brought to the HfO2 effective layer on top of the transistor channel through antibody-(TNF-α) binding events, ΔVT is not exactly the binding-event-induced potential change (ΔΦ) on the effective layer. This is because in this work, ΔVT is the change in the VT measured from the back gate. However, ΔVT and ΔΦ can be related by ΔVT = (CHfO2/CSiO2)ΔΦ, where CSiO2 and CHfO2 are the capacitances of the SiO2 back gate dielectric and the HfO2 effective layer, respectively. More detailed discussion about these potential parameters can be found from the dual-gate transistor model illustrated in Fig. S2. Based on this model, ΔVT can be evaluated using q is the effective charge carried by a TNF-α molecule (the screening effect due to the buffer liquid has been incorporated into q); dSiO2 and kSiO2 are the thickness and dielectric constant of the SiO2 back-gate dielectric layer, respectively; ε0 is the vacuum permittivity; and σTNF is the areal density of TNF-α molecules bound to the antibody receptors functionalized on the effective layer. Therefore, such a calibrated response quantity (S) is proportional to the antibody receptor occupancy at the equilibrium state and it is also independent of the MoS2 transistor performance. These two conditions are critical for the subsequent Langmuir isotherm analysis.The linear regime of an n). The detailed transfer characteristics of these five devices measured at various biodetection stages are displayed in Fig. S3. Although Fig. S3 shows that the transfer characteristics of these five sensors exhibit significant difference in VT, IDS and gm, i.e., a generic S-n curve) for TNF-α detection. This standard curve can be well fitted with Langmuir isotherms (Equation D) of the antibody-(TNF-α) pair is extracted to be 369 ± 48 fM; the maximum sensor response (Smax) is extracted to be 10.7 ± 0.4 V.Equation  and the 2 transistor sensors. In the subthreshold regime of a transistor sensor, the sensitivity of IDS to the variation of electrical potential (or charge) at the effective layer is much higher than that in the linear transport regime of this sensor. Therefore, the responses from the subthreshold regimes of transistor sensors are expected to result in the higher biodetection sensitivity in comparison with those from the linear regimes. 2 transistor sensor, which were measured at various biodetection stages. Here IDS data are plotted in the logarithm scale, and the subthreshold regimes are emphasized. We choose a fixed VG within the subthreshold regimes of all IDS-VG curves  can result in an observable reduction of the SS of this sensor, the SS value does not significantly vary among the subsequent biodetection states, including the inputs of TNF-α samples with incremental concentrations. Therefore, for a given as-functionalized transistor sensor, SS can be approximated as a constant. Based on this observation, a calibrated subthreshold-regime sensor response quantity (S) is derived from Equation IDS(anti) is the drain-source current measured in the “antibody functionalization” state of a sensor biased under a set of fixed VDS and VG; and IDS is the drain-source current measured from a subsequent biodetection state . Similar to the calibrated linear-regime response quantity expressed in Equation ΔVT, independent of the transistor performance, and proportional to σTNF.In the subthreshold regime of a microscale MoSS) measured from five different sensors with respect to TNF-α concentration (n). The detailed transfer characteristics of these five devices measured at various biodetection stages are displayed in Fig. S4. As shown in Fig. S4, the transfer characteristics of these five sensors exhibits significant difference in VT, IDS and SS parameters. However, as shown in S-n curves measured from these devices are consistent with each other and can be well fitted with Langmuir isotherms (Equation D) of the antibody-(TNF-α) pair is extracted to be 424 ± 70 fM, which is consistent with the KD value extracted from the linear-regime sensor responses . The Smax parameter is fitted to be 15.3 ± 0.6 V, which is about 40% larger than that extracted from the linear-regime responses . This observable discrepancy has not been fully understood. However, this could be temporarily attributed to the different back-gate VG levels required for biasing sensors in subthreshold and linear regimes, which could result in different magnitudes of electric field penetrating through few-layer MoS2 channels as well as HfO2 effective layers and leaking into the analyte solution. This could lead to different degrees of the modification of electrical-double-layers around sensors and therefore different degrees of the screening of the charges brought through analyte-receptor binding pairs.Equation . Here, tIDS signals measured from both linear and subthreshold regimes can be mathematically normalized to consistent device-independent response quantities using Equations IDS to the variation of analyte concentration (dn) as well as the noise level of electrically measured IDS signals. This IDS sensitivity is quantitatively defined as the relative change in IDS per change in n  the subthreshold-regime IDS signals from the five sensors shown in Fig. S4. All differential sensitivity values are evaluated at TNF-α concentration of n = 60 fM. This can provide critical information about the sensitivity required for obtaining fM-level detection limits. IDS sensitivities (0.52 ± 0.3%/fM) are statistically higher than the linear-regime IDS sensitivities (0.14 ± 0.02%/fM). Therefore, subthreshold-regime sensor responses are more desirable in achieving high detection sensitivity. However, it should be noted that the ultimate detection limit of a transistor sensor is also limited by the signal-to-noise ratios of electrically measured IDS signals. In addition, for detecting low-abundance molecules, the non-specific adsorption of target molecules could also strongly affect the detection limit. The further analysis of these aspects is beyond the scope of this work but will be addressed in the future research. Finally, it is also noted that the sensitivity data listed in 2 transistors are more sensitive to the fabrication-introduced defects than their linear-regime transconductance properties. Therefore, for our current MoS2 transistors, their linear-regime IDS signals (or linear-regime transconductances) exhibit the higher device-to-device consistency than their subthreshold-regime IDS signals.Although the 2 transistor sensors, a sensor functionalized with anti-human TNF-α antibody is used for detecting interleukin-6 (IL-6) cytokine. Figure S5 shows the transfer characteristics of this sensor measured at various stages, including (1) bare transistor, (2) antibody functionalization, and inputs of IL-6 solutions with concentrations of (3) 600 fM and (4) 6 pM. Figure S5 shows that the presence of IL-6 that is not specific to TNF-α antibody cannot result in prominent change in the transfer characteristics. Such experimentally observed weak sensor responses to IL-6 indicate a negligible nonspecific adsorption of IL-6 molecules on the sensor surface, which may be effectively blocked by the densely-packed self-assembled monolayers of (3-Aminopropyl) triethoxysilane (APTES) on HfO2 effective layers.To evaluate the detection specificity of our MoSi.e., n = 60 fM, 600 fM, 3 pM, and 6 pM). Each of the time-dependent response curves was measured from a different MoS2 transistor sensor and all as-measured IDS responses were normalized using i.e., Equation SS parameters required for normalizing IDS responses were acquired. In addition, the operation points  are also labelled in Fig. S6. In i.e., Equation Seq is the sensor response at the final equilibrium state; kon and koff are association and dissociation rates, respectively; konn + koff relates to the rising slope of the linear regime of the response curve. Seq and (konn + koff) parameters for n = 60 fM, 600 fM, 3 pM, and 6 pM. These Seq values extracted from the real-time binding responses are consistent with the sensor responses directly measured at the equilibrium state, that is, after a long incubation time of ~2 hours  is extracted to be 326 ± 37 fM and the maximum response (Smax) is extracted to be 15.6 ± 0.2 V, which are consistent with those extracted from the equilibrium-state subthreshold-regime responses shown in The biosensor setup shown in kon and koff parameters, the extracted (konn + koff) data are plotted as a function of TNF-α concentration (n) (see kon = (5.03 ± 0.16) × 108 M−1s−1 and koff = (3.44 ± 0.15) × 10−4 s−1. It should be noted that this fit is not sensitive to the dissociation rate (koff) because of its small numerical value. To achieve a more precise quantification of koff, we directly measured the real-time dissociation kinetics of the antibody-(TNF-α) pair. Specifically, two as-functionalized MoS2 transistor biosensors were incubated in solutions with TNF-α concentration of 600 fM and 3 pM, respectively. The incubation time was more than 2 hours so that antibody-(TNF-α) association/dissociation processes reached to the equilibrium state. Afterwards, these fully incubated sensors were rinsed with pure buffer liquid flow and the calibrated sensor responses were recorded as a function of the lapsed time, as displayed in i.e., the desorption equation expressed in Equation Sr represents the sensor response corresponding to the areal density of bound molecule residues after the desorption process. This fit results in koff = (1.97 ± 0.08) × 10−4 s−1, from which the affinity equilibrium constant KD can be also estimated to be KD=koff/kon= 392 fM. This KD value is also consistent with those extracted from the equilibrium-state sensor responses  see . The linkoff = 3.4 ± 0.15 Seq = 13.6 ± 1.0 V, S2 = 4.5 ± 0.2 V, Sr = 2.9 ± 0.3 V, k2 = (2.0 ± 0.16) × 10−3 s−1, and koff = (1.79 ± 0.13) × 10−4 s−1. As reported by several previous works1055The response curve measured from the device incubated with TNF concentration of 3 pM can be hardly fitted with monoexponential Equation 2 transistor sensors, the calibrated sensor responses do not explicitly depend on HfO2 layer thickness (tHfO2). All sensors discussed above have 30 nm thick HfO2 effective layers. To further experimentally verify that the sensor responses of our sensors do not strongly depend on HfO2 layer thickness (tHfO2), we fabricated additional sensors with tHfO2 = 60 nm. Figure S7 (a) in the supplementary information displays the transfer characteristics of an exemplary sensor with tHfO2 = 60 nm, which were measured from a set of incremental TNF-α concentrations. From such transfer characteristics, we extracted calibrated subthreshold-regime responses (S) at VG = −25 V (VT ~ −10 V) and plotted them as a function of TNF-α concentration (n) ). This S-n relationship is consistent with those measured from the sensors with tHfO2 = 30 nm. This result proves that the calibrated sensor response values do not strongly depend on the HfO2 effective layer thickness.Finally, it should be noted that for our current MoS2 transistor biosensors and demonstrated that multiple such sensors can be utilized to enable quantification of low-abundance biomarker molecules as well as the affinities and kinetics of antibody-mediated binding events. In particular, our biosensors exhibited a TNF-α detection limit at least as low as 60 fM. Such a low detection limit can be obtained in both linear and subthreshold regimes of MoS2 transistors. We further observed that the sensors operated in the subthreshold regime showed the higher current sensitivities in comparison with those in the linear regime. Such high subthreshold-regime sensitivities hold significant potential to further lower the TNF-α detection limit. In both transport regimes, the measured current signals can be normalized into response quantities independent of the transistor performance, which can effectively reduce the effect of sensor-to-sensor variation on biodetection results. Based on this calibration method, all sets of our biosensors can generate very consistent sensor responses with respect to TNF-α concentration and therefore a standard curve for TNF-α quantification. From this standard curve, the equilibrium constant of the antibody-(TNF-α) pair was extracted to be KD = 369 ± 48 fM from linear-regime responses (or KD = 424 ± 70 fM from subthreshold-regime responses). Furthermore, the real-time association/dissociation processes of the antibody-(TNF-α) pair were also quantified using multiple sensors. The association/dissociation rates were extracted to be kon = (5.03 ± 0.16) × 108 M−1s−1 and koff = (1.97 ± 0.08) × 10−4 s−1, respectively. This work laid an important foundation for leveraging the excellent electronic properties of emerging atomically layered semiconductors in bio-assay applications as well as advanced the critical research capability in analyzing the biomolecule interactions with fM-level detection sensitivities. Notably, such capability would enable selection of antibodies with a high binding constant with respect to a specific target biomarker molecule, thereby providing a means to further improve the selectivity and fidelity of immunoassay.In conclusion, we presented important device physics and metrics for calibrating the responses of MoS2 transistors were fabricated using a microprinting method previously reported2 channel thicknesses were specifically controlled to be 15–20 nm. Such a MoS2 thickness range has been demonstrated to result in the optimal field-effect mobility values for MoS2 transistors52L) were ~5 μm and the channel widths (W) ranged from 5 to 8 μm. Ti (5 nm)/Au (50 nm) electrode pairs served as drain (D) and source (S) contacts, which were created using photolithography followed with metal deposition and lift-off. The p+-Si substrates were used as the back gates (G). Thermally grown SiO2 layers (300 nm thick) were used as the back-gate dielectrics. Such 300 nm thick SiO2 layers can enable a simple color coding method for us to quickly identify MoS2 flakes with suitable thicknesses The MoS2 effective layer of a MoS2 transistor sensor with anti-human TNF-α antibody receptors for detecting TNF-α molecules. First, an as-fabricated transistor biosensor is immersed in 5% (3-Aminopropyl) triethoxysilane  in ethanol for 1 hour. After the incubation, the sensor is rinsed with phosphate buffered saline (PBS) and blown dry by nitrogen gas. After this step, the HfO2 effective layer is silanized with an APTES monolayer. The device is subsequently immersed in 5% gluteraldehyde (GA) (purchased from Sigma-Aldrich Co. LLC.) in PBS for 2 hours followed by rinsing with PBS. Afterwards, anti-human TNF-α antibody  of 50 ug/ml concentration in DI water is dropped on the sensor and incubated for 1 hour. For studying the equilibrium-state sensor responses, the as-functionalized sensor is incubated with TNF-α solutions with incremental concentrations . The incubation is performed using the setup illustrated in Figure S1 in the supporting information illustrates the protocol for functionalizing the HfO2 transistor biosensor is covered with a polydimethylsiloxane (PDMS) block bearing a microfluidic channel , as illustrated in 2 transistor is biased under a given set of VG and VDS. After the IDS value is stabilized, the analyte solution with a specific TNF-α concentration is injected into the sensor.An as-functionalized MoSHow to cite this article: Nam, H. et al. Multiple MoS2 Transistors for Sensing Molecule Interaction Kinetics. Sci. Rep.5, 10546; doi: 10.1038/srep10546 (2015).


In this study, we investigated the molecular mechanism underlying inhibiting TNF-α-induced chondrocytes apoptosis of MW. MTT assay, DAPI, and flow cytometry demonstrated that MW significantly increased cell activity and inhibited chromatin condensation accompanying the loss of plasma membrane asymmetry and the collapse of mitochondrial membrane potential. Our results also indicated that MW reduced the elevation of [Ca2+]i in chondrocytes by LSCM. Moreover, MW suppressed the protein levels of calpain, Bax, cytochrome c, and caspase-3, while the expressions of Bcl-2, collagen II, and aggrecan were increased. Our evidences indicated that MW treatment inhibited the apoptosis of chondrocytes through depression of [Ca2+]i. It also inhibited calpain activation, which mediated Bax cleavage and cytochrome c release and initiated the apoptotic execution phase. In addition, MW treatment increased the expression of collagen II and aggrecan of chondrocytes.The molecular mechanisms of TNF- Osteoarthritis (OA) is characterized by a progressive loss of joint articular cartilage caused by the interaction of mechanical and biological factors. The abrasion of articular cartilages stimulates an inflammatory reaction in synovial cells and chondrocytes. The local increase in inflammatory stimuli in the articular tissues leads to secondary synovial proliferation, articular exudation, and cartilage dystrophy, resulting in articular cartilage degeneration –4.α (TNF-α), induces inflammation, promotes the release of bone matrix-degrading enzymes, and mediates fibroblastic hyperplasia and histological damage i) by dyeing chondrocytes with fluo-3/AM, exposing them to MW, and observing the change of [Ca2+]i with a laser scanning confocal microscope (LSCM).Our preliminary work demonstrated that MW inhibits the activation of the TNF- pathway . To furtnetworks , 15. In 2, and a radiation area of 15.1 cm2 (45.6 ± 4 mm long and 33.2 ± 3 mm wide) were manufactured by Zhongcheng Kangfu Technology Co., Ltd. . A confocal laser scanning microscope (LSM 710) was obtained from Carl Zeiss . A fluorescence-activated cell sorting (FACS) calibur  was obtained from Becton, Dickinson and Company .KFA-100A millimeter wave therapeutics at wavelengths of 7.5–10.0 mm, a power density of 4 mw/cmα, fluo-3/AM, and a 3--2, 5-diphenyltetrazolium bromide (MTT) colorimetric assay were obtained from Sigma-Aldrich Corporation . Toluidine blue was obtained from Guoyao Group , and a type II collagen immunostaining kit from Boster Inc. . An apoptosis assay, the Annexin V-FITC Apoptosis Detection Kit II, was provided by Becton, Dickinson and Company . A JC-1 mitochondrial membrane potential detection assay was obtained from Invitrogen . Hoechst 33258 was provided by the Beyotime Institute of Biotechnology . The antibodies calpain, Bax, Bcl-2, cytochrome c, caspase-3, collagen II, and aggrecan were obtained from Beijing Boao Sen Biotechnology Co., Ltd. . An HRP secondary rabbit anti-goat antibody was obtained from Zhongshan Golden Bridge Biotechnology Company .Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), and trypsin were purchased from HyClone Laboratories, Inc. . Type II collagenase, TNF- Guidance Suggestions for the Care and Use of Laboratory Animals Welfare, published by the Ministry of Science and Technology of China i changes after TNF-α and TNF-α + MW treatment. As shown in Figures 2+]i fluorescence intensity in the group treated with 20 ng/mL TNF-α (14.22 ± 3.17) was higher than in the control group (7.24 ± 1.30) (P < 0.05). The change of [Ca2+]i in chondrocytes under TNF-α + MW treatment increased by a much smaller amount: 7.94 ± 2.04. There was no significant difference in intracellular calcium in the group treated with MW compared to the control. These results suggest that MW can prevent an influx of [Ca2+]i and may inhibit apoptosis, whereas TNF-α alone can induce a calcium overload.To further explore the change of TNF-The mitochondria-dependent pathway is the most common apoptotic pathway in vertebrate animal cells. Mitochondrial membrane permeabilization, accompanied by the collapse of electrochemical gradient across the mitochondrial membrane, is one of the key events of cellular apoptosis.α, indicating a TNF-α-induced loss of mitochondrial membrane potential in the chondrocytes. The chondrocytes treated with TNF-α + MW exhibited a JC-1-green-bright/JC-1-red-bright signal. As shown in α, and TNF-α + MW groups were 10.04 ± 4.42, 38.43 ± 4.48, and 15.99 ± 6.15, respectively. Compared with the control group, the 10 ng/mL TNF-α group had a greater number of cells with low red fluorescence (FL-2 [LR]) (P < 0.01), indicative of a reduction in the ΔΨ and of the induction of apoptosis. The TNF-α + MW group had significantly fewer cells with low red fluorescence (FL-2 [LR]) (P < 0.01) as compared to the TNF-α group. This indicates that MW treatment inhibited TNF-α-induced apoptosis.To investigate the mechanism of how MW induces apoptosis of chondrocytes, we examined the change in mitochondrial membrane potential after MW treatment using FACS analysis with JC-1 staining. JC-1 is a lipophilic, cationic dye that selectively enters into mitochondria. In unhealthy cells with high mitochondrial potential, JC-1 forms J-aggregates with intense red fluorescence (590 nm). Conversely, in apoptosis, the mitochondrial membrane potential collapses so that the JC-1 does not accumulate within the mitochondria but remains in the cytoplasm in monomeric form, emitting green fluorescence (525 nm). These differences in fluorescence can be detected with FACS analysis using green and red JC-1 channels. As shown in Bcl-2 family proteins, including antiapoptotic members such as Bcl-2 and proapoptotic members such as Bax, are key regulators of mitochondria-mediated apoptosis. Upon apoptosis induction, the proapoptotic protein Bax is translocated from the cytosol to the mitochondria, where it promotes release of cytochrome c, a caspase-activating protein. Calpain-mediated Bax cleavage activity was found in the mitochondrial membrane-enriched fraction. Data from a Western blot analysis showed that the levels of protein expression of calpain, Bax, cytochrome c, and caspase-3 were profoundly reduced with MW treatment, while the pattern of protein expression of Bcl-2, collagen II, and aggrecan was increased Figures .Osteoarthritis (OA) is a multifactorial disease characterized by the breakdown of hyaline articular cartilage and the formation of osteophytes. The chondrocyte is the only cell type present in mature cartilage that is responsible for extracellular signals and the maintenance of cartilage homeostasis. Therefore, the functional changes of chondrocytes play an important role in OA, as they contribute to the degradation of articular cartilage and thus to the pathogenesis of OA , 23. We α, is involved in the synovial inflammatory response, the initiation of chondrocytic apoptosis, the imbalance between chondral damage and repairs, and the eventual acceleration of articular cartilage degeneration i and a downregulation of mitochondrial membrane potential (ΔΨm), resulting in apoptosis. These findings suggest that Ca2+ potentially plays a role in TNF-α-induced apoptosis in chondrocytes. Ca2+ is a factor in excitation-contraction coupling, and the increase of [Ca2+]i is dependent on extracellular Ca2+ entry and Ca2+ release from intracellular stores such as sarcoplasmic reticulum (SR) or mitochondria. Precise control of intracellular Ca2+ homeostasis is vital for the regulation of a chondrocyte's functions. The involvement of a Ca2+ influx has been reported for various apoptotic stimuli. In tributyltin-induced T-cell apoptosis, a rise in Ca2+ concentration is required for postmitochondrial events, namely, the activation of caspases i and results in chondrocytic apoptosis. Conversely, millimeter wave treatment efficiently suppresses apoptosis through the downregulation of intracellular [Ca2+]i, reduces cell membrane permeability and increases ΔΨm, and inhibits the activation of calpain, which in turn mediates Bax cleavage and cytochrome c release and initiates apoptotic execution.Our results show that an 8-hour treatment of TNF-


Inflammation and reactive oxygen species (ROS) play important roles in the pathogenesis of atherosclerosis. Resveratrol has been shown to possess anti-inflammatory and antioxidative stress activities, but the underlying mechanisms are not fully understood. In the present study, we investigated the molecular basis associated with the protective effects of resveratrol on tumor necrosis factor-alpha (TNF-α)-induced injury in human umbilical endothelial cells (HUVECs) using a variety of approaches including a cell viability assay, reverse transcription and quantitative polymerase chain reaction, western blot, and immunofluorescence staining. We showed that TNF-α induced CD40 expression and ROS production in cultured HUVECs, which were attenuated by resveratrol treatment. Also, resveratrol increased the expression of sirtuin 1 (SIRT1); and repression of SIRT1 by small-interfering RNA (siRNA) and the SIRT1 inhibitor Ex527 reduced the inhibitory effects of resveratrol on CD40 expression and ROS generation. In addition, resveratrol downregulated the levels of p65 and phospho-p38 MAPK, but this inhibitory effect was attenuated by the suppression of SIRT1 activity. Moreover, the p38 MAPK inhibitor SD203580 and the nuclear factor (NF)-κB inhibitor pyrrolidine dithiocarbamate (PDTC) achieved similar repressive effects as resveratrol on TNF-α-induced ROS generation and CD40 expression. Thus, our study provides a mechanistic link between resveratrol and the activation of SIRT1, the latter of which is involved in resveratrol-mediated repression of the p38 MAPK/NF-κB pathway and ROS production in TNF-α-treated HUVECs. The pathogenesis of atherosclerosis is a complex pathological process that involves an ongoing inflammatory response in blood vessels, which mediates all stages of atherosclerosis including initiation and progression . Recentl2-), hydrogen peroxide (H2O2), hydroxide (OH-), and hypochlorite (OCl-), is also crucial to the pathogenesis of atherosclerosis. NADPH oxidase, enzymatic activation of cytochrome P450, xanthine oxidase, and inflammatory activities are considered to be the major sources of ROS  vector using Lipofectamine. At 24 h post-transfection, the cells were pretreated with vehicle, resveratrol, or PDTC as indicated before TNF-α stimulation. The luciferase activity was determined by the luciferase assay kit.HUVECs  at 4°C overnight, followed by another incubation with the polyclonal FITC-conjugated goat anti-rabbit IgG (1:50 dilution) for 1 h at room temperature as described previously . CellulaData were collected from three independent experiments, each carried out in triplicate and expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed by means of one-way analysis of variance followed by the least significant difference test using SPSS 16.0 software (USA). A p value of < 0.05 was regarded as statistically significant, and < 0.01 was regarded as highly significant.p < 0.05 vs. control). Therefore, we used 10 μg/L TNF-α as the proper dose to induce the inflammatory response. Since a high concentration of resveratrol (>30 μM) has been shown previously to impair cell viability [p < 0.05 vs. TNF-α alone). Similar results were observed with 20 μM resveratrol. These observations suggest that resveratrol improves the viability of HUVECs impaired by TNF-α treatment.HUVECs were grown as a confluent monolayer with cobblestone morphology , and theiability , we treap < 0.01 vs. control), this induction was downregulated by resveratrol in a dose-dependent fashion , although 10 μM resveratrol did not have any significant effect on CD40 expression at baseline. Thus, we conclude that resveratrol treatment attenuates the increased expression of CD40 triggered by TNF-α stimulation.Increased expression of the CD40 receptor activated by CD40L has been shown to alter the expression levels of specific adhesion molecules, thus promoting the inflammatory response . In addip < 0.05 vs. control). However, this decrease was significantly attenuated by 10 and 20 μM resveratrol treatment . Intriguingly, 20 μM resveratrol treatment increased the expression of SIRT1 at baseline ($p < 0.05 vs. control). Thus, we conclude that resveratrol improves the expression of SIRT1, which was suppressed by TNF-α stimulation.A previous study has suggested that resveratrol activates SIRT1 . Therefop < 0.01 vs. TNF-α + resveratrol).As shown in To further examine the subcellular location and expression of SIRT1 in HUVECs, cells were exposed to TNF-α (10 μg/L) for 24 h in the absence or presence of 10 or 20 μM resveratrol or transfected with SIRT1 siRNA, followed by immunostaining to detect SIRT1 expression. As shown in p < 0.05 vs. control), which was substantially attenuated by resveratrol (10 μM) . A similar inhibitory effect was observed by using PDTC, an inhibitor of NF-κB, and SB203580, a MAPK inhibitor. We also found that cotreatment with Ex527, an inhibitor of SIRT1, impaired the effectiveness of resveratrol (+p < 0.05 vs. TNF-α + resveratrol) in reducing ROS production. Taken together, we argue that resveratrol suppresses TNF-α-triggered ROS generation in HUVECs via potentiating the activity of SIRT1 and that both NF-κB and MAPK signaling are involved in mediating TNF-α-induced ROS generation.To investigate the anti-oxidant effect of resveratrol, we evaluated ROS generation in HUVECs after resveratrol treatment using DCFH-DA staining, and the mean fluorescence intensity was measured by flow cytometry. As shown in p < 0.01 vs. control), which was greatly suppressed by resveratrol at concentrations of 10 and 20 μM . As expected, PDTC, an inhibitor of NF-κB, substantially inhibited the expression of p65. In addition, SB203580, a MAPK inhibitor, also exhibited a similar inhibitory impact on p65 expression as PDTC.NF-κB signaling has been reported previously to be involved in mediating the cellular inflammatory response . To probNext, to investigate the potential link between the suppression of NF-κB and the activation of SIRT1 by resveratrol, HUVECs were transfected with SIRT1 siRNA 24 h prior to the experiments, followed by western blot analysis, PCR, and the luciferase reporter assay to detect the expression levels of p65. As shown in p < 0.01 vs. TNF-α alone). To explore whether SIRT1 is involved in resveratrol-suppressed p38 MAPK phosphorylation, we inhibited SIRT1 by both siRNA-knockdown and Ex527 in HUVECs during resveratrol treatment in the presence of TNF-α. Inhibition of SIRT1 significantly reversed the repressive effects of resveratrol on p38 phosphorylation (p < 0.05 vs. TNF-α + Nc-siRNA). Collectively, we conclude that resveratrol attenuates TNF-α-induced p38 phosphorylation via activating SIRT1.ERK, p38 MAPK, and JNK are all members of the MAPK family. Since SB203580, a MAPK inhibitor, exhibited an inhibitory impact on p65 expression , we nextAs mentioned above, the NF-κB pathway plays a critical role in the inflammatory response. Given that the increased CD40 expression and ROS production elicited by TNF-α stimulation were significantly diminished by treatment with PDTC, a NF-κB signaling inhibitor in , 6 and 8In this study, we focused on the protective effects of resveratrol against inflammatory and oxidative injury in HUVECs induced by TNF-α. Given the important roles of the CD40/CD40L axis in the inflammatory response and ROS production in oxidative stress during the pathological process of atherosclerosis, we explored the mechanisms by which resveratrol reduces CD40 expression and ROS generation induced by TNF-α. Based on previous findings that only low-to-moderate doses of resveratrol are able to attenuate ROS exposure while high doses induce cytotoxicity, the latter of which is caused by mitochondrial dysfunction , in the et al. have found that the expression levels of eNOS and SIRT1 are upregulated in HUVECs treated repeatedly with 1 mM resveratrol for 6 days. Moreover, Cantó et al. have reported that resveratrol activates SIRT1 by the cAMP–Epac1–AMPK pathway [et al. [et al. [Resveratrol is a subtype of phytoalexins that protects against pathological processes through suppressing elevated levels of proinflammatory cytokines in macrophages. For instance, it has been shown that the CD40/CD40L levels in rats with hypercholesterolemia are downregulated by trans-resveratrol administration . Another pathway , suggest pathway . In addi [et al.  and Lin  [et al.  have rep2-), hydroxide (OH-), and peroxynitrite (ONOO-) radicals [et al. have shown that oxidative stress induced by balloon injury could be attenuated by resveratrol in the rat carotid artery through altering the activity of the NF-κB pathway [Resveratrol is also known as a robust scavenger of ROS. In normal bodies, there is a balance between ROS production and clearance. However, chronic inflammation can induce continuous production of high levels of ROS. Therefore, the uptake of exogenous antioxidants to clear ROS is believed to interfere with oxidative stress in a beneficial manner. Previous studies have shown that resveratrol directly attenuates the generation of superoxide Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S8 Table(PDF)Click here for additional data file.S9 Table(PDF)Click here for additional data file.


Polycystic ovarian syndrome (PCOS) is one of the most common endocrine conditions affecting women of reproductive age with a prevalence of approximately 5-10 % worldwide. PCOS is characterized by hyperandrogenism, irregular menstrual cycles, polycystic ovaries, insulin resistance, obesity particularly abdominal phenotype. We evaluated the role of TNF α in PCOS patients and compared to age matched healthy controls.2). Clinical, biochemical characteristics of PCOS and molecular study of TNF alpha gene C850T polymorphism were studied.204 women clinically diagnosed with PCOS and 204 healthy women controls in the age group of 17 to 35 years were evaluated. PCOS were subdiveded into Obese and lean based on BMI . Patients had elevated serum TNF α, Free and Total testosterone, Androstenedione and DHEA when compared to controls (P <0.05). We have demonstrated an association between TNF α –C850T polymorphism and PCOS. Frequency of T allele was 93.5% in PCOS and 73.5% in controls . TT genotype confers a fivefold high risk for PCOS in our population .TNF α contributes to the clinical , biochemical manifestations of PCOS, and –C850T TNF α gene polymorphism is associated with PCOS and could be used as a relevant molecular marker to identify women with risk of developing PCOS in our population.


Tumor necrosis factor (TNF) has pleiotropic functions during both the demyelinating autoimmune disease multiple sclerosis (MS) and its murine model experimental autoimmune encephalomyelitis (EAE). How TNF regulates disability during progressive disease remains unresolved. Using a progressive EAE model characterized by sustained TNF and increasing morbidity, this study evaluates the role of unregulated TNF in exacerbating central nervous system (CNS) pathology and inflammation.Progressive MS was mimicked by myelin oligodendrocyte glycoprotein (MOG) peptide immunization of mice expressing a dominant negative IFN-γ receptor alpha chain under the human glial fibrillary acidic protein promoter (GFAPγR1∆). Diseased GFAPγR1∆ mice were treated with anti-TNF or control monoclonal antibody during acute disease to monitor therapeutic effects on sustained disability, demyelination, CNS inflammation, and blood brain barrier (BBB) permeability.TNF was specifically sustained in infiltrating macrophages. Anti-TNF treatment decreased established clinical disability and mortality rate within 7 days. Control of disease progression was associated with a decline in myelin loss and leukocyte infiltration, as well as macrophage activation. In addition to mitigating CNS inflammation, TNF neutralization restored BBB integrity and enhanced CNS anti-inflammatory responses.Sustained TNF production by infiltrating macrophages associated with progressive EAE exacerbates disease severity by promoting inflammation and disruption of BBB integrity, thereby counteracting establishment of an anti-inflammatory environment required for disease remission. Tumor necrosis factor (TNF) is a pleiotropic cytokine that regulates numerous physiological and pathological processes. Specifically, the pathophysiology of a variety of neurological disorders, including the demyelinating autoimmune disease multiple sclerosis (MS) is associated with TNF . Express−/− and TNFR2−/− mice develop more severe EAE [−/− mice are protected from EAE [−/− mice [EAE has provided insights into the dual pro- and anti-inflammatory activities exhibited by TNF during CNS autoimmunity. Two forms of TNF, a soluble and transmembrane form, interact with two distinct receptors, namely TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2), which differ in expression and ligand affinity. The pleiotropic functions of TNF are predominantly dictated by interaction with these two receptors. Soluble TNF with higher affinity for TNFR1 mediates apoptosis and chronic inflammation . Conversvere EAE , 20, 21,from EAE . A recenfrom EAE . Interesfrom EAE . Similar−/− mice , 20, 21,−/− mice , 22, 23.Collectively, these studies suggest that the protective effects of TNF in EAE are mediated by the interaction of transmembrane TNF with TNFR2 and that blocking these anti-inflammatory properties during the relapsing-remitting phase of MS contributes to the adverse outcome. Contrasting these studies predominantly focusing on the effect of TNF on either disease induction or the acute phase of EAE, the study herein specifically addresses the role of TNF during a chronic, non-remitting form of EAE. We have previously reported that mice, in which astrocytes are unable to respond to interferon gamma (IFN-γ), develop a chronic progressive form of EAE  with manb GFAP/IFN-γR1∆IC (GFAPγR1∆) transgenic mice expressing a dominant negative IFN-γ receptor alpha chain under control of the glial fibrillary acidic protein (GFAP) promoter [b) WT mice were purchased from the National Cancer Institute . FVB/N-Tg (GFAP-GFP) 14Mes/J mice  were backcrossed 11 times into the C57BL/6 background to generate C57BL/6 WT mice expressing green fluorescent protein (GFP) under the GFAP promoter (GFAP-GFP). GFAP-GFP mice were crossed with GFAPγR1∆ mice to generate dual transgenic GFAP-GFP/GFAPγR1∆ mice. All animal experiments were conducted in mice from both sexes in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. All procedures were performed in compliance with protocols (Protocol number 1165) approved by the Cleveland Clinic Institutional Animal Care and Use committee.Homozygous H-2promoter , 33 were35–55 peptide  emulsified at 3 mg/ml in PBS with an equal amount of incomplete Freund’s Adjuvant  supplemented with 5 mg/ml of Mycobacterium tuberculosis, strain H37Ra . Mice were immunized subcutaneously with 200 μl emulsion over the flanks. Mice also received 200 ng of Pertussis toxin (Sigma-Aldrich) in 200 μl of PBS intraperitoneally (i.p.) on the day of the initial immunization and at day 2 post-immunization (p.i.). Mice received a second immunization with 200 μl emulsion in the left side over the flanks 7 days later. Animals were scored daily for clinical symptoms as follows: 0 = no signs of disease, 1 = flaccid tail, 2 = flaccid tail and partial hind limb paralysis, 3 = complete hind limb paralysis, 4 = moribund state, 5 = dead.EAE was induced by immunizing mice with myelin oligodendrocyte glycoprotein (MOG)g for 30 min at 4 °C to separate mononuclear cells from myelin debris. Cells from the 30/70 % interface were collected and washed in RPMI medium. Antibody non-specific binding was prevented with mouse anti-CD16/CD32  and 10 % mixture of normal goat, human, mouse, and rat serum for 10 min on ice. To distinguish astrocytes from microglia and inflammatory cells, recovered cells were stained with anti-CD45 (clone 30-F11) APC and CD11b (M1/70) PerCp . Astrocytes were identified as GFAP-GFP+ CD45-, whereas microglia were defined as CD45int CD11b+. Cells were purified by FACS using a FACSAria (BD Biosciences) and FACS Diva software (BD Biosciences).EAE was induced in GFAP-GFP/GFAPγR1∆ as described above. At d19 and d30 p.i., brains were homogenized using a Papain Neuronal Tissue Dissociation Kit . Cells from homogenates were resuspended in RPMI medium containing 25 mM HEPES (pH 7.2) and adjusted to 30 % Percoll . A 1-ml underlay of 70 % Percoll was added prior to centrifugation at 800×Mice received 0.5 mg of either neutralizing rat anti-murine TNF (clone MD6-XT3.11) or control rat IgG1 anti-β-galactosidase (clone GL113) monoclonal antibody (mAb) i.p. starting at the peak of acute disease (day 19 p.i.) followed by injections every 2 days for a total of four dosages. Anti-TNF mAb was purchased from Bioxcell, West Lebanon, NH. The GL-113 hybridoma, originally obtained from Dr. Robert Coffman  was adapted to BD cell Serum Free Medium . After propagation in a BD CELLine device, the mAb concentration was determined by optical density at 480 nm. All mAbs were diluted to 1 mg/ml in endotoxin free PBS and stored at −20 °C until use.g for 7 min. Supernatants were collected and immediately stored at −80 °C for cytokine analysis (see below). Cells were resuspended in 30 % Percoll and isolated by centrifugation (800×g for 30 min at 4 °C) onto a 70 % Percoll cushion as described above for cell sorting. Non-specific antibody binding was inhibited by incubation with anti-CD16/CD32 (2.4G2 mAb) and a 10 % mixture of normal goat, human, mouse, and rat serum for 15 min on ice. CD45 (30-F11) APC, CD4 (GK1.5) PercP-Cy5.5, CD8a (53−6.7) FITC, and CD11b (M1/70) PE mAbs (BD Biosciences) were used to analyze microglia and infiltrating inflammatory cells (BD Biosciences). Microglia and inflammatory cells were distinguished based on their differential CD45 expression. Major histocompatibility complex (MHC) class II  expression was determined as a measure of macrophage and microglial activation. To detect intracellular cytokines cells were stimulated ex vivo for a total of 4 h at 37 °C with phorbol 12-myristate 13-acetate (PMA)  and ionomycin . Monensin  was added for the last 2 h of incubation. Following permeabilization  intracellular cytokines were detected using anti-IFN-γ  and anti-IL-17  mAb. To determine intracellular TNF, cells were incubated in serum free RPMI at 37 °C for 4 h with Monensin (2 μM) added for the last 2 h. After 4 h, cells were permeabilized with cytofix/cytoperm reagent (BD Biosciences) and stained using PE anti-TNF mAb .Brains from mice perfused with ice-cold PBS were individually homogenized in 4 ml of RPMI medium containing 25 mM HEPES, pH 7.2 using chilled Tenbroeck tissue grinders. Homogenates were centrifuged at 450×IFN-γ and IL-17 were determined by ELISA as previously described  using mAg at 4 °C. Supernatants were mixed with an equal volume of 15 % trichloroacetic acid and centrifuged at 10,000×g for 10 min at 4 °C. After centrifugation, 125 μl of 5 N NaOH was added to 500 μl of supernatant. NaF concentrations were determined by comparison to standards ranging from 125 to 4000 μg on a SpectraMax M2 at an excitation of 485 nm, emission of 530 nm, and gain of 50 . CNS values were normalized to NaF serum levels as follows: (μg florescence tissue/mg protein)/(μg fluorescence sera/μl of blood) [BBB integrity was determined by measuring permeability to sodium fluorescein (NaF) as described . Mice ref blood) . Data ar+ area was calculated as an average of three different fields per group using Image-Pro.Spinal cords from WT and GFAPγR1∆ mice perfused with ice-cold PBS during acute and chronic EAE were collected in OCT embedding compound . Frozen sections were stained with anti-mouse TNF (1:100) , anti-mouse Iba1 (1:200)  or anti-mouse GFAP (1:5000) , and Prolong Gold for nuclear staining  as previously shown . ImmunorSpinal cord frozen sections were also stained with laminin (1:2000) , IgG conjugated with Alexa Fluor-594 (1:200) , and Prolong Gold. Laminin was visualized using goat anti-rabbit Alexa Fluor-488 (Life Technologies).Spinal cords were obtained from mice perfused with ice-cold PBS and fixed in Zn Formalin. Each spinal cord was dissected into six sections, two of each corresponding to the cervical, thoracic, and lumbar regions and embedded in paraffin. To determine the percentage demyelination, 6-μm cross sections were stained with Luxol Fast Blue (LFB), scanned at 40× and digitally imaged at high resolution with an Aperio Scanscope  as described . Aperio Following PBS perfusion, snap-frozen spinal cords were homogenized in Trizol (Invitrogen) using a TissueLyser with stainless beads . RNA was extracted according to the manufacturer’s instructions followed by DNAse I  treatment for 30 min at 37 °C. M-MLV Reverse Transcriptase (Invitrogen), oligo-dT primers (20 μM) , and random primers (20 μM) (Promega) were used to synthesize cDNA. Gene expression analysis was performed by quantitative real-time PCR using a 7500 Fast real-time PCR system . Foxp3 and IL-27 p28 messenger RNA (mRNA) levels were detected by Taqman Gene Expression Assay using Taqman primers  .RNA was extracted from FACS-purified cell populations frozen in 400 μl of Trizol reagent . Removal of DNA contamination and cDNA synthesis were performed as described above. TNF mRNA expression analysis was determined using 4 μl of cDNA and SYBR Green master mix  containing TNF primers (GCCACCACGCTCTTCTGTCT and GGTCTGGGCCATAGAACTGATG).t test or Mann-Whitney Rank Sum test using SigmaStat v3.5. A value of p < 0.05 was considered statistically significant. Graphs were plotted using GraphPad Prism v5.02 software.Data represent the mean ± SEM. Significance was determined using two-tailed Student’s In active MS lesions, TNF is produced by microglia, macrophages, and astrocytes, but not T cells . Similar+ astrocytes or T cells by histology during either the acute or chronic phase in either mouse group (data not shown). TNF primarily co-localized with Iba1+ cells in both groups of mice undergoing acute EAE  of microglia produced TNF during acute EAE in both mouse groups, with only a minor increase at the population level evident by mean fluorescent intensity (MFI) in GFAP∆R1γ mice  and the IL-27-dependent antigen specific T regulatory (Tr-1) cells [+ T cells, including Th1 and Th17 cells were reduced by anti-TNF treatment, increased IL-10 suggested a more prominent function of either regulatory cell type. Both Foxp3 mRNA levels  were similar in anti-TNF treated GFAPγR1∆ mice and controls, confirming a relative increase compared to pathogenic effector T cells. Transcript levels for IL-27p28, required to promote Tr-1 cells, was even increased ~3-fold  cells –59. As oels Fig.  as well old Fig. , suggestDespite preclinical data strongly supporting beneficial effects of suppressing TNF, TNF blockade in MS patients has shown variable success. For example, treating relapsing-remitting patients with a soluble TNFR1 fusion protein that neutralizes TNF increased the relapse rate and neurological deficits . Similar+ cells were slightly increased (Fig. Activation-induced production of TNF by numerous cell types further contributes to the complex regulation of TNF and distinct outcomes of intervention. Both microglia/macrophages and astrocytes can secrete TNF in vitro and in vivo, yet little is known on the regulation in vivo , 41. In -/- mice have reduced BBB permeability compared to WT mice during EAE [The protective effect of anti-TNF Ab treatment on progressive EAE supported that macrophage-derived TNF is a detrimental contributor to disability. Improved clinical disease and survival directly coincided with diminished cellular CNS inflammation and demyelination. Interestingly, TNF blockade decreased the overall numbers of CNS infiltrating immune cells, without affecting a specific population or the ratio of Th1 and Th17 cells. A potential therapeutic mechanism by which TNF blockade reduces CNS inflammation during progressive EAE is the regulation of peripheral immune response, similar to the effects described during induction of acute EAE . Howeverring EAE . Consistring EAE . IL-6 blring EAE , supportring EAE  may refl-/- mice are susceptible to severe EAE, whereas IL-10 administration suppresses acute disease [+ T cells into IL-10 secreting Tr1 cells [An additional mechanism associated with the failure to transition from acute autoimmune mediated-inflammation towards remission in progressive EAE is dysregulation of IL-10, a potent regulator of T cell activation , 55, 70. disease , 71, 72.r1 cells , 73–75. r1 cells . GFAPγR1r1 cells , 25. IntTNF has also been implicated in promoting macrophage polarization to a pro-inflammatory M1 phenotype following spinal cord injury, thereby contributing to tissue damage . Using MOur data demonstrate that sustained TNF production by CNS infiltrating macrophages promotes BBB permeability, inflammation, and demyelination, thereby progressively increasing disability and morbidity. While our studies confirmed the failure of anti-TNF therapy in WT mice undergoing EAE , 22, 23,


Cardiopulmonary bypass can lead to postoperative hemodynamic disorders. Several genetic polymorphisms have been studied in this setting. We investigated the possible existence of a synergism between polymorphisms of plasminogen activator inhibitor type 1 (PAI-1) and tumoral necrosis factor beta (TNF-B) on hemodynamic response after cardiac surgeryWe prospectively studied the association between hemodynamic response and polymorphisms of TNF-B and PAI-1 in 563 patients undergoing elective cardiac surgery during the years 2008 to 2011. We tested the Hardy-Weinberg equilibrium in the sample. V18 SPSS was used.P < 0.001), post hoc Scheffé  vs. 0.06 (0.04 to 0.09) μg/kg/minute, P < 0.001; GG vs. AG, 0.32 (0.11 to 0.65) vs. 0.06 (0.03 to 0.08), P < 0.001)) and at 24 hours , post hoc Scheffé  vs. 0.10 (0.06 to 0.14), P = 0.019; GG vs. AG, 0.27 (0.01 to 0.52) vs. 0.07 (0.04 to 0.09), P = 0.003)). Unfavorable TNF-B  and PAI-1 unfavorable  were grouped, after adjusting for perioperative significant variables. The homozygous GG and 4G alleles were significant for NA 4 hours  and GG-4G allele interaction  , 4G allele  and interaction .We studied 563 patients. We found significant differences in TNF-B polymorphisms regarding norepinephrine requirements at 4 hours (F: 15.9; GG homozygous polymorphism TNF-B is associated with an increased dependence on norepinephrine after cardiopulmonary bypass, showing a synergistic action with the 4G allele of PAI-1.


Indeed, germline mutations in PHF6 are the cause of the Börjeson–Forssman–Lehmann X-linked intellectual disability syndrome (BFLS), while somatic PHF6 mutations have been identified in T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Studies from different groups over the last few years have made a significant impact towards a functional understanding of PHF6 protein function. In this review, we summarize the current knowledge of PHF6 with particular emphasis on how it interfaces with a distinct set of interacting partners and its functional roles in the nucleoplasm and nucleolus. Overall, PHF6 is emerging as a key chromatin adaptor protein critical to the regulation of neurogenesis and hematopoiesis.The importance of chromatin regulation to human disease is highlighted by the growing number of mutations identified in genes encoding chromatin remodeling proteins. While such mutations were first identified in severe developmental disorders, or in specific cancers, several genes have been implicated in both, including the  Ca. Ca14]. We have previously demonstrated PHF6 to endogenously co-purify with multiple constituents of the NuRD chromatin remodelling complex  see A, a tranet al. confirmed a direct PHF6-RBBP4 interaction, and subsequently resolved the crystal structure to show that a PHF6 peptide (aa 162–170) peptide occupies a binding pocket located upon the surface of the RBBP4 β propeller ,8759,87]cription .Aside from these direct studies investigating PHF6 interactions, several phosphoproteomic screens have identified PHF6 peptides, suggesting that it is regulated by phosphorylation ,107,108.2 phase when nucleolar size and the rate of ribosome biogenesis is higher relative to G1 [Two other studies have identified the phosphorylation of three serine residues  during mitosis and in response to T-cell receptor signalling ,107. Intve to G1 ,116. MorPHF6 mutations  within hematopoietic cells. Since female Coffin–Siris patients have a similar type of mutation to those individuals with T-ALL, they may be at greater risk for developing cancer. However, female patients with de novo germline deletions of PHF6 all have skewed X-chromosome inactivation in the blood, which may explain why hematopoietic cancers have so far not been observed. The PHF6 protein is important for both faithful development and tumour suppression, yet only 6.5% of germline ions see  are assoThe PHF6 interactors identified to date suggest that PHF6 is a multifaceted regulator of transcription working with the NuRD chromatin remodelling complex (or UBF) to repress or activate transcriptional initiation, and with the PAF1 complex in controlling transcriptional elongation A. While PHF6 to positively regulate NGC/CSPG5 (via PAF1) to ensure proper cortical neuron migration, and miR-128 to negatively regulate PHF6, which allows for subsequent neuronal maturation, represent the first clear model of a developmental role for PHF6 [NOTCH1 and RUNX1 promoters, genes that have mutations that are co-expressed with PHF6 mutations in T-ALL and AML, respectively [PHF6 promoter in a T-ALL cell line suggests that hematopoietically expressed PHF6 may participate within a feedback regulation network [In conducting future investigations to elucidate the transcriptional roles of such PHF6-containing complexes, it will be important to identify all PHF6 target genes through tissue-specific ChIP-Seq as a starting point to assess which PHF6 targets are PAF1 regulated, which ones require the recruitment of NuRD, and which ones might utilize both complexes. Already, ChIP-Seq experiments for the NuRD constituents CHD4 and MBD3 indicate an enrichment of binding sites in gene bodies and well-defined peaks at transcriptional start site (TSS) regions ,120. Simfor PHF6 . Moreovefor PHF6 . Interesectively . Further network . TherefoIn addition to developmental gene targets, PHF6 may utilize similar complexes to control rDNA transcription, as outlined in RPL5, RPL10, RPL11, RPL22), whereas others  target aspects of the cap-dependent mRNA translation machinery, as has been the subject of recent reviews [In many cancers, it is the hyperactivation of ribosome biogenesis that contributes to the pathology, with large nucleoli correlating to poorer clinical outcomes . Given t reviews ,126. For reviews ,126. It  reviews . In this reviews .In neurons, large nucleoli facilitate the sufficient supply of ribosomes to growing neurites to accommodate local demands for translation . Indeed PHF6 mutations, will certainly augment the dissection of the molecular mechanisms driving BFLS and leukemia. In making use of such models, further characterization of the functional roles of PHF6 may thus facilitate the development of novel therapeutics for these distinct disorders. Since the discovery of PHF6 as the cause of BFLS in 2002, the precise function of the protein has remained elusive. Nonetheless, it is becoming clear that PHF6 is a highly dynamic chromatin adaptor protein, containing two ZaP domains that facilitate its interactions with nucleic acids and a growing number of interaction partners  to regulate transcription. Moreover, several potential phosphorylation sites, its interaction with rDNA, and its ability to shuttle between the nucleolus and nucleoplasm suggest that it may be a key regulator of ribosome biogenesis. The generation of transgenic mouse models with BFLS-like, T-ALL-like, or AML-like