Cisplatin is a potent antitumor agent. However, toxicity and primary and secondary resistance are major limitations of cisplatin-based chemotherapy, leading to therapeutic failure. We have previously reported that mono-sulfonamide platinum complexes have good antitumor activity against different tumoral cell lines and with a different and better cytotoxic profile than cisplatin. Besides, N-sulfonamides have been used extensively in medicinal chemistry as bactericides, anticonvulsant, inhibitors of the carbonic anhydrase, inhibitors of histone deacetylases, and inhibitors of microtubule polymerization, among others.trans-sulfonamide-platinum-complex (TSPC), in two human melanoma cell lines that differ in their TP53 status: SK-MEL-5, TP53 wild type, and SK-MEL-28, TP53 mutated. We performed cytotoxicity assays with both drugs, alone and in combination, cell cycle analyses, western blotting and immunoprecipitation, and fluorescence immunocytochemistry.We aimed to compare the cytotoxic effects of cisplatin and a TP53 mutated. Also, strong accumulation of CDKN1A and CDKN1B in cells nuclei was seen upon TSPC treatment in both cell lines.TSPC had similar antiproliferative activity than cisplatin against SK-MEL-5 (3.24 ± 1.08 vs 2.89 ± 1.12 μM) and higher against SK-MEL-28 cells (5.83 ± 1.06 vs 10.17 ± 1.29 μM). Combination of both drugs inhibited proliferation in both cell lines, being especially important in SK-MEL-28, and showing a synergistic effect. In contrast to cisplatin, TSPC caused G1 instead G2/M arrest in both cell lines. Our present findings indicate that the G1 arrest is associated with the induction of CDKN1A and CDKN1B proteins, and that this response is also present in melanoma cells containing Overall, these findings provide a new promising TSPC compound with in vitro antitumor activity against melanoma cell lines, and with a different mechanism of action from that of cisplatin. Besides, TSPC synergism with cisplatin facilitates its potential use for co-treatment to reduce toxicity and resistance against cisplatin. TSPC remains a promising lead compound for the generation of novel antineoplastic agent and to explore its synergism with other DNA damaging agents. Its mode of action is mediated by its interaction with DNA to form DNA adducts, which activate several signal transduction pathways leading to cell death [Cisplatin -2-(5-(dimethylamino)naphthalene-1-sulfonamido)cyclohexylamino] (dimethylsulfoxide)platinum(II) (hereinafter TSPC),  and SK-MEL-28 (TP53 mutated) [Therefore, we decided to study several mechanistics aspects of TSPC in two melanoma cell lines with a different TP53 status: SK-MEL-5 (mutated) . TP53 ismutated) , whose rmutated) . These fmutated) . It is tIn this study, we show the in vitro antitumor activity of TSPC and CDDP in these melanoma cell lines and mechanistic differences in cell cycle effects. We also aim to explore if these differential effects might be used as a basis for a synergism between both compounds.2). RPMI 1640 and other culture materials were from Lonza Ltd. . TSPC was synthesized and characterized in the Inorganic Chemistry Department of the Universidad Autónoma de Madrid (Spain), as previously reported [Human melanoma cell lines SK-MEL-5 and SK-MEL-28 were obtained from American Type Culture Collection  in February 2010. Low passage cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine, under standard culture conditions  was calculated by nonlinear regression fit of the mean values of the data obtained in at least three independent experiments using GraphPad Prism software version 5.0 .Briefly, cells were seeded in a 96-well flat-bottom plate at 5000 cells/well and cultured for 24 h prior to exposure to CDDP or TSPC at varying concentrations, from 0 to 100 μM for 72 h. For combination assays, cells were treated with CDDP ranging from 0.5 to 10 μM and two different doses of TSPC (1 and 5 μM). Results were expressed as a percentage relative to vehicle-treated control (0.1% DMSO was added to untreated cells). Viability was determined using the WST-1 method  following the manufacter’s instructions. The 50% inhibitory concentration . DNA content and cell cycle analyses were performed by using a FACScalibur flow cytometer and the CellQuest software (Becton Dickinson Biosciences). Apoptosis was studied with the APO-BrdUTunel assay kit  following the experimental protocol provided by the manufacturer, and analyzed with a FACScalibur flow cytometer and the CellQuest software (BD Biosciences). All experiments were performed in triplicate.Cip1), CDKN1B (p27Kip1), CDKN2B (p15INK4b), CDK2, CDK4, CCND1 (Cyclin D1), CCNE2 (Cyclin E2), phospho-CDK1 (P-cdc2) and GAPDH using specific primary antibodies  diluted 1/1000 in blocking solution followed by peroxidase-conjugated appropriate secondary antibody , and visualized by Immun-Star WesternC kit (Bio-Rad) detection system. Results were scanned with Image Quant LAS 4000 Imaging densitometer and quantify with Image Quant TL software . To ensure that control cells gave reproducible baseline protein levels, we carefully maintained cells cultures in a constant semiconfluent and logarithmically growing state throughout each experiment.Cells untreated (control) or treated with equimolar concentrations of either CDDP or TSPC for 24 h were lysed with MCL1 lysis buffer in the presence of protease and phosphatase cocktail inhibitor  following the manufacter’s protocol. Total protein concentrations were determined using the BCA protein assay kit . Protein lysates (30 μg) were subjected to SDS-PAGE on 15% polyacrylamide gel. The separated proteins were transferred on to PVDF membrane followed by blocking with 5% non-fat milk powder (w/v) in TTBS . Membranes were probed for the protein levels of TP53, CDKN1A (p21Pierce Classic IP Kit (Thermo Scientific) was used for immunoprecipitation assays, and manufacturer’s instructions were followed. Thereby, 1 mg of protein was immunoprecipitated using CDK2 primary antibody . The immune complex eluted was subjected to SDS-PAGE and trans-blotted into PVDF membranes as described above. Membranes were probed for the protein levels of CDK2, CDKN1A and CDKN1B.Cells were cultured in coverslip glasses for 24 h with equimolar concentration of either CDDP or TSPC. Untreated cells were used as control. After exposure to the drugs, cells were fixed with 4% formaldehyde and permeabilized with PBS/triton (0.1%). For immunofluorescence labeling, cells were incubated with anti-human CDKN1A (1:1000) or CDKN1B (1:1000) monoclonal antibodies  followed by a secondary antibody conjugated to Alexa Fluor 488 . Nuclei were stained with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) . Images from six to ten fields per sample of at least three independent experiments were taken with a LSM 510 Meta Confocal Microscope .50 for 72 h, or with CDDP 1 μM (SK-MEL-5) or 3 μM (SK-MEL-28) and TSPC 1 or 5 μM in combination assays. After this time, cells were washed with PBS and put in drug-free medium to recover. Viable cell number was assessed with Trypan blue and a Neubauer chamber for 7 days of recovery.To study the effects of duration of platinum exposure on cell proliferation, cells were treated with CDDP or TSPC at their corresponding ICt-test was used to examine differences between groups. For recovery experiments, cell growth was fit to a Gompertz function, typical of cell growth and characterized by a lag period, an exponential and a saturation phase. All the statistical tests were conducted at the two-side 0.05 level of significance.Results are expressed as the mean ± SD, and data are representative of at least three different experiments. Analysis of variance (ANOVA) one way, followed by Dunnett’s TP53 wild-type) and SK-MEL-28 (TP53 mutated). First, we analysed TSPC antiproliferative activity. As shown in Fig. 50 of this compound (3.24 ± 1.08 μM) was similar to the one obtained with CDDP (2.89 ± 1.12 μM) in SK-MEL-5, and significantly lower (p < 0.05) in SK-MEL-28 (5.83 ± 1.06 μM vs 10.17 ± 1.29 μM).Based on our promising previous work with TSPC  we decidp < 0.05) increment of cell number in G1 phase in both melanoma cell lines  CDKN1A (p21Cip1), CDKN1B (p27Kip1), and CDKN2B (p15INK4b).Based on the G1 arrest induced by TSPC in both cell lines, we assessed the effect of this compound on the cell cycle regulatory molecules that play important roles in G1 phase of cell cycle progression. TP53 is the most important tumor suppressor protein associated with the G0-G1 arrest in cell cycle, and a key protein in CDDP mode of action and resistance. Since SK-MEL-28 has a mutation in Western blot analysis showed that TSPC treatment strongly increased the protein levels of CDKN1A and CDKN1B and had no effect on TP53 levels in both cell lines Fig. . The obsWe also assessed the effect of TSPC on the protein levels of CDKs and cyclins involved in G1 phase and G1 to S phase transition of cell cycle. As shown in Fig. In order to assess the mechanism of action of CDDP in the absence of TP53, we also examined the phosphorylation state of CDK1 (cdc2), which have been shown to be associated with S and G2/M arrest and with CDDP mechanism of action. Western blot analysis showed that CDDP treatment strongly induced the protein levels of phospho-CDK1 in both cell lines, while TSPC had no effect Fig. . DensitoSince up-regulation in CKIs levels was observed, and these proteins exert their inhibitory effect and G1 cell cycle arrest by direct binding to CDK2-cyclin E complexes, we next examined CDK2-CKIs binding by immunoprecipitation assay. As shown Fig. The inhibitory effect of CKIs on CDK-cyclin complexes occurs in the nucleus, so we next examined by immunofluorescence and confocal microscopy the localization of CKIs in this cell lines. As shown in Fig. In addition, we determined the percentage of nuclear CDKN1A and CDKN1B staining in cell lines exposed to CDDP or TSPC. As shown in Fig. Once analysed the effect of TSPC on cell cycle and cell growth, we next examined how long this effect would last. Cell treated with TSPC recovered a normal proliferation rate after drug removal. However, cell treated with CDDP did not recover Fig. . Seeing 50: 1 μM (SK-MEL-5) or 3 μM (SK-MEL-28), and TSPC 1 or 5 μM. In all the cases cells recovered in the same way (Fig. Taking into consideration this synergism we studied whether cells treated with both drugs in combination were able to recover after drug removal. For this purpose we treated cells with CDDP at a dose lower than their respective ICway Fig. .cis-isomerism.Platinum complexes are widely used in cancer therapy. The successful clinical applications of CDDP have inspired the synthesis and investigation of numerous platinum compounds as drug candidates. All the commercially available platinum compounds are based in a trans-platinum derivative TSPC were investigated and directly compared with those of CDDP in SK-MEL-5 and SK-MEL-28 melanoma cell lines. The choice of these cell lines were made on the basis of TP53 status, and in our previous cytotoxicity results in a panel of different cell lines [In this study, the cellular and molecular effects of the ll lines .TP53 mutant line. TP53 plays a very important role in CDDP cytotoxicity [TP53 mutant line show some resistance to this drug [TSPC showed to be as active as CDDP in SK-MEL-5 and more active in SK-MEL-28, the toxicity , so it ihis drug , 22. On Upon 24 h incubation with TSPC, cells were arrested in G1 phase that suggest cell cycle delay for a DNA damage response, DNA repair and/or apoptosis. One goal of this study was to define the cell cycle regulatory pathway responsible for the G1 arrest. The results obtained in these two cell lines with different TP53 status show that TSPC inhibits cell proliferation through cell cycle arrest at G1 phase, likely due to the up-regulation of CDK inhibitors CDKN1A and CDKN1B. Progression through the G1 phase of the cell cycle is controlled by CDK4-cyclin D complexes, while CDK2-cyclin E complexes are required for proper G1/S transition and initiation of S . CDKs arOn the other hand, cells incubated with CDDP were arrested in S and G2/M phase. Several reports have previously described that CDDP induces S- and G2/M-phase arrests in a sequential manner . These aThe cell cycle regulatory function of both CDKN1A and CDKN1B is associated with its nuclear localization, but the protein can also localize in the cytoplasm where they could exert different functions . In the 50 dose, was permanent, TSPC treatment was transitory. This is not necessarily a negative result, but it implies that TSPC treatment should be frequent. Thus, in regard to clinical treatment, it would be useful to find an easy and sequentially way of TSPC administration.Recovery experiments showed that while CDDP effect, at least at ICCDDP is one of the most potent antitumor agents known. However, toxicity and resistance are major limitations of CDDP-based chemotherapy , 2. ThusTP53 gene and are inherently resistant to commonly used DNA-damaging agents.In this study we present a new promising TSPC compound with in vitro antitumor activity against SK-MEL-5 and SK-MEL-28 melanoma cell lines, with a different mechanism of action that of CDDP. From our results it can be inferred that TSPC induces cell cycle G1 arrest probably mediated by CDKN1A and CDKN1B inhibitors in a TP53-independent manner. CDK2-cyclin E complexes are required for G1/S transition. By co-immunoprecipitation assays, we show that CDKN1A and CDKN1B bind to CDK2 after TSPC treatment, inhibiting CDK2 activity. Besides, synergy between TSPC and CDDP facilitates its potential use for co-treatment to reduce toxicity and resistance against CDDP. On the other hand, TSPC remains a promising lead compound for the generation of novel drug candidates with different cytotoxicity profiles from those of CDDP. Moreover, these results could have therapeutic implications for TSPC, because the majority of human solid tumors contain a mutant TP-53 or deleted


Cell surface proteins have provided useful targets and biomarkers for advanced cancer therapies. The recent clinical success of antibody-drug conjugates (ADCs) highlights the importance of finding selective surface antigens for given cancer subtypes. We thus attempted to develop stand-alone software for the analysis of the cell surface transcriptome of patient cancer samples and to prioritize lineage- and/or mutation-specific over-expression markers in cancer cells.A total of 519 genes were selected as surface proteins, and their expression was profiled in 14 cancer subtypes using patient sample transcriptome data. Lineage/mutation-oriented analysis was used to identify subtype-specific surface markers with statistical confidence. Experimental validation confirmed the unique over-expression of predicted surface markers  in lung cancer cells at the protein level. The differential cell surface gene expression of cell lines may differ from that of tissue samples due to the absence of the tumor microenvironment.http://compbio.sookmyung.ac.kr/~qsurface.In the present study, advanced 3D models of lung cell lines successfully reproduced the predicted patterns, demonstrating the physiological relevance of cell line-based 3D models in validating surface markers from patient tumor data. Also QSurface software is freely available at The online version of this article (10.1186/s12918-018-0541-6) contains supplementary material, which is available to authorized users. Cell surface proteins have provided major targets and biomarkers for anticancer therapies. In colorectal cancer, the expression of surface proteins such as CDH17, CD138 and members of the integrin family is related with tumor progression . EpidermMore recently, cell surface proteins have been successfully used as targets for antibody-drug conjugates (ADC) as part of cancer therapy –8. ADCs The Cancer Genome Atlas (TCGA) is one of the largest datasets from pan-cancer analyses . The relAs an analysis tool to find differentially expressed genes, cBioPortal , 14 is uIn the present study, we developed stand-alone software, QSurface, to analyze lineage- and/or mutation-specific cell surface transcriptome marker from cancer patients’ samples obtained from TCGA. Selected expression markers were validated at the protein level using lung adenocarcinoma (LUAD) cell lines. Notably, the gene expression of extracellular/membrane proteins exhibits inconsistent patterns between cell lines and patient tissue samples . In the http://cancergenome.nih.gov/) in 2015. The RNASeqV2 data were sequenced using the Illumina HiSeq 2000 and Illumina Genome Analyzer (GA) platforms. We selected 658 tumor samples with matched normal samples obtained from the same patient in 14 cancer types, satisfying the requirement more than 10 samples  data from patients’ tumor and normal tissue samples were downloaded from TCGA website  from the cellular components category to identify cell surface genes from the AmiGO website, October 2016 , 20. A tP-values. We calculated log2 delta as the average difference in the expression levels for lineage-specific cell surface genes between tumor and normal samples and for mutation-specific cell surface genes between mutant and wild-type tumor samples per lineage.To identify differentially expressed cell surface genes, we used log2 delta and t-test 5 cells per well were seeding on a 6well culture plate for monolayer cell culture during 5 days.Three types of cell lines, an STK11 mutant type , STK11 wild type , and STK11-recoverd type  were used for validation experiments. HCC-827 and H1975 cells were obtained from the American Type Culture Collection , respectively. All other STK11 mutant and wild-type cell lines were obtained from the National Institutes of Health, National Cancer Institute . STK11 mutant cell lines and wild-type cell lines were cultured in RPMI 1640  supplemented with 10% fetal bovine serum (HyClone Laboratories) and 1% antibiotics . STK11-recoverd cell lines were cultured in the same medium with added 1μg/ml puromycine. A total of 1~ 3 × 102 and 95% air at 37 °C and the culture medium was refreshed every 2 to 3 days. The culture plates for stem-like cells (SLCs) were coated with a 5-mg/ml solution of poly-2-hydroxyethyl methacrylate (Sigma-Aldrich) in 95% ethanol. The same amount of cells as 2D cell culture was seeded in a 6-well plate for sphere culture.Cancer stem-like cell (CSLC) spheres were cultured in serum-free conditioned DMEM/F-12 medium supplemented with 20-ng/ml EGF, 20-ng/ml basic fibroblast growth factor, and B27 (Thermo Fisher Scientific). The cells were maintained in a humidified atmosphere of 5% COTotal cell extracts were prepared by incubating the cells in lysis buffer  on ice for 30 min. Cell debris was removed by centrifugation, and the total protein levels in the supernatants were quantified using the Bradford method (Bio-Rad). Equal amounts of protein (50 μg) were heated at 95 °C for 5 min, electrophoretically resolved using 12% SDS-PAGE, and then transferred to nitrocellulose membranes (Millipore). The membranes were blocked with TBST  containing 5% skim milk for 1 h and then hybridized as indicated to specific primary antibodies (1:1000 dilution) at 4 °C overnight. The membranes were washed and hybridized to HRP-conjugated secondary antibodies for 1 h at room temperature. Specific bands were visualized using an enhanced chemiluminescence (ECL) detection system  and an LA3000 luminescence image analyzer . Antibodies against MUC4 and SLC7A11 were purchased from Abcam PLC. Antibodies against Mesothelin and GAPDH were purchased from Cell Signaling. GAPDH was used as a loading control. The anti-STK11 antibody was purchased from Santa Cruz.We implemented QSurface, a tool for exploring lineage- and/or mutation-specific gene expression of all potential surface proteins Fig. . Genes fUsers can browse differential gene expression of 29 known target antigens that are currently under phase I or II clinical trials , 21, 22 P-value < 0.01) in the top of the window   Fig. . MSLN (o02) Fig. . Lastly,Cancer cell culture system are classified into two-dimensional (2D) and three-dimensional (3D) cancer models . CompareThis study presents QSurface, rapid and efficient tools to identify novel tumor-specific cell surface markers for advanced cancer therapies. QSurface provides two analyzing method, lineage−/ and mutation-oriented profiles. To identify potential surface genes, QProfile used fold changes to find sensitivity of gene expression on given conditions. By using QSurface, we obtained 3 STK11-mutant specific expression markers, MUC4, MSLN, and SLC7A11 in LUAD. Furthermore, advanced 3D cell line models of lung cancer successfully reproduced the predict patterns by QSurface. And it demonstrates the physiological relevance of cell line-based 3D models with patient tumor data and confirms that QSurface is useful and reliable tools for identifying mutation/lienage-specific cell surface markers.Additional file 1:Table S1. Data description of TCGA RNA sequencing data. Table S2. List of antibody-drug conjugates. Figure S1. Distribution of tumor sample-specific gene expression in 14 cancer types. Totally 20,531 genes, 519 cell surface marker, and significant cell surface hits  are illustrated in grey, yellow, and red, respectively. Figure S2. Western blot analysis of the MUC 4, MSLN and SLC7A11 expression on STK11 mutant, restored and wild type cell lines. (A) gene expression on STK11 mutant, restored and wild-type cell lines on 2D culture status and (B) on 3D culture status. (DOCX 1902 kb)


Zika virus (ZIKV) is unique among human-pathogenic flaviviruses by its association with congenital anomalies and trans-placental and sexual human-to-human transmission. Although the pathogenesis of ZIKV-associated neurological complications has been reported in recent studies, key questions on the pathogenesis of the other clinical manifestations, non-vector-borne transmission and potential animal reservoirs of ZIKV remain unanswered. We systematically characterized the differential cell line susceptibility of 18 human and 15 nonhuman cell lines to two ZIKV isolates (human and primate) and dengue virus type 2 (DENV-2). Productive ZIKV replication  protein expression and cytopathic effects (CPE)) was found in the placental (JEG-3), neuronal (SF268), muscle (RD), retinal (ARPE19), pulmonary (Hep-2 and HFL), colonic (Caco-2),and hepatic (Huh-7) cell lines. These findings helped to explain the trans-placental transmission and other clinical manifestations of ZIKV. Notably, the prostatic (LNCaP), testicular (833KE) and renal (HEK) cell lines showed increased ZIKV load and/or NS1 protein expression without inducing CPE, suggesting their potential roles in sexual transmission with persistent viral replication at these anatomical sites. Comparatively, none of the placental and genital tract cell lines allowed efficient DENV-2 replication. Among the nonhuman cell lines, nonhuman primate (Vero and LLC-MK2), pig (PK-15), rabbit (RK-13), hamster (BHK21) and chicken (DF-1) cell lines supported productive ZIKV replication. These animal species may be important reservoirs and/or potential animal models for ZIKV. The findings in our study help to explain the viral shedding pattern, transmission and pathogenesis of the rapidly disseminating ZIKV, and are useful for optimizing laboratory diagnostics and studies on the pathogenesis and counter-measures of ZIKV. Our findings provided novel implications on the disease pathogenesis, transmission and potential animal reservoirs that could be applied to optimize laboratory testing protocols and infection control strategies for this global health emergency.ZIKV research has so far been mainly focused on the pathogenesis of ZIKV-associated neurological complications. Questions on the pathogenesis of ZIKV's other clinical manifestations, its unique ability among flaviviruses to be transmitted trans-placentally and sexually, and its potential animal reservoirs remain mostly unanswered. Although To investigate on the differential cell line susceptibility to epidemic ZIKV and pre-epidemic ZIKV, we included a clinical isolate of ZIKV (Puerto Rico strain PRVABC59) obtained from a patient in the recent South American epidemic , and another pre-epidemic ZIKV strain isolated from a nonhuman primate in Uganda in 1947 (976 Uganda strain) . An archived clinical isolate of DENV-2 was obtained from Department of Microbiology, The University of Hong Kong, for comparison with ZIKV, as the two viruses are closely related phylogenetically and have similar clinical manifestations. The ZIKV and DENV-2 isolates were amplified by three additional passages in Vero cells and C6/36 cells, respectively, to make working stocks of the viruses.Institutional Review Board approval for use of the vial isolates was obtained and all samples were anonymized. The cell lines used in this study were obtained from sources listed in 14 Briefly, 33 cell lines derived from different tissues or organs and host species were each inoculated with one multiplicity of infection of the ZIKV and DENV-2 isolates for 1 h  was used for the detection of ZIKV and DENV-2 using QuantiNova Probe RT-PCR Kit (Qiagen) in a LightCycler 96 Real-Time PCR System . Five microliters of purified TNA was amplified in a 20 μL-reaction containing 10 μL of 2 × QuantiNova Probe RT-PCR Master Mix, 0.2 μL QN Probe RT-mix, 0.8 μM forward primer, 0.8 μM reverse primer and 200 nM probe. ZIKV detection was performed as previously described, with slight modifications.18 Forward primer (5′-CGY TGC CCA ACA CAA GG-3′), reverse primer (5′-CCA CYA AYG TTC TTTT GCA BAC A-3′) and probe (5′-HEX-AGC CTA CCT TGA YAA GCA RTC AGA CAC TC-IABkFQ-3′) targeting the ZIKV envelope gene were used. For DENV-2 detection, forward primer (5′-GCA TAT TGA CGC TGG GAR AGA C-3′), reverse primer (5′-CGY TCT GTG CCT GGA WWG ATG-3′) and probe (5′-FAM-CAG AGA TCC TGC TGT C-MGB-NFQ-3′) targeting the DENV-2 3'-untranslated region were used. Reactions were incubated at 45 °C for 10 min, followed by 95 °C for 5 min, and then thermal cycled for 50 cycles . A series of 10-fold dilutions equivalent to 1 × 102–1 × 106 copies/reaction mixture were prepared to generate calibration curves and run in parallel with the test samples.Total nucleic acid (TNA) was extracted from culture supernatants of the 33 cell lines infected by ZIKV or DENV-2 at 1, 3 and 5 d.p.i. using EZ1 Virus Mini Kit v2.0  as we previously described.NdeI and XolI sites of expression vector pETH in frame and downstream of the series of six histidine residues. The recombinant ZIKV–NS1 protein was expressed in Escherichia coli, denatured in 8 M urea, and purified with Ni-nitrilotriacetic acid affinity chromatography (Qiagen) according to the manufacturer's instructions. The solubilized protein was refolded by slowly dialyzing with refolding buffers. Expression of the recombinant ZIKV–NS1 protein was confirmed by western blot analysis using mouse anti-His monoclonal antibody . Preparation of DENV-2-NS1 protein was performed as previously described.19Primers (5′-CAT ATG GAT GTG GGG TGC TCG GTG GAC T-3′ and 5′-CTC GAG TGC AGT CAC CAT TGA CCT TAC T-3′) were used to amplify the gene encoding the ZIKV–nonstructural protein 1 (NS1) protein by RT-PCR. The sequence encoding a 352-amino-acid fragment of ZIKV–NS1 protein was amplified and cloned into the 14 Briefly, 30 μg of purified recombinant ZIKV-NS1 protein was mixed with an equal volume of complete Freund's adjuvant (Sigma-Aldrich) and injected subcutaneously into BALB/c mice. Incomplete Freund's adjuvant (Sigma-Aldrich) was used in subsequent injections at 14-day intervals for three times. Serum samples were collected 14 days after the fourth injection. Preparation of specific antibodies against DENV-2-NS1 protein was performed as previously described.19This was performed as we previously described with modifications.14 Cell smears at 1, 3 and 5 d.p.i. were prepared and fixed in chilled acetone at −20 °C for 10 min. The fixed cells were incubated with mouse antiserum against the ZIKV- or DENV-2-NS1 protein, followed by FITC-rabbit anti-mouse IgG . Cells were then examined under a fluorescence microscope. Three representative microscopic fields were chosen and 100 cells were counted for characteristic cytoplasmic apple green fluorescence. The mean percentage of positive cells was rounded up to the nearest multiplicity of 10. Uninoculated cells were used as negative control.This was performed as we previously described with modifications.t-test was used to compare the mean viral load of the different cell lines at 1, 3 and 5 d.p.i. with the mean baseline viral load at 0 d.p.i. All calculations were based on log-transformed viral loads. P-value of <0.01 was considered as statistically significant. Computation was performed using the Predictive Analytics Software v18.0 for Windows.The Student's P<0.01) within 5 d.p.i. was observed in 14/18 cell lines infected with ZIKV-PR or ZIKV-U (P>0.01), but none of the immune cell lines achieved a ⩾2 log increase in mean viral load within 5 d.p.i. The highest mean viral loads (⩾10 lgcopies/mL) were observed in the placental (JEG-3), cervical (HeLa), neuronal (SF268), upper respiratory tract (Hep-2) and intestinal (Caco-2) cell lines. Twelve of these 14 cell lines  showed obvious ZIKV-NS1 protein expression by IF in addition to a high viral load  in only 9/18 cell lines  and DENV-2-NS1 protein expression in the HeLa cell line. Most of these nine cell lines with increased viral loads demonstrated DENV-2-NS1 protein expression  was observed within 5 d.p.i. in 8/15 cell lines (Fifteen nonhuman cell lines were tested . ZIKV-PRll lines . These ill lines . ZIKV-NSll lines . Some ofll lines . CPE werP<0.01) within 5 d.p.i. was observed in only 6/15 cell lines, including the nonhuman primate (Vero and LLC-MK2), pig (PK-15), bat (TP2), hamster (BHK21) and mosquito (C6/36) cell lines  and neuronal (SF268) cell line tropism corroborated with the congenital and neurological manifestations of ZIKV infection. These manifestations include congenital microcephaly and other central nervous system malformations in infected fetuses, and meningoencephalitis, myelitis, and post-infectious Guillain–Barré syndrome in adults.24 ZIKV RNA, full-length viral genome and/or viral particles could be detected in the brain tissue of fetuses with congenital microcephaly.25 Microscopically, flavivirus-like virions were observed together with extensive inflammation, astrocyte and microglial activation, and injury in the cortex and the lateral corticospinal tracts in the fetal brain tissue.25 Moreover, ZIKV could impair growth and deplete neural progenitor cells in human neurospheres and cerebral organoids.27Productive ZIKV replication as defined by ⩾2 log increase in mean viral load, ZIKV-NS1 protein expression by IF, and presence of CPE was observed within 5 d.p.i. in 8/18 human cell lines, which represented cells of placental, neuronal, muscle, retinal, pulmonary, intestinal and hepatic origin. This broad tissue tropism is likely related to the wide distribution of the candidate receptors of ZIKV, including AXL, TYRO3 and DC-SIGN, in different organs.28 Myositis and myocarditis were observed in suckling mice inoculated with ZIKV intracerebrally, and viral RNA could be detected in the muscle and heart of type I interferon receptor-deficient mice infected with ZIKV.30Consistent with ZIKV's tropism for the human rhabdomyosarcoma (RD) cell line, myalgia and arthralgia with periarticular edema are common features in symptomatic ZIKV infection. Fetuses with congenital ZIKV infection may develop arthrogryposis.13 Abdominal symptoms including diarrhea, vomiting, abdominal pain and jaundice with hepatic dysfunction have been reported in patients with ZIKV infection, and are in agreement with the susceptibility of the intestinal (Caco-2) and hepatic (Huh-7) cell lines to ZIKV.31 Respiratory distress may occur in severe ZIKV infection, and this correlates with ZIKV's tropism for respiratory  cell lines.7 Similarly, the clinical features of DENV infection, including encephalopathy/encephalitis (SF268), myalgia and rhabdomyolysis (RD), retinitis (ARPE19), respiratory symptoms (Hep-2 and HFL), diarrhea (Caco-2) and hepatic dysfunction (Huh-7), also correlated with the cell line susceptibility to DENV-2 in our study.33Viral tropism for the retinal (ARPE19) cell line corroborates with the ophthalmological complications of congenital ZIKV infection, including chorioretinal atrophy and pigmentary maculopathy.34 Although a previous study suggested that type III interferons produced by human placental trophoblasts could confer protection against ZIKV infection, it is notable that the study utilized primary human trophoblasts of full-term instead of early gestation placenta.35 Indeed, our in vitro finding concurred with the recent reports of detectable viral particles and RNA in trophoblasts of the maternal and fetal placenta of ZIKV-infected type I interferon signaling-deficient mice and permissibility of placental macrophages and cytotrophoblasts to productive ZIKV infection.36Although neurological manifestations may also be seen in infections caused by other flaviviruses, such as DENV, yellow fever virus, West Nile virus (WNV) and Japanese encephalitis virus (JEV), none of these flaviviruses have been associated with congenital microcephaly and central nervous system malformations. These unique clinical features of ZIKV infection require the virus to be able to infect neural progenitor cells of the fetus in utero during early gestation. A striking difference between the cell line susceptibilities of ZIKV and DENV-2 observed in our study was ZIKV's ability to productively replicate in placental cells (JEG-3). JEG-3 cells infected with ZIKV produced high viral loads (~10 lgcopies/mL), abundant ZIKV-NS1 protein expression and prominent CPE. This finding helped to explain the unique ability of ZIKV to infect the fetus via the placenta and cause congenital anomalies, particularly when the infection occurred during the first trimester.22 ZIKV-infected male patients might develop hematospermia and perineal pain compatible with prostatitis, and might have detectable viral RNA and/or infectious viral particles in their semen.22 In contrast to DENV-2, which did not efficiently replicate in any of the genital tract cell lines, the mean ZIKV load significantly increased in the prostatic (LNCaP) and testicular (833KE) cell lines without inducing CPE. This suggested that the prostate and testes might be important sites of ZIKV replication that led to prolonged virus shedding in the semen of infected men.37 Interestingly, abundant ZIKV-NS1 protein expression was detected in ZIKV-infected LNCaP cells, but not 833KE cells. This finding might imply that ZIKV could replicate more efficiently in the prostate than the testes. Recent studies only reported high viral loads in the testes of ZIKV-infected mice deficient in interferon signaling and/or receptor, but did not report the viral loads in their prostates.38 Further studies should be conducted to investigate the prostate's role as a replication site of ZIKV. The female genital tract cell lines, including HeLa and HOSE6-3, were also susceptible to ZIKV, supporting the hypothesis that non-immune women might acquire the infection through sexual intercourse with their ZIKV-infected male partners.Besides trans-placental transmission, our findings also provided insights to explain the pathogenesis of other non-vector-borne human transmission routes of ZIKV. Probable sexual transmission of ZIKV has been reported.41 The finding of high viral load and abundant ZIKV-NS1 protein expression without inducing CPE in HEK cells is particularly interesting, as it suggests that the kidney might be another site of persistent ZIKV replication. Similarly, DENV-2 infection also caused increased mean viral load and DENV-2-NS1 protein expression without inducing CPE in HEK cells, and viral RNA might be detected in urine of dengue patients.42 An important implication of this finding is that kidney transplant donors in ZIKV-affected regions should be tested for the infection before organ donation, as virus shedding in urine might be prolonged.41 The intestinal (Caco-2) cell line was also highly susceptible to ZIKV, which corroborated with the high viral load detected in the intestines of ZIKV-infected mice.43 Serial testing of fecal samples of ZIKV-infected patients should be performed to determine whether feces represent another possible transmission route of ZIKV. It would also be interesting to investigate ZIKV's ability to productively replicate in salivary gland cells, as high viral load could also be detected in the saliva of ZIKV-infected patients.44We showed that the renal (HEK) and respiratory tract  cell lines were also susceptible to ZIKV. This helped to explain the high viral loads in the urine and nasopharyngeal swabs of ZIKV-infected patients.Aedes albopictus (C6/36) cell line was quite expected, and corroborated with the high non-fatal infection, dissemination and transmission rates of ZIKV in infected A. albopictus.45 A ⩾2 log increase in mean viral load with ZIKV-NS1 protein expression and CPE were observed in the nonhuman primate (Vero and LLC-MK2), pig (PK-15), rabbit (RK-13), hamster (BHK21) and chicken (DF-1) cell lines. It is well known that the transmission of flaviviruses may involve sylvatic cycles between the mosquito vectors and susceptible wild animals, and urban cycles between the vectors and domestic animals.47 Nonhuman primates are known wild animal reservoirs of ZIKV and DENV.46 Our findings of efficient ZIKV replication in pig, rabbit, hamster and chicken cell lines suggest that these mammalian or avian species might have a role in the transmission of ZIKV. Similar to domestic pigs and wild birds for JEV, and domestic and wild birds for WNV, these species may serve as the animal reservoirs or amplifying hosts of ZIKV.46 Moreover, vector-free JEV transmission due to persistence of virus in tonsils and virus shedding in oronasal secretions of pigs has recently been reported.48 Alternatively, these animal species may be susceptible hosts with less importance in the transmission cycle, which is analogous to horses for JEV and many vertebrate species for WNV.46 Systematic sampling of domestic animals in ZIKV-affected regions would help to confirm these findings. Pigs, rabbits and hamsters should be evaluated as animal models for ZIKV infection, as interferon signaling- or receptor-deficient mice are not routinely available in most laboratories. Pig and/or hamster models have been previously reported for DENV and WNV infection.50The differential cell line susceptibility of our nonhuman cell lines to ZIKV may provide insights into the virus' potential animal reservoirs and alternative animal models. The finding of high viral loads and abundant ZIKV-NS1 protein expression without induction of CPE within 5 d.p.i. in the 54 These cell lines may also be used in pathogenesis studies conducted by laboratories without the technical expertise to handle brain organoids, and to evaluate antiviral treatment and vaccines in vitro prior to animal experiments.Our findings are useful for optimizing laboratory diagnostics and studies on the pathogenesis and counter-measures of ZIKV. Some of the laboratories in resource-limited areas affected by ZIKV may not have the capability to routinely perform molecular diagnostic tests. In addition to the few cell lines previously reported, our study has identified a broad range of cell lines that are suitable for growing ZIKV as an alternative diagnostic tool in clinical virology laboratories.


FMR1 gene. This gene encodes the FMRP protein that is involved in neuronal development. Repeat expansion leads to heterochromatinization of the promoter, gene silencing, and the subsequent absence of FMRP. To date, there is no specific therapy for the syndrome. All treatments in clinic practice provide symptomatic therapy. The development of drug therapy for Fragile X syndrome treatment is connected with the search for inhibitors of enzymes that are responsible for heterochromatinization. Here, we report a weak transcriptional activity of the FMR1 gene and the absence of FMRP protein after Fragile X syndrome cell lines treatment with two FDA approved inhibitors of histone deacetylases, romidepsin and vorinostat. We demonstrate that romidepsin, an inhibitor of class I histone deacetylases, does not activate FMR1 expression in patient cell cultures, whereas vorinostat, an inhibitor of classes I and II histone deacetylases, activates a low level of FMR1 expression in some patient cell lines.Fragile X syndrome is the most common cause of inherited intellectual disability in humans. It is a result of CGG repeat expansion in the 5′ untranslated region (5′ UTR) of the FMR1 gene. The normal allele contains less than 55 triplets. FXS corresponds to a fully mutated allele that contains greater than 200 CGG triplets. Expansion leads to methylation of the FMR1 promoter and of the expanded CGG triplet, resulting in silencing of gene expression. FMR1 encodes the FMRP protein that is involved in neuronal development [γ-aminobutyric acid receptors [ FMR1 gene expression. The search for drugs that activate the FMR1 gene is thought to be an important scientific direction.Fragile X syndrome (FXS) is the main cause of inherited intellectual disability in humans caused by CGG repeat expansion in the 5′ UTR of theeceptors . Additio FMR1 leads to deacetylation of histones H3 and H4 in the locus. Moreover, additional markers of silenced chromatin can be observed in the region [ FMR1 gene in embryonic cells HESC depends on the modification of histones without DNA methylation [Heterochromatinization includes DNA methylation and histone modifications. Some authors reported DNA methylation followed by histone modifications, such as changes in lysine in the N-terminus of histones by histone acetyltransferases , 5. The e region . Howeverhylation . FMR1 expression in FXS cell lines [ FMR1 gene in FXS cells. All studied inhibitors are not applicable for drug development given their low effect [FXS therapy development involves the search for chemicals that inhibit enzymes responsible for heterochromatinization. One method involves DNA methyltransferase (DNMT) inhibition in FXS cell lines with 5-aza-2-deoxycytidine (5-azadC). This drug reactivatesll lines , 9. Addiw effect .To date, three of HDAC inhibitors  are approved by the FDA for human treatment as anticancer drugs. Romidepsin is dipeptide that inhibits class I HDACs. Vorinostat and belinostat are hydroxamic acid derivatives that inhibit class I and II HDACs , 12. FMR1 gene expression in FXS patient cell lines.Here, we present study of the ability of romidepsin and vorinostat to activate FMR1 promoter [All cell lines in the study are immortalized B-lymphocytes. The full mutation cell line GM04025 from the Coriell Cell Repository  has a repeat size of 645 triplets and a methylated promoter , 14. Anopromoter . Cells wμM, 50 μM, and 250 μM romidepsin (PubChem CID 5352062) ; 250 μM, 0.5 mM, and 5 mM vorinostat (SAHA) (PubChem CID 5311) .The 10 mM 5-azadC (PubChem CID 451668)  stock solutions were prepared in sterile water and stored at −20°C in aliquots. The following stock solutions were prepared in sterile 100% DMSO and stored at −20°C: 0.5 mM trichostatin A (PubChem CID 444732) ; 15 ∗105 cells/ml in a total volume of 10 ml per flask. Immediately before use, 10 μl of the 10 mM 5-azadC stock solution was thawed and added daily to the flasks and thoroughly resuspended . The concentration of romidepsin and vorinostat was determined experimentally. The controls were B lymphocyte cell lines without treatment or treated with 15 μl 100% DMSO (solvent for these agents). Briefly, 5∗105 сells were harvested after 72 hours from the start of treatment for RNA and protein extraction, washed twice with 1х PBS solution, and stored at −70°C.In the case of romidepsin and vorinostat treatment, cells were counted, split, and seeded at the initial concentration of 7X = (1 − a/b)∗100, where X is the viability of cell culture, a is the number of stained cells, and b is the total number of cells.Viability of cell cultures was determined by Trypan blue staining followed by counting with a hemocytometer. Viability was determined as  FMR1 and GAPDH in cell line GM06895 as described earlier and all X-fold changes presented as values relative to GM06895 [t-test using GraphPad Prism software . Each sample consisted of three biological replicates in a single experiment. Differences were significant if P < 0.05, where P is type I error.RNA was purified from cell cultures using CCR-100 RNA purification kit  followed by reverse transcription with iScript™ Select cDNA Synthesis Kit . Real-time PCR was performed with iQ™ SYBR® Green Supermix  on a CFX96 Touch™ Real-Time PCR Detection System . Primers and normalization were used as described before . Normali GM06895 , 17. Sta GM06895 . Statistμl lysis buffer  supplemented with protease inhibitor . The resulting homogenates were incubated on ice for 30 min and centrifuged at 5000g for 10 min to remove insoluble precipitates. Protein concentrations in samples were determined with the Pierce™ BCA Protein Assay Kit (Thermo Fisher) according to the manufacturer's protocol. All samples were diluted with water to the same protein concentration. Protein extracts were collected and stored at −80°C.Each sample of cells was suspended in 100 Protein samples were loaded in each lane, separated on a 10% SDS-PAGE, and transferred to a PVDF Transfer Membrane (Thermo Fisher). Membranes were stained with Ponceau S to verify loading and transfer efficiency. FMRP and GAPDH were detected by anti-FMRP  and anti-GAPDH  primary antibodies. Secondary antibodies were goat anti-mouse IgG Fc  and goat anti-rabbit HRP , respectively.n = 46 for each cell line. Analysis of FMR1 expression in all cell lines was carried out as described previously [Cell lines used for drug testing were carriers of a normal human karyotype. Karyotypic analysis of cell lines tested in our research revealed 2eviously . All expeviously  correspoμM [μM to 5 μM. In all studies, treatment time varied from 4 hours to 72 hours [ FMR1 mRNA in cells after treatment. Romidepsin did not activate FMR1 gene expression; moreover, the treatment with high concentrations of the drug led to death of all cells. Thus, preliminary experiments indicated that romidepsin cannot be used as a FMR1 gene activator in FXS cell lines because there is no detectable level of FMR1 mRNA after treatment and very high cytotoxicity. In contrast, vorinostat activated FMR1 gene expression at concentrations 5 μM, but high cytotoxicity was observed.Optimal concentrations of romidepsin and vorinostat were selected based on previously published works. Romidepsin was used for function assays, and the optimal concentration ranged from 10 to 500 nM . VorinosμM . Thus, w72 hours  for romi72 hours  for vori FMR1 expression in FXS cell lines as previously reported [ FMR1 gene expression were noted after treatment with trichostatin A (P > 0.05) (P < 0.05). In the FXS cell line CPG7, no detectable FMR1 transcripts were noted in response to vorinostat and trichostatin A treatment . FMR1 gene transcription levels in GM04025 after treatment with trichostatin A and vorinostat are just 6% and 14% of expression in the control cell line GM06895.Cell lines were treated with vorinostat for 72 hours. As a positive control, we treated cells with trichostatin A, a well-known HDAC inhibitor that can activatereported . Two FXS > 0.05) . Regardireatment . The FXSAll cell lines showed statistically significant decreases in viability after trichostatin A and vorinostat treatment . Vorinos FMR1 mRNA.Western blot analysis revealed a minor increase in FMRP levels in control cell lines after treatment with HDAC inhibitors . Patient FMR1 gene expression in FXS cell lines. Treatment with increasing concentrations of romidepsin leads to cell death without FMR1 gene activation. In contrast, 5 μM vorinostat activated FMR1 gene expression in the GM04025 FXS cell line. Romidepsin and vorinostat are both inhibitors of class I HDACs. Vorinostat additionally inhibits class II HDACs [ FMR1 gene by vorinostat and the absence of activation after romidepsin treatment indirectly indicate that class II HDACs but not class I HDACs play a role in FMR1 promoter heterochromatinization in the FXS patients cell line GM04025.In this research, we studied ability of the FDA approved HDACs inhibitors romidepsin and vorinostat to activateII HDACs , 12. Act FMR1 gene expression in the FXS cell line GM04025 at low levels. Similar low levels of reactivation were previously demonstrated with treatment with another HDAC inhibitor trichostatin A [ FMR1 gene activation with trichostatin A or vorinostat produce similar levels of mRNA with no statistically significant differences (P > 0.05).Vorinostat reactivatesstatin A . In GM04 FMR1 expression in normal cell lines in response for 5-azadC treatment possibly means that cells in culture exhibit different methylation statuses of the FMR1 promoter. Two control cell lines had different levels of FMR1 expression before treatment and consistently the same level after 5-azadC treatment. It is possible that treatment results in a demethylated FMR1 promoter and maximal gene expression in all cell lines.Activation of FMR1 gene expression with DNMT inhibitors is increased compared to activation with HDACs inhibitors. This finding was demonstrated in our research and in previous works [The activation ofus works , 10. TheThe very high cytotoxicity for FXS cell lines and low activating ability make HDAC inhibitors an unviable drug option for FXS therapy development. FMR1 gene silencing were proposed. Transcription of 5′ UTR of FMR1 gene leads to R-loop formation in GC-rich DNA and its interaction with RNA followed by RNA:DNA hybrid formation. Such structures are proposed to be a trigger for hypermethylation of FMR1 gene promoter [Also, it should be noted that other ways ofpromoter , 23. ThuInhibitors of histone deacetylases are very weak reactivators of the FMR1 gene in Fragile X syndrome cell lines. Some cell lines activate the gene expression at modest level; other cell lines show absence of reactivation. All inhibitors of histone deacetylases are very cytotoxic. These facts make it be not applicable for anti-Fragile X syndrome drugs development.


For over 60 years, hematopoietic stem cell transplantation has been the major curative therapy for several hematological and genetic disorders, but its efficacy is limited by the secondary disease called graft versus host disease (GvHD). Huge advances have been made in successful transplantation in order to improve patient quality of life, and yet, complete success is hard to achieve. This review assimilates recent updates on pathophysiology of GvHD, prophylaxis and treatment of GvHD-related complications, and advances in the potential treatment of GvHD. Barnes and Loutit demonstrated that when irradiated mice were infused with allogenic marrow and spleen cells, mice recovered from radiation injury and aplasia but they developed diarrhea, weight loss, skin changes, and liver abnormalities, and subsequently died due to “secondary disease” . Secondly, APCs activate donor T cells, also known as an afferent phase. Finally, in efferent phase, cellular and inflammatory factors work together to damage the target organs.Conditioning is crucial to eradicate underlying disease and to support engraftment of donor cells without rejection by recipient . Prior tGraft versus host disease occurs when donor T cells activate and respond to HLA differences on recipient’s tissue . Experimin vivo approaches such as the use of antithymocyte globulin pretransplant  and natural killer (NK) cells, utilize the Fas/Fas ligand (FasL) pathway and perforin/granzyme pathway to lyse the target cells , 33. FurThe broad activity of corticosteroids including induction of T-cell apoptosis, suppression of macrophage activation, and cytokine release explains why these old drugs are still the treatment of choice for first-line treatment of both acute GvHD and cGvHD. Cytokine inhibitors like TNF blocking agents were thought to be more specific but did not result in increased response rates . For almIn the last 10 years, the concept of GvHD pathophysiology has been largely extended, and a more differentiated view has been adapted.Firstly, the mechanism of conditioning-related damage has further been specified. It is now clear that the tissue damage results in release of several danger signals such as uric acid and the metabolites of adenosine triphosphate pathway and its receptor has been shown to be involved in activation of GvHD .Secondly, the concept of LPS-triggered inflammation has been substituted by multiple microbiota derived signals and differential activation of toll-like receptors (TLRs) and NOD-like receptors (NLRs). NOD2/CARD15 has been shown to be involved in triggering the inflammation both in mice , 38 and Finally, and in context with the concept of microbiota as important players, the importance of regulatory immune cells that balance immune reactions is recognized. Regulatory T cells (Tregs) expressing the transcription factor Foxp3 occur as natural, thymus derived T cells and are able to prevent alloreaction . On epitThe skin explant model has long been established as a tool for studying the immunobiology of GvHD Figure  and moreex vivo expanded Treg cells as well as their capacity to prevent GvH reactions , and Reg3α, Levine and colleagues  were ablde novo  can occur independently from the occurrence of GvHD or in patients with minor GvHD. Overall, NRM has decreased in the last 10 years as a result of several improvements such as reduced intensity conditioning; resulting in reduced organ toxicity, improved donor selection and matching, and progress in supportive treatment .Major complications include viral and fungal infections, which can occur independently from GvHD due to the immunodeficiencies induced by HSCT. GvHD and its treatment aggravate and prolong the risk of infectious complications, and many patients suffering from severe GvHD die from infectious complications. Beyond the period of acute GvHD, cGvHD and long-term complications are major causes of NRM and morbidity. Long-term complications include organ toxicities, endocrine deficiencies, and most important secondary cancers. HSCT patients’ survivors therefore need a long-term follow-up in order to allow early detection of complications, and several guidelines summarize the current recommendations , 84.A detailed presentation of infectious complications, organ toxicities, and long-term complications is beyond the focus of this review; we therefore focus on the most relevant targets of complications: endothelial cells and pulmonary complications.Endothelial complications occur clinically throughout the different phases of HSCT. In the early weeks after transplantation, sinusoidal obstruction syndrome (SOS) formally known as veno-occlusive disease (VOD) can result in severe liver damage and eventually multi-organ failure , 86. SOSIn vitro models of endothelial cell cultures reveal that conditioning can induce endothelial apoptosis, which is aggravated by LPS-mediated inflammation and followed allogeneic cytotoxic T-cell damage , and adenoviruses predominate as well as fungal pneumonia, especially in patients with severe immunosuppression (Toxoplasma gondii and Pneumocystis jirovecii causing toxoplasma and pneumocystis pneumonia, respectively, can cause pneumonia during the period of B cell reconstitution while B cell numbers are absent or low. Pneumonias caused by encapsulated bacteria such as pneumococci are also observed (A further central target organ of HSCT-related complications is the lung. Early after transplantation, bacterial and fungal pneumonia are common, mainly due to pression . In addiobserved , 103. Eaobserved  and in eobserved –88. The observed , 105. Eaobserved , 107. Beobserved .Cellular therapy is one approach increasingly used as a second-line treatment. Tregs  and MSCsex vivo T-cell depletion is no longer routinely used in HLA-matched transplantation as it also largely abolishes GvL effects. A more recent report from Finke and colleagues suggested ATG as an in vivo T-cell depletion may be more efficacious in lowering the incidence of severe acute GvHD in matched and mismatched HSCT from unrelated donors while GvL effects seemed less affected (For over 30 years, immunosuppressive drugs have served as a central strategy to reduce GvHD. Drugs such as sirolimus, tacrolimus, and methotrexate are the mainstay in the treatment of GvHD . Completaffected . Hundredaffected . The useaffected . WhetherPathogen recognition receptors like NLRs and TLRs are known to control adaptive immune responses in inflammatory disorders , and thein vitro and used for prophylaxis and treatment of GvHD in experimental and small clinical trials (Regulatory T cells have been expanded l trials , 118. Anl trials . Inductil trials , 121. Bel trials . MSCs arl trials , 124.Among pharmacological agents, drugs with anti-inflammatory effects of corticosteroids but without numerous side effects are urgently needed. Recently, anti-inflammatory JAK2 inhibitors have shown promising effects both in GvHD and in rheumatology. Proteasome inhibitors and histone deacetylase inhibitors originally developed as anticancer drugs now show some promising activity in dampening T-cell responses . In cGvHA major issue in the treatment of aGvHD is that most approaches are initiated too late, when major changes have already severely damaged the target tissue. Therefore, biomarkers allowing early identification of patients at high risk are needed. A handful of biomarkers have been discovered, which might be used to guide treatment in the future .Finally, the practice of stem cell transplantation differs between countries, within the same countries and between transplantation institutes. Approaches aimed at standardization of diagnosis and treatment are urgently needed, some of which have been addressed by several consensus projects , 126.EH and AD designed and revised the review. SG provided the draft, summarized available data, selected the references, and wrote the review. DW, EM and XNW contributed to the review. All authors approved the final version of the manuscript.All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil, which is approved for the treatment of patients with metastatic colorectal cancer refractory to standard chemotherapies. A microRNA analysis of three colorectal cell lines was conducted to investigate causes of FTD resistance. Drug resistant sublines of DLD-1, HCT-116, and RKO cells were developed by continuous administration of increasing doses of FTD for 5 months. The let-7d-5p gene, which maps to chromosome 9q22.32, was downregulated in the FTD-resistant DLD-1 sublines. DLD-1 cells became more resistant to FTD when let-7d-5p was knocked down and more sensitive when let-7d-5p was overexpressed. The FTD-resistant sublines were not cross-resistant to 5-fluorouracil (5-FU); 5-FU sensitivity was affected only slightly when let-7d-5p as overexpressed or knocked down. These data indicate that let-7d-5p increases sensitivity of FTD but not 5-FU and that let-7d-5p is a potential clinical marker of treatment sensitivity. Colorectal cancer (CRC) was the second leading cause of cancer death in both men and women in the 35 Organization for Economic Co-operation and Development countries in North and South America, Europe, and Asia-Pacific in 2015 , with itin vitro in colorectal cell lines [Trifluridine/tipiracil is an oral combination therapy comprising trifluridine (FTD), which is a thymidine analog, and tipiracil, which is a thymidine phosphorylase inhibitor that improves the bioavailability of FTD . FTD is ll lines , and thell lines , 13. miRll lines . Severalll lines .In this study, we analyzed miRNA expression in CRC parental cell lines and FTD-resistant sublines to investigate the relationship of miRNAs and FTD treatment effectiveness. The miRNA let-7d-5p was associated with FTD resistance; both miRNA and mRNA expression differed in FTD-resistant and parental cells. miRNA and mRNA clustered in a locus on chromosome 9 (miRNA at 9p22.32) were downregulated in FTD-resistant cells, one of which was the miRNA let-7d-5p, a member of the let-7 family that is known to target oncogenes and several genes regulating the cell cycle and cell proliferation .50 of each resistant line to that of the respective parent cell line after exposure to various concentrations of FTD for 3 days. Each of the cell lines had become highly resistant to FTD, with IC50 ratios that were 22.5- to > 40-fold higher than the IC50 of the parent cell lines . Probe sets that downregulated with a fold-change greater than two were counted, and Fisher's exact probability test results with p Figure . We alsop Figure . We therqPCR was used to compare let-7d-5p expression in FTD-resistant and parental sublines. We found that let-7d-5p was downregulated in DLD-1/FTD compared with its parental cell line Figure , but tha50 values of 16.8 μM and 7.6 μM, respectively ,   Figure . 5-FU se) Figure . CollectWe showed that let-7d-5p was associated with FTD sensitivity. Let-7 is a well-known tumor suppressor, and its expression is downregulated in several cancers . RestoraLet-7 miRNA regulates the expression of several oncogenes, including RAS and MYC , 21 to iTrifluridine/tipiracil was effective for the treatment of refractory metastatic colorectal cancer in clinical trials, but the mechanism of FTD- induced antiproliferative effects is not clearly understood. We showed that miRNA let-7d-5p was related to FTD-induced antiproliferative effects, and that its expression was inversely correlated with those effects. In contrast to FTD, a change in let-7d-5p expression had only a slight effect on 5-FU-induced antiproliferative effects. In preclinical studies, trifluridine/tipiracil demonstrated antitumor activity against both 5-FU-sensitive and resistant colorectal cancer cell lines . This stTrifluorothymidine-5′-triphosphate (F3dTTP) the active form of FTD is incorporated into DNA much more efficiently than FdUTP, the active form of 5-FU , 9. FdUTLet-7 family members share the same seed sequence, i.e., nucleotides 2–8 at the 5′ end, and often have similar functional consequences. This may explain why other family members, such as let-7a, let-7b, let-7c, let-7e, and let-7f, had expression patterns like those of let-7d in the DLD-1 cell microarrays . We founRAS and MYC. K-N- and HRAS all have let-7d-5p binding sequences in their 3′-UTRs. Only HRAS was upregulated in DLD-1/FTD cells with downregulated let-7d-5p. KRAS was downregulated, and NRAS expression did not change. The data thus indicated that let-7d-5p-mediated RAS expression was compensated by another feedback loop. KRAS is involved in the sensitivity to anticancer agents in CRC, but FTD treatment has been reported to prolong survival independent of the presence of KRAS mutations [KRAS was not involved in causing FTD resistance. In this study, MYC expression in DLD-1 and DLD-1/FTD cells was not different. MYC and let-7 have been reported to participate in a double negative feedback loop [MYC expression is not involved in FTD resistance, then other let-7d-5p targets need to be considered.Clinically, the study findings may have predictive and therapeutic value. First, low let-7d-5p expression was correlated with decreased trifluridine/tipiracil effectiveness. Therefore, assay of let-7d expression in tumor cells may assist in predicting drug effectiveness. Second, let-7d-5p overexpression was found to sensitize FTD-induced antiproliferative effects in both parental and FTD-resistant cell lines to a greater extent than 5-FU. Let-7d-5p expression profiling might be useful for allocating patients to genome information-based chemotherapy, and targeting let-7d with an appropriate delivery system might overcome FTD resistance. A recent report showed that reduced let-7d expression was associated with poor CRC outcomes , suggestutations , suggestack loop , 27. If Gadd45, PTEN, PUMA, and p21 [Aurora B has been considered as a molecular target of anticancer agents . Its upr and p21 . These aThe results indicated that FTD resistance was partially related to Aurora B. Let-7b binds to the Aurora B kinase 3′ UTR and inhibits expression of the kinase mRNA and protein . Much li−1 penicillin, and 100 UmL−1 streptomycin . Cells were grown at 37°C in a humidified atmosphere with 5% CO2. FTD-resistant cells  were established from each parent cell line by repeated, continuous (3- to 5-day) exposure of the cell cultures to escalating concentrations of FTD for about 5 months. Resistant cell lines were maintained as above. Cells were checked for short tandem repeats before use, and all experimental procedures were performed with exponentially growing cells.The RKO, HCT-116, and DLD-1 human colon cancer cell lines were obtained from the American Type Culture Collection  and grown in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum , 100 U mLFTD and 5-FU were purchased from Tokyo Chemical Industry .in vitro antiproliferative effects of FTD and 5-FU were evaluated by crystal violet staining. Cells were fixed with 4% glutaraldehyde for 20 minutes, stained with 0.05% crystal violet in 20% methanol for 20 minutes, and then rinsed in tap water. The plates were dried on paper for 1 hour, and 100 μL of a 1:1v/v mixture of ethanol and 0.1 mol L−1 sodium phosphate was added to each well. Absorbance was measured at 595 nm by an EnSpire multimode plate reader (PerkinElmer). The FTD and 5-FU concentrations that inhibited cell growth by 50% (IC50) were calculated from the regression lines. FTD and 5-FU resistance was estimated by dividing the FTD or 5-FU IC50 of the FTD-resistant cell line by the FTD or 5-FU IC50 of the corresponding parental cell line.Cell lines were seeded at a density of 2,000 cells per well into 96-well plates and precultured for 24 h. They were then exposed to various concentrations of FTD and 5-FU for 72 h. The http://www.ncbi.nlm.nih.gov/geo/) using the SuperSeries accession number GSE96787 and includes both the mRNA (GSE96785) and microRNA expression profiles (GSE96786).Total RNA was isolated from cells of line with an miRNeasy mini kit (Qiagen), following the manufacturer's instructions. Microarray analysis of miRNA and mRNA expression were performed by 3D-Gene microarray (Toray). The raw data are available on the Gene Expression Omnibus Website , and miRNA negative control (5′-UCACAACCUCCUAGAAAGA GUAGA-3′) were obtained from GeneDesign, Inc. . The MiRNA inhibitor of let-7d (mirVana™ miRNA Inhibitors), and miRNA inhibitor negative control were obtained from Applied Biosystems. . DLD-1 cells were transfected with 10 nM of miRNA mimics or 30 nM of miRNA inhibitors using Lipofectamine 3000  following the manufacturer's protocol.AURKB was 5′-CCGG- CCTGCGTCTCTACAACTATTT-CTCGAG-AAATAGT TGTAGAGACGCAGG-TTTTT-3′. The vector was cotransfected into 293T cells along with expression vectors containing the gag/pol, rev, and vsvg genes. Lentivirus was harvested 48 h after transfection, and 5 μg mL−1 polybrene was added. DLD-1 cells were infected with harvested lentivirus and selected with 2 μg mL−1 puromycin for 1 week.The shRNA construct was provided by Lewis Cantley  and cloned into the lentivirus vector pLKO. The oligonucleotide sequence of the shRNA targeting To assay miRNA and mRNA expression, cDNA was first synthesized from extracted total RNA, and qPCR was then performed using a Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR Kit (Clontech). Relative expression was calculated by the CT-based calibrated standard curve method. The calculated values were then normalized against the expression of U6 small nuclear (sn)RNA for miRNA and GAPDH for mRNA.t-test and Fisher's exact probability test as appropriate. Statistical analysis was performed using JMP Pro 9 . Fisher's exact probability test with a p-value of 2 × 10−3 was used to select chromosomal regions with significant differential miRNA and mRNA expression. Probe sets that were downregulated in DLD-1 cells with a fold change of < 50% were counted and analyzed for statistical significance.Statistically significant differences were determined by Student's


Anaplasma ovis is a widely distributed pathogen affecting sheep, goats and wild ruminants. Here, the HL-60 human promyelocytic leukemia cell line was used to isolate A. ovis from PCR-positive sheep and goats in Heilongjiang Province, China. Two weeks after inoculation, morulae were observed in cytoplasmic vacuoles in four different HL-60 cultures. Confocal microscopy using a Cy3-labeled A. ovis-specific probe confirmed that the HL-60 cells were infected with A. ovis. Cells from the 6th HL-60 subculture displayed positive fluorescence when incubated with A. ovis antiserum in the indirect fluorescent antibody assay. PCR amplification and sequencing of 16S rRNA, groEL, gltA, msp2 and msp4 Anaplasma genes revealed that the four A. ovis culture isolates were identical. Phylogenetic analysis showed that the sequences clustered with other A. ovis strains but could clearly be distinguished from other Anaplasma species. When the 18th subculture of infected HL-60 cells was examined by electron microscopy, lysosomes were often observed near the vacuoles. After the 24th subculture, Giemsa staining and PCR indicated that the HL-60 cells were negative for A. ovis. Although A. ovis can infect HL-60 cells for only four months, the ability of the organism to infect and multiply in HL-60 cells provides a tool to study intra-erythrocytic Anaplasma and host cell interactions.The tick-borne bacterium  Anaplasma ovis is an intra-erythrocytic tick-borne bacterial pathogen which mainly affects domestic goats and sheep,1 but has also been reported to be present in deer,2 wild boar 3 and domestic dogs.4A. ovis infections are widely distributed and have been reported in North America, Europe and Asia.5 The organism can cause severe anemia, fever, weight loss, spontaneous abortion, jaundice and mortality in affected sheep or goats, thus resulting in economic losses in many countries.6 In 2007, the first human case of A. ovis, characterized by fever, hepatosplenomegaly and lymphadenopathy, was reported in Cyprus.7A. ovis transmission is not well understood. The acquisition and transmission of A. ovis through different developmental stages of the various tick vectors have not been documented.8 It has been reported that Rhipicephalus bursa and Dermacentor marginatus are the potential vector ticks for A. ovis in Europe and North America, respectively.9 In Asia, in particular in China, D. nuttalli, Hyalomma asiaticum and R. pumilio are the potential vectors.8A. ovis was attempted in transplantable cell lines in 1966, but these cell lines were not easily prepared.10 Similarly to A. ovis, A. marginale is another intra-erythrocytic bacterium and was successfully been cultivated in tick cell lines (IDE8 and BME26).13 These reports have provided good examples for studying A. ovis. However, tick cell lines are not easily managed and are not widely used in laboratories. We reasoned that cultivation of A. ovis in the HL-60 human promyelocytic leukemia cell line might be feasible, owing to the reported susceptibility of humans to infection with A. ovis. Here, HL-60 cells were infected with several isolates of A. ovis derived from infected sheep and goats from different herds, which acquired the infection in Heilongjiang Province in northeastern China.Cultivation of gltA) of A. ovis were performed on all samples . Nested PCR reactions targeting the citrate synthase gene ( samples . PCR proA. ovis-infected blood samples were inoculated into HL-60 cells (ATCC CCL-240) by using previously reported methods.14 Briefly, after the infected blood was quickly thawed and washed, 100 μL blood was added to a HL-60 cell culture with a density of 5 × 105cells/mL. Cultures were maintained in RPMI 1640 medium  with 10% heat-inactivated fetal bovine serum  and 2 mM L-glutamine  in a 37 °C incubator with 5% CO2. Cultures were examined twice per week. To maintain a density of (2–5) × 105 cells per mL, fresh medium or non-infected HL-60 cells were added as required. Two weeks after inoculation, Wright-Giemsa staining was used to detect intracellular morulae in cytospins by light microscopy. After the infection was confirmed, the cell density was also maintained at (2–5) × 105 cells/mL. Wright-Giemsa staining and PCR were performed weekly to monitor the infection.in situ hybridization (FISH) to observe the same cytospin slide with two methods.To identify the specificity of intracellular morulae observed in HL-60 cells, we developed an assay combining Wright-Giemsa staining with fluorescence 15 The FISH probes were designed specifically for A. ovis . To achieve a sufficient signal-to-background ratio, multiple probes were targeted along each individual lncRNA/mRNA sequence of the A. ovis msp4 gene (KX579070). A set of 15–20 probes covering the entire length of the RNA molecule allowed for optimal signal strength, whereby each probe carried multiple fluorophore signals. A non-specific probe was also synthesized and included as a negative control. The probes were labeled with fluorescein isothiocyanate (FITC). The pooled FISH probes were re-suspended in a final concentration of 25 μM in RNase-free storage buffer and protected from light by storage at −20 °C.Cytospins were fixed in methanol and acetone  for 10 min and then fixed in methanol and acetic acid  for 15 min. First, FISH was performed with a commercial kit according to the manufacturer’s instructions  with some modifications based on a patent description to improve the hybridization of the probe specifically to the bacterial and not the host cells.12After examination of FISH results under a microscope with a fluorescent light source, the same cytospin slide was washed with wash buffer for 3 min, followed by PBS for 3 min and Wright-Giemsa B solution for 3 min. Then, the same slide was stained by Wright-Giemsa as previously reported.Cytospin slides prepared from the same infected culture as in FISH and Wright-Giemsa staining were used. The slides were fixed with methyl hydrate and acetone . The following process was similar to the aforementioned FISH assay, except that for confocal laser scanning microscopy, cyanine 3 (Cy3) labeled probes were used, and DAPI was used to counterstain the cell nuclei. In each experiment, a non-specific probe was used as a negative control. An Olympus Fluoview FV1000 automated inverted research microscope was used to observe the result. Images were acquired by using Olympus Fluoview Ver4.0b Viewer software in combination with a × 60 oil immersion objective lens and a numerical aperture of 1.25. The excitation wavelengths used were 405 nm (blue) and 594 nm (red). All experiments were conducted in triplicate.14 for transmission electronmicroscopic examination using a CM-120 electronmicroscope .A 6th subculture and an 18th subculture of infected HL-60 cells and normal HL-60 cells, respectively, were processed, as previously described groEL(1365 bp), gltA (804 bp), msp2 (350 bp) and msp4 (845 bp) were amplified with the primers and PCR conditions presented in http://blast.ncbi.nim.nih.gov/Blast.cgi). Phylogenetic analyses were performed, and phylogenetic trees were constructed by using Mega 5.0 software.16Total DNA was extracted by using a QIAamp DNA Blood Mini Kit. Full length 16S ribosomal RNA gene (1461 bp) and partial sequences of A. ovis-specific PCR-positive samples were used as positive controls, and another Anaplasma species infection was excluded by PCR targeting the 16S rRNA gene of the Anaplasmataceae family.14A. phagocytophilum and A. capra infections were further excluded by using indirect fluorescent antibody assay to detect serum antibodies with a commercial kit  or with our prepared A. capra antigen slides.14 Uninfected cells were stained with positive goat serum as a negative control to assess the background.A 6th subculture of infected HL-60 cells was processed for the preparation of antigen slides. During the current survey on anaplasmosis, serum samples from goats and sheep were collected. The Anaplasma ovis blood samples obtained from sheep and seven obtained from goats were inoculated into different HL-60 cell cultures, thus yielding four Anaplasma ovis isolates (two derived from sheep and another two from goats). Two weeks after the blood was inoculated, typical morulae were observed in the cytoplasm in the host cells. Wright-Giemsa-stained cytospins prepared 30 days post-inoculation from the 6th HL-60 subculture showed numerous small inclusion bodies in the cytoplasm  exhibited no staining with Wright-Giemsa stain  in tick cell lines has promoted understanding of bacterial infectivity, cell tropism and vaccine development.25 Our initial objective was to cultivate A. ovis in one easy-to-handle human cell line, owing to its potential to infect human beings. Although the A. ovis isolate in HL-60 cells was able to be established for only approximately four months, this cultivation can be used to prepare antigen slides to confirm any infection with this bacterium. This study does not clarify the clearance mechanism of A. ovis in HL-60 cells for the later passages. Through in vitro study of A. ovis transient infection in HL-60 cells and continuous infection in other cell lines to compare differences in cell responses, the effects of pathogen–host cell interactions on cell attachment, bacterial invasion and intracellular development of A. ovis can be explored.The successful propagation of


Acute graft-versus-host disease (GvHD) is still a major cause of treatment-related mortality after allogeneic stem cell transplantation. Allo-antigen recognition of donor T cells after transplantation account for the onset and persistence of this disease. MicroRNAs (miRNAs) are molecular regulators involved in numerous processes during T-cell development, homeostasis, and activation. Thus, miRNAs also contribute to pathological T-cell function during GvHD. Given their capacity of fine-tuning T-cell function, miRNAs have emerged as promising therapeutic targets to curtail acute GvHD, but simultaneously maintain T-cell-mediated graft-versus-tumor effects. Here, we review the role of key miRNAs contributing to the pathophysiology of GvHD. We focus on those miRNAs acting in T cells and for which a role in GvHD has been established in preclinical models. Finally, we provide an outlook for clinical application of this new therapeutic target for GvHD prevention and treatment. Acute graft-versus-host disease (GvHD) is still a major complication after allogeneic hematopoietic stem cell or bone marrow transplantation . Allogeneic donor T cells are the main inducers of this too often lethal disease. Donor T cells recognize and respond to allo-antigen, use chemokine receptors and integrins to home to epithelial organs, such as the liver, the intestine, or the skin and orchestrate a significant immune response, which frequently results in severe organ damage. Unspecific prophylactic and therapeutic immunosuppressive treatment is the current clinical practice to improve patients’ outcome. However, the repertory of therapeutic and preventive options is limited and, therefore, new targets to control acute GvHD are urgently needed .via the T-cell receptor (TCR) in alloSCT, whereas central and/or memory T cells are not able to induce acute GvHD  effects  is a critical enzyme in providing essential amino acids for T cell proliferation and exerts immunosuppressive effects. Increased levels of IDO levels prevent acute GvHD . IDO is miR-181a acts as a rheostat for T cell sensitivity to antigen by downregulation of several phosphatases downstream of the TCR . Primaryvia a number of mRNA targets lacking corresponding miR-125b binding sites in mouse, including the genes encoding IFN-γ and IL-10RA (ex vivo. Recently, methods for efficient CRISPR/Cas9-mediated genome engineering of primary human T cells have been reported (in vivo (Understanding the role of miRNAs in physiological T cell function and in the context of disease has the potential to open up new avenues for therapy. Most of this knowledge is derived from the analysis of rodent models and is only beginning to be complemented by studies in human primary T cells . To a la IL-10RA . In termreported . In addi(in vivo , 96, som(in vivo , 69. In (in vivo .Our knowledge of miRNA action is far from complete and a number of miRNAs with a role in T cell function, for instance, miR-148a, miR-182, and miR-326, may ultimately also play a role in GvHD –100. Simex vivo and potentially even in vivo modulation. A better understanding of miRNAs controlling physiologic T-cell function has resulted in efficient translation of manipulation of miRNAs in preclinical models of GvHD. However, despite the generally broad interspecies conservation of miRNAs, translation into clinical treatment of GvHD remains a challenge.MicroRNAs play critical role in many biological processes including T-cell development and differentiation. Given that GvHD is a predominantly T-cell driven disease, miRNAs have emerged as attractive therapeutic targets for All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We demonstrate the feasibility of creating a pair of reference samples to be used as surrogates for clinical samples measured in either a research or clinical laboratory setting. The reference sample paradigm presented and evaluated here is designed to assess the capability of a measurement process to detect true differences between two biological samples. Cell-based reference samples can be created with a biomarker signature pattern designed in silico. Clinical laboratories working in regulated applications are required to participate in proficiency testing programs; research laboratories doing discovery typically do not. These reference samples can be used in proficiency tests or as process controls that allow a laboratory to evaluate and optimize its measurement systems, monitor performance over time (process drift), assess changes in protocols, reagents, and/or personnel, maintain standard operating procedures, and most importantly, provide evidence for quality results.The biomarkers of interest in this study are microRNAs (miRNAs), small non-coding RNAs involved in the regulation of gene expression. Multiple lung cancer associated cell lines were determined by reverse transcription (RT)-PCR to have sufficiently different miRNA profiles to serve as components in mixture designs as reference samples. In silico models based on the component profiles were used to predict miRNA abundance ratios between two different cell line mixtures, providing target values for profiles obtained from in vitro mixtures. Two reference sample types were tested: total RNA mixed after extraction from cell lines, and intact cells mixed prior to RNA extraction. MicroRNA profiling of a pair of samples composed of extracted RNA derived from these cell lines successfully replicated the target values. Mixtures of intact cells from these lines also approximated the target values, demonstrating potential utility as mimics for clinical specimens. Both designs demonstrated their utility as reference samples for inter- or intra-laboratory testing.Cell-based reference samples can be created for performance assessment of a measurement process from biomolecule extraction through quantitation. Although this study focused on miRNA profiling with RT-PCR using cell lines associated with lung cancer, the paradigm demonstrated here should be extendable to genome-scale platforms and other biomolecular endpoints.The online version of this article (10.1186/s12896-018-0423-4) contains supplementary material, which is available to authorized users. Quality measurements during this phase are essential for the success of all subsequent phases of biomarker development.The translation of a biomarker from discovery to clinical practice involves a transition from the research-oriented approach of the discovery laboratory to a procedure in an accredited clinical laboratory. However, most published cancer biomarkers never make it into clinical practice , with rePreclinical studies to develop cancer biomarkers typically involve comparing measurement results from tumor tissue to normal tissue and identifying features that distinguish the two classes. Reference samples, with known differences, enable laboratories to demonstrate their ability to detect potential biomarkers. Ideally, these biomarkers would be measured in one or more biofluids and provide a means for non-invasive screening at the earliest stages for the detection of cancer. Two EDRN laboratories are assessing microRNAs (miRNAs) found in sputum as potential biomarkers, which is the context for designing mixture reference samples described in the present paper.MicroRNAs are a class of small non-protein-coding RNAs that regulate the expression of hundreds of target genes, controlling biological functions involved in differentiation and development . Dysreguoverexpressed and two (miR-126 and miR-486) were underexpressed when comparing lung cancer and normal tissue, providing a potential panel of biomarkers for early detection [Three cell lines with relatively different expression profiles for five miRNAs of interest were assessed as potential mixture components to create a pair of reference samples to act as surrogates for sputum samples measured in a non-invasive screening assay for lung cancer. Lung cancer cell lines from the NCI-H series   were proetection , 14.The miRNA profile of each reference sample need not mimic a particular biological state , but should provide the ability to assess technical performance of the measurement process used to discriminate based on the particular set of miRNAs of interest. Ideally, these reference samples should possess the following properties: 1) express all or most of the biomarkers of interest; 2) be regenerable for use over time as a process control; 3) be amenable to identical processing as clinical samples; 4) contain well-established relative differences in the biomarkers of interest; and 5) be commutable between measurement systems. Such samples could be used to assess repeatability within, and reproducibility between, laboratories for the entire measurement process.In the first part of this study, a “crossover” design was used to assess reproducibility of miRNA profiles and parse out factors contributing to variability see Fig. . Two difTo inform the design for the two reference samples, the miRNA measurements for each cell line were used to predict the ratio of miRNA abundance between samples formed by mixing the component cell lines in different proportions , 16. To Cell Lines were obtained from the American Type Culture Collection (ATCC®), Manassas, VA. H226 (ATCC# CRL-5826), H358 (ATCC# CRL-5807), and H460 (ATCC# HTB-177) were the cell lines selected by the biomarker discovery laboratory (BDL) , 14. TheThe BRL developed the following standard operating procedures for the isolation of total RNA enriched for miRNA from the cell pellets using the Qiagen miRNeasy Mini Kit. Briefly, cells are lysed in a denaturing buffer containing phenol / guanidine-thiocyanate designed to inhibit RNases to ensure the purification of intact RNA from cellular DNA and proteins. Samples are homogenized using the QIAShredder columns. Ethanol is then added to the sample to precipitate the RNA, and the sample is applied to an RNeasy mini spin column, where the RNA binds to the silica-based membrane. After washing contaminants away, RNA is eluted from the membrane in RNase-free water. Each lab performed three separate RNA isolations on different days on each component cell line.For total RNA mixtures, a 3-component design with a 1:1 component and two reciprocal 3:1 components were used. Total RNA isolated by the BRL was quantified using an Agilent BioAnalyzer and mixed by mass in the following proportions: Mix1 (1:3:1) and Mix2 (3:1:1), using H226, H358, and H460, respectively. In silico modeling was used to design the mixtures, selecting the cell line mixing proportion to use for each component that provided a useful distribution of ratios across the dynamic range  in the same proportions as the RNA mixtures: Mix1 (1:3:1) and Mix2 (3:1:1), using H226, H358, and H460, respectively.For miRNA profiling of cell lines, each lab measured the RNA isolated within their lab as well as the RNA isolated by the other lab using RT-PCR. Human (Hsa-) specific kits  for miR-16 (#000391), miR-21 (#000397), miR-126 (#002228), miR-210 (#000512), miR-486 (#001278), miR-375 (#000564) were used for reversed transcription to cDNA. The TaqMan miRNA assays were performed using an ABI 7900 HT  in the BRL and an IQ5 Multicolor Real-Time PCR Detection System  in the BDL. The same RT-PCR protocol was used for miRNA profiling of mixtures from RNA components or cell line components.Yceip denote the average of three Cq measurements from within a single plate for RNA extracted from cell line c  during extraction replicate e  at isolation lab i  and measured at PCR lab p . Under the assumption that miRNA abundance should be additive and linear, we can predict a Cq value for a hypothetical sample reflecting a designed mixture of the component cell line samples using the following equation:Each analyte  was modeled independently according to the following analysis. From the individual cell lines, let p of designed mixture m  hypothetically created by combining the samples from isolation replicate e at isolation lab i at relative concentrations of ϕm1, ϕm2, and ϕm3 for component cell lines H226, H358 and H460, respectively. We refer to each of the four combinations of isolation lab and PCR lab as a different measurement process.where Data collected throughout the described experiments were analyzed in a Bayesian framework to evaluate the uncertainty related to average Cq values or their differences . For each target miRNA in turn, the observed Cq values were analyzed using mixed effects models that include a fixed mean for each of 18 distinct combinations , after integrating across the modeled distribution of all other model parameters. We examine peak shapes formed by plotting marginal likelihood as a function of the value of the parameter of interest to assess whether or not particular factors appear to contribute to bias in these results. This approach is used to assess differences in Cq responses between samples (Mix1 vs. Mix2), laboratories (BDL vs. BRL), or source material (in silico vs. RNA vs. cells). In these plots, a value of 0 along the x-axis indicates “no difference” for the comparison while deviation from zero indicates a possible bias.rjags [To examine the sensitivity of the reported results to modeling choices, the entire analysis was repeated using three model perturbations. All models were fit using Markov Chain Monte Carlo (MCMC) via the R package rjags , 18. MCMrjags . DatasetThe expression profiles of five miRNAs of interest  , 14 and An analysis of variance (ANOVA) was performed on the Cq values in phase 1 (RNA extracted from pure cell lines). Fig. When evaluating log abundance ratios  across cell lines (or cell line mixtures), only the interactions involving cell line will matter. This is because effects that remain constant across cell lines will cancel when taking the difference . From this perspective, the most substantial source of confounding variability is the factor associated with the interaction among analyte, cell line and PCR lab, which contributes, on average, about an order of magnitude less variability than do any of the biologically-relevant factors.Figure ϕ11 = 0.2, ϕ21 = 0.6, ϕ31 = 0.2 were selected for mixture design 1 (Mix1); and ϕ12 = 0.6, ϕ22 = 0.2, ϕ32 = 0.2 for mixture design 2 (Mix2). This design predicted a useful distribution of analyte abundances across the dynamic range as well as ΔCq (Mix1 – Mix2) between the samples ranging from − 1 to 1, i.e., two-fold up and down as shown in Fig. Two different mixture designs were selected on the basis of iterative in silico modeling via Eq. Each measurement process demonstrated a consistent rank order of miRNA with respect to average Cq. Although the smallest difference in that dimension was observed between miR-126 and miR-375, the mixing design still predicted a useful separation between those two miRNAs with respect to ΔCq. The isolation lab differences observed with miR-21 in Fig. Four separate total RNA isolations per cell line from separate expansions were performed in the BRL and each isolation was used to prepare a single Mix1 and Mix2 pair see Fig. . Both PCAn ANOVA was performed on the Cq values in phase 2 (RNA mixtures). In this phase, all isolations occurred at the BRL, which precludes further assessments of variability due to isolation lab. Figure To test the feasibility of using cell lines as surrogate samples, the three component cell lines were combined in the previously provided Mix1 and Mix2 proportions based upon cell counts to create four separate mixture pairs of cell suspensions. The mixture samples were then processed in triplicate by the BRL in the same manner as cells in a clinical specimen see Fig. . A PCR pFigure To characterize potential biases between labs, Fig. The likelihoods of the observed data for miR-21, miR-210, and miR-375, respectively, are largest under models for which biases due to PCR lab are near zero. However, for both miR-126 and miR-486 and for both the in silico mixtures Fig.  and the If the measurements in phases 1 and 2 had been deemed insufficiently reproducible, additional measurements could be obtained to confirm the apparent biases along with efforts to identify differences in how PCR measurements were conducted at each lab for each combination of cell line and either miR-126 or miR-486. For the proof-of-concept purposes of the present study, the exhibited reproducibility was deemed adequate and no further attempts were made to investigate or control the observed variability.Although the source material for each of these measurement scenarios was obtained from different expansions of the cell lines, the direction of fold-change between samples for each target miRNA is consistent and the order of relative abundance is the same among the miRNA targets: miRNA-486 < miRNA-375 < miRNA-126 < miRNA-210 < miR-21 . The screening of candidate cell lines by multiple laboratories was useful in establishing robust differences in expression for a selected set of miRNAs. In silico modeling of these levels informed the final mixture design for both RNA mixtures and mixtures of cells. Identifying cell lines with even greater differences between miRNA profiles might allow for mixtures that provide a broader range of designed-in ratios. RNA mixtures derived from these cell lines can also serve as well-matched reference samples for measurement assurance of the post-RNA isolation stages of gene expression measurement systems.This testing paradigm should be generalizable to other biomarkers and other laboratories and provide a model for establishing both intra- and inter-laboratory measurement assurance and demonstrate translatability of biomarker assays between research development laboratories and clinical reference laboratories.Additional file 1:Intro to Bayesian analysis. (PDF 1844 kb)Additional file 2:Cell line profile Cq values. (CSV 13 kb)Additional file 3:RNA mixture Cq values. (CSV 18 kb)Additional file 4:Cell line mixture Cq values. (CSV 8 kb)Additional file 5:Code for running MCMC. (PDF 41 kb)Additional file 6:Phase1 ANOVA parsed by analyte and cell line. (PDF 78 kb)