Knockdown of ITGB1-DT repressed LUAD cellular proliferation, migration, and invasion. ITGB1-DT interacted with EZH2, repressed the binding of EZH2 to ITGB1 promoter, reduced H3K27me3 levels at ITGB1 promoter region, and therefore activated ITGB1 expression. Through upregulating ITGB1, ITGB1-DT activated Wnt/β-catenin pathway and its downstream target MYC in LUAD. The expressions of ITGB1-DT, ITGB1, and MYC were positively correlated with each other in LUAD tissues. Intriguingly, ITGB1-DT was found as a transcriptional target of MYC. MYC directly transcriptionally activated ITGB1-DT expression. Thus, ITGB1-DT formed a positive feedback loop with ITGB1/Wnt/β-catenin/MYC. The oncogenic roles of ITGB1-DT were reversed by depletion of ITGB1 or inhibition of Wnt/β-catenin pathway. In summary, these findings revealed ITGB1-DT as a prognosis-related and oncogenic lncRNA in LUAD via activating the ITGB1-DT/ITGB1/Wnt/β-catenin/MYC positive feedback loop. These results implicated ITGB1-DT as a potential prognostic biomarker and therapeutic target for LUAD.Lung adenocarcinoma (LUAD) is the main histological type of lung cancer, which is the leading cause of cancer-related deaths. Long non-coding RNAs (lncRNAs) were recently revealed to be involved in various cancers. However, the clinical relevance and potential biological roles of most lncRNAs in LUAD remain unclear. Here, we identified a prognosis-related lncRNA ITGB1-DT in LUAD. ITGB1-DT was upregulated in LUAD and high expression of ITGB1-DT was correlated with advanced clinical stages and poor overall survival and disease-free survival. Enhanced expression of ITGB1-DT facilitated LUAD cellular proliferation, migration, and invasion, and also lung metastasis  Lung cancer is still the most frequently diagnosed malignancy and the leading cause of mortality among all malignancies, accounting for 11.6% of the total cancer cases and 18.4% of the total cancer deaths worldwide . The hisThe identification of critical genes involved in LUAD provided several therapeutic intervention targets, such as EGFR, ALK, HER2, MET, VEGF, and PD-1 . Apart fITGB1-DT is located on chromosome 10p11.22 and is transcribed in the opposite direction from coding gene ITGB1. ITGB1-DT has four exons and encodes transcript (ITGB1-DT) with 1078 nucleotides. ITGB1-DT was previously reported to be related to poor prognosis of clear cell renal cell carcinoma and gastric cancer  LUAD dataset and identified ITGB1-DT as an upregulated and poor-prognosis correlated lncRNA in LUAD. c cancer . HoweverA total of 64 pairs of LUAD tissues and matched normal lung tissues were randomly selected from LUAD patients who received lung lobectomy at Xiangya Hospital. All clinical samples were confirmed by histopathological examination. We obtained written informed consents from all participants. The study protocol was approved by the Ethics Committee of Xiangya Hospital. We conducted this study in accordance with the ethical standards of our hospital and the Helsinki Declaration. The clinicopathological features of these 64 LUAD samples are summarized in 2 at 37°C. Where indicated, LUAD cells were treated with 200 ng/ml Wnt3a  or 25 μM ICG-001  for the indicated time.Human bronchial epithelial cell line 16HBE was purchased from Millipore . Human LUAD cell lines A549 (ATCC CCL-185), NCI-H1975 (ATCC CRL-5908), and NCI-H1299 (ATCC CRL-5803) were acquired from the American Type Culture Collection . 16HBE was cultured in Airway Epithelial Cell Basal Media (ATCC) supplemented with Bronchial/Tracheal Epithelial Cell Growth Kit components (ATCC). A549 was cultured in F-12K Medium  added with 10% fetal bovine serum . NCI-H1975 and NCI-H1299 cells were cultured in RPMI-1640 Medium (Invitrogen) added with 10% FBS. These cells were cultured in 5% CO® Green Realtime PCR Master Mix  and the primers 5′-TGTATGTGATGGCTTTGGAG-3′ (forward) and 5′-AGGTGGGCAGTGGTATGG-3′ (reverse) for ITGB1-DT, 5′-GTTTGTAGGAAGAGGGATAA-3′ (forward) and 5′-CCA GTGTAGTTGGGGTTG-3′ (reverse) for ITGB1, 5′-GTCGG AGTCAACGGATTTG-3′ (forward) and 5′-TGGGTGGAATC ATATTGGAA-3′ (reverse) for GAPDH, 5′-GGAGCAGAA AACAGCAGG-3′ (forward) and 5′-GGAAGGGGTCAGGAG AAA-3′ (reverse) for MALAT1, 5′-CTTCCCCTACCCTCTCAA-3′ (forward) and 5′-CGATTTCTTCCTCATCTTCT-3′ (reverse) for MYC, 5′-GAACGGATTTAGAAGCCTG-3′ (forward) and 5′-GGTCCTCCATTTCAGCCT-3′ (reverse) for HOTAIR, 5′-G CCCCATCAGGCCTCCGTTT-3′ (forward) and 5′-ACCT TGCCTTCTTTGTCTTTGTTGGA-3′ (reverse) for E-cadherin, 5′-TGGACCATCACTCGGCTTA-3′ (forward) and 5′-ACA CTGGCAAACCTTCACG-3′ (reverse) for N-cadherin, and 5′-CCTGAACCTGAGGGAAACTAA-3′ (forward) and 5′-GCA GAAAGGCACTTGAAAGC-3′ (reverse) for Vimentin . Reverse transcription was performed using M-MLV Reverse Transcriptase (Invitrogen) and the extracted RNA. qRT-PCR was conducted on the Applied Biosystems 7300 Fast Real-Time PCR System  using SYBRVimentin . The qua® Max DNA Polymerase  and the primers 5′-CCCAAGCTTGCTTTTCTGGAGTGATAA TG-3′ (forward) and 5′-GCTCTAGAGTAATCAAATAAAACTT ACAGTGGT-3′ (reverse). After digestion by the restriction enzymes HindIII (Takara) and XbaI (Takara), the PCR products were inserted into pcDNA3.1 vector (Invitrogen) and pSPT19 vector  to generate ITGB1-DT overexpression vector pcDNA3.1-ITGB1-DT and ITGB1-DT in vitro transcription vector pSPT19-ITGB1-DT, respectively. Coding sequence (CDS) of MYC was PCR-amplified using the PrimeSTAR® Max DNA Polymerase (Takara) and the primers 5′-CCCAAGCTTGCCAGGACCCGCTTCT-3′ (forward) and 5′-G CTCTAGAGGTGATTGCTCAGGACATTTC-3′ (reverse). After digestion by the restriction enzymes HindIII and XbaI, the PCR products were inserted into pcDNA3.1 vector (Invitrogen) to generate MYC overexpression vector pcDNA3.1-MYC. CDS of ITGB1 was PCR-amplified using the PrimeSTAR® Max DNA Polymerase (Takara) and the primers 5′-GGAATTCAGCG GGAGTCGCGGAACA-3′ (forward) and 5′-GCTCTAGAGG ATTTGCACGGGCAGTAC-3′ (reverse). After digestion by the restriction enzymes EcoRI and XbaI, the PCR products were inserted into pcDNA3.1 vector (Invitrogen) to generate ITGB1 overexpression vector pcDNA3.1-ITGB1. ON-TARGETplus Human MYC siRNA SMART Pool (Cat: L-003282-02-0010), ON-TARGETplus Human ITGB1 siRNA SMART Pool (Cat: L-004506-00-0010), and ON-TARGETplus Human EZH2 siRNA SMART Pool (Cat: L-004218-00-0010) were purchased from Dharmacon . The transfection and co-transfection of vectors and siRNAs were conducted using Lipofectamine 3000 (Invitrogen).ITGB1-DT full-length sequence was PCR-amplified using the PrimeSTARTo construct LUAD cells with ITGB1-DT stable overexpression, ITGB1-DT overexpression vector pcDNA3.1-ITGB1-DT was transfected into A549 and NCI-H1975 cells. Seventy-two hours later, the transfected cells were treated with neomycin to select ITGB1-DT stably overexpressed LUAD cells. Two pairs of cDNA oligonucleotides targeting ITGB1-DT were synthesized and inserted into the shRNA lentivirus expressing vector pLV6/EF-1aF/Puro , which were further used to produce shRNA lentiviruses targeting ITGB1-DT. One pair of cDNA oligonucleotides targeting ITGB1 were synthesized and inserted into pLV6/EF-1aF/Puro, which were further used to produce shRNA lentiviruses targeting ITGB1. Scrambled non-targeting cDNA oligonucleotides were used as negative control (NC). The cDNA oligonucleotides sequences were as follows: for shRNA-ITGB1-DT-1, 5′-GATCCGGAGCAAATTGATT GACAATGTTCAAGAGACATTGTCAATCAATTTGCTCCTT TTTTG-3′ (forward) and 5′-AATTCAAAAAAGGAGCAAAT TGATTGACAATGTCTCTTGAACATTGTCAATCAATTTGC TCCG-3′ (reverse); for shRNA-ITGB1-DT-2, 5′-GATCCGGTC TAGCTGAGTTGACAAGATTCAAGAGATCTTGTCAACTCA GCTAGACCTTTTTTG-3′ (forward) and 5′-AATTCAAAA AAGGTCTAGCTGAGTTGACAAGATCTCTTGAATCTTGTC AACTCAGCTAGACCG-3′ (reverse); for shRNA-ITGB1, 5′-GATCCGGATACTAGTACTTGTGAAGCTTCAAGAGAGCTT CACAAGTACTAGTATCCTTTTTTG-3′ (forward) and 5′-AA TTCAAAAAAGGATACTAGTACTTGTGAAGCTCTCTTGAA GCTTCACAAGTACTAGTATCCG-3′ (reverse); for shRNA-NC, 5′-GATCCGTTCTCCGAACGTGTCACGTTTCAAGAGAACG TGACACGTTCGGAGAACTTTTTTG-3′ (forward) and 5′-AATTCAAAAAAGTTCTCCGAACGTGTCACGTTCTCTTGA AACGTGACACGTTCGGAGAACG-3′ (reverse). To construct LUAD cells with ITGB1-DT stable silencing, the shRNA lentiviruses targeting ITGB1-DT were infected into A549 and NCI-H1299 cells. Ninety-six hours later, the infected cells were treated with puromycin to select ITGB1-DT stably silenced LUAD cells. To construct LUAD cells with ITGB1-DT stable overexpression and concurrent ITGB1 stable silencing, the shRNA lentiviruses targeting ITGB1 were infected into A549 cells with ITGB1-DT stable overexpression. Ninety-six hours later, the infected cells were treated with puromycin and neomycin to select ITGB1-DT stably overexpressed and ITGB1 stably silenced A549 cells.Cell proliferation abilities were evaluated using Cell counting kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) incorporation assays as previously described . Briefly5 indicated LUAD cells resuspended in FBS-free medium were seeded into the upper chambers of transwell inserts (Millipore). For transwell invasion assay, the inserts were pre-coated with matrigel (1:30 dilution) . For transwell migration assay, the inserts were not pre-coated with matrigel. Medium containing 10% FBS was added into the under chambers. After incubation at 37°C for another 48 h, the cells in the upper chambers were removed and the cells adhering to the underside of the membranes were fixed, stained, and counted under a microscope based on at least five random fields.Cell migration and invasion abilities were evaluated using transwell migration and invasion assays as we previously described . BrieflyProtein quantification by Western blot was performed as we previously described . Brieflyin situ detection of ITGB1-DT in LUAD cells, the probes complementary to ITGB1-DT were synthesized by Advanced Cell Diagnostics . The hybridization and fluorescence detection were conducted using the RNAscope Fluorescent Multiplex Detection Kit (ACD) following the manufacturer’s manual. Confocal laser scanning microscopy  was used to detect the subcellular localization of ITGB1-DT in LUAD cells.For Fractionation of cytoplasmic and nuclear RNA in A549 cells was conducted using the Cytoplasmic and Nuclear RNA Purification Kit . Fractionated RNA from the same amounts of cells was used for qRT-PCR analysis.in vitro transcribed from pSPT19-ITGB1-DT using the Biotin RNA Labeling Mix (Roche) and T7 or Sp6 RNA polymerase (Roche), respectively. After DNase I treatment for 30 min at 37°C to remove DNA templates, the in vitro transcribed RNA was purified using the RNeasy Mini Kit . Three micrograms per reaction of purified RNA was denatured for 5 min at 65°C in RNA Structure buffer and slowly cooled down to room temperature. Then, folded RNA was incubated with 1 mg of whole-cell lysates from A549 cells at 25°C for 1 h. The complexes were further isolated by the streptavidin agarose beads (Invitrogen). To harvest the proteins, 50 ml of 1 × SDS loading buffer was added and boiled for 10 min. Retrieved protein was detected by Western blot.Sense or antisense strand of biotinylated ITGB1-DT was RNA immunoprecipitation assays were conducted in A549 cells using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) and a primary antibody against EZH2  according to the manufacturer’s manual. Retrieved RNA was detected by qRT-PCR.ITGB1 promoter, 5′-CCCGTTTGTCCACTGATGTC-3′ (forward) and 5′-CTTAGCCAGTTCCCTTCCAG-3′ (reverse) for ITGB1-DT promoter, and 5′-CTCTAAGTCCTAACCCCTCT-3′ (forward) and 5′-CCACATACGTCCTTTACGATA-3′ (reverse) for negative control.Chromatin immunoprecipitation assays were conducted in indicated LUAD cells using the EZ-Magna ChIP Kit  and primary antibodies against EZH2 , H3K27me3 , or MYC . Retrieved DNA was detected by qRT-PCR with the primers 5′-ATCCTCCGCCTCCTCCTA-3′ (forward) and 5′-TCCTCTGCGCGTCTGATC-3′ (reverse) for To measure the transcriptional activity of β-catenin, TOPFlash (β-catenin-LEF/TCF sensitive luciferase reporter vector) or FOPFlash (LEF/TCF binding site mutated luciferase reporter vector) was co-transfected with pRL-TK  into indicated LUAD cells. pRL-TK expressed Renilla luciferase and was used as endogenous control. The luciferase activities were measured using the Dual-luciferase Reporter Assay System (Promega) 48 h after transfection.in vivo lung metastasis assays, 2 × 106 indicated LUAD cells labeled by luciferase were injected into the lateral tail veins of female BABL/c athymic nude mice (age 4–5 weeks). Seven weeks later, the activities of luciferase in lungs were detected and captured under IVIS Lumina II . The mice were sacrificed and the lungs were excised, fixed in formalin, and subjected to hematoxylin and eosin (H&E) staining to detect lung metastatic nodules. The animal experimental procedures were approved by the Animal Ethics Committee of Xiangya Hospital.For t-test (parametric) was performed. For comparison of the differences between more than two groups, one-way ANOVA followed by Dunnett’s multiple comparisons test, or Kruskal–Wallis test (non-parametric one-way ANOVA) followed by Dunn’s multiple comparisons test was performed. Kaplan–Meier survival analyses were performed using Log-rank test. Gene expression correlation analyses in clinical specimens were performed using Spearman correlation analysis. Analyses of the correlation between gene expression and clinicopathological features were performed using chi-square test. P-values < 0.05 were considered statistically significant.All statistical analyses were conducted using GraphPad Prism v6.0 . For comparison of the differences between two groups, Wilcoxon signed rank test (non-parametric) or Student’s 1. The genes associated with overall survival of LUAD were shown in 2, also revealed that high expression of ITGB1-DT was correlated with poor survival in LUAD 4, Coding Potential Calculator (CPC)5, and PhyloCSF. The CPAT and CPC scores of ITGB1-DT were 0.00838 and -1.00977, respectively, both of which indicated ITGB1-DT as non-coding. Furthermore, the PhyloCSF from UCSC Genome Browser6 also identified the non-coding nature of ITGB1-DT. Therefore, these findings characterized ITGB1-DT as an upregulated and poor prognosis-related lncRNA in LUAD.To identify the lncRNAs associated with overall survival in LUAD, we analyzed TCGA LUAD dataset using the online tool Gene Expression Profiling Interactive Analysis (GEPIA) in LUAD . Analysi tissues . In addi tissues . Kaplan– tissues . Analyzi tissues . To furt tissues . The codDue to the significant correlation between ITGB1-DT and poor prognosis in LUAD, we next investigated the potential roles of ITGB1-DT in LUAD. ITGB1-DT stably overexpressed LUAD cells were constructed via stable transfection of ITGB1-DT overexpression vector into A549 and NCI-H1975 cells . CCK-8 aNext, the potential roles of ITGB1-DT in LUAD cellular migration and invasion were investigated using transwell migration and invasion assays, respectively. Transwell migration assays showed that A549 and NCI-H1975 cells with ITGB1-DT overexpression had more migrated cells than that of control A549 and NCI-H1975 cells . ConversITGB1-DT is located at the antisense strand of ITGB1; (2) ITGB1 has been reported to exert oncogenic roles in multiple cancers, including LUAD , was identified to interact with ITGB1-DT with an interaction probability score of 0.86 (> 0.5 was considered positive). To elucidate whether ITGB1-DT binds to EZH2 in LUAD cells, we conducted RNA pull-down assays using sense or antisense strand of biotinylated ITGB1-DT. The enriched proteins were detected by Western blot. As presented in ITGB1 promoter and H3K27me3 levels at ITGB1 promoter were both reduced in A549 cells with ITGB1-DT overexpression compared with control A549 cells  ing LUAD . Intriguing LUAD . Conversing LUAD . Consisting LUAD . ITGB1 ping LUAD . To furting LUAD . Biocheming LUAD . Multipling LUAD . To inve49 cells . Convers99 cells . To dete99 cells . At the 99 cells , indicatin vivo, the correlation between ITGB1 expression and ITGB1-DT expression in TCGA LUAD tissues was investigated using the online tool starBase (see text footnote 3). The results revealed that the expression of ITGB1-DT was remarkably positively correlated with the expression of ITGB1 in TCGA LUAD tissues (in vivo.To elucidate whether the upregulation of ITGB1 by ITGB1-DT also exists  tissues . Further tissues . Consist tissues . The cor tissues . In our  tissues . TherefoBecause ITGB1 was reported to activate Wnt/β-catenin pathway in multiple cancers, including LUAD, we next investigated the potential influences of ITGB1-DT on Wnt/β-catenin pathway. Nuclear β-catenin levels were upregulated in A549 cells with ITGB1-DT overexpression compared with control A549 cells , and dowITGB1-DT by in silico tool JASPAR8 with a score of 15.5622 (ITGB1-DT promoter region, ChIP assays were performed using MYC specific antibody. The ChIP results revealed that MYC directly bound ITGB1-DT promoter, but not a distal negative control region (ITGB1-DT promoter (ITGB1-DT promoter (Due to the significant positive correlation between ITGB1-DT and MYC in LUAD, we further investigated whether MYC also regulates ITGB1-DT. Intriguingly, a MYC binding site was identified in the promoter region of  15.5622 . To invel region . ITGB1-Dl region , which sl region . Activatl region . In contl region . As expel region , and ITGl region . Overexppromoter , and ITGpromoter . These fTo elucidate whether the roles of ITGB1-DT in LUAD were dependent on the positive ITGB1-DT/ITGB1/Wnt/β-catenin/MYC feedback loop, the expression of ITGB1 was silenced in A549 cells with ITGB1-DT overexpression. CCK-8 assays showed that the pro-proliferative roles of ITGB1-DT were reversed by ITGB1 silencing . Furtherin vivo, luciferase-labeled A549 cells with concurrent ITGB1-DT overexpression and ITGB1 silencing were injected into tail vein of nude mice. Seven weeks later, lung metastases were detected using bioluminescence analyzing. As shown in in vivo. The oncogenic roles of ITGB1-DT in LUAD were dependent on the ITGB1-DT/ITGB1/Wnt/β-catenin/MYC positive feedback loop.To investigate the potential roles of ITGB1-DT/ITGB1/Wnt/β-catenin/MYC feedback loop Through analyzing the available RNA-seq data of LUAD tissues from TCGA database, we found that ITGB1-DT was the most significantly correlated lncRNAs with overall survival of LUAD. The significant correlation between increased ITGB1-DT expression and poor prognosis of LUAD patients was also found in another available online database, Kaplan–Meier Plotter. Third, the correlation between increased ITGB1-DT expression and poor outcome of LUAD was also verified in our own cohort. Furthermore, ITGB1-DT was found to be highly expressed in LUAD tissues compared with normal lung tissues both in TCGA dataset and our own cohort. However, the expression of ITGB1-DT was not correlated with prognosis of LUSC in TCGA LUSC dataset. Thus, our findings suggested ITGB1-DT as a LUAD-related lncRNA and implied that ITGB1-DT may be a potential prognostic biomarker for LUAD. Given that many new lncRNAs may not be annotated in the TCGA RNA-seq dataset, we did not exclude the clinical relevance of other lncRNAs in LUAD. The involvements of these lncRNAs in LUAD need further investigation.in vitro cell model and in vivo animal model. The gain- and loss-of-function assay results revealed that ITGB1-DT facilitated LUAD cellular proliferation, migration, and invasion in vitro and LUAD lung metastasis in vivo. Depletion of ITGB1-DT showed the opposite effects. Thus, to our knowledge, these findings first revealed the critical oncogenic roles of ITGB1-DT in LUAD and suggested ITGB1-DT as a potential therapeutic target for LUAD.To elucidate whether the prognosis-related ITGB1-DT functions as a driver or passenger during LUAD progression, we systematically investigated the potential biological roles of ITGB1-DT in LUAD using ITGB1-DT is located on the opposite strand of ITGB1, we first investigated the potential regulatory roles of ITGB1-DT on ITGB1. Several lncRNAs have been reported to regulate their nearby genes, such as the transcription promoting roles of NDIME on its nearby gene MEF2C and the stabilization of BAZ2B pre-mRNA by lnc-BAZ2B (ITGB1 transcription by ITGB1-DT. Bioinformatic prediction and experimental verification demonstrated that ITGB1-DT physically bound to EZH2 and competitively reduced the binding of EZH2 to ITGB1 promoter. EZH2 is a well-documented epigenetic regulator, which induces H3K27me3 at target gene promoter and represses target gene expression (ITGB1 promoter and H3K27me3 levels at ITGB1 promoter. The positive regulation of ITGB1 expression by ITGB1-DT was supported by the remarkably positive correlation between the expression of ITGB1 and ITGB1-DT in LUAD tissues. Our findings provided new evidences to support lncRNAs as epigenetic regulators.Subcellular localization of lncRNAs determines the potential mechanisms of action of lncRNAs. Cytoplasmic lncRNAs may act as microRNA sponges and attenuate the biological roles of interacted microRNAs . Cytoplanc-BAZ2B . In thispression . ConsistITGB1, also known as integrin β1, has been documented to exert oncogenic roles in various cancers . AKT/WntIn summary, our findings identified ITGB1-DT as an upregulated and poor prognosis-related lncRNA in LUAD, which facilitated LUAD progression through activating the ITGB1-DT/ITGB1/Wnt/β-catenin/MYC positive feedback loop .The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Xiangya Hospital and were conducted in accordance with the ethical standards of Xiangya Hospital and the Helsinki Declaration. We obtained written informed consents from all participants. The animal experimental procedures were approved by the Animal Ethics Committee of Xiangya Hospital.YG, RC, and XZ designed the study. RC, XX, YF, and XZ performed the experiments. YG, XZ, and CZ supervised the project. YG, RC, and XZ analyzed the data. YG and RC drafted the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mycotoxins are toxic fungal secondary metabolites that pose a major threat to the safety of food and feed. Mycotoxins are usually converted into less toxic or non-toxic metabolites through biotransformation that are often made by living organisms as well as the isolated enzymes. The conversions mainly include hydroxylation, oxidation, hydrogenation, de-epoxidation, methylation, glycosylation and glucuronidation, esterification, hydrolysis, sulfation, demethylation and deamination. Biotransformations of some notorious mycotoxins such as alfatoxins, alternariol, citrinin, fomannoxin, ochratoxins, patulin, trichothecenes and zearalenone analogues are reviewed in detail. The recent development and applications of mycotoxins detoxification through biotransformation are also discussed. Alternaria, Aspergillus, Fusarium and Penicillium. Mycotoxins may infer serious risks such as carcinogenic, teratogenic, mutagenic and immunosuppressive effects on animal and human health, and lead to economic loses. Several detoxification strategies, including physical ..19), als21) consists of a chlorinated dihydroisocoumarin linked through a 7-carboxyl group to L-phenylalanine by an amide bond. OTA (21) was hydroxylated into 7-carboxy--5-chloro-8-hydroxy-3,4-dihydro-3R- methylisocoumarin (22) and a dihydrodiol derivative (23) by a Gram-negative bacterium Phenylobacterium immobile  consR)-4-Hydroxyochratoxin A (24), or 4-hydroxyochratoxin A, was isolated from the urine of rats after injection with OTA (21), which indicated that OTA (21) was converted to (4R)-4-hydroxyochratoxin A (24) ((4n A (24)  29]..R)-4-Hyd21) was hydroxylated into (4R)-4-hydroxyochratoxin A (24) and (4S)-4-hydroxyochratoxin A (25) as well as 10-hydroxyochratoxin A (26) by rabbit liver microsomes  w27) was incubated with horse radish peroxidase (HPR), the high level of the hydroquinone metabolite of ochratoxin  was produced  was found to be produced by more than 30 fungal species, particularly by Aspergillus species such as A. flavus, A. parasiticus, A. versicolor and A. nidulans . In. In31) wyl group . 19-Hydr33) was converted into (5S)-5-hydroxy ZEN (34) with hydroxylation of the aliphatic ring of ZEN (33) by the fungus Cunninghamella bainieri (33) labeled with deuterium at specific positions, evidence was provided for the preferential hydroxylation of ZEN (33) at C-8 and, to a lesser extent, at C-5, C-9 and C-10 with rat liver microsomes. The stereochemistry of the aliphatic hydroxylation products of ZEN (33) needs to be further studied [33) was converted into two monohydroxylated metabolites namely 13-hydroxy-ZEN (35) and 15-hydroxy-ZEN (36) with aromatic hydroxylation by human hepatic (liver) microsomes  w studied . In addicrosomes  40]..33) was Oxido-reduction between alcohols and ketones of mycotoxins include: (i) oxidation of the hydroxyl group and (ii) reduction of the carbonyl group .1  was converted into alflatoxicol (37) through reduction of the keto group to the hydroxyl group by several fungi such as Eurotium herbariorum, Rhizopus sp. and non-aflatoxin-producing Aspergillus flavus  at C-3 position by converting DON (38) to 3-keto-DON (39) was revealed from the bacterium Devosia sp.  at C40) is a benzohydrofuran phytotoxin that was produced by the forest pathogen Heterobasidion annosum during the infection process ..40) is a33) was transformed to both α-zearalenol  and β-zearalenol  with reduction by the fungi Candida tropicalis (Saccharomyces cerevisae ..46) belo1  was catalyzed into AFB1-8,9-epoxide (48) by channel catfish liver microsomes (1 (1) was also transformed to AFB1-8,9-dihydrodiol (49) by the fungus Phanerochaete sordia  and stemphyltoxin III  were reduced to alcohols in human colon Caco-2 cells, resulting in the formation of altertoxin I  and alteichin , respectively (50) was also reduced to ATX I (52) in other human tumor cell lines such as HCT 116, HepG2 and V79 ..40) is a19) was reduced to fusarinol (64), also called 5-butyl-2-pyridinemethanol, by the fungus Aspergillus tubingensis (64) was significantly less phytotoxic than fusaric acid (19). In fusarinol (64), the acid has been reduced to an alcohol group. The reduced phytotoxicity of fusarinol (64) indicates the importance of the carboxylic acid in the toxic function of FA (19). The fungus A. tubingensis provides a novel detoxification mechanism against FA (19), which may be utilized to control Fusarium wilt ..KodameaeO-methyltransferase  was converted into alternariol 9-O-methyl ether  by methylation through an alternariol O-methyltranferase. The methyl donor was S-adenosyl-L-methionine (SAM)   63]..7) was c33) was converted into zearalenone 16-methyl ether (69) and zearalenone 14,16-bis (methyl ether) (70) by the fungus Cunninghamella bainieri (69) showed similar estrogenic activity compared with the parent compound ZEN (33). However, zearalenone 14,16-bis (methyl ether) (70) exhibited inactive estrogenic effect . Ined cells  67]..7) could82) is a macrocyclic lactone produced by a number of fungi from the genera Curvularia, Penicillium and Alternaria, and have been reported to possess a variety of biological activities including phytotoxicity [82) was converted into curvularin 11-O-β-d-glucopyranoside (83) and curvularin 4′-O-methyl-11-O-β-d-glucopyranoside (84) with glycosylation and methylglycosylation through the the fungus Beauveria bassiana (Curvularin (toxicity . Curvulabassiana  69]..82) is a38) is the main trichothecene toxin produced by the Fusarium species and a relevant virulence factor in Fusarium head blight (FHB) disease of cereal crops. Trichothecene toxins inhibited eukaryotic protein synthesis and elicited a wide range of pathophysiological effects in humans and animals. DON (38) could be glycosylated to DON 3-O-β-d-glucoside , which was a masked mycotoxin, by a recombinant UDP-glucosyltranferase from rice (38), deepoxy-deoxynivalenol , iso-deoxynivalenol  and iso-deepoxy-deoxynivalenol  were respectively transformed to a series of glucuronides (88–97) by glucuronidation through rat liver microsomes (RLM) or human liver microsomes (HLM)  ies (HLM)  71,72].,72.38) i98) and 4,15-diacetoxyscirpenol  were produced by the fungi from the genus Fusarium such as F. sporotrichioides and F. poae . Fu. Fu103) 31) was converted into T-2 toxin 3-O-α-d-glucoside (105) with glycosylation by the fungi Blastobotrys muscicola and B. robertii (31) was transformed to T-2 toxin-3-O-β-d-glucuronide  by UDP glucuronyl transferase (UDPGT) released from rat liver microsomes (T-2 toxin (robertii  79]. In. In31) wcrosomes  80]..31) was 33) was glycosylated into zearalenone 14-O-glucoside  by Arabidopsis UDP-glucosyltransferases expressed in Saccharomyces cerevisiae (33) was converted to ZEN 14-O-Glc (107) with glycosylation by the fungi Mucor vainieri and Thamnidium elegans . Th. Th33) wnsferase  84]..33) was 21) was converted to OTA methyl ester (110) by the cell cultures of wheat and maize  was 31) was converted to 3-acetyl T-2 toxin (111) with acetylation by bovine rumen fluid in vitro (T-2 toxin (in vitro  85]..31) was Fusairum sp. strain C37410-90 possessed significant esterase activity and hydrolyzed the 3-acetyl deoxynivalenol  to DON (38) (Sphaerodes mycoparasitica incubated with 3-acetyl DON (112) . Th. ThFusai99) is a potent mycotoxin produced by some Fusarium species. 4,15-DAS (99) was hydrolyzed to either 4-monoacetoxyscirpenol  or 15-monoacetoxyscirpenol , and the final product was scirpentriol  with deacetylation in rats  is a1  was transformed to the hydrolyzed FB1 namely aminopentol 1 , with hydrolysis by the fungus Exophiala spinifera 2141.10 (1 (115) was also converted to AP1 (116) either with the enzyme from the bacterium Sphingopyxis sp. MTA144 [Fumunisin B 2141.10  89]. FB. FB1 (FB. MTA144  or with . MTA144 .117) was hydrolyzed into nivalenol  via deacetylation in mice -L-phenylalanine (120) was tested to be more toxic than the parent molecule which indicated that the lactone ring was not considered to be responsible for the toxic activity of OTA (21) . Th. Th31) wtylation .31) was first hydrolyzed into HT-2 toxin (122), which was further converted to 15-acety-tetraol (123), which was finally transformed to T-2 tetraol (124) with hydrolyzation  was 31) was selectively hydrolyzed at C-8 resulting in the removal of isovaleryl group by the fungus Blastobotrys capitulate to neosolaniol (125)   79]..31) was 38) and zearalenone  were converted to their corresponding sulfates DON 3-sulfate (126) and ZEN 14-sulfate (127) (Sphaerodes mycoparasitica . U. U4 , which was also named as aminopentol 1 (AP1), was converted into 2-keto HFB1 (132) or namely 2-keto AP1 through oxidative deamination by the fungus Exophiala spinifera  was converted to dihydrohydroxyaflatoxin B1 (133), which was also named as aflatoxin B2a (AFB2a), via reduction and oxidation by Pleurotus ostreatus GHBBF10  was detoxified to aflatoxin D1 , aflatoxin D2  and aflatoxin D3  through hydrolysis, decarboxylation, and oxidation-reduction by Pseudomonas putida, which was isolated from sugarcane (1 (1) to AFD1 (134) was also entirely elucidated 54) was converted into botryenedial (141) with dehydration by the fungus Botrytis cinerea (141) was less phytotoxic than botrydial (54) . Wh. Wh38) wnization  [71].40) was added in the cell cultures of Pinus sylvestris, formannoxin (40) was transformed into the non-toxic fomannoxin acid (59), which was further glycosylated to fomannoxin acid β-glucoside (145) with time prolonging (When fomannoxin (olonging  44]..40) was 1 (115) comprises at least two enzymatic steps, an initial hydrolysis (deesterification reaction) to form aminopentol 1 , followed by deamination of AP1 (116) to form 2-keto AP1 (132). Two recombinant enzymes carboxylesterase and aminotransferase from the bacterium Sphingopyxis sp. were expressed heterologously. Carboxylesterase catalyzed fumonisin B1 (115) to AP1 (116) with deesterification (116) to 2-keto AP1 (132) with deamination in the presence of pyruvate and pyridoxal phosphate . OT. OT21) wOTA (21)  117]..21) was 65) was transformed to desoxypatulinic acid  with multi-step reactions  to the less toxic desoxypatulinic acid (150) was considered a detoxification process . S. S33) ways rosea ,124.Aspergillus species. They are threats to human and animal health, and are classified by the International Agency for Research on Cancer (IARC) as group 1 carcinogens  we we1 (AFB1 (1) was reduced to hydroxyl group. AFB1 (1) was converted into alflatoxicol (37) by several fungi . Th. Th40) i40) was added in the cell cultures of Pinus sylvestris, formannoxin was transformed into the non-toxic formannoxin alcohol (41) -L-phenylalanine [(i) Hydrolysis: hydrolysis of OTs include degradations of amide and ester bonds. OTA  usually occurs at its C-4, C-5, C-8′, C-9′ and C-10 positons. Hydroxylation as an inactivation pathway in animal is catalyzed by CYP 450. OTA (21) was hydroxylated into 4R-hydroxyochratoxin A in the urine of rats after injection with OTA (21) [21) was hydroxylated into 7-carboxy--5-chloro-8-hydroxy-3,4-dihydro-3R-methylisocoumarin (22) and a dihydrodiol derivative (23) by a Gram-negative bacterium Phenylobacterium immobile [(ii) Hydroxylation: hydroxylation of OTA (OTA (21) . OTA (21immobile .21) was hydroxylated into (4R)-4-hydroxyochratoxin A (24) and (4S)-4-hydroxyochratoxin A (25), which were further glycosylated or esterificated by the cell cultures of wheat and maize [(iii) Hydroxylation and glycosylation or esterification: OTA (nd maize .21) was catalyzed to OTA methyl ester (110) by the cell cultures of wheat and maize [(iv) Esterification: OTA (nd maize .21) was dechlorinated into OTB (27) in the renal microsomes of rabbits pretreated with phenobarbital (PB) [(v) Dechlorination: OTA  .65), a polyketide lactone, exhibits genotoxic, mutagenic, carcinogenic, teratogenic and cytotoxic properties for humans and animals as well as plants [65) can be produced by various fungal species belonging the genera Penicillium, Aspergillus and Byssochlamys, and particularly, Penicillium expansum is considered as the major producer of PAT (65), which is causative agent of the blue mold disease of stored apples [65) through biotransformation by using yeast, bacteria and fungi have been shown good results, and it seems to be attractive since it works under mild and environment-friendly conditions [65) was destroyed, the transformed products became less or non toxic (65) are as follows.Patulin  was added in the cell cultures or cell-free supernatant of Lactobacillus plantarum, it was transformed to E-ascladiol (66) and Z-ascladiol (67). The ascladiol isomers were then further transformed over a 4-week cell-free incubation (4 °C) into the metabolite hydroascladiol (68) [(i) Oxido-reduction: when PAT (iol (68) .65) was converted into desoxypatulinic acid , which was less cytotoxic than PAT (65) to in vitro human lymphocytes, by the yeast Rhodotorula kratochvilovae LS11 [Rhodosporidium paludigenum [150) derived from PAT (65) through hydrolysis of the γ-lactone ring and subsequent enzymatic modifications by reductase and dehydratase [65) conversion into E/Z-ascladiols (66 and 67) and DPA (150), other reported microorganisms included fungi such as Kodameae ohmeri [Pichia caribbica [Rhodotorula mucilaginosa JM19 [Saccharomyces cerevisiae [Sporobolomyces sp. IAM 13,481 [Lactobacillus plantarum [(ii) Multi-step biotransformations: PAT , T-2 toxin (31), DON (38), 3-acetyl DON (112), 15-acetyl DON, 4,15-DAS (99) and nivalenol  due to their high toxicity and prevalent occurrence. DON (38) may be the most commonly occurring trichothecene in nature. T-2 toxin (31) does not occur as much as DON (38), but its toxicity is higher than that of DON (38) [Trichothecenes are chemically tricyclic sesquiterpenoids, characterized by a double bond at the C-9 and C-10 position, an epoxy functional group at the C-12 and C-13 position and variable numbers of hydroxyl and acetoxy groups. Trichothecenes can be divided into four types  according to characteristic functional groups . Among tDON (38) ,160.The double bond between C-9 and C-10, the 12, 13-epoxide ring, and hydroxyl group at C-3 have been considered as the toxicity of trichothecenes in eukaryotic organisms . RemovalDetoxification of trichothecenes via various biotransformation pathways mainly include oxygenation , de-epox38) was transformed to different mono-O-glucuronides by glucuronidation through animal liver microsomes [31) was transformed to T-2 toxin 3-O-β-d- glucuronide (106) by UDP glucuronyl transferase (UDPGT) released from rat liver microsomes [38) is dominantly excreted as a glucuronide [(i) Glycosylation or glucuronidation: Glycosylation of C-3 hydroxy group by plant glycosyltransferase enzymes can convert trichothecenes into less toxic glycosides ,70. DON crosomes . Similarcrosomes . In humacuronide .38) was converted to 3-epi-deoxynivalenol (143) with epimerization by the bacterium Nocardioides sp. WSN05-2 [(ii) Epimerization: DON ( WSN05-2 .38) with de-epoxidaton at the C-12 and C-13 positions by Eubacterium sp. DSM 11798. The converted product was de-epoxydeoxynivalenol  [(iii) De-epoxidation: transformation of DON  .31) was hydrolyzed either into HT-2 toxin (122) by the bacterium Eubacterium sp. BBSH 797 [125) by the fungus Blastobotrys capitulate [(iv) Hydrolysis: T-2 toxin (BBSH 797  or into 33) is a macrocyclic phenolic β-resorcyclic acid lactone. It mainly produced by fungi belonging to the genus Fusarium such as F. graminaearum and F. culmorum. It possesses estrogenic activity in pigs, cattle and sheep [42) and β-zearalenol (43) of ZEN, are also estrogenic and the effects of ZEN (33) and related metabolites on animal reproductive function have been reported [33) also damages the liver and kidneys and reduces immune function which leads to cytotoxicity and immunotoxicity [33) biotransformation relate mainly to the hydrolysis of the lactone ring, reduction of the ketonic carbonyl group, modification of the hydroxyl groups , and reduction of the carbon-carbon double bond. The main transformation pathways of ZEN analogues are as follows.Zearalenone  was transformed to decarboxylated hydrolyzed ZEN, namely 1-- 6′-hydroxy-l’- undecen-l0′- one (154), at degradation rate of 95.7% by Bacillus pumilus ES-21 [(i) Hydrolysis: ZEN (us ES-21  or purifus ES-21 .33) was transformed to zearalanone (44) with reduction of the carbon-carbon bond in ovine [(ii) Reduction of the carbon-carbon double bond: ZEN (in ovine .33) was converted to α-zearalenol (42) and β-zearalenol (43) with reduction of the keto group by the fungi Rhizopus sp. and Aspergillus sp. [Candida tropicalis [(iii) Reduction of the ketonic carbonyl group: ZEN  was transformed to α-zearalenol (42), β-zearalenol (43), zearalanone (44), α-zearalanol (151), β-zearalanol (152) and with multi-step oxido-reductions in human body [(iv) Multi-step oxido-reductions: ZEN (man body .33) was converted to ZEN 14-sulfate (127) by the fungus Sphaerodes mycoparasitica [(v) Sulfation: ZEN (rasitica .33) was converted to ZEN 16-methyl ether (69) and ZEN 14,16-bis (methyl ether) (70) by the fungus Cunninghamella bainieri. ZEN 14,16-bis (methyl ether) (70) showed an inactive estrogenic effect [(vi) Methylation: ZEN (c effect .33) was converted to zearalenone-14-O-glucoside (107) by Arabidopsis UDP-glucosyltransferases expressed in Saccharomyces cerevisiae [33) was also glycosylated into zearalenone 14-O-β-glucoside (107) and zearalenone 16-O-β-glucoside (108) by a barley UDP-glucosyltransferase [107 and 108 of glycosylated ZEN showed nontoxic activity [(vii) Glycosylation: ZEN  and ZEN 16-O-glucoside (108), which can be accumulated in organisms and cannot be eliminated [The reaction types and stereochemistry of conversion depend on the functional groups in the molecular structures of mycotoxins together with the specific enzymes provided by the organisms. Detoxification through biotransformation is now an important strategy to eliminate mycotoxins from food and feed. Indeed, not all converted products are nontoxic. Some converted products are still toxic or even more toxic ,171. Somiminated ,172,173.iminated ,174. ThiBacteria and fungi that can transform mycotoxins to less toxic or nontoxic products serve as a source of enzymes that can be used to decontaminate agricultural commodities or used as feed additives. More and more degradation enzymes of mycotoxins have been purified and identified from microorganisms ,175. TheIn plants, sequential hydroxylation, glycosylation and demethylation of fungal phytotoxins can avoid plant cell death and overcome the fungal invader . DetoxifMany other mycotoxins such as beauvericin ,179, ennIn short, detoxification of mycotoxins through biotransformation provides a reliable reference strategy for the management of mycotoxins in foods and feeds. It will help us to better understand the fate of mycotoxins in animals and humans, as well as to provide basic information for the risk assessment of mycotoxins for food and feed safety. Further investigations, especially the development of methods to utilize the multi-reaction processes as well as to clone and express the genes of detoxification enzymes in organisms, will be necessary for the detoxification of mycotoxins through biotransformation.


Scientific Reports 10.1038/s41598-020-57989-6, published online 22 January 2020Correction to: The Supplementary Information file that accompanies this Article contains errors in Supplementary Figure S1, where the figure keys are incorrect. The correct Figure S1 appears below as Figure


The extended social isolation necessitated by the COVID-19 pandemic will likely have a prolonged negative impact on the health of community-dwelling older adults. We studied the potential to counteract these negative effects, examining the before and after measurements of participants in two obesity intervention studies that were converted from in-person to remote delivery, due to COVID-19. The Veterans Achieving Weight Loss and Optimizing Resilience-Using Protein study  and the Egg-Supplemented Pre-Diabetes Intervention Trial  enrolled obese (BMI = 34.2±3.8 kg/m2) older adults  with prediabetes (fasting blood glucose 100-125 mg/dL and/or HbA1c of 5.7-6.4%). Participants followed a hypocaloric diet, attended weekly support groups, and weighed themselves weekly; VALOR-UP participants also attended a weekly exercise class delivered remotely. Outcomes were assessed at baseline (pre-COVID) and the end of a 4-month period coinciding with stay-at-home orders. Between baseline and 4 months, calorie intakes (3-day diet record) decreased by 402.1±529.3 kcal/day (p<0.05) and body weight by 6% . Physical function  improved by 1.1±1.4 units (p<0.01) and Short Form Health Survey (SF-36) physical and mental composite quality of life remained stable . Thus, despite COVID-19 circumstances, most participants were able to benefit from an intervention to improve function and reduce obesity. The somewhat surprising findings for this small cohort offer promise not only for future lifestyle interventions during COVID-19 isolation but also for other isolated populations, including home-bound older adults.


Memory B cells specific for SARS-CoV-2 spike and nucleocapsid proteins persist in peripheral blood after recovery from COVID-19. + or IgG1+ and continued to rise until 150 days. RBD-specific IgG+ Bmem were predominantly CD27+, and numbers significantly correlated with circulating follicular helper T cell numbers. Thus, the SARS-CoV-2 antibody response contracts in convalescence with persistence of RBD- and NCP-specific Bmem cells. Flow cytometric detection of SARS-CoV-2-specific Bmem cells enables detection of long-term immune memory following infection or vaccination for COVID-19.Lasting immunity following SARS-CoV-2 infection is questioned because serum antibodies decline in convalescence. However, functional immunity is mediated by long-lived memory T and B (Bmem) cells. Therefore, we generated fluorescently-labeled tetramers of the spike receptor binding domain (RBD) and nucleocapsid protein (NCP) to determine the longevity and immunophenotype of SARS-CoV-2-specific Bmem cells in COVID-19 patients. A total of 36 blood samples were obtained from 25 COVID-19 patients between 4 and 242 days post-symptom onset including 11 paired samples. While serum IgG to RBD and NCP was identified in all patients, antibody levels began declining at 20 days post-symptom onset. RBD- and NCP-specific Bmem cells predominantly expressed IgM Coronavirus disease (COVID)-19 is a global health emergency. The causative agent, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is highly contagious and has infected tens of millions worldwide and caused over 1.2 million deaths since its discovery in Wuhan, China in December 2019  activity. These analyses highlight that a decline in serum antibodies in convalescence may not reflect waning of immunity, but rather a contraction of the immune response with the development and persistence of B cell memory.We extensively characterized the SARS-CoV-2-specific Bmem cell compartment using unique sets of fluorescently-labeled recombinant tetramers of the SARS-CoV-2 RBD and NCP antigens in combination with an extensive flow cytometry panel. The SARS-CoV-2-specific Bmem cells were quantified and characterized in 36 samples from 25 patients with COVID-19 or in convalescence. Circulating RBD- and NCP-specific Bmem cell subsets were detected early after infection and persisted over 242 days post-symptom onset. Early after infection, antigen-specific Bmem cells predominantly expressed IgM, followed over time by a predominance of IgG1. RBD-specific Bmem cell numbers were found to positively correlate with circulating TThe antigen-specific B cell response to SARS-CoV-2 was characterized using recombinant forms of the RBD and NCP. Both proteins were generated in Expi293F cells with an AviTag for targeted biotinylation and tetramerization with fluorescently-labeled streptavidins to minimize epitope masking . Two tet+ T-cell lymphopenia due to reduced CD8+ T-cell counts (table S2). All patients exhibited normal absolute numbers of B cells (table S2).The SARS-CoV-2-specific antibody and B cell response was investigated in 25 COVID-19 confirmed patients with samples obtained between 4 and 242 days after symptom onset (table S1). Patients were classified into three levels of disease severity , and RBD- and NCP-specific IgG levels in our patients declined over time in convalescence . Neutralhigh CD27+ plasmablasts, whereas this population was negligible in any of the samples taken >20 days post-onset of symptoms (fig. S1). Bmem cells were defined using IgD and CD27  (+IgM+IgD+) and immunoglobulin (Ig) class-switched cells (CD27+/−IgD-) . Thus, SARS-CoV-2 infection induces robust Bmem cell responses, which are predominantly comprised of IgM+ and IgG1+ Bmem cells with distinct immunophenotypes for those directed against RBD versus NCP.To examine the nature and kinetics of the RBD- and NCP-specific Bmem following SARS-CoV-2 infection, the RBD and NCP proteins were biotinylated and tetramerized with fluorescently-labeled streptavidins. RBD- and NCP-specific B cells were evaluated by flow cytometry in all 36 samples for expression of markers for plasmablasts (CD38), activated (CD71) and resting (CD27) Bmem cells, as well as surface IgD, IgA and IgG1, 2, 3 and 4 subclasses  (Table Sand CD27 . All patctively) . The RBD+/−IgD-) . The lat+/−IgD-) . These dem cells . CompareIg genes  (30). Ir+ RBD- and NCP-specific Bmem cells in samples taken 26 days or more post-symptom onset . Of these subsets, the CD4+ TFH, CD4+ TFR and CD8+ TFH cell numbers showed a significant positive correlation with RBD-specific total, IgM+ and IgG+ Bmem cells across the 36 samples (fig. S5). In contrast, only CD8+ TFH cell numbers showed a significant correlation with NCP-specific total and IgG+ Bmem cells (fig. S6).The numbers and Ig isotype distribution of RBD- and NCP-specific Bmem cell subsets varied between individuals. However, similar trends were still observed for both subsets with higher proportions and absolute numbers of IgG1om onset , fig. S3om onset . Total a240 days . RBD- an240 days , althoug240 days . IgM+ anFH cell numbers. This long-term presence of circulating Bmem cell subsets directed against both the major SARS-CoV-2 neutralization target (RBD) and a non-neutralizing target (NCP) is indicative of persisting immune memory following natural exposure and could provide a means to evaluate protection from severe COVID-19 following vaccination.In summary, RBD- and NCP-specific IgG and Bmem cells were detected in all 25 patients with a history of COVID-19. While specific IgG levels in serum declined with time post-symptom onset, SARS-CoV-2-specific Bmem cell numbers persisted, and RBD-specific Bmem cell numbers correlated with T+ Bmem cells predominated in the first 20 days post-infection, followed by a gradual increase in IgG1+ Bmem cells. The fact that nearly all RBD-specific IgG+ Bmem cells expressed CD27 and their numbers correlated with circulating TFH cells is indicative of long-lived immune memory.We have shown that COVID-19 patients rapidly generate B cell memory to both the spike and nucleocapsid antigens following SARS-CoV-2 infection. Although rare events, numbers of Bmem cells specific for the major neutralization target, RBD, and a major non-neutralizing target, NCP, were both in the same order of magnitude. Using an extensive flow cytometry panel, we show that in line with typical T-cell dependent responses, IgMNone of our patients showed a reduction in total B cell numbers, even those sampled within 14 days of symptom onset. Several studies have reported a decrease in B cell frequencies during COVID-19 infection which normalized in convalescence  and allophycocyanin (APC))  and this population has been shown to contract after ~2 weeks  indicating long-lived memory. There have been some studies reporting that the Bmem cell response to SARS-CoV may not be long-lived . From March to September 2020, 25 individuals with a history of PCR-confirmed COVID-19 disease and 36 healthy controls (sampled in 2019 and Q1 2020) consented to one or two 40 ml donations of blood as well as the collection of clinical data including: basic demographics , clinical details of COVID-19  and co-morbid medical conditions (table S1). This study was conducted according to the principles of the Declaration of Helsinki and approved by local human research ethics committees.Blood samples of patients and controls were processed as described previously , and 30 μg DNA was transfected into 293F cells using the Expi293 Expression system . Supernatants from 25 ml cultures were collected on days 3 (NCP) or 5 (RBD) post-transfection and purified by application to a Talon NTA-cobalt affinity column  with elution in 200 mM imidazole. Eluted proteins were then dialyzed against 10 mM Tris for 48 hours at 4°C. Purified proteins were biotinylated by incubating at room temperature overnight with 1/8 of final volume each of Biomix A  and Biomix B  followed by 2.5 μg of BirA enzyme per milligram of protein. Biotinylated protein was subsequently dialyzed against 10 mM Tris for 36 hours at 4°C, and subsequently stored at -80°C prior to use. Soluble biotinylated NCP protein was tetramerized by the addition of either Brilliant Ultra Violet (BUV)395-conjugated streptavidin, or streptavidin-BUV737, and biotinylated RBD with streptavidin-BV480 or streptavidin-BV650  at a protein:streptavidin molar ratio of 4:1 making 4 unique tetramers: [NCP]4-BUV395, [NCP]4-BUV737, [RBD]4-BV480 and [RBD]4-BV650.Recombinant nucleocapsid protein (NCP) and receptor binding domain of the spike protein (RBD) of SARS-CoV-2 (GenBank: MN908947.3) were produced with a human Ig leader and the Fel d 1 leader sequence, respectively. Each protein construct was C-terminally fused to the biotin ligase (BirA) AviTag target sequence and a 6His affinity tag . The DNA. Briefly, 10 μl of sample was mixed with 2.5 μl of 4X Laemmli Sample buffer (non-reducing)  or reducing buffer (4X Laemmli Sample buffer with the addition of 1.25 μl DTT). Samples under reducing conditions were heated to 85°C for 10 min. 10 μl of ladder (1:1 mixture) of Precision plus protein standard , and reduced or non-reduced sample was loaded on a 4-15% Mini-PROTEAN TGX Stain-Free gel (BioRad) and run for 30 min at 200 V then imaged on the BioRad ChemiDoc Touch imaging system (BioRad).SDS-PAGE analyses were performed as described previously  were coated with 2 μg/ml recombinant SARS-CoV-2 NCP or RBD or with hemagglutinin (HA) from influenza A/Michigan/08/2015 (AM15) overnight at 4°C .Absolute numbers of leukocyte subsets were determined as described previously .Detailed T-cell subsetting was performed with two 11-color flow cytometry panels (table S3) as previously described . In addition, 5 million PBMC were similarly incubated with fixable viability stain 700 (BD Biosciences), antibodies against CD3, CD19, CD27 and IgD, plus BUV395-, BUV737-, BV480- and BV650-conjugated streptavidin controls (tables S3 and S4). Following staining, cells were washed twice with FACS buffer and filtered through a 70 μM filter prior to acquisition on the 5-laser BD LSRFortessa X-20. Flow cytometer set-up and calibration was performed using standardized EuroFlow SOPs, as previously described (tables S3 and S4) , antibodies against CD3, CD19, CD21, CD27, CD38, CD71, IgA, IgD, IgG1, IgG2, IgG3, IgG4, (tables S3 and S4) and 5 μg/ml  each of [NCP]U-test. Correlations were performed using the non-parametric Spearman’s rank correlation. For all tests, p < 0.05 was considered significant.All flow cytometry data were analyzed with FlowJo v10 software . Statistical analysis was performed with GraphPad Prism 8 Software (GraphPad Software). Matched pairs were analyzed with the non-parametric Wilcoxon matched pairs signed rank test. Unpaired groups were analyzed with the non-parametric Mann-Whitney


Several studies indicate that fatty replacement of myocardium occurs after left ventricular myocardial infarction. Magnetic resonance imaging can readily identify the location and morphology of both fatty tissue and myocardial infarction and may have the ability to non-invasively identify fatty replacement within infarcted regions. Fat deposition may impair global and regional cardiac function as well as the electrical activation of the heart.Twenty-five patients  with documented prior myocardial infarctions and ten normal volunteers (age: 63 ± 10 yrs) underwent MR imaging on a clinical 1.5 T scanner using precontrast fat-water separated (Dixon) and late gadolinium-enhanced infarct imaging. Myocardial infarct location and size were assessed using the full width half maximum (FWHM) infarct sizing algorithm applied to the late gadolinium-enhanced images. Fat-water separation was performed using a three-point Dixon reconstruction from in- and opposed-phase gradient-echo images (Echo times (TEs) = 4.8, 7.2, 9.6 ms). Fat segmentation was performed on a slice by slice basis using a signal intensity threshold set two standard deviations above the noise level. Volumes of myocardial late gadolinium-enhancement and fat deposition were compared using a Student's t-test. Precontrast infarct location and detection was compared with late gadolinium-enhanced infarct imaging using a seventeen segment model.Subjects with prior myocardial infarction had a 68% prevalence of fat deposition 17/25). In subjects without a history of myocardial infarction, fat deposition and late gadolinium-enhancement were not detected (0/10). In the patients with fat deposition, the fat volume was 19.6 ± 19 ml (range: 1.7–62.6 ml) vs late gadolinium-enhancement volume of 30 ± 15 ml (range: 9.6–59.2 ml). The volumes of late gadolinium-enhancement and fat deposition were statistically different (p = 0.01). Of the 425 myocardial segments, 156 (37%) had late gadolinium-enhancement and 76 (18%) had fat deposition. 65 segments (15%) had both fat deposition and late gadolinium-enhancement, while 91 segments (21%) had late gadolinium-enhancement and no fat deposition. 11 segments (2.6%) had fat deposition and no late gadolinium-enhancement, but all 11 segments had adjacent myocardial segments with late gadolinium-enhancement. Figure 7/25. In Fatty replacement of myocardium after myocardial infarction is common and can be readily identified using fat/water separation MR imaging. The clinical significance and biologic mechanism of fatty deposition in infarction remain to be determined.


Cytokine storm is suggested as one of the major pathological characteristics of SARS-CoV-2 infection, although the mechanism for initiation of a hyper-inflammatory response, and multi-organ damage from viral infection is poorly understood. In this virus-cell interaction study, we observed that SARS-CoV-2 infection or viral spike protein expression alone inhibited angiotensin converting enzyme-2 (ACE2) receptor protein expression. The spike protein promoted an angiotensin II type 1 receptor (AT1) mediated signaling cascade, induced the transcriptional regulatory molecules NF-κB and AP-1/c-Fos via MAPK activation, and increased IL-6 release. SARS-CoV-2 infected patient sera contained elevated levels of IL-6 and soluble IL-6R. Up-regulated AT1 receptor signaling also influenced the release of extracellular soluble IL-6R by the induction of the ADAM-17 protease. Use of the AT1 receptor antagonist, Candesartan cilexetil, resulted in down-regulation of IL-6/soluble IL-6R release in spike expressing cells. Phosphorylation of STAT3 at the Tyr705 residue plays an important role as a transcriptional inducer for SOCS3 and MCP-1 expression. Further study indicated that inhibition of STAT3 Tyr705 phosphorylation in SARS-CoV-2 infected and viral spike protein expressing epithelial cells did not induce SOCS3 and MCP-1 expression. Introduction of culture supernatant from SARS-CoV-2 spike expressing cells on a model human liver endothelial Cell line (TMNK-1), where transmembrane IL-6R is poorly expressed, resulted in the induction of STAT3 Tyr705 phosphorylation as well as MCP-1 expression. In conclusion, our results indicated that the presence of SARS-CoV-2 spike protein in epithelial cells promotes IL-6 trans-signaling by activation of the AT1 axis to initiate coordination of a hyper-inflammatory response. The mechanisms of SARS-CoV-2 induced excessive inflammatory response associated with disease severity needs to be critically studied for therapeutic intervention. Cytokine storm is typical of macrophage activation; which leads to tissue damage, lung injury, and acute respiratory distress syndrome. Lung epithelial cells; a primary host for SARS coronavirus infection, can generate a cytokine response leading to an expanded pathology. Our present study was designed to understand SARS-CoV-2 and human epithelial cell interactions, by delineating their contribution to inflammatory cytokine systems with special emphasis on ectopic expression of the viral spike protein. We observed that SARS-CoV-2 infection or spike protein expression in human epithelial cells inhibits ACE2 expression leading to the induction of AT1 signaling, and promotion of IL-6/soluble IL-6R release. IL-6 is one of the major mediators of the hyper-inflammatory response, and can exert signaling through two main pathways referred to as classical or trans- signaling to coordinate prominent pro-inflammatory properties. Our experimental observations suggest the potential for IL-6 trans-signaling during SARS-CoV-2 infection of epithelial cells. These observations will aid in the development of new therapeutic modalities for combating COVID-19 associated disease severity. Aggressive tissue damage via increased IL-6 secretion and other cytokines leading to a hyper-inflammatory response are in line with COVID-19 disease severity. We propose that SARS-CoV-2 infection in pulmonary epithelial cells induces IL-6 trans-signaling for secretion of chemokines; like MCP-1, from pulmonary vascular endothelial cells to attract monocytes/macrophages and create a hyper-inflammatory state leading to enhanced disease severity and acute respiratory distress syndrome. The novel human coronavirus causative agent of coronavirus disease-19 (COVID-19) initiates and promotes rapid disease spread and severity. Potential mechanisms for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease progression include a high rate of viral replication, resulting in enhanced host cell cytolysis, as well as, production of inflammatory cytokines and chemokines that perpetuate damage [Acute kidney injury, cardiac damage, and abdominal pain are the most commonly reported side effects of COVID-19 , 4. SARSRecent clinical studies have found that COVID-19 patients with severe illness had elevated levels of certain cytokines including IL-6, TNF-α, IL-10, MCP-1 and IP-10 . The patThe angiotensin converting enzyme (ACE) and its close homologue ACE2 belong to the dipeptidyl carboxypeptidase enzymatic family. ACE converts angiotensin I into the peptide angiotensin II, which binds to and activates the AT1 receptor leading to vasoconstriction. On the other hand, ACE2 cleaves angiotensin II and produces angiotensin 1–7 peptide leading to vasodilation via activation of its G protein coupled Mas receptor . ACE2 isIL-6 is a pleiotropic cytokine with biological functions that affect tissues beyond the immune system and the vasculature . BindingOur present study was designed to understand SARS-CoV-2 and host cell interactions, with special emphasis on the inflammatory cytokine system. Ectopic expression of viral spike protein was also analyzed in understanding potential mechanisms of SARS-CoV-2 interaction with cells. We observed that SARS-CoV-2 infection or spike protein expression in human epithelial cells inhibits ACE2 expression and promotes IL-6/soluble IL-6R release. We further defined the influence of IL-6 trans-signaling that may have upon neighboring endothelial cells.We used Huh7.5 and A549 cells by infection with SARS-CoV-2 or transient transfection with cloned viral spike gene. Microscopically we noted minimal indications of cell death (<10%) 48 h post-infection or transfection. Spike protein expression status was analyzed from cell lysates generated after 48 h . To idenWe examined the possibility that AT1 upregulation may play a role in MAPK and NF-kB activation in virus infected or spike gene transfected cells generated from two distinct human tissues (Huh7.5 and A549). We observed that SARS-CoV-2 infected Huh7.5 and A549 cells expressed an elevated level of phospho-p38 MAPK (Thr180/Tyr182) and phospho-p42/44 MAPK (Thr202/Tyr204) molecules . MAPK reTo verify the involvement of AT1 signaling in MAPK activation, we used the AT1 receptor antagonist Candesartan cilexetil in Huh7.5 and A549 cell lines expressing SARS-CoV-2 spike protein. Candesartan cilexetil binds to the angiotensin II receptor type 1 to block the binding of angiotensin II to its target receptor expressed in vascular and other cell types to regulate blood pressure and fluid retention . Our resin vitro observations in COVID-19 patient samples, we measured twenty SARS-CoV-2 virus infected patient sera for detection of IL-6 and soluble IL-6R levels. We found that both IL-6 and soluble IL-6R concentrations were increased in virus infected samples, as compared to eight uninfected healthy control sera  are activated. Functional activation of STAT3 occurs via phosphorylation of the Tyr705 residue, resulting in translocation of STAT3 to the nucleus . We obseWe have observed that epithelial cells expressing SARS-CoV-2 spike protein were unable to induce STAT3 mediated transcriptional activity, which is necessary to induce monocyte chemoattractant protein-1 (MCP-1) expression . To reviAlarmins are released from deleterious response in the host provoking an uncontrolled inflammatory activity. This group of proteins includes IL-1α and the high-mobility group box 1 protein (HMGB1) . We measin vitro observations are consistent with the recent report that the angiotensin II levels in plasma from SARS-CoV-2 virus infected patients are markedly elevated and associated with viral load and lung injury [We investigated the contribution of SARS-CoV-2 spike protein on the renin–angiotensin system. SARS-CoV-2 infection or ectopic spike protein expression in human epithelial cells inhibited ACE2 expression, leading to increased angiotensin II and AT1 receptor expression. An earlier study suggested that angiotensin II upregulates AT1 receptor expression . Our in g injury . SARS-Cog injury , 39. To Our study demonstrated that SARS-CoV-2 spike protein was associated with the up-regulation of AT1 signaling which led to the induction of transcriptional regulatory molecules, such as NF-κB, c-Fos, and MAPK activation. Cytokine storm during SARS-CoV-2 infection is a possible mechanism of morbidity and mortality, and IL-6 is one of the major inflammatory molecules involved in COVID-19 . Spike pIL-6 signaling is triggered when bound to the transmembrane IL-6 receptor. Alternatively, IL-6 may bind to a soluble form of the IL-6 receptor and activate STAT3 signaling in cells that express gp130, exclusive of transmembrane IL-6R. In general, classic IL-6 signaling is responsible for the anti-inflammatory properties of IL-6, whereas trans-signaling is responsible for the pro-inflammatory actions of IL-6 . While sCoded clinical serum samples from twenty adult patients positive for SARS-CoV-2 infection and eight serum samples from uninfected healthy volunteers were obtained from Saint Louis University Hospital COVID-19 repository . Written or verbal consents were obtained from the patients and volunteers.2. For virus infection, the cells were cultured in DMEM containing 2% FBS in 25 cm2 T-flask or 6-well plate at ~60% confluency. SARS-CoV-2 isolate  from Wuhan, China, was grown in Vero E6 cells. Early passaged virus was used for infection of other cell lines at a multiplicity of infection of 0.5 (moi = 0.5). All live virus related experiments were performed in our Institutional Biosafety Committee (IBC) approved P3 laboratory facility. Infected cells were lysed after 48 h of infection and lysates were used for further experiments.Transformed human lung epithelial cells (A549), liver epithelial cells (Huh7.5), and liver sinusoidal endothelial cells (TMNK-1)  were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone) containing 10% fetal bovine serum (FBS) (Sigma), 100 U of penicillin/ml, and 100 mg of streptomycin/ml (Sigma). The cells were maintained in a humidified atmosphere at 37°C with 5% COCells were sub-cultured in 6-well plate at ~60% confluency and transfected with plasmid DNA , pCMV3-SARS-CoV-2-Spike S1 region or pCMV3-SARS-CoV-2-Spike S2 region , and empty vector construct (500 ng/plate) using Lipofectamine 3000 (Life Technologies) following the manufacturer’s instruction. Cell lysates were prepared after 48 h of transfection for analyses. AP1-luciferase construct was obtained from commercial source (Addgene) and used in luciferase assay.AT1 receptor antagonist Candesartan cilexetil (Tocris) was used in this study. The cells were treated with the Candesartan cilexetil (dissolved in DMSO) at 1 μM concentration for 20 h following earlier reports , 53. ADACell lysates were electrophoresed to resolve proteins by SDS-PAGE, transferred onto a nitro-cellulose membrane, and blocked with 4% non-fat dry milk. The membrane was incubated at 4°C overnight with specific primary antibody, followed by a secondary antibody conjugated with horseradish peroxidase. The protein bands were detected by chemiluminescence (Life Technologies). The blot from the same run was reprobed with β-actin (Sigma) or α-tubulin (Santa Cruz Biotechnology) HRP conjugated antibody to compare protein load in each lane. Commercially available antibodies for ACE2, AT1 receptor, c-Fos, IκBα, ADAM-17, MCP-1 and IL-6Rα were procured from Santa Cruz Biotechnology; phospho-p38 MAPK (Thr180/Tyr182), phospho-p42/44 MAPK (Thr202/Tyr204), phospho-NF-κB (Ser276), SOCS3, phospho-STAT3 (Tyr705), phospho-STAT3 (Ser727), total STAT3 were procured from Cell Signaling Technologies; and SARS-CoV-2 spike protein  were also procured for western blot analyses.Patient serum samples and cell culture supernatants from SARS-CoV-2 spike protein transfected cells were analyzed for the presence of secreted IL-6 (Sigma), soluble IL-6R (Life Technologies) IL-1α (Sigma) and HMGB-1  using ELISA kits following the manufacturer’s instructions. We also analyzed MCP-1 level by ELISA in culture supernatants of LPS stimulated TMNK-1 cells and from SARS-CoV-2 spike expressing A549 cell culture medium in the presence or absence of Tocilizumab (Absolute Antibody).2 (Enzo Life Sciences). A change in fluorescence was measured after addition of substrate at 5 minutes time interval up to 40 minutes in a Synergy Mx Microplate Reader . The rate of enzyme activity was calculated using recombinant human ADAM-17 as a positive control. Total protein estimation was performed using BCA kit (Thermo Fisher) following supplier’s instruction.ADAM-17 enzymatic activity was analyzed by fluorometric cleavage of specific substrate . BrieflyChemotactic activity of MCP-1 was performed by cellular migration assay of THP-1 cells using QCM (5 μm) fluorometric 24-well chemotaxis cell migration assay kit (Sigma).p <0.05.Graph Pad Prism 7 was used to analyze the experimental data. All experiments were performed at least three times for reproducibility. The results are presented as mean ± standard deviation. Non-parametric Mann-Whitney U test and paired two-tailed t-test analyses were performed to compare the mean values between the two groups. Statistical significance was considered as


Achieving a functional cure is an important goal in the development of HIV therapy. Eliciting HIV-specific cellular immune responses has not been sufficient to achieve durable removal of HIV-infected cells due to the restriction on effective immune responses by mutation and establishment of latent reservoirs. Chimeric antigen receptor (CAR) T cells are an avenue to potentially develop more potent redirected cellular responses against infected T cells. We developed and tested a range of HIV- and SIV-specific chimeric antigen receptor (CAR) T cell reagents based on Env-binding proteins. In general, SHIV/SIV CAR T cells showed potent viral suppression in vitro, and adding additional CAR molecules in the same transduction resulted in more potent viral suppression than single CAR transduction. Importantly, the primary determinant of virus suppression potency by CAR was the accessibility to the Env epitope, and not the neutralization potency of the binding moiety. However, upon transduction of autologous T cells followed by infusion in vivo, none of these CAR T cells impacted either acquisition as a test of prevention, or viremia as a test of treatment. Our study illustrates limitations of the CAR T cells as possible antiviral therapeutics. HIV infection is a chronic viral disease, generally treated with combination antiretroviral therapy to suppress HIV replication. However, virus infected cells can persist as a latent reservoir hidden from the immune system –3. LatenCytotoxic T lymphocytes (CTL) have the potential to kill and eradicate virus-infected cells . Howeverin vitro-activated CTL clones skewed to terminal effector T cells. Those cells were trapped in the lung soon after infusion and did not traffic to lymph nodes where the virus was replicating. This study demonstrated the importance of T cell phenotype before infusion permitting the trafficking of transferred cells to intended sites  γ-irradiator. Feeder cells were supplied every 2 weeks and anti-SIV CAR T cells were maintained for one month. For the short-term expansion, ITS01-NGFR transduced cells were maintained in R-10 (IL-2: 200 IU/ml) for 4 days.For the long-term expansion, ITS01-ZsGreen-transduced cells were stimulated with human PBMC and human EBV-transformed B cell lines irradiated with 6000 RAD and 15000 RAD using MARK I [Viral suppression activity of anti-SIV CAR T cells was evaluated by co-culture with SIV-infected cells. CD8 T cells were depleted from rhesus PBMC using a CD8 MicroBead Kit, non-human primate (Miltenyi Biotec) and activated with ConcanavalinA (SIGMA Aldrich) for SIV infection. CD8-depleted rhesus PBMC were infected with tier 1, 2 or 3 SIV four days after activation. One day after infection, SIV-infected cells were washed three times and co-cultured with sorted anti-SIV CAR T cells. Intracellular p27 was stained or p27 concentration in the culture supernatant was measured three days after co-culture. Percentages of viral suppression were calculated based on changes in percentages of p27 positive cells or p27 concentration compared with the CAR T cell negative samples.ZsGreen positive and negative CD4 and CD8 T cells were sorted on a FACS Aria after CAR transduction to rhesus PBMC and used as effector cells. On the day of co-culture, ZsGreen negative effector cells were stained with CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher SCIENTIFIC) to be distinguished from target cells on a flow cytometer. Target cells infected with SIVsmE660-FL14-AK were co-cultured with CD4 or CD8 T cells or a combination of CD4 and CD8 T cells at E:T ratios of 1:1 or 1:1:1. Intracellular p27 was measured 4 days post-infection.ZsGreen expressing anti-SIV CAR transduced cells were stained with anti-CD3-Cy7-APC, CD4-BV421, CD8-QDot655 and SIVmac239 gp140 Env trimer conjugated to PE. Cells were acquired on BD LSR II and the data were analyzed in FlowJo .in vivo proliferation of anti-SIV CAR T cells. Rhesus PBMC were isolated. Cells were washed twice with PBS and stained with LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Thermo Fisher SCIENTIFIC) for 20 min at room temperature followed by staining with the following antibodies: CD3-Cy7-APC , CD4-BV605 , CD8-BV711 , CD28-Cy5PE , CD45RA-Cy7-PE , CD69-ECD , CD95-APC , CCR7-Alexa Flour 700 , HLA-DR-BV785 , CD271 . After surface staining, cells were permeabilized with Foxp3 / Transcription Factor Staining Buffer Set (eBiosciences) for 20 min at room temperature, then intracellular stained with anti-Ki67-PE . Cells were acquired on BD LSR II and BD FACSymphony.Surface markers and intracellular Ki67 were stained for phenotyping and measuring For CAR+IL-15 combination therapy, we developed another flow panel for phenotyping. Dead cells were stained with LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit, for UV excitation (Thermo Fisher). For surface staining, we used CD297-PerCP-eFlour710 , CD45RA-Cy7PE , CD28-Cy5-PE , CXCR5-PE-eFlour610 , CD3-Cy7APC , MBL2-Alexa flour680 , anti-ITS01-APC, HLA-DR-BV785 , CD8-BV711 , CD95-BV650 , CCR7-BV605 , NGFR-VioBlue , CD4-BUV661 , CD69-BUV737 . Ki67-BV480  was used for intra cellular staining after fixation/permeabilization. Cells were acquired on BD FACSymphony. All flow data were analyzed on Flowjo (BD Biosciences).S1 FigSCM and TCM.SIV and SIV-CAR constructs. SHIV-CAR and CD4-MBL CAR had CD28 and CD3 zeta for signal transduction. To prevent virus infection to CAR-T cells, antigen binding moieties consist of domain 1 and 2 from CD4 molecule. For comparison of length of linker for target recognition, we used SHIV-ACR with 35AA and 10 AA linkers for CD4 and 17b CAR. SIVmAb CAR is designed to use 4-1BB and CD3 zeta signaling domain expecting better in vivo persistence resulting from maintenance of T(PDF)Click here for additional data file.S2 FigDesign of in vivo CAR studies in NHP. (A) SHIV-CAR study outline. Animals were treated with/without ART for 4 weeks and CAR T cells were transferred 2 weeks post ART initiation. (B) Comparison of long-term and short-term expanded CAR T cells. PBMC isolated from maximum blood draw were stimulated with anti-CD3 and anti-CD28 antibodies for 3 days and transduced with ITS01-CAR expressing ZsGreen. ITS01-CAR T cells were expanded with irradiated human PBMC and BLCL. Short-term expanded CAR T cells were prepared from PBMC stimulated with IL-2. IL-2 stimulated PBMC were transduced with ITS01-CAR expressing NGFR and cultured for 4 days. ITS01-ZsGreen and ITS01-NGFR CAR T cells were mixed and transferred to the same animals simultaneously and compared in vivo persistence and memory phenotypes. (C) CAR-T cell therapy using dual CAR T cells. PBMC isolated from SIVsmE660-FL14 infected animals were transduced with ITS01-ZsGreen and ITS06.02-NGFR in the presence of IL-2. Dual CAR transduced PBMC were transferred to the original animals. (D) SIV prevention study using Dual CAR T cells. ITS01 and ITS06.02 CAR transduced PBMC were transferred at 1 day before SIV challenge.(PDF)Click here for additional data file.S3 FigTransduced T cells were expanded for 3 weeks (A) or minimally cultured (B). Flow cytometric assessment of the cells is shown: Progressive gating on live, CD3+, transduced (GFP+), CD4 or CD8 (top rows). The bottom rows show the differentiation stages as defined by expression of CCR7 and CD45RA, and activation status as defined by expression of CD69 and HLA-DR.(PDF)Click here for additional data file.S4 FigIn vitro viral suppression activity on SIVmac239 (A) and SIVsmE660-FL14-AK (B) by 7 different CAR T cells are evaluated. 139 CAR is control. CD4-MBL-ZsGreen, ITS01 and ITS06 CARs are transduced to triple CAR T cells. CAR T cells and SIV-infected target cells were co-cultured at E:T ratios of 1:1, 1:5, 1:25 and 1:125. Culture supernatants are collected on day 3, 5, 7, 10 and 13 post co-culture. p27 concentrations in the culture supernatants were measured by ELISA. Error bars represent the average and SD of triplicates.(PDF)Click here for additional data file.S5 FigSCM: Blue, TCM: Green, TEM: Orange, TTEM: Red. Memory phenotypes are based on CCR and CD45RA expression. Each bar represents data from an animal.Percent of each memory fraction in CAR T cells at the time of transfer is demonstrated. T(PDF)Click here for additional data file.S6 FigSCM, yellow: TCM, green: TEM, blue: TTEM. (B) Plasma IL-18 measurement during hetIL-15 administration. Plasma IL-18 levels were determined at the indicated time points. Individual animals are shown.(A) Percent of each memory phenotype is shown in the graphs. Red: T(PDF)Click here for additional data file.


Bromodomain-containing protein 4 (BRD4) is a potential therapeutic target of skin squamous cell carcinoma (SCC). I-BET726 is a novel BRD4 inhibitor. Its potential effect in skin SCC cells was tested in the present study. We show that I-BET726 potently inhibited survival, proliferation, cell cycle progression, and migration in established (A431/SCC-9/SCC-12/SCC-13 lines) and primary human skin SCC cells. I-BET726 induced significant apoptosis activation in skin SCC cells. It was more efficient in inhibiting skin SCC cells than known BRD4 inhibitors . I-BET726 not only downregulated BRD4-regulated proteins , but also inhibited sphingosine kinase 1 (SphK1) and Akt signalings in SCC cells. Restoring Akt activation, by a constitutively active S473D mutant Akt1 (“caAkt1”), partially inhibited I-BET726-induced cytotoxicity in A431 cells. In vivo, I-BET726 oral administration potently inhibited A431 xenograft growth in severe combined immunodeficient mice. Downregulation of BRD4-regulated proteins and inhibition of the SphK1-Akt signaling were detected in I-BET726-treated A431 xenograft tumor tissues. Together, I-BET726 inhibits skin SCC cell growth in vitro and in vivo. The prognosis for the advanced, metastatic, and recurrent skin SCC is poor3. Molecularly targeted therapies are desperately needed for better skin SCC therapy3.Skin squamous cell carcinoma (SCC) causes significant cancer-related human mortalities5. Bromodomain-containing protein 4 (BRD4) is the most studied and a primary member of BET family. BRD4 overexpression and/or hyperactivation is associated with tumorigenesis of hematological and solid tumors5. BRD4 regulates transcription elongation of key genes of cell proliferation, cell cycle progression, and apoptosis, including c-Myc, Bcl-2, and cyclin D15. BRD4 inhibitors have been tested in a number of preclinical cancer studies, showing promising anticancer outcomes. Our previous study has shown that overexpression of BRD4 in human skin SCC cells can promote cell growth in vitro and in vivo6. BRD4 shRNA or knockout (by CRISPR/Cas9 method) potently inhibited skin SCC cell proliferation. Reversely, forced overexpression of BRD4 facilitated skin SCC cell proliferation6. These results suggest that targeting BRD4 could be a novel and efficient strategy against skin SCC cells.Bromodomain and extraterminal (BET) family proteins have emerged as exciting and novel therapeutic proteins for cancer7. I-BET726 competes with tetra-acetylated histones for binding to the bromodomain of BRD47. I-BET726 exhibited over 1000-fold selectivity of BRD4 than the bromodomain-containing homologs7. The compound displayed favorable physicochemical and pharmacokinetic properties along with acceptable safety profiles, suitable for potential clinical development7. Its potential activity in human skin SCC cells has not been studied.I-BET726 (GSK1324726A) is a novel and potent inhibitor of BET family proteins, showing high affinity binding to BRD4I-BET726 was purchased from Adooq . MTT, AZD5153, PD98059 and LY294002, JQ1 and CPI203 were purchased from Sigma . Z-DEVD-fmk, Z-LEHD-fmk, and Z-VAD-fmk were provided by Calbiochem . Antibodies for c-Myc (#9402), Cyclin D1 (#2922), BRD4 (#13440), Bcl-2 (#15707), SphK1 (#12071), phosphorylated (“p”)-Akt (Ser-473) (#9271), Akt1/2 (#9272), p-p44/42 MAPK (Erk1/2) (#9101) and Erk1/2 (#9102), cleaved-caspase-3 (#9664), cleaved-caspase-8 (#9496), cleaved-caspase-9 (#20750), cleaved-poly (ADP-ribose) polymerase (PARP) (#5625), β-tubulin (#15115) and β-actin (#3700) were purchased from Cell Signaling Tech .6. Cells were subjected to mycoplasma and microbial contamination examination every 3–4 months. Authentication by short tandem repeat profiling, population doubling time, and morphology were routinely confirmed as well to verify the genotype. The primary human skin SCC cells were provided by Dr. Wang8. Cells were derived from two written-informed consent SCC patient  and cultured as described previously6. Cultures of primary human skin keratinocytes and fibroblasts were reported previously9. The protocols of using primary human cells were conducted to accordance with the Declaration of Helsinki, with approval by the Ethics Board of Wenzhou Medical University.The established skin SCC cell lines, A431, SCC-9, SCC-12, and SCC-13, were purchased from the Cell Bank of Chinese Academy of Science . The cell culture procedure was described early3 cells per well). After I-BET726 treatment, cell viability was tested by MTT dye assay. MTT optical densities were measured at 550 nm using a microplate reader.Cells were seeded onto 96-well plates (5 × 104 cells per treatment)10. I-BET726 medium was renewed every two days. After 10 days, the number of A431 colonies was counted.Following I-BET726 treatment, A431 cells were placed onto 10-cm dishes (2×104 cells per well). Following the treatments, cells were stained with Hoechst-33342 (Sigma). Nuclei with intensified Hoechst-33342 condensation or fragmentation were labeled as nuclei of apoptotic cells. The nuclei of non-apoptotic cells showed the faint delicate Hoechst-33342 staining. At least 300 cells from five random-selected views (1:100 under microscope) were included to calculate the apoptotic nuclei ratio.Cells were plated onto 24-well plates (2 × 103 cells per well), treated with I-BET726 in the presence of BrdU . BrdU incorporation was examined by an ELISA kit . BrdU enzyme-linked immunosorbent assay (ELISA) OD values at 405 nm were recorded.Cells were seeded onto 96-well plates . After the applied treatment, EdU Apollo-567 assay kit 13. FBS-containing complete medium was added to the lower chamber surface. After incubation for 48 h the migrated cells on the lower surface were stained and counted. Five repeated views of each condition were included to calculate the average number of migrated cells.Skin SCC cells with the applied treatments were initially seeded on the “Transwell” upper chamber , at a density of 10,000 cells in 300 μL serum-free medium (each chamber)Cells with I-BET726 treatment were fixed and stained with propidium iodide  and RNase . A flow cytometer  was employed to analyze DNA content. Cell cycle distribution percentages  were recorded.The cytosolic protein extracts (30 μg per treatment) were incubated with the 7-amino-4-trifluoromethylcoumarin (AFC)-conjugated caspase (−3/−8/−9) substrates (10 μg/mL each) in the caspase assay buffer. AFC intensities were tested by Infinite 200 PRO reader at 400 nm excitation and 505 nm emission.+/+ cells were labeled as apoptotic cells.In brief, following the treatments, cells were stained with Annexin V-FITC (10 μg/mL) and PI (10 μg/mL) (Sigma). Afterwards, cells were detected via fluorescence-activated cell sorting on a FACSCalibur machine (BD Biosciences). Annexin VIn brief, following the indicated treatment, medium LDH was collected and tested by a two-step LDH detection kit , and normalized to total LDH contents.9, the quantified protein lysates (30 μg proteins per treatment) were separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis, transferred to the PVDF blots (Biyuntian). After blocking, the blots were immunoblotted with applied primary antibodies, followed by incubation with HRP-conjugated secondary antibodies. The enhanced chemiluminescence reagents (Pierce) were added to visualize the targeted protein bands. Total gray of each band was quantified by the ImageJ software14.As described16. SphK1 activities were valued as pmol/hour/g protein, normalized to the control.SphK1 activity was detected by a previously described protocol17, the cellular ceramide content was analyzed, and its value was expressed as fmol by nmol of phospholipid. Ceramide content in I-BET726-treated cells was always normalized to that of untreated control cells.Using a previously described protocol18. Cells were initially seeded into six-well plates . The adenovirus was added to cultured cells for 48 h. The stable cell lines were established via selection for 4–5 passages. For virus infection, cells were cultured in polybrene-containing medium.The purified constitutively active S473D mutant Akt1 (“caAkt1”) recombinant adenovirus and empty vectors recombinant adenovirus (Ad-GFP) were both provided by Dr. Cao at Soochow UniversityThe CRISPR/Cas9 BRD4-KO plasmid  was transfected to cultured A431 cells via the Lipofectamine 2000 protocol . The transfected cells were further selected with puromycin after 4–5 passages, and two stable cell lines were established (“BRD4-KO-sL1/2”). Control cells were treated with an empty vector with Cas9 control construct . BRD4 expression in stable cells was verified by western blotting.9 A431 cells  were inoculated via subcutaneous (s.c.) injection to the right flanks of the severe combined immunodeficient (SCID) mice . Within 18–20 days the volume of each tumor was close to 100 mm3, and the A431 tumor xenografts were established. SCID mice were randomly assigned into three groups (10 mice per group). Every 5 days tumor volumes and mice body weights were recorded. All animal studies were performed according to the standards of ethical treatment and IACUC of Wenzhou Medical University. The protocols were approved by the Ethics Committee (2016-R311) of Wenzhou Medical University.As describedt test was applied (Excel 2007). p values < 0.05 were considered statistically different. All the protocols of this study were approved by Ethics Committee of Wenzhou Medical University.The investigators were blinded to the group allocation during all experiments. In vitro experiments in this study were repeated at least three times. Data were presented as mean ± standard deviation (SD). Statistics were analyzed by one-way analysis of variance by SPSS software . To compare difference between two specific groups, a two tailed m). MTT assay results, in Fig. m  potently decreased EdU ratio in A431 cells, further confirming proliferation inhibition. In addition, when analyzing cell cycle progression, we show that I-BET726 (50 nm) disrupted cell cycle progression, causing G1–S arrest in A431 cells  significantly inhibited A431 cell migration in vitro  robustly decreased cell viability . As shown, I-BET726 significantly inhibited viability , were treated I-BET726  was significantly more potent in inhibiting cell viability , CPI203 (500 nm), and AZD5153 (100 nm). These results show that I-BET726 potently inhibits survival, proliferation and migration of established and primary human skin SCC cells.We compared the activity of I-BET726 with other known BRD4 inhibitors, including JQ1ity Fig.  and prolity Fig.  than eve26. The potential effect of I-BET726 on skin SCC cell apoptosis was studied next. As shown, I-BET726 dose-dependently increased the activities of caspase-3 and caspase-9 in A431 cells -treated A431 cells , it was ineffective on cell apoptosis -treated A431 cells -induced apoptosis activation was more potent than other known BRD4 inhibitors: JQ1, CPI203, and AZD5153 -induced A431 cell viability reduction. Therefore, apoptosis activation should be the primary reason of I-BET726-induced cytotoxicity in A431 cells. In SCC-9, SCC-12, and SCC-13 cells I-BET726 treatment  increased TUNEL staining ratio as well -positive nuclei ratio was significantly increased in I-BET726  significantly increased the Annexin V-positive cells , I-BET726  are key oncogenes5. In A431 cells, I-BET726 dose-dependently inhibited c-Myc, Bcl-2, and cyclin D1 protein expression  can still induce significant cytotoxicity , required for transcription elongationion Fig. . I-BET72ion Fig. . Caspaseion Fig. . Importa29. SphK1 overexpression/hyperactivation promotes skin SCC cell proliferation and metastasis29. On the contrary, SphK1 inhibition or silencing shall lead to ceramide production and cell apoptosis29. SphK1 activity was therefore analyzed. We show that I-BET726 dose-dependently decreased SphK1 activity in A431 cells  in the primary skin SCC cells (“C1/C2”) as well  also induced ceramide accumulation  to de-phosphorylate and in-activate Aktlls Fig. . Erk1/2 lls Fig. .18) was transduced to A431 cells, which completely restored Akt activation in I-BET726-treated cells  within 18–21 days, when I-BET726 administration was started (“Day-0”). By recording tumor volumes, we show that oral administration of I-BET726  potently inhibited A431 xenograft growth in SCID mice  were significantly downregulated in I-BET726-treated A431 tumor tissues Fig. . As comp34. In addition, BRD4 phosphorylates P-TEFb and RNA polymerase II to promote transcription elongation34. BRD4 is essential for the transcription of several key oncogenes, including c-Myc, Bcl-2 and cyclin D135. Moreover, BRD4 is important for the activation of oncogenic nuclear factor-kappa B signaling in cancer cells4. Our previous study has shown that BRD4 is overexpressed in skin SCC cells, functioning as a potential key pro-cancerous molecule6. Targeting BRD4, i.e., by AZD5153, can potently inhibit skin SCC cell growth, in vitro and in vivo6.BRD4 binds to the chromatin to re-activate silenced genes in the process of mitosis7, inhibited survival, proliferation, cell cycle progression, and migration in multiple established skin SCC cell lines (A431/SCC-9/SCC-12/SCC-13) and primary human skin SCC cells. I-BET726 provoked apoptosis in skin SCC cells. It was highly potent in killing skin SCC cells, more efficient than the other known BRD4 inhibitors . Significantly, it was non-cytotoxic to normal skin keratinocytes and fibroblasts, where BRD4 levels are extremely low6. In vivo, I-BET726 oral administration inhibited A431 xenograft growth in SCID mice. Downregulation of BRD4-dependent oncogenic proteins  was detected in I-BET726-treated skin SCC cells and A431 xenografts. These results suggest that I-BET726 potently inhibited skin SCC cell progression in vitro and in vivo.In the present study, we show that I-BET726, a novel BRD4 inhibitor37. Previous studies have demonstrated that SphK1 is overexpressed in skin SCC, represents as a novel prognostic marker and potential therapeutic target29.The outcomes for the current treatments of advanced skin SCC have been disappointing. The better skin SCC therapies should include rational inhibition of key molecular targets in multiple pro-survival/growth signalings. The facts that I-BET726 is more efficient than other known BRD4 inhibitors and it could still induce cytotoxicity in BRD4-KO A431 cells suggest the existence of BRD4-independent mechanisms by this compound. SphK1 promotes cancer cell viability, proliferation, and apoptosis resistance, as well as metastasis, and angiogenesisThe novel findings of the study are that in skin SCC cells I-BET726 can significantly inhibit SphK1 activation, followed by pro-apoptotic ceramide accumulation and Akt inactivation. These actions by I-BET726 were independent of BRD4 inhibition. As other BRD4 inhibitors  failed to change SphK1 activity nor Akt signaling. Concurrent inhibition of BRD4, SphK1, and Akt signalings by I-BET726 could probably explain its superior anti-skin SCC activity, better than the known BRD4 inhibitors. SphK1-Akt inhibition was also detected in I-BET726-treated A431 xenograft tissues. Thus, I-BET726 concurrently inhibits BRD4, SphK1, and Akt signalings in skin SCC cells (see the proposed signaling pathway of this study, Fig. 39. These trials highlighted the importance of concurrent targeting multiple oncogenic signalings to efficiently inhibit cancer cell growth and induce apoptosis39. In the current study we show that I-BET726 concurrently inhibited BRD4, SphK1, and Akt signalings in established and primary skin SCC cells. This might be one reason of its superior anti-SCC cell activity. Importantly, we show that Erk inhibition or PI3K-Akt blockage potentiated I-BET726-induced cytotoxicity in SCC cells, suggesting that Erk and PI3K-Akt activation could be key resistance mechanisms of I-BET726 in SCC cells. Although additional studies will be needed to further support this notion.The effects of inhibiting Akt-mTOR in combination with other cascades pathways are being tested in a number of phase I–II clinical trialsI-BET726 potently inhibits BRD4, SphK1-Akt signalings, and human skin SCC cell growth, in vitro and in vivo.Supplementary Figure 1Supplementary Figure 2Supplementary Figure Legends


Receptor interacting serine/threonine kinase 1 (RIPK1) is the central mediator of tumor necrosis factor (TNF) signaling. It regulates both pro-survival/pro-inflammatory and cell death pathways. In order to fulfill this complex regulation, RIPK1 is regulated by several post-translational modifications, including ubiquitination, acetylation, and phosphorylation. In our recent work, we show that the unc-51-like autophagy activating kinase 1 (ULK1) phosphorylates RIPK1 at Ser357 and thus blocks TNF-induced cell death. Receptor interacting serine/threonine kinase 1 (RIPK1) is the central mediator of the signaling cascades downstream of the tumor necrosis factor (TNF) receptor superfamily member 1A . RIPK1 is composed of an N-terminal kinase domain, an intermediate domain containing a RIP homotypic interaction motif (RHIM), and a C-terminal death domain (DD). Upon TNF stimulation, a TNFR1-associated “complex I” is assembled that consists of RIPK1, TNFRSF1A associated via death domain (TRADD), TNF receptor-associated factor 2/5 (TRAF2/5), baculoviral IAP repeat-containing 2/3 , and the linear ubiquitin chain assembly complex (LUBAC). cIAP- and LUBAC-generated ubiquitin chains then establish the platform for the recruitment of further protein complexes such as TAB2-TAB3-MAP3K7/TAK1 and IKBKG/NEMO-CHUK/IKKα-IKBKB/IKKβ, which mediate activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) signaling pathways. Upon dissociation of RIPK1 from complex I (either caused by deubiquitination or by inhibited ubiquitination), different cytosolic RIPK1-dependent complexes are assembled that mediate apoptotic  or necroptotic cell death (necrosome). Generally, kinase-active RIPK1 supports apoptosis and necroptosis, whereas kinase-inactive or scaffolding functions of RIPK1 inhibit these events. If deubiquitinated RIPK1 and inactivated caspase 8 are present, necroptosis is induced. Kinase-active RIPK1 and receptor interacting serine/threonine kinase 3 (RIPK3) are required for the assembly of the necrosome, an amyloid-like signaling complex. The RIPK3 oligomerization then leads to RIPK3 autophosphorylation and transphosphorylation of the mixed lineage kinase domain like pseudokinase (MLKL). Ultimately, phosphorylated MLKL translocates to the plasma membrane and induces pore formation.1 Of these sites, Ser14/15, Ser20, Ser161, and Ser166 were phosphorylated only in samples subjected to the kinase reactions  and inhibitor of nuclear factor kappa B kinase subunit beta  directly phosphorylate RIPK1 within complex I.2 The identified sites include Ser25 and Ser166 in the kinase domain, and Ser296, Ser331, and Ser416 in the intermediate domain , which acts downstream of the TAB2-TAB3-MAP3K7/TAK1 complex.7 In the case of MK2, the major phospho-acceptor sites are Ser320 and Ser335, respectively  and the cell death-regulating kinase RIPK1.d Ser389 .10 Of th