SARS-coronavirus 2 (SARS-CoV-2), pathogen of coronavirus disease 2019 (COVID-19), is constantly evolving to adapt to the host and evade antiviral immunity. The newly emerging variants N501Y.V1 (B.1.1.7) and N501Y.V2 (B.1.351), first reported in the United Kingdom and South Africa respectively, raised concerns due to the unusually rapid global spread. The mutations in spike (S) protein may contribute to the rapid spread of these variants. Here, with a vesicular stomatitis virus (VSV)-based pseudotype system, we demonstrated that the pseudovirus bearing N501Y.V2 S protein has higher infection efficiency than pseudovirus with wildtype (WT) and D614G S protein. Moreover, pseudovirus with N501Y.V1 or N501Y.V2 S protein has better thermal stability than WT and D614G, suggesting these mutations of variants may increase the stability of SARS-CoV-2 S protein and virion. However, the pseudovirus bearing N501Y.V1 or N501Y.V2 S protein has similar sensitivity to inhibitors of protease and endocytosis with WT and D614G. These findings could be of value in preventing the spread of virus and developing drugs for emerging SARS-CoV-2 variants. SARS-coronavirus 2 (SARS-CoV-2), pathogen of COVID-19, can cause infection of multiple organs throughout the body, mainly respiratory, digestive, circulatory, genitourinary and nervous systems. Although most symptoms of COVID-19 patients are mild, some may have severe pneumonia, even multiple organs failure, particularly in older people and those pre-existing chronic diseases (The coronavirus disease 2019 (COVID-19) pandemic has become a great threat to the health of human beings. As of May 23, 2021, more than 160 million confirmed cases and approximately 3.4 million deaths have been reported worldwide (diseases .SARS-CoV-2 uses spike (S) protein to bind host receptor angiotensin-converting enzyme 2 (ACE2), followed by membrane fusion and the release of viral RNA genome into the cytoplasm . As a tyRNA viruses have a high mutation rate in the process of host transmission. Some mutations may alter viral pathogenicity, virulence, and/or transmissibility . The mutThe SARS-CoV-2 N501Y.V1 lineage has 17 non-synonymous mutations and deletions, and many of them locate in the S protein. N501Y.V2 lineage includes ten mutations in the S protein, and three of them are located in the receptor binding domain (RBD) . As a common mutation of N501Y.V1 and N501Y.V2, N501Y mutation was first reported in a mouse model and hereafter identified in populations of many countries and regions . In the SARS-CoV-2 N501Y.V1 and N501Y.V2 lineages rapidly replaced the original strain, suggesting the mutations of these variants may be related to increased transmissibility. Preliminary studies have shown that N501Y.V1 and N501Y.V2 lineages have high transmission efficiency and immune escape from neutralizing antibodies than the original strain . HoweverExpression vector pcDNA3.1-2S (GenBank: MT_613044) with codon-optimized S gene of SARS-CoV-2 was obtained from Genscript Biotechnology Company. In order to effectively integrate S protein into pseudovirion, the last 19 amino acids of S protein were replaced with HA tag according to previously reported with modifications . The S g2 at 37°C.Human cell lines , Africa monkey kidney Vero cell, and mouse cell lines (MC38 and LLC-JSP) were obtained from American Type Culture Collection. Cells stably expressing human ACE2 and/or TMPRSS2  were constructed with lentiviral mediated gene transduction. H460 cell line was cultured in RPMI (HyClone), and the others were cultured in DMEM (HyClone). All above cells were maintained in the medium containing 10% fetal bovine serum  and 100 U/mL of Penicillin-Streptomycin solution (Gibco). The cells were cultured in an incubator with atmosphere of 5% CO6 293T cells were seeded into 10 cm cell culture dish. The next day, when the cells reached 70–90% confluent, 293T cells were transfected to express the S gene with lipofectamine 3000 (Invitrogen). 12 h post transfection, the cells were infected with G*△G-VSV dual reporter virus, a GP-deleted replication defective recombinant VSV virus containing firefly luciferase and eGFP reporter genes . 6 h post infection, the cell supernatant was discarded, and the cells were gently washed twice with PBS. Next, 10 mL fresh DMEM medium was added into the cell culture dish. 24 h post infection, the supernatant containing pseudovirions was harvested, then centrifuged at 3500 g for 5 min to remove the cell debris, and stored at -80°C in 1.5 mL aliquots until use.Pseudovirions bearing SARS-CoV-2 S protein were generated according to published protocols . BrieflyPseudovirions were quantified by RT-qPCR. Virus RNA was extracted from 200 μL of supernatant containing pseudovirions with Viral RNA/DNA Mini kit, and reversed to cDNA by PrimeScript™ RT reagent Kit (Takara). Pseudovirions quantification was performed using FastStart Essential DNA Green Master (Roche) following the manufacturer’s protocols. The copy numbers of pseudovirions were calculated by the VSV-P protein gene. Primers are listed in 4/100 μL cells were seeded into 96-well plates. The next day, cells were infected with 100 μL of media containing pseudovirions. 16 or 24 h post infection, cells were lysed with 60 μL diluted passive lysis buffer (Promega) at room temperature for 10 min. The infection efficiency of the pseudovirus was detected by measuring the activity of luciferase using Luciferase Assay System (Promega). For analysis of pseudovirus entry into 293T-hACE2 cells at indicated time points, images were captured in each well with a Nikon inverted microscope ECLIPSE Ts2 epifluorescence microscope. Each group contained 4 replicates, and each experiment was repeated at least two times.Before the infection assay, the pseudovirions were normalized to the same amount by quantitative RT-qPCR. The day before virus infection, 2x10Pseudovirions incorporated with S protein of SARS, SARS-CoV-2 WT, D614G, N501Y.V1 and N501Y.V2 lineages were diluted with DMEM and incubated in a 37°C or 42°C water bath. At indicated time points, 100 μL DMEM containing the pseudovirions was taken out and added into the wells with 293T-hACE2. 24 h post infection, the infection efficiency of pseudovirus was detected by measuring the activity of luciferase. The relative infection efficiency of pseudovirus was calculated by dividing the relative luminescence unit (RLU) values at each time point by the mean viral RLU at 0 h.In order to verify the expression of S protein in cells, the SARS-CoV-2 S expression vectors were transfected into 293T cells with lipofectamine 3000 (Invitrogen). 40 h post transfection, cells were lysed by RIPA Lysis Buffer (Beyotime) for 30 min on ice. In order to analyze incorporation of S protein into pseudotyped particles, VSV pseudovirions was concentrated with PEG8000. Briefly, medium containing pseudovirions was loaded by 6% PEG8000 and 0.1M NaCl, and shaked on the ice overnight. Next, the mixture was centrifugated  for 120 min at 4°C. After that, the supernatant was discarded, and the residual volume was mixed with 50 uL RIPA.All the samples were heated at 96°C for 10 minutes, and then the expression of S protein was detected by SDS-PAGE. After protein transfer, the nitrocellulose membrane was blocked by 5% milk for 1 h. Then the membranes were blotted with primary antibodies, followed by incubation with a secondary antibody and visualized by Bio-Rad ChgmiDoc MP Bio-Rad Laboratorigs. The primary antibodies used for western blot analysis were as follows: mouse anti-SARS-CoV/SARS-CoV-2 (COVID-19) spike [1A9] , mouse anti-β-actin monoclonal antibody , mouse anti-VSV matrix protein . We used a horseradish peroxidase-linked anti-mouse IgG antibody  as secondary antibody.For experiments involving endocytosis and protease inhibitors, 293T-hACE2 or 293T-hACE2-TMPRSS2 cells were pretreated with either endocytosis inhibitors  or protease inhibitors  for 2 h. Then the pseudovirions was added into the corresponding wells of plates. 24 h post infection, cells were lysed, and the infection efficiency of viruses was detected by measuring the luciferase activity using Luciferase Assay System.The GraphPad Prism 7 software package was used for all statistical analyses. The unpaired two-tails Student’s t-test was used to determine the statistical significance. The values were presented as mean ± SEM and p < 0.05 was defined as statistically significant .The purpose of this study was to gain insights into the differences of N501Y.V1 and N501Y.V2 lineages with previous WT and D614G. For this, we first constructed S genes expression vectors of N501Y.V1 variant  and N501Y.V2 RBD mutations   is at the similar level, except for a slightly weakened band in N501Y.V1  and 42°C (possible body temperature in fever). The infectivity of the five pseudoviruses decreased faster at 42°C than 37°C  and 293T-hACE2-TMPRSS2. Cathepsins inhibitor E64d treatment absolutely inhibited the infection of WT, D614G, N501Y.V1 and N501Y.V2 RBD pseudoviruses into 293T-hACE2 cells  and the serine protease TMPRSS2 were required for coronavirus S protein priming in target cells . We nextPrevious studies have shown that SARS-CoV-2 enters target cells mainly through endocytosis . Next, wAs RNA virus, SARS-CoV-2 has a high mutation rate during transmission in the population. Moreover, some mutations are adaptive because they alter pathogenesis, virulence, and/or transmissibility . Particuvia promoting the binding affinity with ACE2 ; The Chinese Academy of Medical Sciences Initiative for Innovative Medicine ; National grand Foreign Experts projects (G20190001633 and G20190001639).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


These results uncovered the crucial transcriptional mechanism that mediates the reciprocal controls of Notch and myogenesis.Muscle precursor cells known as myoblasts are essential for muscle development and regeneration. Notch signaling is an ancient intercellular communication mechanism that plays prominent roles in controlling the myogenic program of myoblasts. Currently whether and how the myogenic cues feedback to refine Notch activities in these cells are largely unknown. Here, by mouse and human gene gain/loss-of-function studies, we report that MyoD directly turns on the expression of Notch-ligand gene Dll1 which activates Notch pathway to prevent precautious differentiation in neighboring myoblasts, while autonomously inhibits Notch to facilitate a myogenic program in Dll1 expressing cells. Mechanistically, we studied  The formation of skeletal muscle tissue during development and regeneration is orchestrated by controlling the expression levels of muscle-specific transcriptional factors including MyoD gene. Previous studies have identified the key function of Notch signaling, an evolutionarily conserved cell-cell communication pathway, in blocking the expression of MyoD and the generation of functional muscle cells. Therefore, at the beginning of myogenesis, the activity of Notch pathway needs to be downregulated in order to promote MyoD expression thus the induction for expression of muscle structural genes. Here, we identified a key regulatory mechanism by which MyoD directly induces the transcription of Dll1 gene which encodes a classical Notch ligand. Interestingly, Dll1 can dampen Notch cell-autonomously to promote muscle cell differentiation while activate Notch in neighboring cells to block their myogenic differentiation. The discovery of the MyoD–Dll1–Notch gene control axis fills the knowledge gap for our understanding of the molecular regulation of myogenesis. Skeletal muscle accounts for around 40% of adult human body weight. Myogenesis involves a series of events that begins with the specification of muscle lineage by master transcriptional regulators, including Pax7 and Muscular Regulatory Factor (MRF), followed by the expression of a vast number of genes that establish muscle structure and function –6. In adNotch signaling is an ancient intercellular communication mechanism that determines the cell-fate for various tissue types in metazoan –15. AlteIn skeletal muscle, Notch is popularly known as a potent inhibitor of myogenic differentiation –31. Genecis-regulatory element whereby MyoD controls Dll1 expression. Employing heterologous cell-mixing experiment, we report the cis-inhibitory and trans-activation roles of Dll1 on Notch transduction whereby divergent myogenic programs can be established among the subpopulations of myoblast. These results provide an insight to the evolutionarily conserved control mechanism of myogenesis for mouse and human.Currently, it remains largely unknown whether and how the core myogenic program feedback to restrain Notch activity; and how does this reciprocal regulation determine the differentiation dynamics of myoblast. Here, by gene gain/loss-of-function studies in human and mouse cells, we unraveled the crucial role of MyoD in controlling Dll1 expression and Notch activity. Strikingly, deletion of MyoD in mouse or human myoblast abolished Dll1 expression, whereas forced expression of MyoD robustly induced Dll1 transcription in fibroblast. Utilizing a novel line of mutant mouse, we also probed the Notch ligands play either redundant or divergent functions depending on the biological context , 47. To DLL1 was the highest induced and peaked at 36 hours post human myoblast differentiation  or abundantly (Cdh15) expressed by quiescent muscle stem cells . Possibly due to nonsense-mediated decay of resultant messenger RNA, expression of DLL1 in DLL1KO myoblasts was reduced by 87%, compared to control clones transduced with only Cas9  in mouse MyoGKO cells (Heyl were more pronounced (83% lower)  . Validat% lower) , top row% lower)  were resMyoDKO myoblasts, myosin+ cells were spotted from the bulk CRISPR-treated MyoGKO myoblasts , myosin+ cells were readily detected among a few clones after myogenic induction (e.g. A4 clone)  despite of a fusion defect  . InteresO clones . Similarn defect , showed ol group .Dll1 gene in the bulk CRISPR-treated cells  MyoG. Consistent with our previous report that MyoD is sufficient to induce the expression of Mymx and Mymk , syncytiER) [During the fate conversion of fibroblasts, MyoD induces a pan-myogenic program exemplified by the upregulation of MyoG expression , right –63. We cER)  with treER protein in response to TMX treatment  [As a bHLH transcriptional factor, MyoD activates the expression of its target genes through binding to E-box motifs (CANNTG) . Using FCANNTG) , we discCANNTG)  also disa); when recruited by gRNA, the positioning of dCas9 to these E-box motifs should block MyoD binding thus repressing the expression of Dll1 (b). Consistent with our hypothesis, expression of gRNA that recruits dCas9 to each of these three E-box motifs significantly inhibited Dll1 expression by an average of 65% in human myoblasts  at downstream of MyoD that cooperatively induces Dll1 expression also needs to be identified. Although MyoG appeared as a tempting candidate, the observation that MyoD can robustly induce Dll1 expression from MyoGKO cells indicates that other factor(s) must exist in regulation of this process.In addition to the gene gain- and loss-of-function experiments, we also examined the , NeuroD , during Institutional Animal Care and Use Committee (IACUC) at the University of Georgia. Dll1Δ232 mouse model was generated by oviduct electroporation as previously reported [All animal procedures were approved by the reported . BrieflyDll1Δ232 allele was confirmed by genotyping F1 generation mice.Dll1 genotyping PCR was performed using genomic DNA extracted from the toe clipping with the primers, forward: AGAACCTCTGTTCGTGCCTG and reverse: GCGTCTAGGACAAAAGGGCT. For Sanger sequencing, PCR products were first gel purified and cloned into pCRII Topo vector  and sequenced with T7 or SP6 primers. Germline transmission of the Human myoblasts (ID: hSkMC-AB1190) were immortalized as previously described . Human min vitro was a gift from Feng Zhang (Addgene plasmid # 52961). The guide RNAs that target the coding regions of human and mouse MyoD and MyoG genes were individually cloned into the Lenti-CRISPR v2 vector and sequenced to verify the correct insert. The sequences for gRNA used in this study are provided.Lenti-CRISPR v2 vector  used forMouse Dll1 intron 4 gRNA1: GCACGTAGCCTTCTACCAATMouse Dll1 intron 4 gRNA2: GGGGCTTTGTGCATAGAATTMouse MyoD gene: GTCAAGTCTATGTCCCGGAGMouse MyoG gene gRNA1: ACACCTTACATGCCCACGGCMouse MyoG gene gRNA2: CCACACTGAGGGAGAAGCGCHuman MyoD gene: CGTCGAGCAATCCAAACCAGHuman MyoG gene gRNA1: ACCACCAGGCTACGAGCGGAHuman MyoG gene gRNA2: CCACACTGAGGGAGAAGCGCHuman DLL1 gene gRNA1: ACCCAGAGGCACCTGACGGTHuman DLL1 gene gRNA2: TGACGAACACTACTACGGAGLentivirus was produced by transfecting Lenti-X 293T cells  using FuGENE6 transfection reagent  with pLenti-V2, psPAX2 and pMD2.G plasmids. 48 hours post transfection, lentivirus was collected to infect human or myoblast myoblasts. psPAX2 vector was a gift from Didier Trono (Addgene plasmid # 12260). pMD2.G vector was a gift from Didier Trono (Addgene plasmid # 12259).Retroviral expression vector pMXs-Puro  was used for gene cloning and expression in human and mouse myoblasts. The open reading frames for red fluorescent protein Cherry and mouse Dll1 were cloned into pMXs-Puro vector by In-Fusion cloning. The identities of the DNA inserts in the plasmids were Sanger sequencing verified. MyoD-pCLBabe plasmid  was a giDifferentiation index was calculated as the percentage of the nuclei number within muscle cells (MF20+) divided by the total nuclei number in the imaging area. Fusion index was calculated as the nuclei number in myotubes (≥ 3 nuclei) as a percentage of the total number of nuclei inside muscle cells.ΔΔCt method was used to analyze gene expression after normalization to expression of Gapdh or 18S rRNA. Primer sequences used in this study are provided.RNA was extracted by using TRIzol Reagent (Thermo Fisher Scientific). Before used for reverse transcription, RNA quality and concentration were assessed by using a spectrophotometer . cDNA was synthesized by reverse transcription using random oligos with M-MLV reverse transcriptase . To analyze gene expression changes, the Real-time PCR was performed using QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) with SYBR Green Master Mix (Roche) and qPCR primers. The 2Primers for DLL1 qPCR-F: GATTCTCCTGATGACCTCGCAPrimers for DLL1 qPCR-R: TCCGTAGTAGTGTTCGTCACAPrimers for JAG1 qPCR-F: GTCCATGCAGAACGTGAACGPrimers for JAG1 qPCR-R: GCGGGACTGATACTCCTTGAPrimers for DLL3 qPCR-F: CACTCCCGGATGCACTCAACPrimers for DLL3 qPCR-R: GATTCCAATCTACGGACGAGCPrimers for DLL4 qPCR-F: GTCTCCACGCCGGTATTGGPrimers for DLL4 qPCR-R: CAGGTGAAATTGAAGGGCAGTPrimers for JAG2 qPCR-F: TGGGCGGCAACTCCTTCTAPrimers for JAG2 qPCR-R: GCCTCCACGATGAGGGTAAAPrimers for HEY1 qPCR-F: AACTGTTGGTGGCCCTGAATPrimers for HEY1 qPCR-R: CAATTGACCACTCGCACACCPrimers for HEYL qPCR-F: ATGAGTCCTGGGAGAGACCCPrimers for HEYL qPCR-R: GCCAGTCAGTCATTGCTCCTPrimers for HES1 qPCR-F: TTTTTGGCGGCTTCCAAGTGPrimers for HES1 qPCR-F: GGTGGGCTAGGGACTTTACGPrimers for MYOD1 qPCR-F: CGACGGCATGATGGACTACAPrimers for MYOD1 qPCR-R: TATATCGGGTTGGGGTTCGCPrimers for MYOG qPCR-F1: GCCAACCCAGGGGATCATPrimers for MYOG qPCR-R1: CCCGGCTTGGAAGACAATCTPrimers for MYH3 qPCR-F: ATTGCTTCGTGGTGGACTCAAPrimers for MYH3 qPCR-R: GGCCATGTCTTCGATCCTGTCPrimers for GAPDH qPCR-F: CACCAGGTGGTCTCCTCTGAPrimers for GAPDH qPCR-R: CAAGGGGTCTACATGGCAACTPrimers for 18S qPCR-F: GTAACCCGTTGAACCCCATTPrimers for 18S qPCR-R: CCATCCAATCGGTAGTAGCGPrimers for Dll1 qPCR-F: CAGGACCTTCTTTCGCGTATGPrimers for Dll1 qPCR-R: AAGGGGAATCGGATGGGGTTPrimers for Hey1 qPCR-F: GCACGCCACTATGCTCAATGPrimers for Hey1 qPCR-R: GGGGACCTAGACTACCAGCAPrimers for Heyl qPCR-F: CCACTGGCGCAGATGAGTTAPrimers for Heyl qPCR-R: ATCCTGTTGGCTTGGGATGGPrimers for Hes1 qPCR-F: TTTTTGGCGGCTTCCAAGTGPrimers for Hes1 qPCR-R: GGTGGGCTAGGGACTTTACGPrimers for Gapdh qPCR-F: TCTCCTGCGACTTCAACAGCPrimers for Gapdh qPCR-R: AGTTGGGATAGGGCCTCTCTTPrimers for 18s qPCR-F: ACCGCAGCTAGGAATAATGGAPrimers for 18s qPCR-R: GCCTCAGTTCCGAAAACCAProtein lysate was prepared by using RIPA buffer  supplemented with complete protease inhibitor (Sigma). Lysates were centrifuged at 16,000 x g for 15 minutes. The resultant supernatant was mixed with 4x Laemmli sample buffer . Total 20 μg protein lysates were separated by SDS-PAGE gel electrophoresis. The proteins were transferred to a polyvinylidene fluoride membrane and blocked in 5% milk at room temperature, and incubated with following primary antibodies diluted in 5% milk overnight at 4°C. Gapdh , α-Tubulin , myosin , MyoD , MyoG . The HRP-conjugated secondary antibodies: Donkey anti-sheep IgG-HRP , Goat Anti-Mouse IgG (H+L)-HRP Conjugate  and Goat Anti-Rabbit IgG (H + L)-HRP Conjugate  were diluted at 1:5,000. Signal detection was performed using Western Blotting Luminol Reagent .Lenti-SAM v2 was a gift from Adam Karpf (Addgene plasmid # 92062). Lenti-SAM v2 plasmid was used for gRNA cloning after the removal of VP64 expression cassette. The gRNA sequences that target the control and E-box motif regions of human DLL1 intronic region were provide below.Human DLL1 intron 4 gRNA1: CTGCCCCAGCGCAACAATGCHuman DLL1 intron 4 gRNA2: ATGGAGCAGCTGTCCTGCCCHuman DLL1 intron 4 gRNA3: AGAGTGGACAGCTGGTATGCImmunostaining was performed as previously reported . Brieflyt test with a two-tail distribution. Comparisons with P values < 0.05 were considered significant.Experiments were repeated at least three times. All quantitative results were analyzed with student’s S1 FigA–C) qPCR results of myogenic markers (A), DLL1  in human myoblasts with retroviral expression of NICD. Note that inhibitory effect of NICD on DLL1 expression was rescued by MyoD or MyoG expression (C). Cells were differentiated for 72 hours. n = 3. Data are means ± SEM. **P < 0.01, ***P < 0.001. (D) MyoG and myosin immunostaining results of human myoblasts after differentiation for 72 hours. Note that myogenic differentiation and fusion defects of NICD can be rescued by co-expression of MyoD or MyoG. Scale bar, 100 μm.((TIF)Click here for additional data file.S2 FigA) Human MyoD gene structure and sequencing results that confirmed biallelic frame-shifts of MyoD ORFs in one isolated MyoDKO clone similar to our previous report [B) Immunostaining results of MyoD (top) and myosin (bottom) of human WT and MyoDKO myoblasts. MyoD staining confirmed the depletion of MyoD proteins in MyoDKO cells. Scale bar, 100 μm. (C) Western blotting results that showed the absence of MyoG and myosin expression in human MyoDKO myoblasts at 48 hours post differentiation.  qPCR results of human WT and MyoDKO myoblasts with retroviral expression of MyoD or MyoG. Cells were differentiated for 48 hours. n = 3. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.(s report . Arrow p(TIF)Click here for additional data file.S3 FigA) Human MyoG gene structure and an example of sequencing results similar to our previous report [B) Immunostaining result of MyoG and myosin to show the complete depletion of MyoG proteins and relatively mild defect of differentiation for one clonally derived human MyoGKO myoblasts. Scale bar, 100 μm. (C) Quantifications of myoblast fusion. (D) qPCR result to show that DLL1 expression was not significantly affected upon deletion of MyoG from human myoblasts. Data are means ± SEM. **P < 0.01, ***P < 0.001. ns, not significant.(s report . (B) Imm(TIF)Click here for additional data file.S4 FigA) Schematic of experiment design. (B) Immunostaining results of mouse primary myoblasts to show the expression levels of MyoD/MyoG before and after CRISPR treatments. Arrows point to cells that show cytoplasmic staining signals which are likely non-specific signals. (C) qPCR results of Dll1 in CRISPR treated mouse primary myoblasts. n = 3. Data are means ± SEM. ***P < 0.001.((TIF)Click here for additional data file.S5 FigA) Representative gel electrophoresis result of genotyping PCR of WT and Dll1Δ232/Δ232 mice. (B) Summary of Dll1Δ232/Δ232 genotyping results. (C) Schematic of experiment design and the timeline of treatments and tissue collections. (D) H&E staining results of cross-sections from control and injured tibialis anterior muscles. Arrows point to small regenerating myocytes. Scale bar, 200 μm.((TIF)Click here for additional data file.


To investigate whether hUC-MSCs attenuated severe burn-induced ALI and the effects were based on TSG-6 secreted from hUC-MSCs.  A rat model was established and evaluated as follows: cytokine expression was measured by ELISA, and both inflammatory cell infiltration and lung injury were assessed by immunohistochemistry assay. α, IL-1β, and IL-6) were remarkably decreased, but the anti-inflammatory cytokine IL-10 increased after hUC-MSC administration (p < 0.05). These significant positive effects after hUC-MSC transplantation did not occur in the burn+siTSG-6 group.  In vitro, TSG-6 levels in serum from the burn group were significantly increased compared with those from the sham group. In vivo, TSG-6 levels of lung tissues and serum in the burn+hUC-MSC group were significantly increased compared with those in the burn group. Both in lung tissues and in serum, increased levels of proinflammatory cytokines (TNF- The intratracheal implantation of hUC-MSCs has been an effective treatment for severe burn-induced ALI via promoting TSG-6 secretion and inhibiting inflammatory reaction in lung tissue. Our previous study further testified the potential role and mechanism of hUC-MSCs secreting TSG-6 that attenuated severe burn-induced excessive inflammation via inhibiting activation of P38 and JNK signaling ways [Acute lung injury (ALI) and its severe manifestation acute respiratory distress syndrome (ARDS) are common forms of hypoxic respiratory failure in patients with a mortality rate of 25–40% . ALI/ARDing ways , 12. AboAll experimental procedures were in accordance with the international guidelines for biomedical research in experimental animals issued by the Committee of International Organizations of Medical scientific (CIOMS). We approved the certification of the institutional animal care and use committee of the Fourth Medical Center of PLA General Hospital and obtained the informed consent of all subjects. The primary human umbilical cord mesenchymal stem cells (hUC-MSCs) were purchased from ScienCell Research Laboratories . The hUC-MSCs were examined using flow cytometry and are positive for CD105, CD90, CD44, CD29, and HLA-I and negative for CD34, CD45, HLA-DR, CD31, and vWF as described in our previous publication . They ha2 and incubated with 3 ml mesenchymal stem cell medium serum free (MSCM-sf)  for 1 day, which were subsequently cultured continuously with 3 ml of MSCM-sf containing 10% sham serum or burn serum at 24 hours postsevere burn. Currently, the levels of inflammatory mediators, oxidative stress factors, and other factors were very high. TSG-6 secretion from hUC-MSCs after 10% burn serum continuous culture and the supernatant of the cells were assayed by using the ELISA kit.The hUC-MSCs were plated into 6-well plates with a cell density of 5000 cells/cmWe selected the lentiviral expression vector containing the TSG-6 siRNA sequence  for specific targeting TSG-6, including Lenti-TSG-6-siRNA, and its negative control vector, Lenti-GFP-siRNA. TSG-6 siRNA lentivirus vectors were generated by ligating the vector PGC-LV. TSG-6 siRNA oligonucleotide sequences are forward 5′-CCGGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTG-3′ and reverse 5′-AATTCAAAAATTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAA-3′.The sequences of control siRNA are forward 5′-CCGGGCAAGCTGACCCTGAAGTTCATCTCGAGATGAACTTCAGGGTCACGTTGCTTTTTG-3′ and reverse 5′-AATTCAAAAAGCAAGCTGACCCTGAAGTTCATCTCGAGATGAACTTCAGGGTCACGTTGC-3′.2 and incubated with 3 ml or 10 ml MSCM-sf without antibiotics for 1 day. And then, these cells were transfected with 1 ml medium of Lenti-TSG-6-siRNA or Lenti-GFP-siRNA according to the protocol provided by the manufacturer. After 12 hours, the medium was changed, and these cells were incubated with MSCM-sf without antibiotics for 3–4 days sequentially. The efficiency of TSG-6 gene knockdown in hUC-MSCs was evaluated using GFP-fluorescence observation and Western blot. Then, hUC-MSCs in T-75 culture bottles were harvested with 0.25% trypsin and resuspended at 5 × 106 cells in 1 ml sterile PBS for subsequent implantation.The recombinant lentivirus was produced by cotransfecting HEK293T cells with pGC-LV-GFP-siRNA or pGC-LV-TSG-6-siRNA, pHelper 1.0, and pHelper 2.0 plasmids using Lipofectamine 2000 . The hUC-MSCs were transfected with the prepared lentivirus (Lenti-TSG-6-siRNA or Lenti-GFP-siRNA). The hUC-MSCs were plated into either 6-well plates or T-75 culture bottles with a cell density of 5000 cells/cmAll studies adhered to procedures consistent with the International Guiding Principles for Biomedical Research Involving Animals issued by the Council for the International Organizations of Medical Sciences (CIOMS), and all experimental protocols were approved by the Institutional Animal Care and Use Committee at the Fourth Medical Center of PLA General Hospital and were performed in accordance with the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition. Six-week-old male Wistar rats (180–220 g) were obtained from the local animal facility and housed at the Institute of Animal Experiments of the Fourth Medical Center of PLA General Hospital in stables with a temperature of 22°C, a relative humidity of 55%, and a day/night cycle of 12/12 hours, with food and water ad libitum throughout the experiment.Wistar rats were randomly divided into 5 groups: (1) sham, (2) burn, (3) burn+hUC-MSCs (burn and implantation with hUC-MSCs), (4) burn+vehicle (burn and implantation with hUC-MSCs transfected with Lenti-GFP-siRNA), and (5) burn+siTSG-6 (burn and implantation with hUC-MSCs transfected with Lenti-TSG-6-siRNA). Rats in each group were divided equally into three subgroups of 6 rats according to the indicated time points of d1, d3, and d7 for the samples collected.6 (1 ml) hUC-MSCs after burn. The rats in the burn+vehicle and burn+siTSG-6 groups received intratracheal implantation of 5 × 106 (1 ml) hUC-MSCs transfected with Lenti-GFP siRNA or Lenti-TSG-6-siRNA at the same time. The rats in the sham and burn groups received intratracheal implantation of 1 ml PBS. All rats in each group were clinically evaluated. The burn wound and rats in the sham group were treated as previously described [The rats were anesthetized with Avertin (20 mg/ml and 300 mg/kg)  via intraperitoneal injection. The dorsal and abdominal hair was completely shaved. Rat models with 50% TBSA full-thickness burn injury were established via placing the backside and abdomen into hot water (94°C) for 12 s and 6 s, respectively , 13, 14.escribed . The bloAt each indicated time point, blood samples from the inferior vena cava were extracted, and lung tissue samples were collected. And then, rats were euthanized with an overdose of pentobarbital sodium intravenously.Both the lung tissue and blood samples in all groups were taken from the aorta ventralis at d1, d3, and d7 after injury. Subsequently, one piece of lung specimen was stored in liquid nitrogen for TSG-6 detection using the ELISA kit; also, another piece was fixed using 4% paraformaldehyde for immunohistochemical analysis and H&E staining. The detection range of the kit is from 0.312 ng/ml to 20 ng/ml. 100 mg lung tissue was rinsed with PBS, homogenized in 1 ml of PBS, and stored overnight at −20°C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000 g at 2–8°C. The supernatant was removed and assayed immediately. The standard was diluted following the manufacturer's instruction. Use the stock solution to produce a 2-fold dilution series. Mix each tube thoroughly before the next transfer. The undiluted standard serves as the high standard (20 ng/ml). The sample diluent serves as the standard zero.After fixation with 4% paraformaldehyde for 1 week at room temperature, the specimens were embedded in paraffin and sectioned into five-micrometer-thick sections. Then, the slices were deparaffinized with dimethyl benzene, rehydrated, and then stained with H&E in accordance with standard procedures. Infiltrations of neutrophils and macrophages were detected via incubating with specific antibodies , the corresponding secondary antibody, and the PAP (peroxidase-antiperoxidase) complex in turn and finally stained with DAB . Myeloperoxidase (MPO) exists in the cytoplasm of neutrophils and can be used as an indicator of neutrophils in lung tissue. The positive reaction of neutrophils is brown-yellow colorization. CD68 is a macrophage-specific antigen which is direct evidence for the identification of macrophages. The positive reaction of macrophages is brown colorization. Randomly, five different visual fields under a light microscope (400x) of each respective section were chosen, and CD86- and MPO-positive cells were counted by an experienced scientist in a blinded manner.β-actin antibody   were used for the protein expression assay.Protein from hUC-MSCs was isolated using RIPA buffer (Sigma Life Science), containing PMFS (Sigma Life Science) and complete Mini Protease inhibitor cocktail (Roche). Cells were centrifuged at 3000g for 10 min to collect the nuclei, and then, the supernatants were further centrifuged at 17,000g for 20 min to separate the mitochondrion. The cytoplasm was diluted in Laemmli buffer and denaturized at 95°C for 10 min. Protein was quantified using a commercial bicinchoninic acid (BCA) kit . Equal amounts of protein were subjected to SDS-PAGE gel as previously described [μg at 2–8°C. The supernatant was removed. The double-antibody sandwich ELISA kits  were used to detect the levels of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10, MPO, and TSG-6 in lung tissues and serum according to the manufacturer's protocols.100 mg lung tissue was rinsed and homogenized and then stored overnight at −20°C. The samples were performed to break the cell membranes; the homogenates were centrifuged for 5 minutes at 5000p < 0.05.All data were expressed as the mean ± SD and analyzed using SPSS 18.0 . ANOVA followed by the Tukey Cicchetti test for multiple comparisons or nonparametric Kruskal-Wallis test as appropriate was used for statistical analysis. Statistical significance level was set at To investigate whether TSG-6 secreted from hUC-MSCs influenced their therapeutic effect or not, we firstly examined the levels of TSG-6 secretion from hUC-MSCs after treatment with serum of sham or burn. Compared with the sham group, TSG-6 levels in the supernatant of burn serum were significantly increased in vitro at 24 hours postburn . The hUCp < 0.05). Furthermore, the data showed that the therapeutic effect of hUC-MSCs on neutrophil infiltration in the lung was sharply decreased after TSG-6 knockdown (p < 0.05) (p < 0.05). Furthermore, the data showed that the therapeutic effect of hUC-MSCs on macrophage infiltration in the lung was also sharply decreased after TSG-6 knockdown (p < 0.05)  . As shownockdown . The qua < 0.05) . It sugg < 0.05) –4.α, IL-1β, IL-6, and MPO and the anti-inflammatory cytokine IL-10 in lung tissues at d1, d3, and d7 after severe burn using ELISA. As shown in α (β (p < 0.05). It is worth noting that the level of the anti-inflammatory cytokine IL-10 (p < 0.05).We also tested the levels of the proinflammatory cytokines TNF-own in α , IL-1β   .During the last few years, human MSCs as a cellular therapy for the ALI/ARDS have ignited much interest of researchers, and the therapeutic effects have been demonstrated in different animal models –20. StudPatients with ALI caused only by severe burns or scald were not uncommon in the clinic, especially in children. In the present study, we established a severe burn-induced ALI in a rat model to investigate the therapeutic effects of hUC-MSCs and their possible mechanism. We found that intratracheal implantation of hUC-MSCs increased the survival rates and significantly attenuated severe burn-induced ALI via inhibiting pulmonary inflammation, and the therapeutic effects of hUC-MSCs were strongly reduced when the expression of TSG-6 was inhibited via RNA interference. It indicated that the therapeutic effects of hUC-MSCs in severe burn-induced ALI rats were associated with the soluble factor TSG-6 secreted by hUC-MSCs itself.We created a rat model with a classical pulmonary inflammatory state via severe burn , 14, 25.2β-Smad 2/3 pathway activation [ALI is an uncontrollable pulmonary inflammatory disease characterized by the release of cytokines and neutrophil accumulation. Neutrophils are the primary cells being convened to the site of inflammation and releasing inflammatory cytokines. Macrophages are another predominant cells existing in either a proinflammatory (M1) or anti-inflammatory (M2) state, acting as a trigger of the inflammatory response, and contributing to both the initiation and resolution of inflammation. MSC treatment could regulate macrophages with the anti-inflammatory phenotype and simultaneously enhance the phagocytic activity of macrophages . In our tivation .MPO is an enzyme stored in neutrophils and macrophages and released into the extracellular fluid in the setting of the inflammatory process. Since MPO is an important enzyme in the inflammatory process, there is an ongoing interest in the use of MPO as a biomarker for assessing the extent of inflammatory response in our present study. After a severe burn, a remarkable increase in MPO in lung tissues was detected in the burn group, and the peak level was shown at d1. As we expected, treatment with hUC-MSCs clearly reduced the MPO activity at d1, d3, and d7. Previously, Li et al. found that intravenous infusion with hUC-MSCs significantly inhibited LPS-stimulated MPO activity in rat lung tissues . Reducedα, IL-1β, and IL-6 are the primary mediators of the acute phase response. Previous studies showed that the decreased neutrophil recruitment into the lung tissue and suppressed expression of TNF-α, IL-1β, and IL-6 can improve the outcomes of ALI [α and IL-1β through paracrine secretion [α, IL-1β, and IL-6 significantly increased. The difference is simply that the peak level occurs at different times. For TNF-α and IL-1β, the peak level appeared on d1, while the peak level of IL-6 appeared on d3. When hUC-MSCs were intratracheally implanted after burn, the concentration of proinflammatory cytokines in lung tissues decreased significantly, which was mostly in agreement with previous studies demonstrating that BM-MSCs can reduce TNF-α and IL-6 secretion by lung macrophages via the paracrine pathway or direct contact with host cells [The proinflammatory cytokines TNF-s of ALI . Severalecretion . Consistst cells . Our resInterleukin-10 (IL-10), one of the most important anti-inflammatory cytokines, is known to reduce the synthesis of proinflammatory cytokines. IL-10 treatment is supposed to be a promising therapeutic strategy to reduce lung injury –32. Someα-stimulated gene-6 (TSG-6), a 30 kDa protein generated by activated macrophages, modulates inflammation; however, its mechanism of action and role in the activation of macrophages were not fully understood. From our earlier report [κB activation. TSG6 also prevented the expression of proinflammatory proteins  while increasing the expression of anti-inflammatory proteins  in macrophages. This shift was associated with suppressed activation of proinflammatory transcription factors STAT1 and STAT3. Thus, TSG-6 functions by converting macrophages from a proinflammatory to an anti-inflammatory phenotype secondary to suppression of TLR4/NF-κB signaling and STAT1 and STAT3 activation.According to the above findings, it can be inferred that hUC-MSC administration improved ALI by balancing the homeostasis of the cytokine network. Furthermore, we would like to know more about how hUC-MSCs did work. In our previous study, we found that the TSG-6 levels were significantly elevated in the systemic inflammatory response in burn rats. TSG-6 expression is induced as a result of an inflammatory response. Our data showed that severe burn-induced ALI could upregulate the expression of TSG-6, which was in agreement with our previous study . The ever report  and somer report , we coul6) as suggested in our previous study [It was well known that delivering locally has the advantage of reaching the target organ directly with relatively small dosage, faster and stronger effectiveness, and slighter systemic adverse reactions. In a clinical setting, physicians prefer to use medicine through intratracheal administration or aerosol inhalation for patients with lung diseases, especially for severe cases. Thus, we chose the intratracheal implantation as the route of medication. While increasing the dose of drugs might increase the efficacy of the treatment, we could not exclude systemic adverse reactions and more waste of resources caused by larger doses. Taking these factors into consideration, we selected the optimal dosage of hUC-MSCs  were immediately administered to prevent shock. And the burn wound was then treated with a 1% tincture of iodine and kept dry to prevent infection.To the best of our knowledge, this is the first report about the therapeutic evaluation of hUC-MSCs in severe burn-induced ALI. TSG-6 secreted from hUC-MSCs played a key role in severe burn-induced ALI via inhibiting the inflammatory reaction in lung tissue. It suggests that intratracheal implantation of hUC-MSCs is an effective treatment for a severe burn-induced ALI rat model. Meanwhile, knockdown of TSG-6 mRNA expression in hUC-MSCs did not completely abrogate the anti-inflammatory effects suggesting that some other unknown mechanisms were involved. It means that additional experiments are required to determine the relative contribution of these factors to the beneficial effects of hUC-MSCs in lung injury.


About 10–20 percent of patients with breast cancer are diagnosed with triple-negative breast cancer (TNBC). These tumors are named for their lack of expression of estrogen receptor (ER), progesterone receptor (PR), and amplification of human epidermal growth factor receptor 2 (HER2). These genes are targeted by therapies in other breast cancer patients. However, most TNBC patients recur within 5 years. Understanding how and why these tumors metastasize will help clinicians better treat these underserved cancer patients. TNBC tumors are highly infiltrated by tumor-associated macrophages (TAMs) that promote tumorigenesis and metastasis. Our study elucidates how the tumor co-opts macrophages recruited to the tumor through extracellular vesicles (EVs), further increasing tumor metastasis. Expression of tumor CCL5 regulates EV secretion and cargo that further alters macrophage phenotype to drive tumor metastasis. Together, our data suggest a more extensive role of EVs in the biology of tumor metastasis as well as their potential use as biomarkers.Purpose: To understand how tumor cells alter macrophage biology once they are recruited to triple-negative breast cancer (TNBC) tumors by CCL5. Method: Mouse bone marrow derived macrophage (BMDMs) were isolated and treated with recombinant CCL5 protein alone, with tumor cell conditioned media, or with tumor extracellular vesicles (EVs). Media from these tumor EV-educated macrophages (TEMs) was then used to determine how these macrophages affect TNBC invasion. To understand the mechanism, we assayed the cytokine secretion from these macrophages to determine how they impact tumor cell invasion. Tumor CCL5 expression was varied in tumors to determine its role in regulating macrophage biology through EVs. Results: Tumor EVs are a necessary component for programming naïve macrophages toward a pro-metastatic phenotype. CCL5 expression in the tumor cells regulates both EV biogenesis/secretion/cargo and macrophage EV-education toward a pro-metastatic phenotype. Analysis of the tumor EV-educated macrophages (TEMs) showed secretion of a variety of factors including CXCL1, CTLA-4, IFNG, OPN, HGF, TGFB, and CCL19 capable of remodeling the surrounding tumor stroma and immune infiltrate. Injection of tumor cells with macrophages educated by metastatic tumor cell EVs into mice increased tumor metastasis to the lung. Conclusion: These results demonstrate that tumor-derived EVs are key mediators of macrophage education and likely play a more complex role in modulating tumor therapeutic response by regulating the tumor immune infiltrate. Of the women in the United States that develop breast cancer, 15–20% will have basal-like or triple-negative breast cancer (TNBC) . Thus, iTumor cells are driven to metastasize in part through interaction with cells in the microenvironment. Tumor-associated macrophages (TAMs), generally characterized as alternatively activated macrophages, play an essential role in driving metastasis ,5. PreviTAMs are recruited to mammary tumors through induction of a variety of cytokines and chemokines including C-C Motif Chemokine Ligand 2 (CCL2), CCL3, CCL5, Colony Stimulating Facotor-1 (CSF-1/M-CSF), and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF or CSF2) ,11,12,13One possible mechanism connecting tumor and macrophage education is through EVs secreted from the tumor cells and taken up by the immune system. EVs are small micro-vesicles with a double lipid bilayer, secreted by most cell types, and of endocytic or membrane origin . Small EA number of studies suggest that small EVs, including exosomes, modulate stromal and immune response within the tumor microenvironment ,25. TumoOur results indicate that tumor EVs alone can educate macrophages to a TAM phenotype that drives invasion and metastasis of TNBC. The absence of EVs, either through removal by ultracentrifugation or reduced secretion through Rab27a knockdown reduce the ability of tumors to program macrophages. Furthermore, we show that macrophage education occurs through an indirect mechanism involving stimulation of tumor cells by CCL5. CCL5 expression within the tumor increases the secretion of tumor EVs and alters the ability of EVs to educate macrophages. This results in TAMs that reflect the metastatic state of the tumor cells. Clinically, the genes we identified in this study are highly correlated to CCL5, and cluster in a subset of TNBC patient tumors within The Cancer Genome Atlas (TCGA) cohort. Taken together, these results indicate that tumor EVs play an integral role in education of non-resident macrophages to a pro-metastatic TAM phenotype in TNBCs, driving tumor metastasis.MDA-MB-231, 293T, and L929 were obtained from ATCC. MDA-MB-231 1833   were purchased from Open Biosystems. Lentiviruses were produced as described above. BM1 cells were transduced and selected in 3 μg/mL of puromycin and LMB cells with 10 μg/mL of puromycin for 14 days. Five shRNA targets against Rab27a were used for both human and mouse Rab27a, with a non-targeting shRNA used as control.Lentiviral all-in-one plasmids containing Cas9 as well as guide RNAs were purchased from Applied Biological Materials. Lentivirus was produced as described above. BM1 cells were then infected, then selected for 14 days using 3 μg/mL of puromycin. Transduced cells were assayed for CCL5 expression using a CCL5 ELISA from Ray Biotech as well as by qPCR. Media samples were concentrated 10× to ensure even low levels in KO cells could be measured.Ccl5 in mouse LMB cells, sequences were determined using Broad’s online sgRNA tool, and sgRNA sequences were ordered from IDT and inserted into sgRNA vectors obtained from Broad. Cells were first transduced with dCas9-KRAB or dCas9-VPR and selected with 10 μg/mL blasticidin followed by sgRNA transduction and selection with 10 μg/mL puromycin for 14 days each. RNA levels of Ccl5 were measured in cells and EVs using ddPCR with Gapdh as a loading control.For dCas9-KRAB knock-down and dCas9-VPR over-expression of Hs GAPDH-F: TGCACCACCACCTGCTTAGCHs GAPDH-R: GGCATGGACTGTGGTCATGAGHs CCL5-F: CCAGCAGTCGTCTTTGTCACHs CCL5-R: CTCTGGGTTGGCACACACTTMm Ccl5-F: TTTGCCTACCTCTCCCTCGMm Ccl5-R: CGACTGCAAGATTGGAGCACTMm Slpi-F: GGCCTTTTACCTTTCACGGTGMm Slpi-R: TACGGCATTGTGGCTTCTCAAMm Mmp12-F: CTGCTCCCATGAATGACAGTGMm Mmp12-R: AGTTGCTTCTAGCCCAAAGAACMm Ccl7-F: GCTGCTTTCAGCATCCAAGTGMm Ccl7-R: CCAGGGACACCGACTACTGMm Tnfr2-F: ACACCCTACAAACCGGAACCMm Tnfr2-R: AGCCTTCCTGTCATAGTATTCCTMm Grn-F: ATGTGGGTCCTGATGAGCTGMm Grn-R: GCTCGTTATTCTAGGCCATGTGHs Grn F: ATCTTTACCGTCTCAGGGACTTHs Grn R: CCATCGACCATAACACAGCACHs TNFR2 F: CGGGCCAACATGCAAAAGTCHs TNFR2 R: CAGATGCGGTTCTGTTCCCHs CCL5 Sequencing F: TTAGGGGATGCCCCTCAACTHs CCL5 Sequencing R: CTGAGACTCACACGACTGCTGHs Rab27a-F: GCTTTGGGAGACTCTGGTHs Rab27a-R: TCAATGCCCACTGTTGTGATAAAMm Rab27a-F: TCGGATGGAGATTACGATTACCTMm Rab27a-R: TTTTCCCTGAAATCAATGCCCAmCCL5i sgRNA targetsAGAGATCTTCATGGTACCCGGTACCCGCGGCAGAGGCTGTATCTCCCACAGCCTCTGCCGCTTCATGGTACCCGCGGCAGGGCAGCTGCAGAGATCTTCAmCCL5a sgRNA targetsTTATGACAGCAACAAGTGTTTGGAAACTCCCCAAGTCCTGGTGGAAACTCCCCAAGTCCTTGTGGAAACTCCCCAAGTCCCCCCCCAGCCCCAGGACTTGRKIP (derived in lab from serum of rabbits exposed to an RKIP peptide), Rabbit anti-CD63 antibody , Rab27a , F4/80 , Foxp3 clone:FJK-16s , FITC-CD63 , CD9-PerCP-Cy 5.5 , CD81-Pacific Blue , Alix Ab (3A9) , APC-Fire IgG , Exosome-human CD63 isolation/detection reagent , CCL5 ELISA , Mouse Cytokine Array Two thousand tumor cells were plated in 200 µL of media per well in a 96-well culture plate . Six replicates per condition were used for all assays. Cells were then monitored using an Incucyte Zoom , with 20× phase-contrast images taken every four hours. Percent confluence was calculated using Incucyte Analysis Software and exported as a CSV file for either individual time and data points or average data points per time point. Exported data was graphed as individual points (when available) using Graph Pad Prism and statistical analysis performed as described below.20,000 tumor cells were plated in 200 µL of media per well in a 96-well culture plate  and allowed to adhere overnight. Six replicates per condition were used for all assays. Each well was precisely scratched using an Incucyte Woundmaker Tool . Wells were washed to remove scratched cells and debris, and 200 µL of fresh media per well was added. Cells were then monitored using an Incucyte Zoom , with 20× phase-contrast images taken every four hours. Migration was calculated as  using the Incucyte Scratch Wound Analysis Software Module and exported as a CSV file for either individual time and data points or average data points per time point. Exported data was graphed as individual points (when available) using Graph Pad Prism and statistical analysis performed as described below.4 BM1 cells or 1 × 105 E0771-LMB cells were plated in 24-well trans-well inserts with 8 μm pores  coated with growth factor depleted basement membrane extract  ..Ccl7, TnRab27a, a known regulator of the EV secretion machinery  into the inguinal fat pads of athymic nude mice . We Because we saw no differences in macrophage number recruited to tumors when tumor Rab27a expression was reduced, we next sought to determine how EV education of macrophages alters their phenotype in vivo. We isolated macrophages from control and shRab27a BM1 tumors D. We the in human BM1 or mouse LMB TNBC cells (CCL5 expression through either CRISPR-Cas9 (BM1 cells) or CRISPR-dCas9-KRAB (LMB cells)  C decreasB cells) . When we present .CCL5 regulates the number of EVs that tumor cells secrete, we also wanted to test if it regulates the cargo of the EVs and subsequently their impact on macrophages. When we treated macrophages with the same number of control and highCCL5 tumor EVs (quantitated by NTA), there was an approximately 50% increase in TEM-driven tumor cell invasion (CCL5 expression through either CRISPR-Cas9 (BM1 cells) or CRISPR-dCas9-KRAB (LMB cells)  [CCL5. Our previous work had established OPN and SLPI as markers of macrophages activated by CCL5 [Knowing that invasion A. These B cells) C decreasB cells) B. To detne array . We genepression . We theno tumors    and SLPIin (OPN) ,46,47,48 by CCL5 , and bot by CCL5 ,46,47,48CCL5high BM1 tumors  and LMB EV-educated macrophages (TEMs) into the inguinal fat pad of C57Bl/6 mice A. Co-inj1 tumors .To determine whether the metastatic state of the tumor cells influences the ability of the educated macrophages to promote metastasis, we evaluated non-metastatic cells as a source of EVs . To geneAlthough co-injection of TNBC tumor cells with in vitro-generated TEMs altered the function of tumor associated macrophages, recruitment of macrophages to the tumor did not change. When we stained for the macrophage marker F4/80, we found that the total number of macrophages in the tumor were the same between control tumors and those co-injected with TEMs E,G. ThisBecause EV programming of macrophages resulted in the secretion of cytokines known to recruit T-regs , we also immunostained tumors for the T-reg marker Foxp3. Co-injection of metastatic tumor cells (LMB) with TEMs generated by metastatic tumor EVs significantly increased the number of Foxp3+ T-regs compared to control F,H. ConvCCL5 expression  has been implicated in T-reg polarization [CCL19 and IFNG are highly correlated with CCL5 expression in patient breast tumors, these may be most relevant to T-reg infiltration of tumors in response to CCL5. Our data suggest that tumor EVs not only impact TAM phenotypes directly, but also alter their ability to attract and interact with other cells that contribute to evasion of immune detection.Several of the proteins that are regulated in TEMs and TAMs by tumor EVs and ironment ,52,53. Eironment . In addirization ,56,57,58CCL5 and tumor EVs as key potential targets for tumor immunotherapy. Previous work has established that tumors infiltrated with T cells, T-regs, and macrophage recruitment may respond well to checkpoint blockade therapy [CCL5 altered EVs are highly correlated with expression of CD8, CD4, CD25, LAG3, Foxp3, and IFNG, as well as macrophage markers, in TNBC patients. This suggests that tumor cells EV protein or RNA markers may also be prognostic for response to checkpoint blockade therapies. Because of the strong evidence of a biological role these EVs play in altering the immune microenvironment, it furthers their potential for use as biomarkers of tumor outcome as well as potential response to immunotherapy.These results highlight the importance of  therapy ,60. The Overall, our results show that tumor-associated macrophages, which play a key role in enabling tumor cells to invade surrounding tissue, intravasate into vessels, and metastasize, rely on tumor EVs from metastatic cells to be programmed toward a pro-metastatic phenotype. As such, EVs can play an even more pivotal role in metastatic progression than previously realized. Targeting tumor EVs will not only be essential to reduce the formation of the pre-metastatic niche, but also block the education of the primary tumor microenvironment including macrophages to reduce tumor burden and tumor cell dissemination.


Streptococci, and a similar study revealed that sB2M-9, derived from native B2M, functions as an antibacterial chemokine that binds Staphylococcus aureus. An investigation of sB2M-9 exhibiting an early lymphocyte recruitment in the human respiratory epithelium with bacterial challenge may uncover previously unrecognized aspects of B2M in the body’s innate defense against Mycobactrium tuberculosis. B2M possesses antimicrobial activity that operates primarily under pH-dependent acidic conditions at which B2M and fragmented B2M may become a nucleus seed that triggers self-aggregation into distinct states, such as oligomers and amyloid fibrils. Modified B2M can act as an antimicrobial peptide (AMP) against a wide range of microbes. Specifically, these AMPs disrupt microbe membranes, a feature similar to that of amyloid fibril mediated cytotoxicity toward eukaryotes. This study investigated two similar but nonidentical effects of B2M: the physiological role of B2M, in which it potentially acts against microbes in innate defense and the role of B2M in amyloid fibrils, in which it disrupts the membrane of pathological cells. Moreover, we explored the pH-governing antibacterial activity of B2M and acidic pH mediated B2M amyloid fibrils underlying such cytotoxicity.Beta2-microglobulin (B2M) a key component of major histocompatibility complex class I molecules, which aid cytotoxic T-lymphocyte (CTL) immune response. However, the majority of studies of B2M have focused only on amyloid fibrils in pathogenesis to the neglect of its role of antimicrobial activity. Indeed, B2M also plays an important role in innate defense and does not only function as an adjuvant for CTL response. A previous study discovered that human aggregated B2M binds the surface protein structure in  B2M), a small gene composed of four exons at chromo-some 15q21.1, encodes a full-length, 119-residue protein that becomes a 99-residue mature B2M (pI = 6.02) with a secondary structure of seven beta-pleated sheets (https://www.ncbi.nlm.nih.gov/gene/567 accessed on 11 May 2021). B2M is homologous in sequence with the homology region of IgG and HLA, indicating a common evolutionary origin [Human beta-2 microglobulin  in mice. They also observed that mice had a ability to fight pulmonary infection with tuberculosis when this mechanism was absent [M. tuberculosis [M. tuberculosis infection. Furthermore, Cogen et al. also suggested that an unknown mechanism exists in B2M-dependent bacterial clearance and survival during infection by murine Klebsiella pneumoniae bacteremia [The role of B2M in adaptive immunity has been extensively studied, but its role in innate defense has been neglected. Roch et al. discovered that five earthworm defense proteins exhibited compositional similarity with mouse B2M, reflecting an evolutionary function of B2M in innate defense among invertebrates . The ant. aureus . Due to . aureus . In addis absent . Rolph erculosis . The hetcteremia . These fcteremia . The altcteremia . A 9-kDacteremia . The mecS. aureus. sB2M-9 and other defense molecules, but not native B2M, possibly account for the formation of bacterial clumps. However, these bacterial clumps last only for a few hours. A subsequent abrupt increased in the growth of the bacteria (relative to that of a control group) indicates that sB2M-9 realizes its antibacterial activity by temporarily inhibiting the growth of bacteria and not by killing them. In addition, the bacterial clumps enhance THP-1 monocyte migration, to a level that is greater than even that of bacteria migration itself without forming clumps, indicating the role of sB2M-9 as a chemokine. Thus, sB2M-9 is suggested to exhibit early lymphocyte recruitment in the milieu of REC with bacterial infection, in addition to the classic B2M-restricted mechanism; moreover, it may be responsible for resistance to tuberculosis in the lungs. Such evidence indicates that hitherto unrecognized aspects of B2M in the innate defense against M. tuberculosis in the lungs.Many functional peptides derived from their precursor sequences are known to be released through a proteolytic mechanism. For example, hCAP18, a human antimicrobial peptide (AMP) precursor, harbors an active domain, namely LL-37 in its C-terminal sequence . A recenE. coli and L. monocytogenes [Cole et al. characterized the antibacterial activity of a panel of cationic polypeptides in human airway fluid, and they discovered that B2M plays a putative role in the suppression of ytogenes . Their aytogenes . This siB. subtilis, P. aeruginosa, and L. monocytogenes in human broncho-alveolar-lavage fluid [L. monocytognes. Notably, as indicated by synthetic peptides in a bacterial assay, the AMP activity of B2M is located within a fragment of 18 amino acids at the C-terminal region . In contrast to the structure of sB2M-9, as reported by our previous study [https://web.expasy.org/compute_pi/ accessed on 11 May 2021), the theoretical pI values for these two deduced small fragments of B2M are similar and fairly high  releases an octapeptide with antimicrobial activity [In addition, a panel of chemokines including CXC and CC chemokines, has been characterized as exhibiting antimicrobial activity . Moreoveactivity . This fiactivity . AMP seqactivity . The sheWe recently analyzed a panel of cationic peptides cultured in A549 medium infected with IL-1β by proteomic approach, thioredoxin was especially prominent in addition to some other immune responsible factors, such as cytokines and chemokines . ThioredAMPs can have a pH dependent mode of action in a wide range of multicellular organisms . In geneAntibacterial activity-possessing B2M was initially observed in human amniotic fluid by Kim et al. . Such acKlebsiella pneumonia (a gram-negative strain), implying an affinity for Fc-binding in gram-positive bacteria. Eventually, this effect triggers monocytes migration to the bacterial clumps. AMPs can self-aggregate to facilitate their insertion into bacterial membrane through a pore-forming mechanism [A study reported the binding of IgG to staphylococcus and to the gram-positive streptococcus groups A, C, and G . The rolechanism . Because2+ [In essence, a variety of potential factors can contribute to the formation of B2M amyloid fibrils, including the concentration of free-form B2M, an acidic pH, the truncation and cleavage by proteolysis, sequence mutation, the presence of metals , posttranslational modification (PTM), and lipid interaction . These f2+ , amino a2+ . The eff2+ . Thus, B2+ ,71. The 2+ . Althoug2+ ,73. In a2+ . Only ag2+ . The mol2+ . This ph2+ . Thus, aBecause a beta-sheet rich molecule such as B2M exhibits biological effects that differ depending primarily on the concentration and pH environment, a sB2M-9 concentration of 5 μg/mL is present in the culture medium of human REC, which is close to that in human nasal fluid and other biological tissues ,74. ThisIn addition to sB2M-9 shedding, a fragment with a high pI value hidden in B2M that governs antimicrobial activity could be released in response to a microbial challenge. Moreover, intact or fragmented B2M, which is the more numerous, self-aggregates to form oligomers and amyloid fibrils that may either protect cells against microbes or, conversely, wreak havoc in the body if the cleavage process is dysregulated .


The global spread of antibiotic-resistant infections has meant that there is an urgent need to develop new antimicrobial alternatives. In this study, we developed a strategy to boost and/or synergize the activity of conventional antibiotics by combination with antimicrobial peptides tagged with the bulky non-natural amino acid β-naphthylalanine  to their N- or C-terminus. A checkerboard method was used to evaluate synergistic effects of the parent peptide and the Nal-tagged peptides. Moreover, boron-dipyrro-methene labeled vancomycin was used to characterize the synergistic mechanism of action between the peptides and vancomycin on the bacterial strains. These Nal-tagged antimicrobial peptides also reduced the antibiotic-induced release of lipopolysaccharide from Gram-negative bacteria by more than 99.95%. Our results demonstrate that Nal-tagged peptides could help in developing antimicrobial peptides that not only have enhanced antibacterial activities but also increase the synergistic effects with conventional antibiotics against antibiotic-resistant bacteria. The emergence of multidrug-resistant pathogens emphasizes the need for novel and alternative therapeutics to fight against infections . Some AM2) was developed based on the C-terminal region of Bothrops asper myotoxin II (2), a shortened version of PEM-2-W5K/A9W, only had antimicrobial activities in the low salt LYM broth media  outer membrane of bacteria may hinder the development of AMPs , 2019. REscherichia coli and to boost synergistic effects with vancomycin against drug-resistant Gram-positive bacteria  outer leaflet of Gram-negative bacteria . It was bacteria .The aim of this study was to test if the bulky non-natural amino acid end tagging strategy of short AMPs can be used in combination with various antibiotics to fight against antibiotic-resistant bacterial strains. These results could provide important insights into the synergistic mechanism between AMPs and traditional antibiotics.S1, S1-Nal, and S1-Nal-Nal  were purEnterococcus faecium , Acinetobacter baumannii strains including 14B0091, 14B0097, and 14B0100, and E. coli strains including BCRC 13B0198 and BCRC 13B0207 were purchased from Bioresources Collection & Research Center1 . Escherichia coli and A. baumannii strains were incubated in MHB, and E. faecium BCRC 15B0132 were incubated in TSB overnight at 37°C with 150 rpm shaking overnight. The turbidity of bacteria was measured by the absorbance of optical density at 600 nm  with UV/Visible spectrophotometer .E. coli and A. baumannii strains were incubated in MHB, and E. faecium BCRC 15B0132 were incubated in TSB overnight at 37°C. The cell cultures were regrown to mid-log phase and then diluted to a final concentration of 5 × 105 CFU/mL. 99 μl of each diluted microbes were transferred into each well of a 96-well plate, into which 1 μl of peptides or antibiotics has previously been added and the final concentration of the peptides and antibiotics would be 64–0.13 μg/mL. After incubation for about 16 h at 37°C, the MIC value of peptides or antibiotics was determined by inspecting the visible growth. The MIC value was defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism. All experiments were repeated three times independently.The minimal inhibitory concentrations (MICs) of peptides and antibiotics were determined by using microbroth dilution technique, as described by the guidelines of the 5 CFU/mL into each well of a 96-well plate. After incubation for about 16 h at 37°C, the MIC value of peptide/antibiotic combination was determined by inspecting the visible growth. MIC values obtained were used to evaluate the effects of combination between peptides and antibiotics by calculating the Fractional Inhibitory Concentration Index (FICI) according to the following formula:The synergistic effects of peptides in combination with antibiotics was assessed by using the microbroth dilution checkerboard assay . Each weThe calculated FICI was interpreted as synergistic (FICI ≤ 0.5), no interaction (0.5 < FICI ≤ 4), or antagonistic (FICI > 4) . The exp7 CFU/mL and incubated with S1, S1-Nal, and S1-Nal-Nal peptides at 0.5 × MIC for 30 mins. Then, 2 μg/mL BODIPY-labeled vancomycin were treated for 30 mins. Bacteria treated with BODIPY-labeled vancomycin only served as a control. After treatment, the cell pellet was obtained by centrifugation and washed with PBS to remove free BODIPY-labeled vancomycin. The pellet was resuspended and loaded on the glass slides  and visualized under confocal laser scanning microscope  equipped with 64 × oil objective lens . All experiments were repeated three times independently.The fluorescence experiments were performed with slight modification as described previously . Briefly30H26N2O13) by cytoplasmic esterases (600 of 1.0 with PBS containing 10% (vol/vol) broth. The microbes were incubated with 3 μM calcein-AM for 1 h at 37°C to form calcein-AM loaded cells. The calcein-AM loaded cells were collected by centrifugation , and resuspended to 107 CFU/ml by PBS. Cobalt was added into cells and was used to quench the fluorescence of calcein released into the extracellular environment. The cells were treated with 0.5 × MIC of peptides in the sterile black-wall 96-well plate and measured the fluorescence intensity for 60 mins at an excitation wavelength of 485 nm and an emission wavelength of 510 nm on a fluorescence plate reader  (2O served as negative controls. The intensity of membrane permeabilization (%) was calculated as the absolute percent calcein leakage by peptides with respect to non-treated calcein-AM loaded cells  is a non-fluorescent dye and enters into the bacteria through diffusing across the cell membrane, and hydrolyzed to fluorescent calcein (Csterases . Briefly States) . Bacteried cells . All expEscherichia coli BCRC 13B0198 were cultured at the mid-log phase and diluted to 104 CFU/ml, and then treated with peptide alone at 1 × MIC or combination with vancomycin (both at 0.5 × MIC) at 37°C for 6 h. The cells were filtered by using a pyrogen-free 0.2 μm pore filter  and the endotoxin level was determined by limulus amebocyte lysate (LAL) PYROCHROME® test . The kinetic turbidity was analyzed by using a microplate reader . The experiments were repeated three times independently.p < 0.05 was considered to indicate a statistically significant difference.The statistical results are performed as the mean ± SEM and were analyzed using one-way ANOVA analysis of variance. Statistical analysis was performed using GraphPad Prism version 8.0 , where E. faecium BCRC 15B0132 (VRE) and Gram-negative bacteria including A. baumannii BCRC 14B0091, A. baumannii BCRC 14B0097, A. baumannii BCRC 14B0100, E. coli BCRC 13B0198, and E. coli BCRC 13B0207 were assessed by MIC assay. As shown in E. coli BCRC 13B0207, which is sensitive to ciprofloxacin. S1 peptide also had limited or no antibacterial activities (>64 μg/ml). On the other hand, peptides S1-Nal and S1-Nal-Nal with the addition of bulky non-natural amino acid β-naphthylalanine at the C-termini demonstrated enhanced activities against all bacterial strains. S1-Nal had the MICs of 64 μg/ml against E. faecium, 8 μg/ml against A. baumannii, and 32 μg/ml against E. coli. S1-Nal-Nal had the MICs of 32 μg/ml against E. faecium, 2 μg/ml against A. baumannii BCRC 14B0091, 4 μg/ml against A. baumannii BCRC 14B0100, 8 μg/ml against A. baumannii BCRC 14B0097, and 16 μg/ml against E. coli. These results indicated that the addition of only one or two bulky non-natural amino acid end tags could boost the antimicrobial activity.The antibacterial activities of S1, S1-Nal, S1-Nal-Nal, vancomycin, ciprofloxacin, and tetracycline against Gram-positive bacterium including vancomycin-resistant E. faecium, A. baumannii, and E. coli membranes by measuring the peptide-induced leakage of the fluorescent dye calcein from bacterial cells -labeled vancomycin was used to study the mechanism of synergistic effects of S1, S1-Nal, and S1-Nal-Nal by measuring the uptake of vancomycin in  strains . The res2 strain . Similar strains , 4.Escherichia coli BCRC 13B0198 cells were treated with S1, S1-Nal, and S1-Nal-Nal or the combination of vancomycin and the peptides. The LPS concentration decreased tremendously with the S1-Nal, and S1-Nal-Nal treatment alone or the combination of S1-Nal, S1-Nal-Nal, and vancomycin treatment  from Gram-negative bacteria into the bloodstream of the host and has been shown to be associated with the deterioration of the patients . Herein,reatment .E. faecium BCRC15B0132 strain. S1-Nal or S1-Nal-Nal also displayed synergistic effects with vancomycin and ciprofloxacin against the Gram-negative A. baumannii BCRC14B0091, A. baumannii BCRC14B0097, A. baumannii BCRC14B0100, E. coli BCRC13B0198, and E. coli BCRC13B0207 strains. However, lack of synergy with tetracycline for all peptides was found against the A. baumannii BCRC14B0100, E. coli BCRC13B0198, and E. coli BCRC13B0207 strains.End-tagging peptides with hydrophobic moieties have been developed to increase salt resistance and potency of AMPs. For example, tryptophan and/or phenylalanine stretch , as wellS. aureus and P. aeruginosa isolates  to one of the peptides’ termini. The described Nal-tagged peptides  enhanced the antibacterial activity of the plain peptides and generated a better synergistic effect when combined with conventional antibiotics. Increasing the membrane permeabilization against the bacterial strains was also observed. Additionally, these Nal-tagged AMPs reduced the antibiotic-induced release of LPS from Gram-negative bacteria by more than 99.95%. Only one or two Nal end-tags were needed to have these biological impacts on the peptides, making this a potential strategy for developing new antimicrobial agents.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.C-LW, Y-HC, and K-LP performed the experiments and analyzed the data. C-LW and J-WC wrote the manuscript. B-SY and J-WC planned the study and revised the manuscript. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


OR7E156P was intricately linked to the HIF1A pathway. Hypoxia could induce OR7E156P expression, whereas OR7E156P silencing decreased HIF1A protein levels under hypoxic conditions. Hypoxia promoted glioma cell invasion and DNA synthesis, and HUVEC tube formation, whereas OR7E156P silencing partially reversed the cellular effects of hypoxia. HIF1A overexpression promoted, whereas OR7E156P silencing inhibited tumor growth; the inhibitory effects of OR7E156P silencing on tumor growth were partially reversed by HIF1A overexpression. miR-143 directly targeted OR7E156P and HIF1A, respectively. miR-143 inhibition increased HIF1A protein levels, promoted glioma cell invasion and DNA synthesis. Moreover, they enhanced HUVEC tube formation, whereas OR7E156P silencing partially reversed the cellular effects of miR-143 inhibition. HIF1A targeted the promoter region of miR-143 and inhibited miR-143 expression. Altogether a regulatory axis consisting of OR7E156P, miR-143, and HIF1A, was identified which is deregulated in glioma, and the process of the OR7E156P/miR-143/HIF1A axis modulating glioma cell invasion through ZEB1 and HUVEC tube formation through VEGF was demonstrated.Gliomas are characterized by high incidence, recurrence and mortality all of which are significant challenges to efficacious clinical treatment. The hypoxic microenvironment in the inner core and intermediate layer of the tumor mass of gliomas is a critical contributor to glioma pathogenesis. In this study, we identified an upregulated lncRNA, OR7E156P, in glioma was identified. The silencing of OR7E156P inhibited cell invasion and DNA synthesis  Gliomas are the most common cerebral malignancies . GliomasNon-coding RNAs (ncRNAs) can be divided according to their length: short ncRNA, mid-size ncRNA, and long non-coding RNA (lncRNA); they can also be grouped according to their function, loci, and post-transcriptional modification. The oncogenic role of short ncRNAs such as miRNAs have been a long-lasting research topic . ContrasHypoxia is one of the most propitious microenvironmental conditions during tumor development, including gliomas , 19. In in vitro effects of OR7E156P upon the invasion and proliferation, and HUVEC  tube formation ability were examined under normoxia or hypoxia, and the dynamic effects of OR7E156P and HIF1A on tumor growth in nude mice in vivo were examined. Next, miRNAs which could simultaneously bind to OR7E156P and HIF1A were selected, and the putative bindings were subsequently examined. The dynamic effects of OR7E156P and miR-143 on HIF1A levels, glioma cell invasion and proliferation, and HUVEC tube formation were examined under conditions of normoxia or hypoxia. The binding of HIF1A to miR-143 promoter region and HIF1A regulation of miR-143 were ultimately examined. A network formed by lncRNA OR7E156P, miR-143, and HIF1A that might modulate hypoxia-related glioma growth and metastasis was conclusively demonstrated.In this study, datasets from TCGA and microarray expression profiles from GEO were analyzed for lncRNAs abnormally-expressed in glioma tissues, and lncRNA OR7E156P was selected. The specific effects of OR7E156P on glioma cell invasion, DNA synthesis, and HUVEC tube formation were examined. The signaling pathways potentially associated with OR7E156P were analyzed and HIF1A signaling was identified. Then, the A total of ten glioma tissues from the tumor core region (core), ten glioma tissues from the tumor intermediate region (intermediate), and ten tissue samples from the peritumoral brain edema (PTBE) region were harvested from patients who underwent surgical resection at Xiangya Hospital. The sampling procedure was conducted with the approval of the Ethics Committee of Xiangya Hospital. All the tissue samples were stored at -80°C pending further laboratory investigations.® CRL-8621™), as a normal control, was procured from ATCC  and cultured in Eagle’s Minimum Essential Medium . Human glioma cell line T98G (CRL-1690™) was obtained from ATCC and cultured in Eagle’s Minimum Essential Medium (ATCC) and supplemented with 10% fetal bovine serum . Human glioblastoma cell line U87-MG was procured from the Shanghai Institutes for Biological Sciences Cell Resource Center  and cultured in high-glucose Dulbecco’s modified Eagle medium  supplemented with 10% FBS. Human glioblastoma cell line LN229 was obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Science  and cultured in DMEM/F12 1:1 medium  supplemented with 10% FBS. Human glioma cell line U251-MG was obtained from Sigma-Aldrich  and cultured in DMEM (GIBCO) supplemented with 10% FBS. Primary umbilical vein endothelial cells  were obtained from ATCC . All the cells were cultured in 5% CO2 saturation at a temperature of 37°C.The human fetal glial cell line SVG p12 (ATCCThe primary human glioma cell-line was established and cultured from high-grade glioma patients’ tumor . Fresh hThe knockdown of lncRNA OR7E156P was conducted through the transduction of lentivirus containing short hairpin RNA (shRNA) specific for lncRNA OR7E156P . The sequences of OR7E156P shRNA listed in HIF-1α-overexpressing plasmid (HIF-1α-cDNA) was constructed through the insertion of the full-length HIF-1α cDNA into the pcDNA3.1 expression vector. The overexpressing plasmid was verified by DNA sequencing.-ΔΔCt method. The specific primer sequences used are listed in The total RNA was extracted from target cells or tissues using a Trizol reagent (Invitrogen) as stipulated by the manufacturer’s instructions and then was reverse transcribed using the First-strand cDNA synthesis kit (Promega). The expression levels were then examined using the SYBR Green PCR Master Mix (Qiagen) as per the manufacturer’s instructions. Normalization was performed using Tubulin (for lncRNAs and mRNAs) or U6 (for miRNAs). Relative expression values were calculated using the 22O2 for 15min and further incubated overnight with mouse polyclonal primary antibody of HIF-1α . The sections were subsequently incubated with 45µl secondary antibody horseradish peroxidase-conjugated goat polyclonal anti-mouse IgG H&L (HRP)  at 37°C for 30 min. Slices were stained with 3, 3’-diaminobenzidine (DAB) working solution for 3 min, then washed in water for 10 min. The slices were counterstained with hematoxylin. After rewashing the slices in water for 10 minutes, the sections were finally dehydrated and mounted. The slices were ultimately observed under a microscope (Leica Microsystems).Hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining were performed as previously described . The gli® ELISA Development Systems (R&D System) as per manufacturer’s instructions and aforementioned methods  in a DuoSet methods .The protein levels of HIF-1α, VEGF, ZEB1, Snail, and Vimentin in target cells were detected by Immunoblotting. Target cells were resuspended in an equal volume of a RIPA lysis buffer . The susThe predicted miR-143-5p binding to OR7E156P or 3’UTR of HIF1A was verified using a Luciferase reporter assay. The wild-type or mutated OR7E156P or 3’UTR of HIF1A was cloned to the downstream of the Renilla psiCHECK2 vector , named wt-OR7E156P or wt-HIF1A 3’UTR or mut-OR7E156P or mut-HIF1A 3’UTR. 293T cells were subsequently co-transfected with two types of luciferase reporter vectors and miR-143-5p mimics/miR-143-5p inhibitor and the luciferase activity was examined using the Dual-Luciferase Reporter Assay System (Promega). The sequences of miR-143-5p mimics and miR-143-5p inhibitor listed in For HIF-1α modulating miR-143 transcriptional activity, wildtype (psicheck-2-pro-miR-143) and mutant reporter constructs (psicheck-2-pro-miR-143-mut) were structured following the aforementioned methods . A miR-1293T cell lysates were used for RIP. The Imprint RNA Immunoprecipitation Kit  was used in RNA immunoprecipitation with the AGO2 antibody , which is a key component of the microRNA-containing RISC complex. AGO2 were used as positive controls and IgG as the negative controls. The levels of miR-143, OR7E156P, and HIF1A in the precipitates were determined using a real-time PCR.5. The medium with serum was fixed in the bottom chamber. The cells were incubated at a temperature of 37°C for 48 h. Non-invaded cells in the top chambers were removed with cotton swabs. Invaded cells on the lower membrane surface were fixed in 100% methanol for 10 min, air-dried, stained with crystal violet solution, and then quantified under a microscope.Target cells were planted in medium without serum on the top side of polycarbonate Transwell filters coated with Matrigel at a density of 5 × 10DNA synthesis is based on the method of incorporating thymidine analogue 5-ethoxy 2-deoxyuridine (EdU) into genomic DNA using the Click-IT EdU Alexa Fluor 488 kit following the aforementioned method , 33. ApoGlioma cells were infected with Lsh-NC or Lsh-OR7E156P, exposed to normoxia, and the culture medium were collected and used as a conditional culture medium (U251-MG CM and U87-MG-CM). HUVECs were then seeded into 96-well microtiter plates coated with Matrigel (80 μl/well), incubated for 1 h, and then suspended in above-mentioned conditional culture medium, respectively. The formation of tube-like structures was examined 4-6 h later. Tube formation was analyzed by quantifying the number of branches or tube length per high-powered field using a ImageJ software .5 cells in a 200-μl total volume into their right dorsolateral flanks. After 5 weeks, the mice were sacrificed and the tumor volume was calculated as per the following formula: tumor volume (mm3) = length × width2/2. The tumor tissues were subsequently formalin-fixed and paraffin-embedded alongside standard procedures for histological observation.4 weeks old Babl/c nude mice were purchased from the SLAC experimental animal center . All animal procedures stringently followed the ethical guidelines imposed by the Xiangya Hospital. For tumor establishment, glioma cells were infected or co-infected with Adv-HIF1A (Adv-NC as control) and/or Lsh-OR7E156P (Lsh-NC as control) and then the mice were injected subcutaneously with 2.5× 10t-test. A P value of less than 0.05 was deemed statistically significant.The results from at least three independent experiments were processed with GraphPad  and expressed as mean ± standard deviation (S.D.). Statistical significance was evaluated by a one-way analysis of variance (ANOVA) followed by a Tukey’s multiple comparison test or by a Student’s http://xena.ucsc.edu/) and the GSE42658, to identify lncRNAs related to glioma carcinogenesis using bioinformatics analysis. A differential expression analysis was performed in 10 cases of para-cancer control tissues and 197 cases of GBM tumor tissues from TCGA-GBM microarray dataset (AffyU133a) using an R language toolkit limma, and 8 differentially expressed ncRNAs were yielded , among which 3 were down-regulated  and 5, up-regulated  (P < 0.05), among which 24 were down-regulated and 37 up-regulated (http://betastasis.com/glioma/rembrandt/), OR7E156P expression was higher in glioma subtypes than that in non-cancerous tissue samples  from Xena . Lsh-NC was infected as a negative control. The silencing efficiency was verified by a qRT-PCR and it was established that Lsh1-OR7E156P exhibited better efficiency  was used to analyze the association between OR7E156P and HIF signaling pathway; as shown in 2) significantly induced HIF1A protein levels, especially in U251-MG and U87-MG cells; the expression of OR7E156P showed to be dramatically upregulated within glioma cells than that within HEB cells, more upregulated in U251-MG and U87-MG cells. Next, U251-MG and U87-MG cells were infected with Lsh-OR7E156P, exposed to 1% or 20% oxygen saturations, and the protein levels of HIF1A were then examined. Hypoxia significantly induced the protein levels of HIF1A in both glioma cell lines compared to those in cells exposed to conditions of normoxia , while OR7E156P knockdown significantly decreased hypoxia-induced increase in HIF1A protein  significantly promoted the tube formation by HUVECs, whereas OR7E156P silencing partially inhibited HUVEC tube formation  and/or Lsh1-OR7E156P (Lsh-NC as control). As illustrated in Mechanistically, it is proposed that lncRNAs could act as miRNA “sponges”, inhibiting normal miRNA targeting activity on mRNA , 35. NexTo further select the candidate miRNAs, U251-MG cells were infected with Lsh-OR7E156P and examined for miR-17, miR-20a, miR-153, miR-519d, and miR-143-5p expression; as illustrated in Next, a luciferase reporter assay was utilized to verify the putative bindings. According to the M&M section, two different types of OR7E156P or HIF1A 3’UTR luciferase reporter vectors were constructed, wild-type and mutant-type. These vectors were co-transfected in 293T cells with miR-143 mimics/inhibitor and the luciferase activity was determined. 2 and determined the dynamic effects of OR7E156P and miR-143 on glioma cell invasive capacity and DNA synthesis capacity. OR7E156P knockdown significantly downregulated, whereas miR-143 inhibition further upregulated hypoxia-induced cell invasion and DNA synthesis; miR-143 inhibition dramatically attenuated the effects of OR7E156P knockdown on hypoxia-induced cell invasion and DNA synthesis . The patients/participants provided their written informed consent to participate in this study. This study was performed according to the recommendations of the Declaration of Helsinki and was approved by the ethics committee of XiangYa Hospital, approval No.201703740 .HZ and PD performed the mainly investigation and wrote the manuscript. RP performed the bioinformatic analysis. GP, JY, and DL performed the molecular experiments. YL and XM collected the samples and assisted with the experiments. YWL administrated the whole project and editing the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by National Natural Science Foundation of China, (Grant No: 81702490) and Natural Science Foundation of Xinjiang Uygur Autonomous Region (2017D01C247).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


P < 0.001). Most cases upstaged by 3D MR-S were those positive (stage 1 or higher) among the stage 0 cases detected by 2D MR-S. Axial 3D MR-S, compared with 2D MR-S, understaged four cases, which was due to the imaging range of the axial 3D MR-S. We concluded that a single unilateral sagittal 3D MR-S was sufficient and axial 3D MR-S was unnecessary for SS staging. T1- and T2-weighted images are essential for investigating the salivary glands in patients with SS. Therefore, we also concluded that bilateral sagittal 3D MR-S of the parotid glands in addition to T1- and T2-weighted imaging is necessary, sufficient, and most efficient for precise MR imaging examination of the salivary glands, including diagnosing SS.We investigated the sensitivities of 2-dimensional (2D) magnetic resonance sialography (MR-S) and unilateral sagittal and axial 3-dimensional (3D) MR-S using a surface coil and their combination in diagnosing patients with Sjögren's syndrome (SS). We retrospectively analyzed the 3D and 2D MR-S results of 78 patients with SS. We evaluated the sensitivities of multiple high-signal-intensity spots and staging on MR sialograms and analyzed the efficient imaging methods and cross section for diagnosing patients with SS. The sensitivities of MR-S for detecting abnormal findings  were as follows: 65 cases : 73.2–90.8]) for unilateral sagittal 3D MR-S; 62 cases (79.4% [95% CI: 68.8–87.8]) for axial 3D MR-S; 66 cases (84.6% [95% CI: 74.7–91.8]) for combined unilateral sagittal and axial 3D MR-S; and 32 cases (41.0% [95% CI: 30.0–52.7]) for bilateral sagittal 2D MR-S. The ratio of the abnormal finding of MR-S was tested using the two-tailed Fisher's exact test. Unilateral sagittal, axial, and combined unilateral sagittal and axial 3D MR-S showed significantly higher sensitivity than bilateral sagittal 2D MR-S, respectively ( Magnetic resonance (MR) imaging has become widely used for assessing salivary gland diseases and is useful for the diagnosis of Sjögren's syndrome (SS). X-ray sialography, whose apple tree appearance has been adopted for some SS criteria , 2, cannThree-dimensional (3D) MR-S techniques facilitate the reliable prediction of ductal dilatation, stenosis, and damage better than the two-dimensional (2D) techniques –16. MoreThe procedures in this study adhered to the Helsinki Declaration of 1975, as revised in 1983. This retrospective study was approved by our institutional review board (no. 2012-24-1). Informed patient consent was obtained via an opt-out system on our institutional website displaying information about the study objectives and procedures.2 of salivary gland tissue. Therefore, positive histopathological findings, the chief complaint of oral dryness, and anti-SS-A/Ro and/or anti-SS-B/La antibodies formed the basis of a definitive diagnosis of SS according to the criteria proposed by the American-European Consensus Group [We retrospectively reviewed the MR images of 78 patients  clinically diagnosed with SS based on the chief complaint of xerostomia, positive histopathological findings of labial gland biopsy, and positive serologic evidence of anti-SS-A/Ro and/or anti-SS-B/La antibodies. Autoimmune disease specialists diagnosed all the patients with SS; the patients were negative for human immunodeficiency virus and sarcoidosis. We adopted labial gland biopsy as a criterion for all patients and graded the histopathological findings using focal scores according to previously established criteria [us Group , and revWe performed MR imaging with a 1.5- or 3.0-Tesla MR unit  using the surface coils with a loop diameter of 11 cm. Transverse T1-weighted images  and fat-suppressed T2-weighted images  were obtained using the spin-echo and fast-spin-echo techniques, respectively. The section thickness of the transverse T1- and T2-weighted images was 5 mm with an intersection gap of 1 mm, acquisition matrix of 512 × 358, and field of view (FOV) of 210 mm. MR-S was performed using a high-resolution technique and a heavily T2-weighted 3D fast-spin-echo sequence  according to a previous report that a heavily T2-weighted 3D fast-spin-echo sequence is the most suitable for the evaluation of thin salivary gland ducts  [Two oral and maxillofacial radiologists with 11 and 19 years of experience, respectively, evaluated the coded and randomly presented unilateral sagittal 3D MR-S, axial 3D MR-S, and bilateral sagittal 2D MR-S for the presence or absence of MHS based on a previous report . Both raP values were two-sided, and statistical significance was set at P < 0.05. Interobserver agreement between the two reviewers in the imaging analysis was evaluated using the kappa coefficient (ƙ). The strength of agreement was classified as slight (0.01 to 0.20), fair (0.21 to 0.40), moderate (0.41 to 0.60), substantial (0.61 to 0.80), or excellent (0.81 to 1.00). The sample size calculation for this study was based on Fisher's exact test for a power of 80% with reference to the accuracy of MR-S in a previous report [R , was used for statistical analyses.Categorical variables are presented as counts and percentages. We determined the sensitivity of the MHS , 0.96 , and 0.85 , respectively. All the values showed high agreement (excellent).P = 0.000000106).The MR-S staging on 98 sides of 78 patients with SS is shown in P = 0.00000043, P = 0.00000889, and P = 0.00000014, respectively). We checked the T1- and T2-weighted images and assessed the side with strong HD findings or tendencies if MHS was not observed bilaterally on bilateral 2D MR-S  were upstaged from 2D MR-S. Most of them converted to stage 1 or more from stage 0 designated by 2D MR-S: unilateral sagittal 3D MR-S, 33 cases; axial 3D MR-S, 30 cases; and unilateral sagittal + axial 3D MR-S, 33 cases, respectively. In other words, because 2D MR-S often provides false-negative results, additional 3D MR-S imaging is necessary. A few 3D MR-S cases were understaged from the stage for SS on 2D MR-S: 3 cases by axial 3D MR-S and one case by unilateral sagittal 3D MR-S. The axial 3D MR-S results for some of these cases were due to the imaging range of MR-S, as described above. In the one unilateral sagittal 3D MR-S case, MHS could not be detected on the opposite side, which was due to the imaging range of MR-S. In addition, in the longitudinal parotid gland, the MHS can be easily distinguished from the thin parotid duct on sagittal 3D MR-S. Conversely, in axial 3D MR-S, the MHS and relatively thick parotid ducts are densely packed and overlap, making it difficult to distinguish between them, which is considered to result in understaging. Some cases of understaging by unilateral sagittal and axial MR-S may be misled as upstaging on 2D MR-S because of the partial volume effect on 2D MR-S with poor spatial resolution.A limitation of this study was that we could not compare the sensitivities for detecting abnormal findings of the MHS for unilateral and bilateral 3D MR-S. Since we did not perform bilateral sagittal 3D MR-S and axial 3D MR-S at our hospital because of the limited imaging duration, the imaging side for sagittal 3D MR-S was decided during the examination by the oral and maxillofacial radiologists.Another limitation of this study is the further improvement of the MR-S imaging technique, which should be considered in future studies. The development of imaging methods using phantoms of a wide range of imaging thickness and shorter duration of the imaging acquisition while maintaining the MR-S image quality and evaluating the diagnostic accuracy of these improved MR-S techniques is warranted.We concluded that a single unilateral sagittal 3D MR-S was sufficient and axial 3D MR-S was not necessary for SS staging. T1- and T2-weighted images are essential for investigating the salivary glands in patients with SS. Therefore, we also concluded that bilateral sagittal 3D MR-S of the parotid glands in addition to T1- and T2-weighted imaging is necessary, sufficient, and most efficient for precise MRI examination of diagnosing SS.